Coumarin-based fluorescent probe for the rapid detection of peroxynitrite ‘AND’ biological thiols

A coumarin-based novel ‘AND’ logic fluorescent probe ROS-AHC has been developed for the simultaneous detection of ONOO⁻ and biological thiols. ROS-AHC was shown to exhibit only a very small fluorescence response upon addition of a single GSH or ONOO⁻ analyte. Exposure to both analytes, however, resulted in a significant fluorescence enhancement.

A coumarin-based novel ‘AND’ logic fluorescent probe ROS-AHC has been developed for the simultaneous detection of ONOO⁻ and biological thiols.

Peroxynitrite (ONOO⁻) is a short-lived reactive oxygen and reactive nitrogen species (ROS and RNS) produced intracellularly by the diffusion-controlled reaction of nitric oxide (NO˙) with superoxide (O₂˙⁻).^1–3 Despite playing a key role as a physiological regulator,⁴ it is commonly known for its high reactivity towards most types of biomolecules, causing deleterious effects and irreversible damage to proteins, nucleic acids, and cell membranes.^5,6 ONOO⁻ is therefore a central biological pathogenic factor in a variety of health conditions such as strokes, reperfusion injuries or inflammatory and neurodegenerative diseases (Parkinson''s disease, Alzheimer''s disease).^7–9 Conversely, biothiols such as glutathione and cysteine are endogenous reducing agents, playing a central role in the intracellular antioxidant defence systems.^10–12 Glutathione (GSH), in particular, is the most abundant biothiol in mammalian cells, and exists as both its reduced GSH form, and as the oxidised disulphide form GSSG.^13–15 Peroxynitrite and biothiols such as GSH are intimately linked, as abnormal levels of highly oxidising ONOO⁻ can perturb the delicate GSH/GSSG balance, causing irreversible damage to key processes such as mitochondrial respiration.¹⁶ Thus, abnormal levels of GSH are common in cells undergoing oxidative stress, in which the regulation of and interplay between ONOO⁻ and GSH is closely associated with physiological and pathological processes.^17,18 One such example is drug-induced liver injury (DILI), in which upregulation of ONOO⁻ occurs in hepatotoxicity. Treatment with GSH could be used to remediate this type of cell injury by depletion of ONOO⁻.^19–22One of our core research interests lies in the development of dual analyte chemosensors capable of detecting two distinct analytes such as biological reactive oxygen species and biothiols.^23–26 Although a wide range of single-analyte probes exist for the detection of ROS and thiols separately,^27–30 ‘AND’ logic sensors for their simultaneous detection are still rare.^31–33 We are therefore interested in developing such probes, containing two distinct sensing units, one for each analyte, working simultaneously or in tandem to elicit a fluorescence response.³⁴ This approach allows the monitoring of multiple biomolecular events and factors involved in specific disease pathologies, in order to achieve optimal predictive accuracy for diagnosis and prognostication.³⁵Using these principles, our group has recently focused on developing a range of ‘AND’ logic based sensors exploiting a variety of sensing units and mechanisms of fluorescence. Two such probes are shown below: GSH-ABAH (Fig. 1a), an ESIPT probe with a 4-amino-2-(benzo[d]thiazol-2-yl)phenol (ABAH) core, employing a maleic anhydride thiol-acceptor group;³¹ and JEG-CAB (Fig. 1b), a coumarin-based probe, this time with a salicylaldehyde homocysteine-reactive unit.²⁴ Both of these sensors employ a benzyl boronate ester as their peroxynitrite-reactive unit.Open in a separate windowFig. 1(a) GSH-ABAH, previously reported probe for simultaneous detection of ONOO⁻ and GSH. (b) JEG-CAB, previously reported probe for simultaneous detection of ONOO⁻ and GSH. (c) AHC – a core fluorescent unit that enables the synthesis of ‘AND’ based fluorescent probe for the detection of ONOO⁻ and GSH (d) ROS-AHC, a novel probe detailed in this work for simultaneous detection of ONOO⁻ and GSH.Herein, we set out to develop an ‘AND’ logic gate based fluorescence probe for simultaneous detection of ONOO⁻ and GSH. 3-Amino-7-hydroxy-2H-chromen-2-one (AHC) was chosen as a suitable coumarin fluorophore core for the development of an ‘AND’ logic based sensor, as its free phenol and amine functional groups provided a good opportunity for independent derivatization (Fig. 1).^36–39Previous literature reports show that protection of AHC with a maleic anhydride group results in quenching of the coumarin''s fluorescence intensity due to photoinduced electron transfer (PeT) processes. This fluorescence is rapidly restored in the presence of biological thiols, however, due to their fast addition to this functional group.⁴⁰ Therefore, we suggested that functionalization of the free phenol of this sensor using a benzyl boronic ester should further block the fluorescence, whilst serving as reporter unit for ONOO⁻. The greatly increased reactivity of peroxynitrite over other ROS towards boronate esters^41–43 should allow this functionality to act as a peroxynitrite-selective reporter, leading to an ‘AND’ logic based probe for the detection of ONOO⁻ and biological thiols (Fig. 1, Scheme 1). ROS-AHC was synthesized in 5 steps, starting with a 4-step synthesis of compound 1 adapted from literature procedures,^40,44 followed by the addition of the benzyl boronic pinacol ester (see Scheme S1 ESI^†).Open in a separate windowScheme 1Fluorescence ‘turn on’ mechanism of ROS-AHC in the presence of ONOO⁻ and GSH.The UV-Vis behaviour of ROS-AHC before and after exposure to both GSH and ONOO⁻ was evaluated in pH 7.40 buffer solution, showing a maximum absorption peak at 340 nm for both the unreacted probe and the probe following exposure to GSH, shifting to 350 nm with the addition of ONOO⁻ to the probe and 365 nm after sequential additions of GSH and ONOO⁻ to the probe (Fig. S1 ESI^†). Fluorescence experiments were then carried out. As expected, ROS-AHC was initially non-fluorescent, with a small fluorescence increase upon addition of ONOO⁻ (6 µM) (Fig. 2 and S2 ESI^†). Incremental additions of GSH (0–4.5 µM) resulted in a much larger increase in fluorescence intensity (>69-fold, see Fig. 2 and S3 ESI^†), demonstrating the need for both GSH and ONOO⁻ in order to achieve a significant ‘turn on’ fluorescence response.Open in a separate windowFig. 2Fluorescence spectra of ROS-AHC (5 µM) with addition of ONOO⁻ (6 µM), wait 5 min then incremental addition of GSH (0–4.5 µM), 5 min incubation before measurements in PBS buffer solution (10 mM, pH = 7.40). Fluorescence intensities were measured with λ_ex = 400 nm (bandwidth 8 nm). The green line represents the highest intensity after addition of GSH (4 µM).Similar fluorescence experiments were then carried out in reverse order, with the addition of GSH (6 µM) to ROS-AHC resulting in only a small increase in fluorescence intensity (Fig. 3 and S4 ESI^†). As before, incremental addition of the second analyte, in this case ONOO⁻ (0–5.5 µM), resulted in a large increase in fluorescence intensity (>46-fold, Fig. 3 and S5 ESI^†), confirming that ROS-AHC requires both GSH and ONOO⁻ for a full fluorescence ‘turn on’ response.Open in a separate windowFig. 3Fluorescence spectra of ROS-AHC (5 µM) with addition of GSH (6 µM), wait 5 min then incremental addition of ONOO⁻ (0–5.5 µM) with 5 min incubation before measurements in PBS buffer solution (10 mM, pH = 7.40). Fluorescence intensities were measured with λ_ex = 400 nm (bandwidth 8 nm). The orange line shows the highest intensity after addition of ONOO⁻ (5 µM).Subsequently, the selectivity of this probe towards both analytes was evaluated. A range of amino acids were evaluated (Fig. S6 ESI^†), with only thiol-containing analytes (glutathione, cysteine and homocysteine) eliciting significant fluorescence response, whilst non-thiol amino acids led to no changes in fluorescence intensity. A broad screen of ROS analytes was also carried out, demonstrating excellent selectivity for ONOO⁻, even over H₂O₂ (Fig. S7 ESI^†).The time-dependent response of ROS-AHC with both ONOO⁻ and GSH was also examined (Fig. S8 and S9 ESI^†). After initial addition of GSH or ONOO⁻ to the probe, subsequent addition of the second analyte triggered a rapid and significant increase in fluorescence, achieving maximum fluorescence intensity within 78 s in both cases. Furthermore, LC-MS experiments confirmed the formation of the suggested non-fluorescent intermediates, as well as the final fluorescent species shown in Scheme 1 (Fig S10, S11 and S12^†).In summary, we have developed a coumarin-based dual-analyte ‘AND’ logic fluorescent sensor, ROS-AHC, for the simultaneous detection of ONOO⁻ and biological thiols. ROS-AHC has shown high sensitivity and selectivity towards both ONOO⁻ and biological thiols.     

A simple two-photon turn-on fluorescent probe for the selective detection of cysteine based on a dual PeT/ICT mechanism

Herein, a simple two-photon turn-on fluorescent probe, N-(6-acyl-2-naphthayl)-maleimide (1), based on a dual PeT/ICT quenching mechanism is reported for the highly sensitive and selective detection of cysteine (Cys) over other biothiols. The probe was applied in the two-photon imaging of Cys in cultured HeLa cells, excited by a near-infrared laser at 690 nm.

N-(6-acyl-2-naphthayl)-maleimide (1) is a simple two-photon fluorescent probe with selectivity for cysteine, based on a thiol-Michael-addition-transcyclization cascade and dual PeT/ICT quenching mechanism.

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are structurally similar biothiols, but their biological functions are quite different from one another.^1–6 Among these biothiols, Cys functions as one of the twenty-one amino acids for peptide and protein synthesis, and Cys deficiency is also associated with certain disease symptoms.^7–10 Methods for the selective detection and differentiation of Cys among different biothiols, including high performance liquid chromatography (HPLC),¹¹ capillary electrophoresis,¹² electrochemical assay,¹³ UV-vis spectroscopy,¹⁴ and fluorescence-based methods,^15–17 are important for its biological studies. Recently, fluorescent probes have attracted much attention as vital chemical biology tools due to their high sensitivity, convenient operation, and real-time imaging capabilities.^18–20 A number of Cys-selective fluorescent probes have been reported,²¹ which utilize Cys-selective recognition groups such as aldehydes,^11,22 acrylates,²³ thioesters,²⁴ and electron-deficient aromatic halides^25–27 in their structures. However, many of them have relatively long response times and low sensitivity due to a slow cyclization process. In addition, fluorescent probes with high selectivity for Cys over Hcy are difficult to achieve because they differ by only one methylene group.²⁸ Recently, we reported that N-(N′-butyl-1,8-naphthalimide-4-yl)-maleimide, containing a single maleimide group as the recognition group, is a fast, sensitive, and selective fluorescent probe for Cys based on a dual photo-induced electron transfer (PeT) and photo-induced intramolecular charge transfer (ICT) quenching mechanism.²⁸ Different from many other maleimide-based fluorescent probes that only undergo a PeT mechanism,¹⁵ the additional ICT quenching mechanism keeps the 1,8-naphthalimide (NAP) fluorophore in the thiol-Michael adduct in a low fluorescence emission state due to the strong electron-withdrawing effects of the succinimide group at its 4-position. Then, a subsequent transcyclization step, involving the formation of a six-membered thiomorpholinone ring and cleavage of a five-membered succinimide ring, converts the non-fluorescent thiol-Michael adduct into the major fluorescent product, in which the ICT quenching is removed, resulting in a drastic fluorescence turn-on response.²⁸ A similar transcyclization process and the simultaneous removal of ICT quenching allowed us to design a NAP-based turn-on fluorescent probe for γ-glutamyltranspeptidase²⁹ and a coumarin-based turn-on fluorescent probe with dual recognition groups and dual cyclization for the selective detection of Cys.³⁰ In addition, another NAP-based dual PeT/ICT probe was recently reported by Meka and Heagy for the detection of hydrogen sulfide, although two recognition groups instead of one were adopted in their probe to achieve the dual quenching mechanism.³¹Our previous work and that of other groups has demonstrated that the combination of PeT and ICT mechanisms is particularly suitable for the design of fluorescent probes with a significant fluorescence turn-on response.^30–33 However, many of these probes have a short excitation wavelength in the UV or visible range, which is not optimal for biological applications due to enhanced phototoxicity and/or autofluorescence.^34,35 Considering that two-photon fluorescence imaging has advantages such as the excitation process being carried out by a near-infrared (NIR) laser that has a reduced cell toxicity and low fluorescence background,³⁶ in this work, we aimed to introduce a similar dual PeT/ICT quenching mechanism to the known two-photon fluorophore 6-acyl-2-naphthylamine^37–39 in order to design a simple maleimide-based two-photon fluorescent probe, 1, for the selective detection of Cys over Hcy and GSH. It was also tested to determine whether it is a turn-on fluorescent probe with high sensitivity and selectivity, which reacts with Cys via a fast two-step thiol-Michael addition and transcyclization cascade reaction.²⁸ The structure of probe 1 is shown in Fig. 1. It has a maleimide group at its 2-position, which promotes the PeT quenching effect. It also has an additional electron-withdrawing methylcarbonyl group at its 6-position to ensure a pull–pull ICT quenching effect.Open in a separate windowFig. 1Design rationale of the fluorescent probe 1 for the selective turn-on detection of Cys over Hcy and GSH.Probe 1 was conveniently synthesized from 6-acyl-2-naphthylamine (3)³⁹ in a two-step process with a total yield of 38% (see Scheme S1 in the ESI^†). First, the amine 3 was reacted with maleic anhydride to form the maleic amide acid 4. Then, the amide acid 4 was cyclized to afford the maleimide 1 in the presence of acetic anhydride (see the ESI^† for more details).We then investigated the absorption and fluorescence emission response of the probe towards just 1 equiv. of Cys. The time-dependent absorption spectra upon the addition of 1 equiv. of Cys are shown in Fig. 2a. Probe 1 has a maximum absorption peak at 292 nm. Upon addition of Cys, the maximum absorption peak shifts to 314 nm, a red-shift of 22 nm. Notably, an isosbestic point can be seen at 295 nm after 2 min, indicating the formation of an intermediate within 2 min, which is then converted into the final product. The UV spectral changes supported the presence of a proposed cascade reaction sequence for the fast formation of a thiol-Michael adduct intermediate, which then underwent a relatively slow intramolecular transcyclization process to give the final product. From time-dependent fluorescence emission studies (Fig. 2b), probe 1 was found to have almost no fluorescence emission due to dual PeT and ICT quenching effects. Upon the addition of 1 equiv. of Cys, a drastic turn-on fluorescence response (a >3000 fold increase) was observed at 446 nm (see Fig. S1b in the ESI^†). The fluorescence intensity at 446 nm reached its maximum value after around 30 min indicating that the cascade reaction finished in about 30 min (Fig. 2b, and S2a in the ESI^†). The pseudo-first-order reaction kinetic constant based on the fluorescence enhancement was calculated as 0.123 min⁻¹ (half-time = 5.64 min, Fig. S2b in the ESI^†), indicating an overall fast cascade reaction. Fluorescence titration experiments using an increasing amount of Cys from 0 to 4.0 equiv. over 30 min showed a steady increase in the fluorescence intensity and the maximum intensity was reached at exactly 1.0 equiv. of Cys. Further Cys addition did not increase the fluorescence intensity, indicating that probe 1 reacts with Cys in a 1 : 1 molar ratio (Fig. 2c and S3 in the ESI^†), which was also supported by the Job plot (see Fig. S4 in the ESI^†). From the linear relationship of the fluorescence intensity at 446 nm versus the Cys concentrations, the detection limit of probe 1 (2 μM) for Cys was calculated as 1.4 nM (S/N = 3, Fig. 3d), indicating that 1 is a highly sensitive probe for Cys. Moreover, the probe showed excellent selectivity for the detection of Cys over many other species (Fig. 2e, and S5 in the ESI^†), including the structurally similar thiols Hcy, GSH, and N-acetylcysteine (NAC). The fluorescence intensity at 446 nm for 1 equiv. of Cys was significantly higher (12.2-fold, 9.1-fold, and 17.7-fold, respectively) than that of 10 equiv. of Hcy, GSH, or NAC. To further confirm the reaction mechanism, the reaction product, 2, from the reaction of probe 1 with Cys, was isolated and its structure was confirmed using ¹H nuclear magnetic resonance (NMR) spectroscopy, ¹³C NMR spectroscopy, 2D-rotating-frame nuclear Overhauser effect spectroscopy (ROESY), and high-resolution mass spectrometry (HRMS) (see the ESI for more details^†). The fluorescence quantum yields of probe 1 and product 2 were measured as 0.002 and 0.782, respectively (see the ESI for more details^†). Therefore, the formation of the transcyclization product 2 was determined to be responsible for the observed fluorescence turn-on response. For the other thiols, the transcyclization steps of the thiol-Michael adducts were much slower, resulting in the observed high selectivity. Overall, we have shown here that probe 1 is a highly sensitive and selective turn-on fluorescent probe for Cys.Open in a separate windowFig. 2(a) Time-dependent UV-vis spectra of probe 1 (10 μM) upon the addition of 1 equiv. of Cys (a spectrum was recorded every 2 minutes); (b) time-dependent fluorescence emission spectra of probe 1 (2 μM) upon the addition of 1 equiv. of Cys (a spectrum was recorded every 3 minutes); (c) time-dependent fluorescence emission intensity at 446 nm of probe 1 (2 μM) upon addition of Cys (0 to 4 equiv.); (d) a linear relationship of the fluorescence intensity at 446 nm versus the Cys concentration (0.2–2.0 μM); (e) fluorescence response of probe 1 (2 μM) at 446 nm toward various species in PBS buffer (10 mM, pH 7.4): (1) blank; (2) Cys; (3) Hcy; (4) GSH; (5) NAC; (6) valine; (7) glycine; (8) isoleucine; (9) lysine; (10) leucine; (11) histidine; (12) asparagine; (13) methionine; (14) proline; (15) serine; (16) alanine; (17) threonine; (18) arginine; (19) glutamine; (20) aspartic acid; (21) glutamic acid; (22) tyrosine; (23) tryptophan; (24) phenylalanine; (25) glucose; (26) H₂O₂; (27) Na⁺; (28) K⁺; (29) Ca²⁺; (30) Mg²⁺; (31) Fe³⁺; (32) Fe²⁺; (33); Cu²⁺; (34) Zn²⁺ (All measurements were made in 10 mM PBS buffer, pH 7.4, 25 °C, and λ_ex = 314 nm).Open in a separate windowFig. 3Two-photon fluorescence images (b, e, h, k) of HeLa cells collected at 410–510 nm (blue to cyan-blue, λ_ex = 690 nm), the corresponding bright field view (a, d, g, j), and overlap of the fluorescence channel and the bright field view (c, f, i, l) after different treatments: (a–c) the cells were pretreated with 0.5 mM of N-ethylmaleimide (NEM) for 30 min and then incubated with 10 μM of probe 1 for 30 min; (d–f) cells were first pretreated with 0.5 mM of NEM for 30 min, then after addition of 1 mM of Cys were incubated for 30 min, and finally, incubated with 10 μM of probe 1 for 30 min (scale bar = 10 μm); the conditions for (g–i) and (j–l) were similar to those of (d–f), except that 10 μM of Hcy and 10 μM of GSH were used instead of 10 μM of Cys.Encouraged by the fast, selective, and sensitive in vitro fluorescence response of probe 1 for the detection of Cys, we further evaluated its potential use as a two-photon imaging agent for Cys in biological systems, such as in living cells. The fluorescence response of probe 1 towards Cys at different pH values was evaluated and a suitable pH range for Cys detection was determined to be 7.0 to 10.0, which is a good range for cell imaging applications because physiological conditions have a pH of around 7.4 (see Fig. S7 in the ESI^†). HeLa cells were then pretreated with N-ethylmaleimide (NEM, 0.5 mM) for 30 min to remove the endogenous cellular thiols, and incubated with Cys (1 mM), Hcy (1 mM), or GSH (1 mM), respectively for 30 min to increase the specific thiol levels. The samples were then further incubated with probe 1 (10 μM) for 30 min and were then washed with PBS buffer before two-photon fluorescence cell images and the corresponding bright-field view images were taken (Fig. 3(d–l)). Control images were also taken for samples pretreated with NEM (0.5 mM) and then incubated with probe 1 (10 μM) (Fig. 3a–c). Only cells pretreated with NEM and then Cys showed a distinctive blue fluorescence (Fig. 3e). The above cell imaging studies clearly demonstrated that probe 1 is capable of the selective detection and imaging of intracellular Cys over Hcy and GSH in living cells by two-photon fluorescence imaging with low background fluorescence interference.     

A fast-responsive two-photon fluorescent probe for monitoring endogenous HClO with a large turn-on signal and its application in zebrafish imaging

A novel fast-responsive two-photon fluorescent probe NS-ClO was constructed for imaging endogenous HClO in living cells, tissues and fresh zebrafish with a large turn-on signal (about 860 times) and Stokes shift (about 90 nm). The probe NS-ClO for the recognition of HClO in vivo exhibited fast response (about 1 min) and good selectivity; thus, it might be a useful tool to understand the role of HClO in various physiological processes.

Fast-responsive two-photon fluorescent probe NS-ClO for imaging endogenous HClO in vivo with a large turn-on signal (about 860 times) and Stokes shift (about 90 nm), fast response (about 1 min) and good selectivity.

Hypochlorous acid (HClO) is a weak acid with oxidizing properties in the reactive oxygen species (ROS) family and it is generated from living immunological cells by oxidising hydrogen peroxide (H₂O₂) and chloride with the help of myeloperoxidase (MPO).¹ HClO has an important influence on various physiological processes including immune defence against microorganisms and the lethal effect on pathogens in living biosamples. When the balance of HClO in the body is destroyed, many molecules such as DNA, RNA, fatty acids, cholesterol, and proteins can react with HClO, which is related to different diseases including neurodegenerative disorders and cancers.^2–5 Further research between the HClO level and the pathophysiological process is very necessary. Therefore, it is important to develop a practicable method for monitoring HClO in a physiological atmosphere.In the past few decades, many efficient and functional methods including colorimetric methods, chemiluminescence methods, coulometry, radiolysis, and electrochemical and chromatographic methods were applied to monitor HClO.^6–11 Although the mentioned methods exhibit fast responses and are selective to HClO over other molecules, sophisticated equipment and complex operating techniques are needed in the processes. Also, the living biosystem can often be damaged in the operation. Hence, they are not suitable for detecting HClO in living cells, tissues, and body. During the last few years, an organic molecular probe has been the most useful detection tool and it is efficient for the real-time visualization of small bioactive molecules in the living biosystem with high selectivity and sensitivity; this facilitates comprehensive exploration and manipulation in the physiological atmosphere.^12–21Recently, many fluorescent probes, acting as an inevitable tool for monitoring HClO in the living biosystem, have been constructed.^22–26 Although most of the previous probes were used to image exogenous HClO, it is still difficult to perform endogenous imaging of HClO in living cells. Especially, an organic fluorescent probe with a large turn-on Stokes shift and intensity, two-photon excitation, fast response, and good selectivity and stability is still scarce. Therefore, it is worth to develop a two-photon fluorescent probe for imaging HClO specifically in vivo with a large Stokes shift and a turn-on signal.In this work, the modified organic fluorescent probe NS-ClO was constructed for imaging endogenous HClO specifically with a large turn-on signal (about 860 times) and Stokes shift (about 90 nm) (Scheme 1). This turn-on fluorescent probe NS-ClO with good properties including two-photon excitation, fast response (about 1 min) and good selectivity was studied; this method might be useful to monitor the functions of HClO in various physiological processes (Table S1^†).Open in a separate windowScheme 1The structure of NS-ClO and the proposed sensing mechanism for HClO.The sulfur atom in phenothiazine is very reactive towards HClO. The fluorescence property of phenothiazine was changed by oxidising a sulfur atom to sulfoxide, as depicted in Scheme 1. Benzothiazole is a very common electron-withdrawing group and is stable under oxidation and reduction conditions, which ensured that the constructed probe is stable in excessive HClO. Herein, the probe NS-ClO was developed by introducing benzothiazole into phenothiazine with the sulfur atom as a recognition site to HClO in one step easily. The characterization of the probe NS-ClO by ¹H NMR, ¹³C NMR and HRMS was performed, and the details are provided in the ESI.^†The spectral properties of probe NS-ClO were studied. There was almost no fluorescence intensity of probe NS-ClO at 450 nm in PBS buffer (pH = 7.4) and DMF (v/v = 19/1) at an ambient temperature without the addition of HClO (Fig. 1). When different concentrations of HClO were added to the reaction system, a maximal absorption band appeared at around 360 nm and strong fluorescence emission was observed, as shown in Fig. S1^† and ​and1;1; also, a large Stokes shift (about 90 nm) was seen. Therefore, PBS buffer (pH = 7.4) containing DMF (v/v = 19/1) was considered as the best solvent for this experiment. In addition, we also found that the two-photon probe NS-ClO was stable in the presence of excess HClO (20 equiv.) when the time was extended to 8 min (Fig. 1c and d).Open in a separate windowFig. 1Reaction-time profiles of NS-ClO (5 μM) in the absence or presence of NaClO: (a) NaClO (8.0 equiv.); (b) NaClO (10.0 equiv.); (c) NaClO (20.0 equiv.); (d) NaClO (20.0 equiv.).When the probe NS-ClO was excited at 360 nm, there was almost no fluorescence (φ = 0.03) using a fluorescein (φ_r = 0.90 in 0.1 N NaOH) solution.²⁷ However, when different amounts of HClO were added, the obvious turn-on fluorescence enhancement (about 860-fold) exhibited a quantum yield of 0.57 at 450 nm with a detection limit of 0.75 μM (Fig. S2^†). Therefore, this two-photon probe NS-ClO exhibited a fast response and large turn-on enhancement. In addition, the possible sensing mechanism was studied by mass spectrometry. When the probe NS-ClO (20 μM) was dissolved in PBS buffer (pH = 7.4) and DMF (v/v = 19/1), excess of HClO was added to the previous solvent. The spectra show a clear peak at m/z 377.0774, corresponding to the NS-ClO-adduct (Fig. S4^†); this was in good agreement with the possible sensing mechanism reported in a previous work²⁸ (Scheme 1).Another important factor, i.e., the pH of PBS buffer was examined, which may have significant impact on the response to HClO when PBS buffers having different pH values were used to examine the fluorescence intensity of this probe. There were almost no changes in the absence of HClO when the pH value changed from acidic to basic (about 1.0 to 10.0). With the addition of HClO (5.0 equiv.), the fluorescence intensity gradually increased when the pH was changed from 1.0 to 8.5 and rapidly decreased from 8.5 to 10.0. The main reason is that the oxidizing properties of HClO decline significantly in alkaline conditions. However, we found that the probe NS-ClO could detect HClO in PBS buffer (pH = 7.4) containing DMF (v/v = 19/1) at the physiological pH (7.4) with about 860-fold enhancement. In other words, this probe can be suitable for biological applications (Fig. 2 and ​and33).Open in a separate windowFig. 2The fluorescence spectra of NS-ClO (5 μM) in pH 7.4 PBS buffer (containing 5% DMF) in the absence or presence of NaClO (0–20 equiv.).Open in a separate windowFig. 3The pH effects of fluorescence spectra of NS-ClO (5 μM) in pH 7.4 PBS buffer (containing 5% DMF) in the absence (●) or presence (■) of NaClO (5.0 equiv.).In order to investigate selectivity, the two-photon probe NS-ClO was reacted with distinct biologically reactive analytes including biological thiols, reactive oxygen species (ROSs), reactive nitrogen species (RNSs) and anions. As listed in Fig. 4, the fluorescence enhancement is basically unchanged with the addition of different species (GSH, Cys, Hcy, F⁻, Cl⁻, Br⁻, ·OH, ONOO⁻, DTBP, TBHP, NO, H₂O₂, NO₂⁻, Co⁺, Cu²⁺, and Ni⁺). However, when we added HClO to the detection system, the fluorescence enhancement increased significantly within 1 min. This result indicated that the NS-ClO probe can be applied to monitor HClO with good selectivity compared to other different species (Scheme 2).Open in a separate windowFig. 4Fluorescence spectra of NS-ClO (10 μM) in pH 7.4 PBS buffer (containing 5% DMF) for various relevant species (50 μM). 1: None; 2: GSH; 3: Cys; 4: Hcy; 5: F⁻; 6: Cl⁻; 7: Br⁻; 8: ·OH; 9: ONOO⁻; 10: DTBP; 11: TBHP; 12: NO; 13: H₂O₂; 14: NO₂⁻; 15: Co²⁺; 16: Cu²⁺; 17: Ni²⁺; 18: ClO⁻.Open in a separate windowScheme 2Synthesis of the two-photon fluorescent probe NS-ClO.Encouraged by the above-mentioned excellent results, we inferred that the highly sensitive and selective two-photon probe NS-ClO could be suitable for imaging HClO in the living biosystem. First, the results of MTT assays proved that the HeLa cell survival rate is very high after treatment with different concentrations of NS-ClO. That is to say, the probe NS-ClO exhibited low cytotoxicity to HeLa cells after one day even at high concentrations (30.0 μM) (Fig. S5^†) and could be applied to monitor HClO in living cells. We investigated the applicability of probe NS-ClO for monitoring exogenous HClO in HeLa cells. As depicted in Fig. 5, living HeLa cells are initially treated with probe NS-ClO (10 μM) for 30 min and washed three times with PBS buffer for removing excess probe NS-ClO. The experimental data indicated that the HeLa cells incubated with probe NS-ClO exhibited almost no fluorescence in the blue channel (Fig. 5b). However, when the living HeLa cells were treated with probe NS-ClO for 30 min and NaClO (30 μM) for another 30 min, the fluorescence signal emerged obviously in the blue channel (Fig. 5e). Therefore, the probe NS-ClO with good membrane permeability can be used for imaging exogenous HClO in living HeLa cells.Open in a separate windowFig. 5Imaging of exogenous HClO in HeLa cells stained with the probe NS-ClO (10 μM). (a) Bright-field image of HeLa cells co-stained only with NS-ClO; (b) fluorescence images of (a) from blue channel; (c) overlay of the bright-field image (a) and blue channel (b). (d) Bright-field image of HeLa cells co-stained with NS-ClO and treated with NaClO. (e) Fluorescence images of (d) from blue channel; (f) overlay of the bright-field image (d) and blue channel (e).The above-mentioned data proved that the developed probe NS-ClO can be used for exogenous imaging. Therefore, the endogenous detection of HClO was completed subsequently in murine live macrophage cell line RAW 264.7. According to previous reports,²⁹ lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) are added to stimulate macrophages to produce endogenous HClO. As depicted in Fig. 6, the living RAW 264.7 macrophage cells pre-loaded with probe NS-ClO (10 μM) show almost no fluorescence signal in the blue channel (Fig. 6b). However, when the living RAW 264.7 cells were exposed to LPS (2 μg mL⁻¹) and PMA (2 μg mL⁻¹) together and then treated with probe NS-ClO (10 μM), obvious fluorescence enhancement was obtained (Fig. 6e). The result demonstrated that the constructed two-photon probe NS-ClO is suitable for monitoring endogenous HClO in living RAW 264.7 macrophage cells.Open in a separate windowFig. 6Imaging of endogenous HClO in RAW 264.7 cells stained with the probe NS-ClO. (a) Bright-field image of RAW 264.7 macrophage cells co-stained with NS-ClO. (b) Fluorescence images of (a) from blue channel; (c) overlay of (a) and (b). (d) Bright-field image of stimulated RAW 264.7 macrophage cells co-stained with NS-ClO, PMA and LPS. (e) Fluorescence images of (d) from blue channel; (f) overlay of the bright-field image (d) and blue channels (e).Previous research indicates that the probe NS-ClO can be used for imaging HClO in vitro and in vivo. With these data and advantages of TPM in hand, two-photon fluorescence imaging of HClO in living mouse tissues by TPM was performed with probe NS-ClO. Living tissue slices of mouse liver of about 400 μm thickness were prepared. Initially, the prepared tissues were washed with PBS buffer and treated with the probe NS-ClO (10.0 μM) for 30 min at 37 °C. After scanning by TPM, there was no fluorescence signal in the blue channel (Fig. 7a). On the contrary, when fresh tissues were pre-treated with the probe NS-ClO (10.0 μM) for 30 min and incubated with NaClO (10.0 μM) for another 30 min, an obvious fluorescence signal was observed from 5 to 80 μm depth (Fig. 7b). These excellent merits suggest that the turn-on probe NS-ClO can be used for tissue imaging with two-photon excitation.Open in a separate windowFig. 7(a) Two-photon fluorescence images of a fresh mouse liver slice incubated with NS-ClO probe (10.0 μM) for 30 min in PBS buffer exhibiting no fluorescence at the emission window of 0–80 nm. (b) Two-photon fluorescence images of a fresh mouse liver slice pretreated with NS-ClO (10 μM) and NaClO (10 equiv.) in PBS buffer at the depths of approximately 0–80 μm. (c) The three-dimensional image of (b). Excitation at 800 nm with fs pulse.Because the transparent nature of zebrafish appears in all stages of embryonic growth, the imaging of zebrafish was considered to be a very physiological vertebrate model for the detection of HClO.³⁰ Due to the advantages of two-photon excitation, further investigation of probe NS-ClO to monitor HClO in living zebrafish was carried out. When a 5 day-old zebrafish was treated with probe NS-ClO, no fluorescence appeared in the blue channel (Fig. 8b). However, after further treatment with NaClO for 20 min, the fluorescence signal at around two zygomorphic areas around the yolk extension and eyes of the living zebrafish obviously emerged, as shown in Fig. 8e. The result indicated that the developed probe NS-ClO can be used for zebrafish imaging.Open in a separate windowFig. 8Imaging of HClO in zebrafish stained with the probe NS-ClO (a) Bright-field image of zebrafish costained with NS-ClO; (b) fluorescence images of (a) from blue channel; (c) overlay of (a) and (b). (d) Bright-field image of zebrafish costained with NS-ClO and treated with NaClO. (e) Fluorescence images of (d) from blue channel; (f) overlay of the bright-field image (d) and blue channels (e).In conclusion, a fast-responsive fluorescent probe with two-photon excitation, a large turn-on signal (about 860 times) and a large Stokes shift (about 90 nm) for the detection of HClO in vivo was developed. The ideal probe NS-ClO exhibited good properties including excellent selectivity, high sensitivity (about 1 min) and low cytotoxicity. In addition, the two-photon probe NS-ClO could be used for the detection of HClO in living cells, tissues and fresh zebrafish in vivo. Therefore, the probe NS-ClO can be developed into other functional two-photon probes for the recognition of other analytes and can be applied to investigate the biological and pathological functions of HClO in living biosamples.     

A novel fluorescent probe for imaging the process of HOCl oxidation and Cys/Hcy reduction in living cells

A new on-off-on fluorescent probe, CMOS, based on coumarin was developed to detect the process of hypochlorous acid (HOCl) oxidative stress and cysteine/homocysteine (Cys/Hcy) reduction. The probe exhibited a fast response, good sensitivity and selectivity. Moreover, it was applied for monitoring the redox process in living cells.

A new on–off–on fluorescent probe, CMOS, was designed and applied to detect the process of HOCl oxidation and Cys/Hcy reduction.

Reactive oxygen species (ROS) are indispensable products and are closely connected to various physiological processes and diseases.¹ For instance, endogenous hypochlorous acid (HOCl) as one of the most important ROS, which is mainly produced from the reaction of hydrogen peroxide with chloride catalyzed by myeloperoxidase (MPO), is a potent weapon against invading pathogens of the immune system.^2,3 However, excess production of HOCl may also give rise to oxidative damage via oxidizing or chlorinating the biomolecules.⁴ The imbalance of cellular homostasis will cause a serious pathogenic mechanism in numerous diseases, including neurodegenerative disorders,⁵ renal diseases,⁶ cardiovascular disease,⁷ and even cancer.⁸ Fortunately, cells possess an elaborate antioxidant defense system to cope with the oxidative stress.⁹ Therefore, it is necessary and urgent to study the redox process between ROS and antioxidants biosystems.Fluorescence imaging has been regarded as a powerful visual methodology for researching various biological components as its advantages of high sensitivity, good selectivity, little invasiveness and real-time detection.^10,11 To date, amounts of small molecular fluorescent probes have been reported for detection and visualization of HOCl in vivo and in vitro.^12–22,29 The designed strategies of HOCl sensitive probes are based on various HOCl-reactive functional groups, such as p-methoxyphenol,¹³p-alkoxyaniline,¹⁴ dibenzoyl-hydrazine,¹⁵ selenide,¹⁶ thioether,¹⁷ oxime,¹⁸ hydrazide,¹⁹ hydrazone.²⁰ But, many of these probes display a delayed response time and low sensitivity. And, only few fluorescent probes can be applied for investigating the changes of intracellular redox status.²¹ Besides, it''s worth noting that most of the redox fluorescent probes rely on the organoselenium compounds.²² Even though these probes are well applied for detection of cellular redox changes, excessive organic selenium is harmful to organisms and the synthesis of organoselenium compounds is high requirement and costly. Additionally, almost all the reports have only investigated the reduction effects of glutathione (GSH) as an antioxidant in the redox events. While, there are the other two important biothiols, cysteine (Cys) and homocysteine (Hcy), which not only present vital antioxidants, but also are tightly related to a wide variety of pathological effects in biosystem, such as slowed growth, liver damage, skin lesions,²³ cardiovascular,²⁴ and Alzheimer''s diseases.²⁵ However, the fluorescent probes for specially studying internal redox changes between HOCl and Cys/Hcy are rarely reported. In this respect, a novel redox-responsive fluorescent probe, CMOS, was designed and synthesized in this work, and we hope that it can be a potential tool for studying their biological relevance in living cells.Based on literature research, the aldehyde group has excellent selectivity in identification of Cys/Hcy, and the thiol atom in methionine can be easily oxidized to sulfoxide and sulfone by HOCl.^26,27 Considering these two points, we utilized 2-mercaptoethanol to protect the 3-aldehyde of 7-diethylamino-coumarin as the recognition part of HOCl, meaning that two kinds of potential recognition moieties are merged into one site. Fluorescent probe CMOS can be easily synthesized by the acetal reaction in one step (Scheme S1^†). A control molecule CMOS-2 was also prepared by 3-acetyl-7-diethylaminocoumarin (CMAC) similarly. The structure of all these compounds have been convinced by ¹H NMR, ¹³C NMR, and HR-MS (see ESI^†).As shown in Scheme 1a, we estimated that both CMOS and CMOS-2 can be rapidly oxidized in the appearance of HOCl. The oxidation product CMCHO of CMOS, which has the aldehyde moiety, can further react with Cys/Hcy to obtain the final product CMCys and CMHcy, respectively. In contrast, the oxidation product CMAC of CMOS-2 cannot combine with Cys/Hcy or other biothiols anymore (Scheme 1b).Open in a separate windowScheme 1Proposed reaction mechanism of CMOS and CMOS-2 to HOCl and Cys/Hcy.In order to confirm our design concept, the basic photo-physical characteristics of CMOS, CMCHO, CMOS-2 and CMAC were tested (Table S1, Fig. S1^†). Under the excitation wavelength 405 nm, CMOS and CMOS-2 exhibited strong fluorescence centred at 480 nm in PBS buffer solution, while the fluorescence of CMCHO and CMAC was weak around this band. The emission properties of CMOS and CMCHO were also investigated at the excitation wavelength 448 nm under the same experimental conditions as well (Fig. S2^†). After careful consideration, we chose 405 nm as the excitation wavelength in the follow-up experiments in vitro and in vivo.Next, the sensitivity of CMOS and CMOS-2 to HOCl and Cys/Hcy were investigated. As we expected, both the CMOS and CMOS-2 exhibited good response to HOCl. The fluorescence intensity of CMOS and CMOS-2 decreased gradually with addition of NaOCl (Fig. 1a, S3a^†), indicating that the fluorescence was switched off obviously in the presence of HOCl. The variation of intensity displayed good linearity with concentration of HOCl in the range of 0–20 μM (R² = 0.993, Fig. S4^†), and the detection limit of CMOS to HOCl was calculated to be 21 nM (S/N = 3). Subsequently, when Cys/Hcy was added to the final solution in Fig. 1a, the fluorescence intensity increased gradually within 180 min (Fig. 1b, S5^†). However, the fluorescence cannot be recovered by addition thiols to the CMOS-2 solution with excess HOCl (Fig. S3b^†). These results indicate that the probe CMOS can response to HOCl and Cys/Hcy in a fluorescence on-off-on manner, and can be used for monitoring the redox process with high sensitivity.Open in a separate windowFig. 1(a) Fluorescence responses of CMOS (2 μM) to different concentrations of NaOCl (0–200 μM). (b) Fluorescence responses of the CMOS solution (2 μM) with HOCl (200 μM) to Cys/Hcy (5 mM). (20 mM PBS buffer/CH₃CN, 7 : 3, v/v, pH = 7.4, λ_ex = 405 nm).To further identify the recognizing mechanism of probe CMOS, high performance liquid chromatography (HPLC) and mass spectral (MS) analysis were used to detect the redox process. Initially, probe CMOS displayed a single peak with a retention time at 3.7 min (Fig. 2a, S6^†) while reference compound CMCHO produced a single peak with a retention time at 2.5 min (Fig. 2b, S7^†). Upon the addition of HOCl to the solution of CMOS, the peak at 3.7 min weakened while 2.5 min and 2.2 min appeared (Fig. 2c). According to corresponding mass spectra, the new main peak at 2.5 min is related to compound CMCHO (Fig. S8^†). The other new peak of 2.2 min corresponds to the compound C3, which can be predicted as an intermediate in the oxidation process (Fig. S8^†).²⁸ The addition of Cys to the solution of CMCHO also caused a new peak with a retention time at 2.1 min, which has been confirmed to be the thioacetal product CMCys (Fig. S9^†). The possible sensing mechanism is depicted in Fig. S10.^†Open in a separate windowFig. 2The reversed-phase HPLC with absorption (400 nm) detection. (a) 10 μM CMOS. (b) 10 μM CMCHO. (c) 10 μM CMOS in the presence of 50 μM HOCl for 30 s. (d) 10 μM CMCHO in the presence of 1 mM Cys for 30 min. (Eluent: CH₃CN containing 0.5% CH₃COOH; 100% CH₃CN (0–7 min), 0.5 ml min⁻¹, 25 °C; injection volume, 5.0 μL).To study the selectivity of CMOS towards HOCl, we performed fluorescence response to different reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS). As shown in Fig. 3a, CMOS exhibited significant change of fluorescence intensity only in the presence of HOCl, while other ROS and RNS, such as singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radical (HO·), superoxide anion (O₂⁻), nitric oxide (NO), tert-butylhydroperoxide (t-BuOOH) and tert-butoxy radical (t-BuOO·) had no obvious fluorescence emission changes. Additionally, RSS which are abundant in biological samples, showed no influence in this process under the identical condition. The detection of reducing process was also investigated. As displayed in Fig. 3b, only cysteine and homocysteine induced excellent fluorescence recovery towards other reducing materials, such as RSS and various amino acids. Furthermore, the selectivity of CMOS-2 was also studied in the same condition. As expected, CMOS-2 could selectively detect HOCl, and not alter fluorescence intensity under various kinds of biothiols (Fig. S11^†). Therefore, our design strategy for the on–off–on probe is confirmed by results obtained above, with which CMOS can be utilized for detecting the redox process between HOCl and Cys/Hcy with high selectivity.Open in a separate windowFig. 3(a) Fluorescence response of CMOS (2 μM) to different ROS, RNS and RSS (200 μM). Bars represent emission intensity ratios before (F₀) and after (F₁) addition of each analytes. (a) HOCl; (b) KO₂; (c) H₂O₂; (d) ¹O₂; (e) HO·; (f) t-BuOOH; (g) t-BuOO·; (h) NO₂⁻; (i) NO₃⁻; (j) NO; (k) GSH; (l) Cys; (m) Hcy; (n) Na₂S; (o) Na₂S₂O₃; (p) Na₂S₂O₈; (q) NaSCN; (r) DTT; (s) Na₂SO₃. (b) Fluorescence response of the solution added HOCl in (a) to different RSS and amino acids. Bars represent emission intensity ratios before (F₂) and after (F₃) addition of each analytes (5 mM). (a) Cys; (b) Hcy; (c) Na₂S; (d) Na₂S₂O₃; (e) Na₂S₂O₈; (f) NaSCN; (g) DTT; (h) Na₂SO₃; (i) Ala; (j) Glu; (k) Gly; (l) His; (m) Ile; (n) Leu; (o) Met; (p) Phe; (q) Pro; (r) Ser; (s) Trp; (t) Vc; (u) GSH. (20 mM PBS buffer/CH₃CN, 7 : 3, v/v, pH = 7.4, λ_ex/λ_em = 405/480 nm).Subsequently, the influence of pH on probe CMOS was measured. The fluorescence intensity of CMOS and CMCHO perform no significant variances in wide pH ranges (pH = 4–11, Fig. S12a^†). Fluorescence intensity changes could be observed immediately when HOCl was added into the solution of probe CMOS, especially in alkaline condition (Fig. 4a). Considering the pK_a of HOCl is 7.6,²⁹CMOS is responsive to both HOCl and OCl⁻. Alkaline condition was also benefit for the fluorescence recovery of CMOS from Cys/Hcy (Fig. S12b^†). It is reasonable to consider that thiol atom displays higher nucleophilicity in alkaline condition. From the stop-flow test, the UV-visible absorbance of probe CMOS sharply decreased at the wavelength of 400 nm (Fig. 4b). The response time was within 10 s and the kinetic of the reaction was fitted to a single exponential function (k_obs = 0.67 s⁻¹). The ability of instantaneous response is extremely necessary to intracellular HOCl detection.Open in a separate windowFig. 4(a) Fluorescence responses of CMOS to HOCl under different pH values. Squares represent emission intensity ratios after (F₁) and before (F₀) addition of 200 μM HOCl (λ_ex/λ_em = 405/480 nm). (b) Time-dependent changes in the absorption intensity of CMOS (1 μM) before and after addition of HOCl. (20 mM PBS buffer/CH₃CN, 7 : 3, v/v, pH = 7.4, λ_abs = 400 nm).With these data in hand, we next applied CMOS for fluorescence imaging of the redox changes with HOCl and Cys/Hcy in living cells. After incubation with 5 μM CMOS at 37 °C for 30 min, intense fluorescence was observed of the SKVO-3 cells in the optical window 425–525 nm (Fig. 5a and d), indicating the probe can easily penetrate into cells. Treating the cells with 100 μM NaOCl led to remarkable fluorescence quenching as the probe sensed the HOCl-induced oxidative stress (Fig. 5b and e). After 3 min, the cells were washed with PBS buffer three times, and added 5 mM Cys/Hcy for 1 h, respectively. Then the fluorescence was recovered obviously (Fig. 5c and f). Experimental results clearly declare that the probe CMOS was successfully used to detect the process of HOCl oxidative stress and Cys/Hcy reducing repair in living cells.Open in a separate windowFig. 5Fluorescence imaging of the process of HOCl oxidative stress and thiols repair in CMOS-labeled SKVO-3 cells. Fluorescence images of SKVO-3 cells loaded with 5 μM CMOS at 37 °C for 30 min (a and d). Dye-loaded cells treated with 100 μM NaOCl at 25 °C for 3 min (b and e). Dye-loaded, NaOCl-treated cell incubated with 5 mM Cys (c), 5 mM Hcy (f) for 1 h. Emission intensities were collected in an optical window 425–525 nm, λ_ex = 405 nm, intensity bar: 0–3900.     

Coumarin-based fluorescent ‘AND’ logic gate probes for the detection of homocysteine and a chosen biological analyte

With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO⁻ reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO⁻ (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

With this research we set out to develop of a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys.

Homocysteine (HCys) is a non-proteinogenic amino acid, formed from the de-methylation of methionine,¹ which is then converted into cysteine (Cys) via a vitamin B₆ cofactor. Typical physiological concentrations of HCys range between 5–15 μmol L⁻¹.² However, elevated levels of HCys (>15 μmol L⁻¹), which is known as hyperhomocysteinemia (hHCys),³ have been associated with pregnancy disorders, Alzheimer''s disease, cardiovascular disease and neurodegenerative diseases (NDs).^4–6 It is believed that the main cause of HCys induced toxicity is through the non-enzymatic modification of proteins. This is achieved through irreversible covalent attachment of the predominant metabolite of HCys, homocysteine thiolactone (HTL), to lysine residues; a phenomenon known as ‘protein N-homocysteinylation’ that results in the loss of a proteins structural integrity leading to loss of enzymatic function and aggregation.⁷A number of fluorescent sensors have been developed for the detection of HCys to help improve our understanding of its role in biological systems.^8–11 However, these fluorescent probes have focused on the detection of a single biomarker (HCys), however, processes associated with HCys induced toxicity often involve more than one biochemical species. For example, it has been reported that peroxynitrite (ONOO⁻) and nitric oxide (NO˙) play a significant role in HCys-mediated apoptosis in trigeminal sensory neurons¹² and HCys has been reported to induce cardiomyocytes cell death through the generation of ONOO⁻.¹³ The production of ONOO⁻ is believed to be the result of an increased production of superoxide (O₂˙⁻) by HCys activating the enzyme NADPH oxidase.^14–16 This increased production of O₂˙⁻ leads to a reduction in the bioavailability of NO˙ by increasing the formation of ONOO⁻ (NO˙ + O₂˙⁻ → ONOO⁻).¹⁷ The reported ONOO⁻ concentrations in vivo are believed to be approximately 50 μM but, higher concentrations of 500 μM have been found in macrophages.^18,19 Furthermore, hypoxia has been reported to facilitate HCys production in vitamin-deficient diets²⁰ where hypoxia leads to an upregulation of nitroreductase (NTR) – a reductive enzyme upregulated in cells under hypoxic stress.^21,22 Therefore, the development of tools that enable an understanding of the relationship of HCys with these biologically important species would be highly desirable.To achieve this, a number of fluorescent probes have been developed that are capable of detecting two or more analytes.²³ These include AND logic gate based-fluorescence probes, which require both analytes to work in tandem to produce a measurable optical output.^24–28 In our group, we have developed several ‘AND’ reaction-based probes for the detection of ROS/RNS and a second analyte.^29–32 These ‘AND’ logic scaffolds have been used to detect two analytes within the same biological system.^24,33Owing to the pathological role of HCys, we set out to develop the first example of a fluorescent probe for the detection of HCys and biological related analyte. Aiming towards that target, we became interested in a previously reported coumarin-based fluorescent probe developed by Hong et al.CAH, with a salicylaldehyde (Fig. 1).³⁴ Salicylaldehyde is a known reactive unit towards HCys/Cys, therefore we believed CAH could be used as a scaffold for the development of ‘AND’-based systems for the detection of HCys/Cys and a second analyte.³⁴ In the presence of HCys, CAH exhibited a ‘turn-on’ fluorescence response which is attributed to the nucleophilic nature of the nitrogen and sulfur atoms resulting in thiazine ring formation (Scheme S1, Fig. S1 and S2^†).^34–36Open in a separate windowFig. 1(a) CAH – a core fluorescent unit that enables the synthesis of ‘AND’ based fluorescent probe for the detection of HCys/Cys and a second analyte. (b) JEG-CAB enables the detection of HCys/Cys and (ROS/RNS) while (c) JEG-CAN enables the detection of HCys/Cys and NTR.We believed that CAH was a useful core unit that can be used to introduce the chosen reactive chemical trigger on the phenol for the detection of the corresponding analyte with HCys/Cys. Owing to the relationship between HCys and ONOO⁻/NTR, we set out on the development of a HCys AND ONOO⁻ probe and a HCys AND NTR probe.Therefore, we set out to prepare JEG-CAB and JEG-CAN, which are able to detect HCys/Cys and peroxynitrite (ONOO⁻) or nitroreductase (NTR), respectively (Scheme 1). For JEG-CAB, a benzyl boronate ester was introduced as a ONOO⁻ reactive unit.³⁷ For JEG-CAN, a p-nitrobenzyl group was installed as it is known to be an effective substrate for NTR.^38–40Open in a separate windowScheme 1Synthesis of target probe JEG-CAB and JEG-CAN.To afford CAH, compound 2 was synthesized by refluxing umbelliferone and acetic anhydride at 140 °C. Compound 2 was then dissolved in trifluoroacetic acid at 0 °C followed by the addition of hexamethylenetetramine (HMTA). The mixture was heated to reflux overnight and the solvent was then removed. The intermediate was then hydrolyzed in H₂O for 30 min at 60 °C. Upon isolating CAH, it was then alkylated using 4-bromomethylphenylboronic acid pinacol ester and K₂CO₃ in DMF at r.t. to afford JEG-CAB in 51% yield. JEG-CAN was produced by alkylating CAH using 4-nitrobenzyl bromide and K₂CO₃ in DMF at r.t. to give 49% yield (Scheme 1).We then evaluated the ability of JEG-CAB to detect ONOO⁻ ‘AND’ HCys in PBS buffer solution (10 mM, pH 7.40). The maximum absorption of JEG-CAB at 336 nm shifted to 323 nm with the addition of HCys and then slightly shifted to 328 nm following the addition of ONOO⁻ (Fig. S3^†). As shown in Fig. 2, JEG-CAB was initially non-fluorescent, but the addition of HCys (1 mM) led to a small increase in fluorescence intensity, the subsequent additions of ONOO⁻ (0–24 μM) led to a significant increase in fluorescence intensity (>17-fold, see Fig. S5^†). These results demonstrated the requirement for both ONOO⁻ ‘AND’ HCys to obtain a significant turn ‘‘on’’ fluorescence response.Open in a separate windowFig. 2Fluorescence spectra of JEG-CAB (15 μM) with addition of HCys (1 mM) and incubated for 40 min then measured. Followed by incremental additions of ONOO⁻ (0–24 μM). The data was obtained in PBS buffer solution (pH 7.40, 10 mM) at 25 °C. λ_ex = 371 (bandwidth 20) nm. Dashed line represents JEG-CAB and Hcys addition only. Blue line represents highest intensity after addition of ONOO⁻.The addition of HCys and ONOO⁻ were then performed in reverse where JEG-CAB exhibited a negligible increase in fluorescence intensity upon addition of ONOO⁻ (16 μM). However, in a similar manner to that shown in Fig. 2, a large increase in fluorescence intensity was produced after the subsequent addition of HCys (0–5.5 mM) (Fig. 3 and S6^†). LC-MS experiments were carried out to ascertain the reaction mechanism and the results confirmed the sequential formation of the thiazine ring in the presence of HCys followed by boronate ester cleavage in the presence of ONOO⁻ or vice versa (Scheme S2 and Fig. S19–S21^†).Open in a separate windowFig. 3Fluorescence spectra of JEG-CAB (15 μM) with addition of ONOO⁻ (16 μM) and followed by incremental additions of HCys (0–5.5 mM) measurements were taken after 40 min of both additions. The data was obtained in PBS buffer solution (pH 7.40, 10 mM) at 25 °C. λ_ex = 371 (bandwidth 20) nm. Dashed line represents JEG-CAB and ONOO⁻ addition only. Blue line represents highest intensity after addition of HCys.As expected, probe JEG-CAB was shown to have excellent selectivity with ONOO⁻ against other ROS in the presence of HCys (1 mM) (Fig. S9 and S10^†). Furthermore, JEG-CAB exhibited a high degree of selectivity towards a series of amino acids where only HCys and Cys led to a fluorescence response in the presence of ONOO⁻. This is due to the formation of stable five or six-membered thiazine rings (Fig. S7 and S8^†).³⁴We then evaluated the changes in the fluorescence of JEG-CAN with both HCys and NTR in PBS buffer solution (10 mM, pH 7.40, containing 1% DMSO). As shown in Fig. 4, addition of HCys led to a small increase in fluorescence intensity. However, subsequent addition of NTR (4 μg mL⁻¹) led to a large time dependant increase in fluorescence intensity. To ensure both analytes were required, NTR and NADPH was kept constant (4 μg mL⁻¹ and 400 μM respectively) resulting in a 3.4 fold fluorescence increase (Fig. 5). We attribute the large initial increase to background fluorescence of NADPH.⁴¹ NTR then facilitates reduction of the nitro group of JEG-CAN releasing the core probe CAHvia a fragmentation cascade (Scheme S3^†).^38,42 Subsequent addition of HCys (2.0 mM) led to a 2 fold increase in fluorescence intensity. Again, LC-MS experiments confirmed the proposed reaction mechanism (Fig. S22^†).Open in a separate windowFig. 4Fluorescence spectra of JEG-CAN (15 μM) with initial addition of HCys (2 mM) and incubated for 60 min. Followed by addition of nitroreductase (4 μg mL⁻¹) and NADPH (400 μM) and measured over 90 min in PBS buffer solution (pH = 7.40, 10 mM, containing 1% DMSO). λ_ex = 363 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line represents JEG-CAN and HCys addition only. Blue line represents highest intensity after addition of NTR.Open in a separate windowFig. 5Fluorescence spectra of JEG-CAN (15 μM) with initial addition of nitroreductase (4 μg mL⁻¹) and NADPH (400 μM) and incubated for 60 min. Followed by addition HCys (2 mM) and measured over 90 min in PBS buffer solution (pH = 7.40, 10 mM, containing 1% DMSO). λ_ex = 363 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line represents JEG-CAN and NTR addition only. Blue line represents highest intensity after addition of HCys.Kinetic studies for JEG-CAN with both NTR and HCys were carried out (Fig. S11–S18^†) where it is clear that JEG-CAN exhibits a dose dependant fluorescence increase in response of both HCys and NTR.Unfortunately, the probes failed to give good data in cells, which could be due to their short excitation wavelengths or the extremely low intracellular HCys concentrations (5–15 μM). We are now pursuing the development ‘AND’ logic fluorescence probes with longer excitation and emission wavelengths suitable for in vitro and in vivo applications.In summary, we have developed two coumarin-based ‘AND’ logic fluorescence probes (JEG-CAB and JEG-CAN) for the detection of HCys/Cys and ONOO⁻ or NTR, respectively. CAH is a useful platform that enables easy modification for the development of ‘AND’-based fluorescent probes for the detection of HCys/Cys and a second analyte. Both JEG-CAB and JEG-CAN were shown to be ‘AND’-based fluorescent probes.     

Design of a fluorogenic PNA probe capable of simultaneous recognition of 3′-overhang and double-stranded sequences of small interfering RNAs

We developed a new fluorescent peptide nucleic acid (PNA) probe, COT probe, capable of simultaneous recognition of 3′-overhang and double stranded sequences of target small interfering RNA (siRNA).

We developed a new fluorescent peptide nucleic acid (PNA) probe, COT probe, capable of simultaneous recognition of 3′-overhang and double stranded sequences of target small interfering RNA (siRNA).

Much attention has been paid to the design of RNA-binding molecules capable of binding to non-coding RNAs that can regulate gene expression.¹ This class of molecules have great potential as drug candidates by modulating the expression of genes related to various diseases.² In addition, the molecules that show the fluorescence response upon binding to non-coding RNAs have been useful probes for the analysis of non-coding RNAs.^3–5 For instance, the fluorescent intercalators were shown to be useful for detection of small interfering RNAs (siRNAs), typically 21-mer double-stranded RNAs (dsRNAs) containing 3′-overhanging dinucleotides,⁶ as well as the analysis of siRNA delivery into the cells by means of carriers.^4,5 They can provide simple and easy-to-use analysis for target siRNAs with a view toward the development of siRNA therapeutics.In this context, we have recently reported on new class of peptide nucleic acid (PNA)-based fluorescent probes for siRNA analysis.⁷ PNA dinucleotide that can recognize 3′-overhanging dinucleotide through Watson–Crick base pairing was conjugated with a fluorescent intercalator, thiazole orange (TO).⁸ Our probe thus targeted both 3′-overhanging nucleotides and the dsRNA region near the overhangs of target siRNAs, which is characteristic compared to other siRNA-binding fluorescent probes that simply aim dsRNA region. The PNA–TO conjugate was further attached with pyrene unit at C-termini for enhancing the binding ability and selectivity to target siRNAs. The resulting probe, Py-AA-TO, exhibited light-up response of the TO unit upon selective binding to the target siRNAs containing the overhanging nucleotides that are complementary to the PNA units of the probes, which rendered it useful for selective detection of target siRNAs. Moreover, the use of Py-AA-TO as an affinity-labeling agent for the siRNAs enabled the selective visualization of siRNAs encapsulated in the carriers in the living cells.⁷ One of the key issues is to enhance the binding ability of the probe for target siRNAs with a view toward the goal of sensitive siRNA analytical assays suitable for practical use.In this work, we developed a new fluorescent PNA probe with the improved binding affinity and selectivity for siRNAs, where the probe can simultaneously recognize 3′-overhang and dsRNA sequences near the overhang of target siRNAs (Fig. 1A). In case of Py-AA-TO, TO unit linked with PNA dinucleotide was designed to intercalate into the dsRNA region of target siRNAs, which results in little selectivity to dsRNA sequence. Instead, we explored triplex-forming PNAs (TFPs) as the dsRNA-binding units so as to achieve sequence-selective recognition of dsRNA region.⁹ TFPs are fundamentally composed of homopyrimidine PNA oligomers and can strongly and selectively form triplex structures with the homopurine sequences of target dsRNA tracts at acidic pH, by forming T (U)·A–U and C⁺·G–C base triples through Hoogsteen base pairing (Fig. 1A). Besides the sequence-selective recognition property, TFPs feature the strong binding to target dsRNAs, where even 6-mer oligomers show the dissociation constant (K_d) less than 100 nM.⁹ This affinity is superior to against ds-nucleic acids (K_d = 1.0–10 μM).¹⁰ Therefore, we expect that the integration of TFP as a dsRNA-binding unit into siRNA-targeting probe enables to achieve stronger affinity compared to Py-AA-TO as well as the sequence-selective binding to dsRNA region of target siRNAs. Specifically, a TFP possessing TO as a base surrogate was utilized in the probe design. This class of TFPs named as triplex-forming forced intercalation (tFIT) probes exhibit the significant light-up response of the TO unit upon triplex formation with dsRNA tracts, as demonstrated in our previous work.¹¹ tFIT probes were shown to be useful for analyzing dsRNA sequences at single-base pair resolution. Here, such a tFIT probe was directly attached to C-terminus of PNA dinucleotide capable of recognition of 3′-overhang sequence of target siRNA. The resulting probe which we call COT (combination of overhang recognition and triplex formation) probe was discussed based on the examination of the binding and fluorescence sensing of target siRNAs.Open in a separate windowFig. 1(A) Schematic illustration of COT probe binding for simultaneous recognition of 3′-overhang and dsRNA sequences of target siRNA. (B) Chemical structures of COT probe. (C) Target siRNA sequence against red fluorescent protein gene (X = dT) used in this study. The sequence that can be recognized by COT probe was indicated by dots. We also showed the sequences of control siRNAs having no overhangs (no overhang siRNA: X = none) or mismatched overhanging nucleotides (mismatched siRNA: X = dA).COT probe (, Fig. 1B) was designed for targeting siRNA sequence that can knockdown red fluorescent protein (RFP) gene (Fig. 1C).¹² Overhang recognition unit (italic base) would recognize 3′-overhanging dTdT in the antisense strand while tFIT unit (underlined base) would recognize the purine-rich sequence of the sense strand. TO base surrogate was placed so as to face the uracil nucleotide in dsRNA region because it can function as a universal base that non-discriminatorily binds to all four kinds of base pairs in the triplex.¹¹ Furthermore, two lysine residues were introduced to the C-terminus of the probe in order to increase the solubility as well as the binding affinity to target siRNA through electrostatic interaction.¹³ We also designed the control probe that lacks the overhang recognition unit (; Fig. S1^†). These probes were manually synthesized by solid-phase synthesis, purified by reverse phase HPLC, and characterized by MALDI-TOF-MS (ESI^†). Besides target siRNA, we examined a control siRNA with noncognate dsRNA sequence and 3′-overhanging dTdT (noncognate siRNA;⁶ 5′-CGU ACG CGG AAU ACU UCG AdTdT-3′/3′–dTdTG CAU GCG CCU UAU GAA GCU -5′). In addition, we used another control siRNAs having fully-matched dsRNA sequence with no overhang (no overhang siRNA) or 3′-overhanging dAdA that was mismatched with the overhang recognition unit of COT probe (mismatch siRNA), as shown in Fig. 1C.First, UV melting experiments were performed in order to evaluate the binding of COT probe to target siRNA, in 10 mM sodium acetate (pH 5.5) buffer solutions containing 100 mM NaCl and 1.0 mM EDTA (Fig. S4^†). We monitored absorbance change at 300 nm, where the triplex-duplex transition of tFIT unit can be selectively detected.^11a,14 As the temperature increased from 10 °C to 80 °C, the absorbance remarkably decreased. This is most likely due to deprotonation of N3 protonated PNA cytosines (C⁺) involved in Hoogsteen base pairs with G–C (C⁺·G–C triplets) in the triplex structures. The melting temperature (T_m) was obtained as 66 ± 0.5 °C, which indicates the tFIT unit can form a thermally stable triplex with dsRNA region of target siRNA. Triplex formation is also supported by the results of circular dichroism (CD) spectral change of target siRNAs upon addition of the probe (Fig. S5^†). We found the T_m value was significantly reduced for noncognate siRNA containing unrelated dsRNA sequence (ΔT_m > 30 °C) whereas the mismatches (mismatch siRNA) or deletion of 3′-overhang sequence (no overhang siRNA) led to little influence on triplex stability (Table S2^†). This result indicates that tFIT unit of COT probe retains the sequence selectivity for dsRNA region of target siRNA.Next, we examined the fluorescence response of COT probe for target siRNA in pH 5.5 buffer at 25 °C (Fig. 2). In the absence of target siRNA, the probe (100 nM) showed negligible fluorescence (Φ_free < 0.01), because of non-radiative energy loss by free rotation of the benzothiazole and quinoline rings in the TO base surrogate.⁸ The addition of the equimolar target siRNA caused the remarkable light-up response of the TO unit, in which the light-up factor (I/I₀, where I and I₀ denote the fluorescence intensities in the presence and absence of a target, respectively) was more than 104-fold. The fluorescence quantum yield of COT probe bound to target siRNA reaches 0.29. The light-up property is almost comparable to those of tFIT probes for dsRNA tracts.^11b In contrast, almost no response was found at pH 7.0 where cytosines are hardly protonated (Fig. S6^†). This is consistent with that the COT probe binding involves triplex formation of tFIT unit by Hoogsteen base pairing of C⁺ with G. Fluorescence titration experiments were carried out to estimate the binding affinity of COT probe for target siRNA (inset of Fig. 2). The resulting titration curve was well fitted by a 1 : 1 binding isotherm, which gave the dissociation constant (K_d) of 340 ± 43 nM (n = 3). Significantly, this affinity is one order of magnitude larger than that of Py-AA-TO (K_d = 3.5 ± 0.40 μM).^7a It suggests that the use of tFIT unit as dsRNA-binding unit leads to the improved binding affinity for target siRNAs.Open in a separate windowFig. 2Fluorescence spectra of COT probe (100 nM) in the absence and presence of siRNAs (100 nM) at pH 5.5. Inset: fluorescence titration curve for the binding of COT probe (500 nM) to target siRNA (0–6.0 μM) at pH 5.5. Excitation: 509 nm. Analysis: 534 nm. Temperature, 25 °C.Fluorescence response of COT probe was found to be very sensitive to the overhang sequence as well as dsRNA sequence of target siRNAs. As shown in Fig. 2, the light-up response is much pronounced for target siRNAs over three kinds of control siRNAs under the identical conditions. As for noncognate siRNA, very small response can be attributed to little formation of triplex structure at 25 °C (Table S2^†). On the other hand, it was shown that both mismatch siRNA and no overhang siRNA were able to form a thermally stable triplex with the probe (Table S2^†). Thus, we reasoned small response for these control siRNAs resulted from high flexibility of the TO unit in the resulting complexes.^11a Considering that TO unit is located close to the terminal of triplex-forming region, the intramolecular rotation of TO unit would be less restricted in the complex between the probe with these siRNAs. Accordingly, the fluorescence response of COT probe allows to discriminate not only the overhanging sequence but also dsRNA sequence near the overhang of target siRNAs.The observed abilities of COT probe for target siRNA were compared with those of control probe having no overhang recognition units. While the control probe could form the triplex structure (Table S2^†), its light-up response upon binding to siRNAs was very low (Fig. S7^†). This can be attributed to weaker binding affinity (K_d = 2.3 ± 0.36 μM) as well as the increased flexibility of the TO unit in the resulting triplex discussed above, due to the loss of overhang recognition. Also, we found relatively large response for mismatch and no overhang siRNAs whereas the response for noncognate siRNA was very weak. This resulted in the reduced selectivity for target siRNA over control siRNAs compared to COT probe. These results indicate that the overhang recognition in COT probe is crucial for high affinity and selectivity to overhang and dsRNA sequences for target siRNAs.In order to understand more details about COT probe binding to target siRNAs, we characterized the binding kinetics by stopped-flow experiments (Fig. 3). The absorbance change at 260 nm was monitored upon mixing COT probe with target siRNA, which enables to estimate the association rate constant (k_on) by a nonlinear least-squares regression analysis.^11a,15 We then calculated the dissociation rate constant (k_off) by the equation (k_off = K_d × k_on), where we assume two-state binding for the biomolecular complexes. The obtained kinetic parameters were summarized in †). Meanwhile, k_off values were comparable (k_off/s⁻¹; COT probe, 1.0; control probe, 1.2). Therefore, the overhang recognition is highly likely to be important for the rapid association for the binding to target siRNA, which would be responsible for strong binding affinity of COT probe over control probe.Open in a separate windowFig. 3Stopped-flow kinetics trace for COT probe (3.0 μM) binding to equimolar target siRNA at 25 °C. The fitting curve is the bold line, and the corresponding residual plot is presented below the kinetics trace.Dissociation constants, and kinetic parameters for COT probe binding to the various kinds of siRNA at 25 °C^a

Photoswitchable probe with distinctive characteristics for selective fluorescence imaging and long-term tracing

A photo-switchable and high-contrast bio-imaging indicator 4,4′-(1E,1′E)-(4,4′-(cyclopentene-1,2-diyl)bis(5-methylthiophene-4,2-diyl))bis(methan-1-yl-1-ylidene)bis(azan-1-yl-1-ylidene)bis(2-(benzo[d]thiazol-2-yl)phenol) (BMBT) has been demonstrated, by integrating photochromophore with excited-state intramolecular proton transfer (ESIPT) moiety. The ability of reversible emission switching enables arbitrarily selective labeling or concealing of cells simply by controlling light irradiation. Besides, when the emission was switched on, BMBT is demonstrated to exhibit unique characteristics of aggregation induced emission (AIE), providing a high on–off ratio for favorable bio-imaging. Thus, the non-labeling and easily-controlled selective imaging, as well as good biocompatibility indicates BMBT to be a favorable cell probe with great potentials for functional bio-imaging fluorophore.

In this work, a photoswitchable probe was synthesized by integrating a photochromophore with an excited-state intramolecular proton transfer (ESIPT) moiety. It was explored to be a favorable fluorophore for selective fluorescence imaging and long-term tracing.

Although there are many conventional fluorescent probes used for fluorescent imaging in the past few years,^1–5 such as rhodamine,⁶ cyanine dye,⁷ quantum dots,^8,9 and lanthanide probes,¹⁰ these fluorescent probes can only respond irreversibly to one event.^11,12 In comparison, photochromophores,¹³ which can reversibly response with UV and visible light,¹⁴ are more valuable fluorescent probes for regional optical marking of interested cells.^15,16 Because of their favourable characteristics, such as excellent thermal stability, good fatigue resistance and fast response time, diarylethenes derivatives have drawn wide spread concern of researchers.^17–20 Furthermore, the special optical properties of diarylethenes enable them to be suitable for long time and real time monitoring in bioimaging.The significance of targeted imaging of fluorescence probe for bio-samples in vitro or in vivo is well-known for their high sensitivity, non-invasiveness, real-time detection and especially selectivity.^21–24 However, the synthesis of these targeting materials is usually complicated. Moreover, for the same type of cells, no selectivity is demonstrated by these complex targeting agents.^21–24 Therefore, easy-prepared and easy-controlled non-labeling fluorescence imaging agents have always been pursued by both the industry and scientific communities. Among the above-mentioned photochromophores, diarylethenes are expected to be promising photochromic imaging candidates due to their favorable characteristics described in preceding paragraph. Prospectively, they can be used for selective long-term tracing if the photostability can be ensured.In our previous work, a series of AIE-active excited-state intramolecular proton transfer (ESIPT) complexes have been demonstrated to be good bio-imaging candidate with many advantages such as simple preparation, good biocompatibility, high quantum yields, fast cell staining as well as long-term anti-photobleaching.^25–27 Sometimes, ESIPT compounds exhibit dual emission, originated from keto and enol state, respectively. This caused extremely fast four-level photophysical cycle (E–E*–K*–K–E), mediated by intramolecular H-bonds immediately after photoexcitation, enables two emissions.^28,29 Herein, we have demonstrated a new type of multifunctional bio-imaging materials based on facile synthesis design concept, by introducing a photochromic diarylethene moiety to enable regional emission turn-on, and introducing an ESIPT moiety to allow good photo stability for long-term tracing. To the best of our knowledge, this is the first example with integrating abilities of non-labeling selectively long-term regional tracing.The new diarylethene derivative (BMBT, see the molecular structure in Scheme 1) has been synthesized (Fig. S1^†) via condensation reaction of 5-amino-2-(benzo[d]thiazol-2-yl)phenol with 4,4-(cyclopentene-1,2-yl)-bis(5-methyl-thiophene-2-formaldehyde), according to a previous reported procedure.^30,31 The molecular structures and purities were confirmed by ¹H NMR spectroscopy and mass spectroscopy (Fig. S6 and S7,^† see the synthesis details and full molecular characterizations in the ESI^†).Open in a separate windowScheme 1Structure and photochromic process of BMBT.The photoirradiation-induced changes in absorption and fluorescence spectra at room temperature are investigated in THF/DMEM mixture (1.0 × 10⁻⁵ mol L⁻¹). The open-ring isomer mainly exhibits two absorption peaked at 304 and 380 nm, respectively (Fig. S2^†). This is ascribed to the internal charge transfer and π–π transition of 2-(2′-hydroxy-phenyl)benzothiazole (HBT),^10,30 coupled with the CT inside the HBT unit from the hydroxyphenyl ring to the benzothiazole ring (Scheme 1). BMBT exhibits two emissions; one blue emission peak around 458 nm and a red emission peak around 600 nm, corresponding to the enol and keto emission, respectively (Fig. 1). Upon irradiation with ultraviolet light at 365 nm, the absorption band at longer wavelength region centered at 595 nm increased obviously with irradiation time (Fig. S2^†). This is caused by the formation of the closed-ring isomer (see the photochromic reaction in Scheme 1). Correspondingly, due to spectroscopic overlap between this longer-wavelength absorbance with the red emission ranged from 560–700 nm, the relative intensity of the red emission substantially decreases with the UV irradiation (Fig. 1), because of efficient energy transfer. Upon further visible light (λ = 520 nm) irradiation, the closed-ring isomer transfers back to the initial open-ring isomer, and thus the longer-wavelength absorption decreases and the red emission restores. This indicates good reversibility of the photochromic reaction.Open in a separate windowFig. 1Fluorescence emission changes of BMBT in THF/DMEM (1 : 200, vol : vol) mixture upon irradiation with 365 nm light (λ_ex = 385 nm) and visible light (λ_ex = 520 nm).Excitingly, BMBT exhibit prominent characteristics of Aggregation-Induced Emission (AIE).^31–33 The fluorescence intensity of BMBT in THF solution was relatively weak, while the powder or nanoparticles of the material dispersed in DMEM buffer exhibited a significantly enhanced emission (Fig. 2). When the DMEM buffer fraction was increased gradually from 0% to 20%, the fluorescence intensity only slightly enhanced. When the DMEM fraction was further increased to 40%, the emission exhibits a significant enhancement. The total intensity at the blue enol emission increased more than 10 times at 100% DMEM fraction as compared with that in THF solution. This AIE property enables high signal-to-noise ratios for favorable bioimaging (Fig. S3^†).Open in a separate windowFig. 2Fluorescence spectra of 1 × 10⁻⁵ M BMBT in the THF/DMEM mixture at different water fractions (λ_ex = 385 nm).Based on the good reversibility of light response and AIE characteristics, the practical application of the BMBT as bioprobe was further investigated. The biological imaging of BMBT was observed by using confocal laser scanning microscopy (CLSM). Blue luminescence in the cytoplasm of HeLa cells was observed after incubation with a THF/DMEM (1 : 200, vol : vol) solution of BMBT (20 μM) for 30 min at 37 °C (Fig. 2 inset). The overlay of luminescent images and bright-field images confirmed that BMBT was located mainly in the cytoplasm of cells rather than the membrane and nucleus (Fig. S3^†). Intense intracellular luminescence with a high signal-to-noise ratio (I₁/I₂ > 7) was detected between the cytoplasm (regions 3 and 1) and nucleus (region 2), also implying weak even few nuclear uptake of BMBT (Fig. S4^†). Besides, BMBT has a low cytotoxicity with the cellular viabilities estimated to be greater than 85% after 24 h incubation with the highest cultural concentration of 50 μM BMBT (Fig. S5^†).The luminescence switching of BMBT can also be achieved while alternating UV and visible light illumination in fixed HeLa cells. Cells (shown in red circle, Fig. 3a) were irradiated with 488 nm light (0.5 mW) for 3 min, the blue fluorescence of the irradiated cells was lamped off while the surrounding cells remained almost unchanged. Such fluorescence quenching is likely ascribed to the intramolecular fluorescence resonance energy transfer of BMBT, due to the intensified short-wavelength absorption band (270–450 nm) of the closed-open form of BMBT with the blue emission band (420–560 nm).³⁴ Upon irradiation with 405 nm light (1.25 mW) the fluorescence of all the selected cell was rapidly recovered within 1 min, caused by decrease of the relative intensity of the 270–450 nm absorption. The fluorescence can be repeatedly erased and recovered many rounds without significant fluorescence quenching, which was hardly achieved by conventional fluorophores (Fig. 3b).^35,36Open in a separate windowFig. 3(a) CLSM image (above) and the overlay image (bottom) of fixed HeLa cells incubated with 20 μM BMBT for 30 min at 37 °C (1) and (5) in original state; (2) and (6) irradiated by 488 nm light (0.5 mW) for a single cell; (3) and (7) all cells, and; (4) and (8) recovered by 405 nm light (1.25 mW). (b) Fluorescence switching of fixed HeLa cells by alternating UV (405 nm, 1.25 mW, 10 s/time) and visible (488 nm, 0.5 mW, 3 min/time) light illumination (λ_ex = 405 nm).This characteristic of selectively opto-marking or de-marking of cells may be used for non-invasive and dynamic tracing of the interested objects in vitro. That is, BMBT can arbitrarily opto-label or de-label interested cells without affecting cell proliferation. As shown in Fig. 4, the cell marked in the red circle was treated with visible light illumination (488 nm, 0.5 mW, 3 min), and its fluorescence was effectively quenched. Cells division of the remaining “bright” cells was observed under the microscope field of vision for a long-term tracing with time up to 36 h. Multiple new cells were produced, as indicated by upper white arrow. Even, as indicated by bottom white arrow, the tacked cells were observed to be doubled. The high brightness of the fluorescence in the proliferated cells indicated the good photo stability of BMBT. This indicated that BMBT can be used as a cell marker for arbitrarily selective erasing the fluorescence of the designated cell. It can also be used for selective lighting their emission by making them as the remaining bright cells after selective photo-erasing or selective photo-recovering after full erasing. This long-term tracking with non-labeling and selective optically marking or de-marking is seldom reported by other photochromophore-based bioimaging agents.³⁷ Herein, the excellent anti-photo bleaching characteristic in the long-term tracing is attributed to high photo stability of the HBT moiety.^26,27Open in a separate windowFig. 4Cells was treated with visible (488 nm, 0.5 mW, 3 min) light illumination for erasing the fluorescence. The remaining bright cells were incubated for another 36 hours, which were observed to be amplified normally (λ_ex = 405 nm).     

2′-Deoxy-5-(hydroxymethyl)cytidine: estimation in human cancer cells with a simple chemosensor

A pyrrole-based rhodamine conjugate (CS-1) has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells with a simple chemosensing method.

A new chemosensor, CS-1, has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells.

2′-Deoxy-5-(hydroxymethyl)cytidine (5hmC) is found in both neuronal cells and embryonic stem cells. It is a modified pyrimidine and used to quantify DNA hydroxymethylation levels in biological samples^1–3 as it is capable of producing interstrand cross-links in double-stranded DNA. It is produced through an enzymatic pathway carried out by the Ten-Eleven Translocation (TET1, TET2, TET3) enzymes, iron and 2-oxoglutarate dependent dioxygenase.^4–7 In the DNA demethylation process, methylcytosine is converted to cytosine and generates 5hmC as an intermediate in the first step of this process which is then further oxidized to 5-formylcytosine (fC) and 5-carboxycytosine (caC) of very low levels compared to the cytosine level.⁸ Though the biological function of 5hmC in the mammalian genome is still not revealed, the presence of a hydroxymethyl group can regulate gene expression (switch ON & OFF). Reports say that in artificial DNA 5hmC is converted to unmodified cytosine when introduced into mammalian cells.^9,10Levels of 5hmC substantially vary in different tissues and cells. It is found to be highest in the brain, particularly in nervous system and in moderate percentage in liver, colon, rectum and kidney tissues, whereas it is relatively low in lung and very low in breast and placenta.^11,12 The percentage of 5hmC content is much less in cancer and tumor tissues compared to the healthy ones. The reason behind this loss is the absence of TET1, TET2, TET3, IDH1, or IDH2 mutations in most of the human cancer cells which means decrease of methylcytosine oxidation.^13–15 This loss of 5hmC in cancer cells is being used as a diagnostic tool for the detection of early-stage of malignant disease. Few analytical methods^16–19 such as glucosyltransferase assays, tungsten-based oxidation systems, and TET-assisted bisulfite sequencing (TAB-Seq) or oxidative bisulfite sequencing (oxBS-Seq) protocols are now developed to differentiate 5hmC from other nucleotide which are naturally occurred. There are also few methods such as liquid chromatography/tandem mass spectroscopy (LC/MS-MS), which determine the level of 5hmC in mammalian cancer cell.^20–22 However, these procedures are highly toxic and expensive due to requirement of catalyzation through enzymes or heavy metal ion and these techniques require expertise, facilities, much time and costs even beyond standard DNA sequencing. As a result, these detection techniques are currently inappropriate for the high-throughput screening of genome-wide 5hmC levels (performance comparison is shown in Table S1, ESI^†).Among all reputed methods fluorescence detection method using chemosensors is significantly important due to its indispensable role in medicinal and biological applications.^23–27 Chemosensors have been effectively explored to monitor biochemical processes and assays through in situ analysis in living systems and abiotic samples with much less time and cost.In this contribution we prepared and characterize (Scheme S1 and Fig. S1–S3, ESI^†) a pyrrole–rhodamine based chemosensor (CS-1) which shows efficient and selective fluorescence signal for 5hmC in aqueous medium (Scheme 1). A transparent single crystal of CS-1 (Fig. 1) was obtained by slow evaporation of the solvent from a solution of CS-1 in CH₃CN. It crystallizes as monoclinic with space group P2₁/n (Fig. S4 and Table S2, ESI^†).Open in a separate windowScheme 15hmC-induced FRET OFF–ON mechanism of the chemosensor CS-1.Open in a separate windowFig. 1ORTEP diagram of CS-1 (ellipsoids are drawn at 40% probability level).Spectrophotometric and spectrofluorimetric titrations were carried out to understand the CS-1–5hmC interaction with 1 : 1 binding stoichiometry (Fig. S5, ESI^†) upon adding varying concentrations of 5hmC to a fixed concentration of CS-1 (1 μM) in aqueous medium at neutral pH. Upon the addition of increasing concentrations of the 5hmC, a clear absorption band (K_a = 4.47 × 10⁵ M⁻¹, Fig. S6, ESI^†) appeared to be centered at 556 nm with increasing intensity (Fig. 2a). On the other hand, for the fluorescence emission spectra of CS-1 (Fig. 2b), upon irradiation at 325 nm, an emission maxima at 390 nm was observed, which was attributed to the fluorescence emission from the donor unit i.e. the pyrrole moiety of CS-1. When 5hmC were added, due to rhodamine moiety CS-1 showed a 95-fold increase in fluorescence at 565 nm (K_a = 4.61 × 10⁵ M⁻¹, Fig. S7, ESI^†) with the detection limit of 8 nM (Fig. S8, ESI^†). The binding of 5hmC induces opening of the spirolactam ring in CS-1, inducing a shift of the emission spectrum. Subsequently, increased overlap between the emission of the energy-donor (pyrrole) and the absorption of the energy-acceptor (rhodamine) greatly enhances the intramolecular FRET process,^28,29 producing an emission from the energy acceptor unit in CS-1.Open in a separate windowFig. 2(a) UV-vis absorption spectra of CS-1 (1 μM) upon gradual addition of 5hmC up to 1.2 equiv. in H₂O–CH₃CN (15 : 1, v/v) at neutral pH. (b) Fluorescence emission spectra of CS-1 (1 μM) upon addition of 1.2 equiv. of 5hmC in H₂O–CH₃CN (15 : 1, v/v) at neutral pH (λ_ex = 325 nm).In order to establish the sensing selectivity of the chemosensor CS-1, parallel experimentations were carried out with other pyrimidine/purine derivatives such as 5-methylcytosine, cytosine, cytidine, thymine, uracil, 5-hydroxymethyluracil, adenine and guanine. Comparing with other pyrimidine/purine derivatives the abrupt fluorescence enhancement was found upon addition of 5hmC to CS-1 while others do not make any fluorescence changes under UV lamp (Fig. 3, lower panel). Furthermore, the prominent color change from colorless to deep pink allows 5hmC to be detected by naked eye (Fig. 3, upper panel). The above observation shows consistency with the fluorescence titration experiments where no such binding of CS-1 with other pyrimidine/purine derivatives was found (Fig. S9, ESI^†).Open in a separate windowFig. 3Visible color (top) and fluorescence changes (bottom) of CS-1 (1 μM) in aqueous medium upon addition of 1.2 equiv. of various pyrimidine/purine derivatives (λ_ex = 325 nm) in H₂O–CH₃CN (15 : 1, v/v) at neutral pH.pH titration reveals that CS-1 becomes fluorescent below pH 5 due to the spirolactam ring opening of rhodamine. However, it is non-fluorescent at pH range of 5–13. Upon addition of 5hmC to CS-1 shows deep red fluorescence in the pH range of 5–8 (Fig. S10, ESI^†). Considering the biological application and the practical applicability of the chemosensor pH 7.4 has been preferred to accomplish all experiments successfully.In ¹H NMR titration (Fig. S11, ESI^†), the most interesting feature is the continuous downfield shift of aromatic protons on the pyrrole moiety of CS-1 upon gradual addition of 5hmC. This may be explained as the decrease in electron density of the pyrrole moiety upon binding with 5hmC through hydrogen bonding. Xanthene protons to be shifted downfield upon spirolactam ring opening indicates the probe to coordinate with 5hmC and electrons are accumulated around 5hmC. In ¹³C NMR titration the spiro cycle carbon peak at 65 ppm was shifted to 138 ppm along with a little downfield shift of the aromatic region of CS-1 (Fig. S12, ESI^†). This coordination led to the spiro cycle opening and changes to the absorption and emission spectra, further evident by mass spectrometry (Fig. S13, ESI^†), which corroborates the stronger interaction of CS-1 with 5hmC.The experimental findings were validated by density functional theory (DFT) calculations using the 6-31G+(d,p) method basis set implemented at Gaussian 09 program. Energy optimization calculations presented the conformational changes at the spirolactam position of CS-1 while 5hmC takes part to accommodate a probe molecule. After CS-1–5hmC complexation the energy is minimized by 19.45 kcal from the chemosensor CS-1, indicating a stable complex structure (Fig. 4 and Table S3, ESI^†). This theoretical study strongly correlates the experimental findings.Open in a separate windowFig. 4Energy diagram showing the energy differences between CS-1 and CS-1–5hmC complex.The desirable features of CS-1 such as high sensitivity and high selectivity at physiological pH encouraged us to further evaluate the potential of the chemosensor for imaging 5hmC in live cells (Fig. 5). A549 cells (Human cancer cell A549, ATCC no. CCL-185) treated with CS-1 (1 μM) exhibited weak fluorescence, whereas a deep red fluorescence signal was observed in the cells stained with CS-1 (1 μM) and 5hmC (10 μM), which is in good agreement with the FRET OFF–ON profile of the chemosensor CS-1 in presence of 5hmC, thus corroborating the in-solution observation (Fig. S14, ESI^†). Cytotoxicity assay measurement shows that the chemosensor CS-1 does not have any toxicity on the tested cells and CS-1–5hmC complex does not exert any significant adverse effect on cell viability at tested concentrations (Fig. S15, ESI^†). As far as we are aware, this is the first report where we are executing the possible use of the pyrrole–rhodamine based chemosensor for selective recognition of 5hmC in living cells. These findings open an avenue for future biomedical applications of the chemosensor to recognize 5hmC.Open in a separate windowFig. 5Confocal microscopic images of A549 cells treated with CS-1 and 5hmC. (a) Cells treated with only CS-1 at 1 μM concentration. (b) Bright field image of (a). (c) Cells treated with CS-1 and 5hmC at concentration 10 μM. (d) Bright field image of (c). All images were acquired with a 60× objective lens with the applied wavelengths: For (a) and (b), E_ex = 341 nm, E_em = 414 nm, filter used: DIDS; for (c) and (d) E_ex = 550 nm, E_em = 571 nm, filter used: Rhod-2.The concentration of 5hmC was also quantified from A549 human cancer cells. Lysate of 10⁷ A549 cells was added to 1 μM of CS-1 and the fluorescence signal was recorded. Presence of 5hmC in these cancer cells was detected with the help of CS-1–5hmC standard fluorescence curve (Fig. 6) using the selective detection ability of the chemosensor CS-1.Open in a separate windowFig. 6(a) Calibration curve obtained for the estimation of 5hmC. (b) Estimation of the concentration of 5hmC (red point) from the calibration curve.From the standard curve it was found that the concentration of 5hmC in the tested sample was 0.034 μM present in 16.7 mm³ A549 cell volume (†). Assay of 5hmC was further validated from multiple samples of A549 human cancer cells using CS-1. Increasing fold of fluorescence signals was also statistically validated after calculating the Z′ value (Table S5, ESI^†). All tested samples shows the Z′ score value more than 0.9, indicating an optimized and validated assay of 5hmC.Quantification of 5hmC in human cancer cell A549

A fluorescent calix[4]arene with naphthalene units at the upper rim exhibits long fluorescence emission lifetime without fluorescence quenching

We synthesised a new compound with four naphthyl groups in the upper rims of calix[4]arene (1). Compared to the monomer unit, compound 1 has redshifted absorption and fluorescence, together with high fluorescence quantum yield and long fluorescence lifetime, which is extremely rare because long fluorescence lifetime emission tends to reduce the quantum yield. Single-crystal X-ray analysis and quantum calculations in the S₁ state revealed π–π through-space interactions between naphthalene rings.

Macrocyclic structure brings long fluorescence lifetime emission without fluorescence quenching and TD-DFT calculations revealed π–π interactions between the naphthalene rings.

In 1954, Förster and Kasper first reported dimerised aromatic compounds in the excited state,¹ which were termed ‘excimers’ by Stevens and Hutton.² Excimers have attracted much attention in various fields such as organic solar cells,^3–5 organic electronics,⁶ chemical sensors,⁷ and biotechnology^8–12 because of their unique photophysical properties. In particular, with the development of time-resolved fluorescence imaging and stimulated emission depletion microscopy (STED) microscopes, there is a growing demand for fluorescent dyes with both high brightness and long fluorescence lifetime.^9–12 Excimers with long emission lifetime are promising candidates for next-generation imaging probes.¹³Many progresses have been made towards understanding the relationship between the molecular structure of organic dyes and their fluorescence intensity. However, there is little knowledge on the relationship between the fluorescence lifetime and molecular structure. Also, only a few fluorescent probes have achieved both high intensity and long lifetime.^13–15 In recent years, it was reported that bright and long-lived fluorescence can be obtained from the excimer state^13,16–19 that was conventionally thought to be the cause of quenching due to the low-energy excimer trap states with forbidden radiative transition and activated non-radiative process.^20–24 However, most of these reports were in the solid state, where the molecular movement is suppressed and the luminescence occurs in a single crystal.^16–19 In contrast, there are few reports of dyes with long fluorescent lifetime in solution systems with free molecular movement. The reported substances also suffer from low synthesis yields in the ring-forming reaction and difficulty in introducing functional groups (such as hydrophilic substituents).¹³ In general, the yield of the ring formation step is extremely low in the synthesis of cyclophanes.^13,25–27 Therefore, there remains the need for new molecules that can be easily synthesised and chemically modified.In this study, we adopted the calixarene skeleton as the macrocyclic structure. Calixarenes have been used in supramolecular chemistry,²⁸ analytical chemistry,²⁹ biochemistry,³⁰ material science,³¹ and catalysts³² because of their easy molecular modification. Nevertheless, almost all studies introducing fluorescence sites do so at the lower rim of calixarene, while few reports considered incorporating fluorophores at the upper rim. Further, no researchers have investigated the fluorescence lifetime, and there was also no reported computational investigation of the excited state.^33–35Specifically, we synthesised a fluorescent molecule in which naphthyl group was introduced into the upper rim of calix[4]arene. The new molecule showed both a long fluorescence wavelength and a high quantum yield. After measuring the conformation of tetranaphthylcalix[4]arene in a single crystal, structural optimisation of the ground state and excited state (S₁) was performed by time-dependent density functional theory (TD-DFT) at the DFT-D3-CAM-B3LYP/6-31G(d) level. The calculation takes into account effects such as dispersion force.^36–39 Molecular orbital calculation confirmed that the π orbitals of the naphthalene rings of tetranaphthylcalixarene have a binding interactions in LUMO of the S₁ state.The synthesis of tetranaphthylcalix[4]arene (1) was carried out by deprotecting the tert-Bu groups of tetra-tert-butyl(tetrahydroxy)calix[4]arene, followed by introduction of substituents by Williamson ether synthesis of phenolic hydroxyl groups, and iodation by silver tetrafluoroacetate and iodine. The iodination was followed by Suzuki–Miyaura cross-coupling reaction with naphthylboronic acid pinacol ether (ESI^†).The target substance was characterised by ¹H NMR, ¹³C NMR, HRMS of ESI-TOF-MS, and single-crystal X-ray diffraction (Fig. S7–S9 and Table S3, ESI^†).We also synthesised the phenylnaphthalene derivative 2 (Fig. 1), which is the unit molecule of 1. To compare its photophysical properties with that of 1, first we measured the absorption spectra. At 1 × 10⁻⁴ mol L⁻¹ in chloroform solution, 1 and 2 have their maximum absorption wavelengths at 297 and 294 nm, and the molar absorption coefficients were 3.3 × 10⁵ and 1.0 × 10⁵ mol⁻¹ L cm⁻¹, respectively (Fig. 2). Their fluorescence spectra were measured in chloroform solution at 1 × 10⁻⁴ mol L⁻¹ (Fig. 3). 1 has a broader fluorescence peak with maximum intensity at 389 nm, which is redshifted by 25 nm from that of 2. Furthermore, the fluorescence spectrum of the powder after grinding in a mortar was also measured and it was found that the fluorescence wavelength was increased by 22 nm (Fig. S14, ESI^†). We also measured the absorption and fluorescence spectra at a lower concentration of 1 × 10⁻⁵ mol L⁻¹ (Fig. S15, ESI^†), and there was almost no change in the wavelength or shape of the peak. Therefore, the spectral changes in tetranaphthylcalix[4]arene from the unit model molecule are due to intramolecular rather than intermolecular interactions. Furthermore, the temperature dependence of fluorescence was investigated by measuring fluorescence by changing the temperature from 20 °C to 80 °C (Fig. S16^†). As the temperature rose from 20 °C to 80 °C, a blue shift of the fluorescence wavelength of about 10 nm was observed, and the half-value width of the peak narrowed. These results indicate that as the temperature rises, the intramolecular interaction in the excited state weakens and the light emission becomes closer to that of 2.Open in a separate windowFig. 1Molecular structures of tetranaphthylcalix[4]arene 1 and the unit model molecule 2.Open in a separate windowFig. 2Absorption spectra of tetranaphthylcalix[4]arene 1 and the unit model molecule 2. Both spectra were measured in chloroform at 1 × 10⁻⁴ mol L⁻¹, and normalised to the maximum absorption in the longer wavelength region.Open in a separate windowFig. 3Fluorescence spectra of 1 and 2. Both spectra were measured in chloroform solution at 1 × 10⁻⁴ mol L⁻¹, and normalised by the maximum intensity. 1: λ_ex = 310.5 nm, 2: λ_ex = 326.5 nm.Single-crystal X-ray crystal structure analysis of 1 revealed that two of the four naphthyl groups facing each other had an intramolecular stacking structure, with a distance of 3.54 Å between them (Fig. 4 and Table S3, ESI^†).Open in a separate windowFig. 4(a) Side and (b) top views of the structure of 1 determined by single-crystal X-ray crystallographic analysis. The structures were drawn by ORTEP program with the thermal ellipsoids set at 50% probability.The macrocyclic structure improved the fluorescence quantum yield from 0.38 in 2 to 0.46 in 1 († and 11,40 azadioxatriangulenium (ADOTA⁺) (25 ns),^14,41 and SeTau425 NHS (26.2 ns).¹⁵ To investigate this characteristic property, the fluorescence emission rate constant k_f and the nonradiative decay rate constant k_nr were determined by the following equation:1where Φ_f is the fluorescence quantum yield and τ_f is the fluorescence lifetime. The values are summarized in †). First, the structure of the ground state was optimised. The distance of the naphthalene rings facing each other, which is the focus of this study, was slightly smaller in the crystalline state (3.59 Å) compared to 3.49 Å of the ground state structure. Similar calculation was carried out for the ground state structure of 2. Then, the absorption spectra of both compounds were predicted by TD-DFT calculations. The absorption wavelength of 1 is longer, which qualitatively agrees with the experimental results (Fig. 2, S18, Tables S1 and S2, ESI^†). Thus, it was possible to explain the change in absorption spectrum by calculating the molecular orbitals. In particular, for the first two excited states S₁ and S₂, the main constituent orbitals are HOMO to LUMO+2 and HOMO to LUMO+3 (Table S1, ESI^†). These results indicate that the redshift of absorption in 1 involves the orbital of the intramolecular naphthalene units facing the molecule (Fig. S19, ESI^†). Furthermore, structural optimisation of the S₁ state was performed for compound 1 using the same level of theory and basis functions. The results further reduced the interplanar distance of naphthalene rings in the S₁ state to 3.18 Å (Fig. 5a). In the frontier orbitals calculated for the S₁ geometry, there were clear binding interactions between the naphthalene rings according to the LUMO (Fig. 5b and S20, ESI^†) and a small oscillator strength value (0.0019) of the HOMO ← LUMO transition in agreement with the lower value of k_f compared with that of 2. An oscillator strength value of HOMO ← LUMO transition of 2 with the optimized structure in S₁ state is 0.700. Therefore, the reason why 1 has both a high fluorescence quantum yield and a long fluorescence lifetime is that a decrease in k_nr due to suppression of molecular motion by a rigid macrocyclic structure contributes more significantly to the fluorescence enhancement than the decrease in k_f by intramolecular electronic interaction in the excited state. Subsequently, our group has been developing new imaging dyes using this molecular skeleton having fluorescence sites that have a long conjugation length and therefore can be excited by visible light.Fluorescence quantum yields, lifetimes, and kinetic constants of 1 and 2 at 390 nm in THF (λ_ex = 340 nm)

An aluminium fluorosensor for the early detection of micro-level alcoholate corrosion

The detection of the dry alcoholate corrosion of aluminium is vital to design a corrosion resistive aluminium alloy for the storage and transportation of biofuel (methanol or ethanol). By synthesizing an Al³⁺ fluorescent probe operable in an alcoholic medium, we quantified the alcoholate corrosion in terms of the fluorometrically estimated soluble alkoxide (Al(OR)₃) generation under nitrogen atmosphere. With time, a linear increase in corrosion with specific aluminium dissolution rate constants ∼2.0 and 0.9 μg per day per cm² were estimated for aluminium and Al-7075 alloy, respectively. During open atmosphere monitoring, the adsorbed moisture converted small extent of Al(OR)₃ to the insoluble Al(OH)₃ at the alloy surface which retarded the alcoholate corrosion appreciably.

The monitoring of aluminium alcoholate corrosion using a fluorescent probe operable in alcohol.

Switching over from conventional fossil fuel to biofuel is of current interest owing to the maximum utilization of eco-friendly non-conventional energy.¹ Commercially produced less polluted biofuels such as methanol and ethanol, mixed with fossil fuels have an acceptable performance capacity for the gasoline engine.² Moreover, in comparison to the gasoline, methanol and ethanol have much higher octane rating or compression ratio to resist the knocking for better thermal efficiency.³ Since most of the fuel tanks/pipes are made of aluminium or its alloys owing to its high strength-to-density ratio, the aluminium corrosion due to the formation of alkoxide (alcoholate or dry corrosion) during storage or even transportation of such bio-alcohols may cause leakage in the fuel tanks and in worst cases enough threat is speculated for fire and explosion.⁴ Mechanical overloads, alloy impurities even at elevated temperatures are further contenders for accelerating the alcoholate corrosion.⁵ However, a prolong exposure to the moisture retards the alcoholate corrosion by forming a protective layer of hydrated aluminium oxide in the metallic surface but moisture impurity in the fuel may damage the gasoline engine.⁶ Hence, a maintenance optimization is crucial in critical engineering disasters by detecting alcoholate corrosion as in its nascent state with minimizing the chance of water contamination.^6,7Several electrochemical and mechanical methods have been exploited for decades to propose aluminium alcoholate and other corrosions;⁶ yet the early detection of the alcoholate corrosion is still a challenging task due to the lack of sensitive analytical methods.^6,8 Here, the fluorescence technique may act as a better alternative owing to its simplicity and high sensitivity.⁹ Till date, a large number of fluorescent probes for Al³⁺ have been exploited in the biological or environmental domain,¹⁰ but has never focused on alcoholate corrosion studies. Based on this requirement, we synthesized a fluorescent probe, namely HMBDC ((6Z)-6-(2-hydroxy-3-(hydroxymethyl)-5-methylbenzylideneamine)-2H-chromen-2-one), to detect alcoholate corrosion with μg-level detection ability along with its retarding signature in the presence of moisture in a judicious way. Such novel method may lead to an early detection of alcoholate corrosion in a simpler way.The non-fluorescent phenolic Schiff-base molecule containing a coumarin moiety (HMBDC) was prepared by condensing an equimolar mixture of 6-amino coumarin (6-ACO) and 2-hydroxy-3-(hydroxymethyl)-5-methylbenzaldehyde (HHMB) in dry ethanol (Scheme 1 and Fig. S1^†) (c.f. ESI^† for details). Among various organic solvents, the interaction of HMBDC with Al³⁺ was observed only in the alcoholic medium according to the UV-vis studies (Fig. S2^†). In methanol, the absorption intensity at ∼353 nm for HMBDC (5 μM) decreased gradually with the continuous addition of Al(NO₃)₃ until saturated at ∼8-equiv., giving rise to a new peak at ∼406 nm, where an isosbestic point at ∼384 nm assures the formation of Al³⁺/HMBDC complex (1) (Fig. 1A). Upon optimization of the complex formation affinity in various ethanol/methanol mixed media, highest reactivity with the lowest saturated Al³⁺ concentration (∼5 equiv.) compared to that obtained in pure methanol was observed in a 4 : 1 methanol/ethanol-mixed medium (Fig. S3^†). Most probably, more effective H-bonding interaction of the dimeric ethanol/methanol¹¹ with 1 induces greater complex (1) stability, although the complex formation reactivity was much less in pure ethanol compared to the methanol medium (Fig. 1 and S2^†).Open in a separate windowScheme 1Synthesis of HMBDC and its complexation with Al³⁺ in an alcohol solvent.Open in a separate windowFig. 1(A) UV-vis absorption and (B) fluorescence spectra of HMBDC (5 μM) in the presence (red) and absence (black) of increasing concentration of Al(NO₃)₃ (0–40 μM) in anhydrous methanol at 25 °C. The intensity changes with increasing Al³⁺ concentrations are indicated by arrows. (C) Al³⁺ concentration dependent relative increase in the fluorescence intensity with respect to its absence in methanol (red) or methanol/ethanol (4 : 1) (blue). (D) Fluorescence intensity ratios in the presence and absence of various ions or mixture of ions in the mixed solvent (25 μM each; blue) or methanol (40 μM each; other colors) or are shown by bar-diagram.In spite of the stronger H-bonding interactions of 1 with water compared to the methanol or ethanol, a complete dissociation of 1 in the presence of 20% (v/v) water in methanol (Fig. S4^†) suggests that, in addition to the solvent assisted H-bonded structural stability of 1, alcohol molecule may also participate in the coordination with Al³⁺ to form 1. Indeed, the possible methanol coordination is reflected in the ESI-MS⁺ analysis (Fig. S5B^†). In addition, the Job''s plots in the absorption studies showed that the HMBDC formed 1 : 1 stoichiometric complex with Al³⁺ (Fig. S6^†). To elucidate the probable structure of 1, we carried out the DFT-based theoretical calculation by considering the 1 : 1 stoichiometric Al³⁺/HMBDC complex with or without methanol coordination. A stable structure of 1 was obtained when the oxygen atom of the methanol molecule coordinates with Al³⁺ and other two coordination sites of Al³⁺ are occupied by the phenolic-oxygen and imine-nitrogen of HMBDC (Scheme 1, Fig. 2 and S7^†). Facile coordination of those hard donor sites of HMBDC towards harder Al³⁺ is susceptible towards alcohol assisted stabilization of 1. The UV-vis absorbance at ∼402 nm for 1 computed from the time-dependent DFT (TD-DFT) calculations in methanol medium, where the HOMO (90) → LUMO (92) excitation nicely matched with the experimental absorbance at ∼406 nm (Fig. 1 and ​and2).2). However, monitoring of the ¹H-NMR peak characterized for aldimine proton is a useful strategy to identify the bonding of the imine-N to Al³⁺.¹² We observed that the aldimine proton peak intensity for HMBDC in CD₃OD was quenched to a great extent with a considerable down-field shift from 8.80 to 8.88 ppm in the presence of Al³⁺ (Fig. S8^†); the down-field shift is expected owing to the imine-N and Al³⁺ coordination, but intensity quenching does not follow the previous trend in the aprotic polar medium.¹² The generation of a partial positive charge at the N-centre upon its binding with the Al³⁺ may enhance the acidity of the aldimine proton to become labile for participating in the H/D exchange in a protic medium (CD₃OD), as reported previously for other allied systems.¹³ These results strongly suggest the imine-N and Al³⁺ bonding in 1. On the other hand, Al³⁺ induced large decrease in the IR intensity at ∼3300 cm⁻¹ for phenolic-OH also supports the phenoxide coordination (Fig. S9^†).Open in a separate windowFig. 2Frontier molecular orbital profiles including various UV-vis absorption parameters of HMBDC (left panel) and HMBDC/Al³⁺ complex (right panel) based on TD-DFT (B3LYP/6-31G(d)).The electronic distribution in the molecular orbital diagram (MO) of the HMBDC evaluated from the DFT calculation showed an intra-molecular photo-induced electron transfer (PET) from coumarin to the HHMB moiety, which makes the HMBDC non-fluorescent (Fig. 2). Al³⁺ induced an instantaneous increase in the fluorescence intensity for HMBDC (5 μM) in the alcoholic medium (methanol/ethanol or their mixture) due to the formation of 1 (Fig. 1B and S10^†). A gradual fluorescence intensity increase at ∼506 nm (λ_ex = 406 nm) of ∼30-fold for 8 equiv. of Al³⁺ and ∼40-fold for 5 equiv. of Al³⁺ was observed in the methanol and 4 : 1 (v/v) methanol/ethanol medium, respectively (Fig. 1 and S10B^†). According to the HOMO and LUMO electronic distributions for 1 in the DFT studies, the PET process in HMBDC was highly restricted upon its binding with Al³⁺ in 1, causing for the large increase in the fluorescence intensity (Fig. 2). However, the better fluorescence response (lower intensity-saturated Al³⁺ concentration and larger intensity increase) in the mixed medium than pure methanol may be associated with greater stability of 1, as described in the previous section (Fig. S2^†). The fluorescence intensity increase remains invariant using other soluble Al(iii)-salts (Fig. 1D and S11^†), which eliminates the role of counter anions for the increasing intensity. To ascertain the Al³⁺ selectivity, we performed similar fluorescence studies with other potentially interfering cations but failed to produce any noticeable fluorescence (Fig. 1D and S12^†). However, a linear intensity increase with the increase in the concentration of Al³⁺ up to 6 equiv. in methanol and 4 equiv. in the 4 : 1 methanol/ethanol mixed medium can be useful for a ratiometric detection of unknown concentration of Al³⁺ (Fig. 1C), where the limit of detection¹⁴ (LOD) of Al³⁺ with HMBDC in the methanol medium was found to be ∼0.5 μM (c.f. details in ESI^†). Most importantly, HMBDC recognized Al³⁺ selectively from the mixture of various other cations, and also in presence of other soluble Al(iii) salts, particularly, aluminium alkoxide (ethoxide) with similar accuracy (Fig. 1D and S12^†). Therefore, the Al(iii) sensing ability for an alcoholate corrosion with an aluminium alloy must not be perturbed due to the interference of other leached cations.The dry alcoholate corrosion of aluminium or its alloy while forming soluble alkoxide (Al(OR)₃) can be detected upon incubation in an anhydrous alcoholic medium. However, under a condition of prolonged incubation, the contamination of trace amounts of moisture may also trigger the conversion of Al(OR)₃ to Al(OH)₃, followed by the hydrated alumina (Al₂O₃·xH₂O) coating on the metallic surface.⁶ The formation of hydrated alumina can also be possible via the decomposition of Al(OR)₃.⁶ To characterize the alcoholate corrosion as an exclusive process to the maximum limit, we minimized those wet-processes by allowing the corrosion under inert conditions. A previously grazed aluminium-sheet (dimension ∼3.5 × 1.5 × 0.2 cm³; surface area ∼12.5 cm²) was incubated for 18 days in 100 mL anhydrous methanol or methanol/ethanol (4 : 1) mixed solvent under nitrogen atmosphere by purging nitrogen every 24 h, where the small change in the solution volume if required was adjusted by injecting an appropriate amount of the nitrogen-saturated anhydrous solvent. The amount of Al(OR)₃ (R = -Me, -Et) generated in the medium was estimated by monitoring the HMBDC (5 μM) fluorescence. After 10-fold dilution of the medium with the parent solvent, the amount of Al(OR)₃ formed or the alcoholate corrosion was estimated in every 3 days interval according to the amount of Al³⁺ obtained from the time-dependent fluorescence responses (Fig. S13^†) as per the linear calibration plots in Fig. 1C multiplied by the dilution factor. A linear increase in the normalized fluorescence intensity from ∼3.5 to 16.8 and ∼7.3 to 36.1 was observed with an increase in the incubation time period from 3 day to 18 day for methanol and methanol/ethanol (4 : 1) media (Fig. 3A and S13^†), respectively, which correspond to the linear increase in the Al³⁺ amount in the medium from ∼3.2 to 16.6 μmol for either solvents (Fig. 3C). Indeed, the weight-loss of ∼0.47 mg i.e., ∼17.5 μmol was found to be closely similar with that of the increase in Al³⁺, revealing that not only the dry corrosion leads to the generation of Al³⁺ (Al(OR)₃) as the only product, but also HMBDC is highly effective for an accurate estimation of the alcoholate corrosion. In addition, the nice correlation between the weight-loss and Al(OR)₃ amount also reveals that the decomposition of alkoxide into insoluble alumina is negligibly small during the whole corrosion time-course.Open in a separate windowFig. 3(A and B) Extent of the fluorescence intensity increase due to corrosion-induced leached Al³⁺ (F(x)/F(0)) of HMBDC (5 μM) and (C and D) amount of Al³⁺ in the corrosion medium according to fluorescence response are plotted with various incubation times of pure aluminium sheet or its alloy (Al-7075) in different mediums/atmosphere conditions: nitrogen atmosphere in methanol (red) and methanol/ethanol (4 : 1) (blue); open atmosphere in methanol (green) and methanol/ethanol (4 : 1) (purple). The data at nitrogen conditions are only fitted linearly. (A and B) The fluorescence intensity of HMBDC (5 μM) were monitored after the 10-fold dilution of the corrosion medium with the same solvent. (C and D) The amount of Al³⁺ estimated as the amount obtained from the normalized intensity with comparing the linear plots in Fig. 1C multiplied by the dilution factor 10. The actual amount of alcoholate corrosions for the mixed medium under open atmosphere are depicted by solid circle (purple).However, under open atmospheric conditions maintained by air purging (average relative humidity ∼70%; average temperature 28 °C) in every 24 h interval while maintaining other similar experimental conditions and analysis protocol, the specific corrosion rate (∼2.0 μg per day per cm²) up to 12 days, was found to be closely similar to that detected under the nitrogen atmosphere (Fig. 3C and S13^†). The results also indicate that the early stage of the alcoholate corrosion process (at least up to 12 days) for pure aluminium is not affected significantly by the atmospheric moisture content, although the final corrosion amount after 18 days incubation in normal atmosphere was slightly lower (∼84%) for the mixed medium compared to that obtained for pure methanol (Fig. 3C). The decrease in the Al(OR)₃ amount can be affected by two processes: (a) Al(OR)₃ to insoluble Al(OH)₃ conversion due to the adsorbed moisture; (b) actual retardation of the corrosion rate due to the surface deposition of Al(OH)₃. The extent of the conversion of Al(OR)₃ to Al(OH)₃ in the corrosion medium under the open air condition can be assessed by estimating the fluorescence intensity at every 3 day time interval in the absence of aluminium sheet (from day-3 to day-18) with the addition of same amount of Al(OEt)₃ (3.2, 5.7, 8.0, 11.7, 14.2 and 16.4 μmol (final added amount) at day 0 (beginning of day 1), 3, 9, 12 and 15, respectively, in 100 mL mixed medium) as that of the alkoxide amount detected due to the corrosion under nitrogen condition (Fig. S14^†). In comparison to the actual added Al(OEt)₃, any decrease in the Al(OEt)₃ amount upon such incubation should be added with the corrosion induced formation of Al(OR)₃ amount under nitrogen condition for respective time interval to obtain the actual alcoholate corrosion. The actual corrosion was found to be slightly higher than that estimated from the corrosion-induced Al(OR)₃ formation (Fig. 3C, solid symbol). According to the LOD of Al³⁺, the detection of the alcoholate corrosion amount as minimum as ∼0.1 μg mL⁻¹ can be possible by monitoring the fluorescence response of HMBDC.Alcoholate corrosion in a widely used aluminium alloy, Al-7075 (composition: Al, 90%; Zn, 5.5%; Mg, 2.5%; Cu, 1.5 and Si, 0.5%) was also studied. The previously grazed alloy sheet with same dimension and surface area as that of the pure aluminium sheet was incubated in 100 mL anhydrous methanol or 4 : 1 methanol/ethanol under nitrogen as well as normal atmospheric conditions. The amount of the alcoholate corrosion in every 3 days interval up to 30 days was estimated by evaluating the fluorescence response of HMBDC (Fig. 3B and S15^†). In comparison to the pure aluminium sheet, the increase in corrosion from ∼1.5 to 4.0 μmol evaluated from the increase in the normalized fluorescence intensity (1.65 to 5.90 in methanol; 2.64 to 10.40 in methanol/ethanol (4 : 1) mixture) with the increase in the incubation time from day-3 to day-12 follows a similar linear relation regardless of the solvent compositions and atmospheric conditions (Fig. 3B and D), while the intrinsic rate of corrosion ∼0.95 μg per day per cm² was more than 2-fold slower (Fig. 3C and D). The lower rate constant value for the alloy compared to pure aluminium indicates that the contamination of other metals in the alloy resists the early stage alcoholate corrosion process. However, under normal atmospheric condition, the corrosion amount vs. time relation deviates from the linearity after 12 days. Importantly, after 30 days of incubation, a large reduction in the Al(OR)₃ amount from ∼11.38 to 6.64 μmol was estimated for the mixed medium, but the change was only from ∼13.20 to ∼12.33 μmol for pure methanol (Fig. 3D). By determining the hydration-induced conversion amount of Al(OR)₃ to Al(OH)₃ according to the procedure, as described before (Fig. S16^†), the actual alcoholate corrosion was found to decrease from ∼11.38 to 7.70 μmol by changing the condition from nitrogen to open atmosphere after 30 days (Fig. 3D, solid symbol). Our study reveals that in comparison to pure methanol, the formation of Al(OH)₃ under open atmospheric condition retards the alcoholate corrosion largely due to the presence of more hygroscopic ethanol.¹⁵ The deposition of Al₂O₃·xH₂O onto the alloy-surface is responsible for resisting the further alcoholate corrosion⁶ (Fig. 3D). In fact, the generation of more surface pits owing to the higher extent of the alcoholate corrosion in methanol over the mixed medium was also detected by naked eye (Fig. S17^†). The surface morphology in the SEM studies showed that the alloy surface was little bit smoother after the corrosion in the mixed medium (Fig. S18^†), justifying our proposition for the surface deposition of Al₂O₃·xH₂O. On the other hand, cyclic voltammetric studies in the corrosion medium exposed to normal atmospheric conditions identified an irreversible cathodic peak at ∼−0.7 V due to the formation of insoluble Al(OH)₃ in addition to the conversion from Al to Al³⁺, but such irreversible peak was not observed for the medium exposed to nitrogen (Fig. S19^†). Moreover, the formation of white gelatinous precipitate of Al(OH)₃ in the mixed medium was clearly visible by naked eye under normal atmospheric conditions (Fig. S17B^†). All those results strongly support that the initiation of the wet-process by forming Al(OH)₃ inhibits the alcoholate corrosion rate.In conclusion, a phenolic Schiff-base consisting of a coumarin unit as a fluorescent sensor for Al³⁺ operable only in the alcoholic medium is synthesized to monitor dry alcoholate corrosion. The photo-induced electron transfer process in the probe molecule exhibits Al³⁺ induced large increase of fluorescence intensity, lifted by its complexation with Al³⁺, which was further stabilized by the coordination and H-bonding interaction with the solvent molecule. The alcohol specific complex formation and subsequent fluorescence generation was suitably tuned to monitor the alcoholate corrosion by fluorometrically estimating aluminium alkoxide formation with a sensitivity of ∼10 μg L⁻¹. However, the simultaneous participation of small extent of the wet-process (Al(OR)₃ to Al(OH)₃ conversion) and its deposition in metal surface, particularly for the alloy, inhibits the dry alcoholate corrosion. The alloy specific detection of the early stage alcoholate corrosion is in progress to obtain suitable material useful as a biofuel container.     

Ultrasensitive biosensing platform based on luminescence quenching ability of fullerenol quantum dots

An ultrasensitive biosensing platform for DNA and ochratoxin A (OTA) detection is constructed based on the luminescence quenching ability of fullerenol quantum dots (FOQDs) for the first time. As the surface of FOQDs is largely covered by hydroxyl groups, stable colloidal suspension of FOQDS in aqueous solution can be obtained, which is very advantageous for application in biosensing compared to nano-C₆₀. FOQDs can effectively quench the fluorescence of dyes with different emission wavelengths that are tagged to bioprobes to an extent of more than 87% in aqueous buffer solution through a PET mechanism. Moreover, the nonspecific quenching of the fluorescent dyes (not bound to bioprobes) caused by FOQDs is negligible, so the background signal is extremely low which is beneficial for improving the detection sensitivity. Based on the π–π stacking interaction between FOQDs and bioprobes, such as single-stranded (ss) DNA and aptamers, a nucleic acid assay with a detection of limit of 15 pM and a highly sensitive OTA assay with a detection limit of 5 pg mL⁻¹ in grape juice samples are developed through the simple “mix and measure” protocol based on luminescence quenching-and-recovery. This is the first demonstration of constructing biosensors utilizing the luminescence quenching ability of FOQDs through a PET mechanism, and the pronounced assay performance implies the promising potential of FOQDs in biosensing.

An ultrasensitive DNA biosensor based on the fluorescence quenching ability of FOQDs towards FAM–ssDNA through π–π stacking interactions between ssDNA and FOQDs.

Carbon nanostructures, such as two-dimensional graphene and its derivatives,^1–5 one-dimensional carbon nanotubes (CNTs)^6–9 and zero-dimensional carbon dots^10–17 have attracted increasing attention in biosensing and biological related studies due to their unique electronic and optical properties. In the last decade, the luminescence quenching ability of carbon nanostructures has been extensively studied and various kinds of biosensing platforms have been constructed accordingly,^18–21 with which the single-stranded DNA (ssDNA) or polypeptide probes are generally assembled onto carbon nanostructures via π–π stacking interactions.^22–24 In recent years, several biosensing platforms have been constructed based on the luminescence quenching ability of nano-C₆₀ towards fluorescent dyes and the π–π stacking interaction between single-stranded DNA (ssDNA) and nano-C₆₀.^25–27 However, their relatively poor dispersibility in aqueous solution has restricted their wide use in biosensing applications. Fullerenols, also named polyhydroxylated fullerenes, are the main derivative of fullerenes with good water-solubility and excellent biocompatibility.^28–30 They have attracted great attention in recent years and can work as antioxidative agents,^31,32 free radical scavengers,^33,34 drug delivery vehicle^35,36 and so forth. Most recently, Yang et al. reported that the intrinsic fluorescence of serum proteins (bovine serum albumin (BSA) and γ-globulins) could be effectively quenched by fullerenol through a dynamic mechanism, which was used to characterize the fullerenol–protein interactions.³⁷ However, its applicability in biosensing is relatively unexplored. Inspired by the above findings, we expect that fullerenol quantum dots (FOQDs) could be used in other potential label-free nanoplatforms for homogeneous biosensing with wide applications.We herein propose a novel and effective homogeneous fluorescent biosensing platform based on the luminescence quenching ability of FOQDs towards different fluorescent dyes for the first time. As an initial trial, a ssDNA chain was used as the probe for DNA detection. As shown in Scheme 1, the FAM attached ssDNA (FAM–ssDNA) is spontaneously assembled onto FOQDs by π–π stacking interaction, resulting in the luminescence quenching of FAM caused by FOQDs. The hybridization reaction between the probe ssDNA and its complementary target ssDNA results in the formation of a double-stranded DNA (ds-DNA) with helical structure, in which conformation the exposure of nucleobases to FOQDs is greatly reduced. As a consequence, the π–π stacking interaction between FAM–ssDNA and FOQDs is largely weakened and the distance between FAM and FOQDs is enlarged. Under this circumstance, the luminescence of FAM is expected to be recovered, which is linearly related to the concentration of target ssDNA and enables the quantification of target ssDNA.Open in a separate windowScheme 1Schematic illustration of the DNA biosensor based on fluorescence quenching ability of FOQDs towards FAM–ssDNA through π–π stacking interaction between ssDNA and FOQDs.To realize the above design, the commercially available FOQDs with a diameter of 2 nm (Fig. 1A) were used to develop a label-free nanoplatform for DNA biosensing. The FT-IR spectra of FOQDs shown in Fig. 1B indicated the typical absorptions of fullerenols including an intense broad O–H band around 3400 cm⁻¹ corresponding to –C–O–H, and three characteristic bands around 1080, 1376 and 1593 cm⁻¹ assigned for C–O, C–O–H and C C absorption, respectively.^38,39 XPS results further confirmed that FOQDs had abundant hydroxyl groups. As shown in Fig. 1C, C 1s spectrum of FOQDs can be divided into three peaks. The C 1s binding energies observed for non-oxygenated carbon is centered at 284.7 eV and those for mono-oxygenated carbon and di-oxygenated carbon are centered at 286.6 and 288.3 eV, respectively.⁴⁰ As depicted in EDS, a great deal of C elements and O elements were dispersed on the surface of FOQDs (Fig. 1d). The presence of Na elements may be ascribed to NaOH which was used to react with fullerene to synthesize FOQDs.⁴¹ It was clearly demonstrated that the surface of FOQDs was largely covered by hydroxyl groups which rendered a hydrophilic surface reducing the particle-to-particle interaction and, thus, stable colloidal suspension of FOQDS in aqueous solution was obtained.Open in a separate windowFig. 1(A) TEM image of the FOQDs. (B) FT-IR spectrum of the FOQDs. (C) XPS spectra of the C 1s of the FOQDs. (D) EDS spectra of the FOQDs.Firstly, the concentration of FAM–ssDNA used in the assay was optimized. It was observed in Fig. S1A (ESI^†) that with the increasing concentration of FAM–ssDNA, the fluorescence intensity also increased. Furthermore, at the concentration of 20 nM, FAM–ssDNA exhibited enough fluorescence to be utilized for construction of this biosensor. Although a higher concentration of FAM–ssDNA could increase the output fluorescence signal, the high concentration can also compromise the sensitivity of the biosensor. Therefore, 20 nM FAM–ssDNA was used in the following experiment. After incubating FAM–ssDNA with aqueous dispersion of FOQDs in Tris–HCl buffer (10 mM, 5 mM MgCl₂, pH = 7.4), the FAM–ssDNA was absorbed onto the surface of FOQDs through π–π stacking interaction and the luminescence quenching phenomenon of FAM was observed (Fig. 2A). The absorption spectrum of the aqueous dispersion of FOQDs exhibited negligible absorption in the visible range (Fig. S1B, ESI^†), suggesting that there was no spectra overlap between the luminescence emission spectra of FAM and the absorption spectra of FOQDs and thus no FRET occurred between FAM and FOQDs. As reported by Li et al., nano-C₆₀ could effectively quench the luminescence of FAM through PET mechanism.¹⁸ In our present study, we may suggest that FOQDs, as polyhydroxylated nano-C₆₀, can also quench the luminescence of FAM via PET mechanism. A control experiment showed that the luminescence quenching degree of fluorescein (without attached ssDNA) caused by FOQDs was almost negligible compared with that of FAM–ssDNA (Fig. 2B), indicating the occurrence of assembling of ssDNA chains onto FOQDs through π–π stacking interaction which brought FAM and FOQDs in short range. On the other hand, as the zeta potential of the FOQDs in our present study was measured to be −25 mV, the electrostatic interaction between the negatively charged DNA chains and FOQDs can also be excluded. In the FAM–FOQDs PET pair, the fluorescence quenching degree was dependent on the concentration of FOQDs and the quenching degree up to 87% was achieved in the presence of 0.050 mg mL⁻¹ FOQDs. Besides, the time dependence of the quenching process showed that the interaction between the ssDNA with the FOQDs reached equilibrium in only a few minutes (Fig. S2, ESI^†).Open in a separate windowFig. 2(A) Luminescence quenching of FAM–ssDNA (20 nM) in the presence of various concentrations of FOQDs (0, 0.007, 0.013, 0.017 mg mL⁻¹, 0.034 mg mL⁻¹, 0.050 mg mL⁻¹, 0.067 mg mL⁻¹, 0.083 mg mL⁻¹). (B) Luminescence spectra of fluorescein (20 nM) in the absence and presence of 0.050 mg mL⁻¹ FOQDs, 20 nM ssDNA, 20 nM target ssDNA, respectively. All experiments were per-formed in Tris–HCl buffer (10 mM, 5 mM MgCl₂, pH = 7.4) under excitation at 480 nm.We then investigated the performance of this FAM–DNA–FOQDs PET system for homogeneous DNA sensing. Before the introduction of FOQDs into the solution of FAM–ssDNA, different concentrations of complementary target ssDNA were added first to form double helix structure by hybridization. The recovery time and temperature were optimized for better assay performance. The time dependence of the recovery process showed that the maximum recovery was observed at 60 min (Fig. S3A, ESI^†). And the temperature for maximum recovery was obtained at 25 °C (Fig. S3B, ESI^†). As expected, the fluorescence of FAM was restored in a complementary target ssDNA-concentration dependent manner, which was shown in Fig. 3A. It was explained that the formation of dsDNA with double-helical structure decreased the exposure of nucleic acid bases of ssDNA to FOQDs and thus weakened the π–π stacking interaction between ssDNA and FOQDs. In this way, the distance between FAM–ssDNA and FOQDs was enlarged to block the PET from FAM to FOQDs and the fluorescence recovery of FAM was observed. The relative fluorescence intensity ((F − F₀)/F₀, where F and F₀ represent the emission intensity in the presence and absence of complementary target ssDNA, respectively) was linearly related to complementary target ssDNA concentration in the range from 0.05 nmol L⁻¹ to 20 nmol L⁻¹, with the detection limit of 15 pM (calculated as the concentration corresponding to three times of the standard deviation of the background signal from seven independent measurements) (Fig. 3B). Compared with DNA biosensing platform using other carbon nanostructures, such as graphene oxide (100 pM)⁴² and nano-C₆₀ (25 pM),²⁵ the sensitivity of the present sensor is significantly improved. Besides such an impressive sensitivity, the biosensing platform also offered pronounced specificity, that is, unambiguous discrimination between different sequences. As illustrated in Fig. S4 (ESI),^† the biosensor showed distinctly differed response towards the single-base mismatched target ssDNA and double-base mismatched target ssDNA under identical assay conditions, which caused negligible alteration of the FAM emission as compared to complementary target ssDNA.Open in a separate windowFig. 3(A) The fluorescence recovery trend line in accordance with different concentrations of target ssDNA (0.05, 0.2, 1, 5, 10, 20, 50, 80, 100, 200 nM). F₀ represents the fluorescence intensity in the absence of target ssDNA. (B) The linear relationship between the fluorescence recovery (at 520 nm) and the concentration of target ssDNA within the range from 0.05–20 nM, data were presented as average ±SD from three independent measurements. Experiments were conducted in the presence of 20 nM FAM–ssDNA and 0.05 mg mL⁻¹ FOQDs in Tris–HCl buffer (10 mM, 5 mM MgCl₂, pH 7.4) under excitation at 480 nm.In order to validate the universality of the biosensing platform, we then used aptamer as the probe^43–45 and constructed another biosensor for OTA detection (Fig. S5, ESI^†). As indicated in Fig. S6 (ESI),^† the output fluorescence intensity of TAMRA–OTA aptamer with a concentration of 40 nM was enough to develop the OTA biosensor. Similar to the ssDNA probe, the OTA aptamer was assembled onto the surface of FOQDs due to the π–π stacking interaction, resulting in the fluorescence quenching of TAMRA-labelled OTA aptamer (Fig. S7, ESI^†). The introduction of OTA into the TAMRA–OTA aptamer-FOQDs PET system, which specifically bound with OTA aptamer accompanied by its conformational change, led to the detachment of TAMRA-labelled OTA aptamer from the FOQDs. The recovery time and temperature were also optimized for better assay performance for OTA detection. The time dependence of the recovery process showed that the maximum recovery was observed at 60 min (Fig. S8A, ESI^†). And the temperature for maximum recovery was obtained at 25 °C (Fig. S8B, ESI^†). And the restoration of the fluorescence intensity of TAMRA was observed (Fig. 4A). The relative fluorescence intensity at 580 nm was linearly related to OTA concentration within the range from 0.01 to 1 ng mL⁻¹ (Fig. 4B), with a detection limit of 3 pg mL⁻¹. The specificity of this fluorescent OTA aptasensor was also examined. As illustrated in Fig. S9 in the ESI,^† it clearly showed that other mycotoxins including AFB₁, FB₁, ZEN, DON in the concentration of 10 ng mL⁻¹ caused negligible response compared with 1 ng mL⁻¹ OTA under the same experimental procedures by this OTA biosensor, which indicates that our present aptasensor exhibits excellent selectivity towards OTA. To prove the applicability of this OTA biosensor in practical samples, this biosensor was used to detect OTA in grape juice samples. In this paper, OTA detection was also realized in 100-fold diluted grape juice sample with Tris–HCl buffer under the same experimental procedures as that in the aqueous buffer solution. It can be seen from Fig. S10^† that the degree of fluorescence restoration of TAMRA was linear related to the concentration of OTA in the range from 0.02 to 1 ng mL⁻¹, with a detection limit of 5 pg mL⁻¹. Standard addition experiments were conducted to examine the feasibility of this OTA biosensor in practical OTA-free grape juice samples. The satisfactory recoveries from 94% to 110% and the relative standard deviations (RSDs) were within 1.3–4.2 in Table S1^† convincingly demonstrates that this biosensor based on the luminescence quenching ability of FOQDs towards TAMRA has great potential in practical application. To evaluate the reproducibility of the OTA biosensor, five standard samples containing different concentrations of OTA (0.03, 0.05, 0.08, 0.2, 0.6 ng mL⁻¹) were prepared by spiking standard OTA into the OTA-free grape juice samples. As shown in Table S2,^† the intra-day and inter-day RSD were less than 4.1% and 4.5% (n = 11), suggesting that this OTA biosensor had a good reproducibility. Liu et al. have developed a OTA fluorescence biosensor based on fluorescence energy transfer (FRET) between a cationic conjugated fluorescent polymer and FAM with a detection limit of 0.11 ng mL⁻¹.⁴⁶ It has also been reported that OTA detection was realized based on FRET between quantum dots and MoS₂ nanosheets with a limit of detection of 1.0 ng mL⁻¹.⁴⁷ Compared to the previously reported OTA aptamer-based biosensors, the sensitivity of the present biosensor is greatly improved, which shows great potential to detect lower concentration of OTA in practical samples.Open in a separate windowFig. 4(A) The fluorescence recovery spectra with the addition of increasing concentration of OTA (0, 0.01 ng mL⁻¹, 0.05 ng mL⁻¹, 0.3 ng mL⁻¹, 0.6 ng mL⁻¹, 1 ng mL⁻¹). (B) The linear relationship between the fluorescence recovery (at 580 nm) and the concentration of OTA within the range from 0.01–1 ng mL⁻¹, data were presented as average ± SD from three independent measurements. Experiments were conducted in the presence of 40 nM TAMRA–OTA aptamer and 6.7 μg mL⁻¹ FOQDs in Tris–HCl buffer (10 mM, 5 mM KCl, 5 mM CaCl₂, pH 8.5) under excitation at 550 nm.In conclusion, ultrasensitive biosensing platform based on the luminescence quenching ability of zero-dimensional FOQDs was established for the first time. The fluorescence of FAM and TAMRA can both be effectively quenched under the π–π stacking interaction between ssDNA/aptamer and FOQDs which shorten the distance between fluorescent dyes and FOQDs. And then ultrasensitive detection of biomolecule DNA and small molecule OTA was realized. This work demonstrates that FOQDs can be used as prospective fluorescence quenchers and has extensive applied prospect in bioanalysis in the future.     

Colour-tunable ultralong organic phosphorescence upon temperature stimulus

A new class of single-component molecular crystal with colour-tunable ultralong organic phosphorescence (UOP) was designed and synthesized through alkyl chain engineering. Forming a more rigid environment at 77 K, the colour-tunable UOP from yellow-white to blue-green is achieved through dual-emission of crystal and amorphous states.

A new class of single-component molecular crystal with colour-tunable ultralong organic phosphorescence (UOP) was designed and synthesized through alkyl chain engineering.

Ultralong phosphorescence, a kind of phosphorescence that can be observed by the naked eye after removing the excitation source, has received great attention in the fields of sensing,¹ displays,² imaging,³ anti-counterfeiting⁴ and so on during the past years. Unfortunately, it is limited to inorganic materials because of the weak spin–orbit coupling (SOC) and strong non-radiative transition of pure organic materials.⁵ Compared with inorganic materials, however, organic materials have some excellent merits, such as inexpensive cost, relative safety to the environment, and soft preparation conditions.⁶ The realization of UOP becomes very significant. As mentioned above, UOP can be achieved by promoting intersystem crossing (ISC) through enhancing SOC and suppressing non-radiative transitions. In view of these factors, various strategies such as polymerization,⁷ H-aggregation,⁸ crystallization,⁹ host–guest doping,¹⁰ and freezing conditions¹¹ have been explored to achieve UOP with the unremitting efforts of scientific researchers.Intelligent-response organic luminescent materials can change their luminescent properties such as colour, lifetime, intensity, etc. after being stimulated by the external factor, such as mechanical forces, temperature, pH, light, solvent, etc., which have caught great attraction. The sensitivity of luminescent molecule to excitation wavelength, temperature or oxygen can be applied in sensors, optical recording and so on.^12–14However, the luminescence of these materials with intelligent-response are mostly limited to fluorescence or room temperature phosphorescence (RTP) with relatively short lifetimes.^15–18 Few samples display persistent luminescent feature.¹⁹ Most organic compounds can only tune their afterglow properties by changing molecular side groups or multi-component doping.^20–23 Therefore, the development of single component UOP materials with intelligent response remains a challenge. Inspired by the alkyl-chain engineering²⁴ and freezing conditions, herein we speculate that temperature response UOP might be induced by controlling the activity of the alkyl chain to regulate non-radiative transition rate. By means of reducing the temperature to restrict the molecular motions at amorphous state, colour-tunable UOP with temperature-response can be realized by dual-emission of molecules at both amorphous and crystalline states.In our previous study, MCzT crystals showed yellow UOP at RT.²⁵ The crystals showed the same UOP at 77 K with that at RT (Fig. 1a). Here, 9-(2-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)ethyl)-9H-carbazole (MTOD) was designed and via alkyl chain attaching carbazole with a triazine core. The target molecule was characterized by ¹H NMR and ¹³C NMR (Fig. S1–S3^†). Its melting point reaches 130 °C. (Fig. S4^†) MTOD achieved a lifetime of up to 860 ms under ambient conditions. Surprisingly, the UOP changed from yellow-white to blue-green after the removal of the UV-lamp for several seconds at 77 K (Fig. 1b), demonstrating colour-tunable property of UOP with a low temperature stimulus.Open in a separate windowFig. 1Molecular structures and UOP photographs of MCzT (a) and MTOD (b).In order to explore the reasons for the colour-tunable UOP of MTOD at low temperature. The photophysical properties of MTOD in the crystal state were first investigated under ambient conditions. As shown in Fig. 2a, the photoluminescence (PL) spectrum of MTOD shows two main emission peaks at 375 and 413 nm and a shoulder at 435 nm. From the lifetime decay profiles (Fig. S5a^†), it was confirmed that they were all assigned to the fluorescence. Notably, the crystals of MTOD presented yellow afterglow after turning off the UV lamp. From the delayed phosphorescence spectrum, the main emission of MTOD was located at 414, 556 and 600 nm with lifetimes of 681.91, 860.56 and 860.59 ms, respectively (Fig. 2b and S5b^†). Among these, the emission around 414 nm is assigned to triplet–triplet annihilation (TTA) fluorescence originating larger π–π overlaps of carbazole groups from the crystal data (Fig. S6^†).²⁶ Remaining two emission peaks are attributed to UOP emission. From the phosphorescence excitation-emission spectra of MTOD, UOP can be efficiently excited from 260 to 380 nm, with the optimal excitation at 360 nm (Fig. S7^†).Open in a separate windowFig. 2Photophysical properties of MTOD at room temperature and at 77 K. (a) Steady-state photoluminescence (PL, blue dashed line) and phosphorescence (red solid line) spectra at RT and 77 K. Inset: photographs taken after removing excitation. (b) Time-resolved phosphorescence decay of the emission bands at 556 and 600 nm at room temperature, respectively. (c) Time-resolved phosphorescence decay of the emission bands at 478, 564 and 612 nm at 77 K, respectively. (d) PL (blue dashed line) and phosphorescence (red solid line) spectra at molten state.Subsequently, we measured the PL and phosphorescence spectra of MTOD at 77 K (Fig. 2a). The steady-state PL spectrum showed four peaks at 361, 376, 410 and 436 nm, with little change compared with its corresponding spectrum at RT. However, a new phosphorescence peak appears at about 480 nm with an intense emission than others, which displays an ultralong lifetime of 2.5 s. Obviously, the new emission peak plays an indispensable role in the colour-changed UOP at 77 K. The yellowish-white UOP observed by naked eyes at 77 K is generated by the combination of three phosphorescence peaks. However, as time goes by, only the blue-green afterglow at about 480 nm due to the shorter lifetime of long-wavelength phosphorescence can be observed.To find out the origin of this new peak at 480 nm, the PL and phosphorescence spectra of MTOD at molten state were measured as amorphous emission. As shown in Fig. 2d, the PL peak is located at 380 nm and the phosphorescence spectrum shows a broad band with a main peak at 484 nm. It is suggested that the new phosphorescence emission peak at 77 K may be attributed to amorphous state. Taken together, we deduce that the colour-tunable property of the crystal is possibly due to the presence of an amorphous state at low temperature.The X-ray single crystal diffraction of MTOD crystal was taken to explore the mechanism of UOP at room temperature. Abundant intermolecular and intramolecular interactions (Fig. 3a, S6, S8 and S9a^†) in the crystals strongly restrict the torsional molecular configuration. The dihedral angle between the triazine and carbazole groups is about 75° (Fig. S10^†). In the crystal of MTOD, the single molecule is limited by multiple intermolecular interactions, including C–H⋯N (2.681 Å), π-H⋯π (2.791, 2.876 Å), π-H⋯N (2.703, 2.711 Å) (Table S3^†). The rich intermolecular interactions are beneficial to limit molecular motions to suppress non-radiative transitions of excited molecules, leading to UOP. However, the amorphous molecules around the crystals with weak restriction displayed negligible phosphorescence at room temperature due to the strong motions of alkyl chains.Open in a separate windowFig. 3Intermolecular interactions (a) at room temperature and (b) at 100 K.Comparatively, the single crystal of MTOD molecule at 100 K was measured in order to explore the colour-changing mechanism of UOP at low temperature (Fig. 3b and S9b^†). By comparison, MTOD crystals exhibit more intermolecular interactions at 100 K and the distance become shorter. Molecular conformation of MTOD changed slightly, the dihedral angle between triazine and carbazole changed from 74.95° to 75.32° (Fig. S10^†). These increased interactions can constrain the molecules more effectively and the stronger restriction of alkyl chain and carbazole will further suppress non-radiative transitions, resulting in the much longer phosphorescence lifetime of over 1.0 s at low temperature. Compared with the molecules in the crystalline state, freezing condition can provide a more rigid environment to minimize the movement of the alkyl chain, greatly reducing the non-radiative transition rate at amorphous state, resulting in the lifetime of short wavelength phosphorescence at 478 nm up to 2.5 s.According to the above results, the photophysical process of colour-tunable phosphorescence can be described by Jablonski diagram as shown in Fig. 4. Upon photoexcitation, both electrons in amorphous and crystalline molecules transforms to lowest singlet states (S₁). Then, the electrons in S₁ would further transform to the lowest triplet (T₁) through ISC. At room temperature, amorphous molecules show strong molecular motions to facilitate the non-radiative transitions. However, crystalline molecules due to closely arrangement can exhibit phosphorescence through radiative decay. At 77 K, both excitons in amorphous and crystal states are dominated by radiative transitions, leading to colour-tunable UOP.Open in a separate windowFig. 4(a) Jablonski diagram of the relevant photophysical processes illustrating amorphous and crystalline UOP process at room temperature (top) and 77 K (bottom). (b) Phosphorescence spectra of MTOD at different temperature and (c) corresponding coordinates in CIE.In view of the interesting luminescent phenomenon, we have investigated a series of phosphorescent spectra of MTOD crystals at different temperatures ranged from 183 to 273 K. As shown in Fig. 4b, as the temperature increases, the phosphorescence intensity of the amorphous molecules gradually decreases. The colour variations of the MTOD crystals in response to the environmental temperatures are shown in the Commission International de l’Eclairage (CIE) coordinate diagram (Fig. 4c). As the temperature was gradually changed from 183 to 273 K, the UOP changed from green to yellow with good linearity of the CIE coordinates. This demonstrated that MTOD crystals may have potential in low temperature sensing.In conclusion, we synthesized a colour-tunable single-component UOP compound through alkyl chain engineering. Combined the spectral and single crystal analyses, it is indicated that colour-tunable UOP comes from dual-emission of molecules at amorphous and crystalline states. Low temperature provides better rigid effect on UOP of amorphous molecules than crystals, resulting in the UOP colour changed from yellow-white to blue-green. More interestingly, red-shifted UOP of MTOD crystals with the increase of temperature can be achieved, demonstrating its potential for temperature sensing. This study will provide a platform for the design of single-component UOP molecules with tunable colour emission and broadens its application field.     

A highly selective quinolizinium-based fluorescent probe for cysteine detection

A novel fluorescent quinolizinium-based turn-off probe has been developed for selective detection of cysteine. The probe showed high selectivity and sensitivity towards cysteine over other amino acids including the similarly structured homocysteine and glutathione with a detection limit of 0.18 μM (S/N = 3). It was successfully applied to cysteine detection in living cells with low cytotoxicity and quantitative analysis of spiked mouse serum samples with moderate to good recovery (96–109%).

A novel fluorescent quinolizinium-based turn-off probe for selective detection of cysteine has been developed.

Biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are biomolecules that play important roles in a variety of biological processes, such as cellular growth, redox homeostasis and immune system regulations.^1–5 Among the three biothiols, Cys is the essential amino acid involved in various physiological processes, in which it serves as a biomarker for different dysfunctions and diseases.⁶ The deficiency of Cys can lead to adverse symptoms such as liver damage, psoriasis and lethargy, while high levels of Cys can cause a wide range of disorders such as Alzheimer''s and cardiovascular diseases.^7–10 Therefore, it is of importance to develop effective and selective approaches for Cys detection under physiological conditions.In the past decades, various techniques had been established for the detection of Cys, such as high performance liquid chromatography (HPLC),^11,12 capillary zone electrophoresis (CZE),^13–15 mass spectrometry (MS).^16,17 However, these methods require specialized equipment and sophisticated sample preparations, which restrict their applications on routine detection. In comparison, fluorescence spectroscopy is considered as a powerful technique for detection of Cys due to its high selectivity, operation simplicity, and non-invasiveness.^18–20 Nowadays, a variety of fluorescent probes have been developed based on the characteristic redox properties and strong nucleophilicity of the thiol group on Cys.^21–38 However, due to the structural similarity of Cys, Hcy and GSH, selective fluorescent detection of Cys in biological samples still remains a challenge.^39,40 Therefore, development of fluorescent probes for highly selective Cys detection is important.Cys-triggered addition–cyclization–cleavage reaction with acrylate, which was first reported by Yang and Strongin in 2011,⁴¹ is the most widely used response mechanism for the design of Cys-selective fluorescent probes.^5,18,20,21 Upon the addition of Cys, nucleophilic attack of Cys on acrylic ester followed by intramolecular cyclization releases the fluorophore''s hydroxyl and a seven-membered ring amide. The high selectivity of this reaction towards Cys over Hcy and GSH is attributed to the kinetic difference of the intramolecular cyclization.Various Cys-responsive fluorescent probes have been developed based on the incorporation of acrylate group on common fluorephores, such as BODIPY, rhodamine, coumarin and fluorescein.^42–50 However, the use of these dyes might suffer from low water solubility, which results in decreased sensitivity of detection and difficulty in biological applications.²² In comparison, quinoliziniums are cationic aromatic heterocycles with improved water solubility, which enable their applications in cell imaging with good biocompatibility.^51,52 Compared with these common fluorescent scaffolds, studies on the applicability of quinolizinium compounds as fluorescent chemosensors remain largely elusive (Scheme 1).Open in a separate windowScheme 1(a) Common fluorophores used for construction of thiol detection probes. (b) Novel fluorescent quinolizinium-based probe for cysteine detection.In 2017, we have developed a new series of fluorescent quinolizinium compounds with tunable emission properties in visible light region (λ_em = 450 to 640 nm) and large Stokes shifts (up to 6797 cm⁻¹).⁵³ The application of this class of fluorescent quinoliziniums in live cell imaging was demonstrated by incubation with HeLa cells, in which the subcellular localization of the quinoliziniums could be switched by modifying the substituents. Based on this work, we envision that the fluorescent quinoliziniums would be amenable for the design of fluorescent probes for Cys detection in biological samples.Herein we introduce a novel fluorescent quinolizinium-based turn-off probe 1 for highly selective detection of Cys over Hcy, GSH and other amino acids. The acrylate group was incorporated on the phenyl ring of the quinolizinium, which served as the moiety for the reaction with Cys. Cys triggered the change in fluorescence intensity of probe 1 due to the conjugated addition–cyclization reaction with the acrylate group. The probe exhibited highly selective detection for Cys and good biocompatibility, which could be successfully applied to detection of Cys in living cells and quantitative analysis of Cys concentrations in mouse serum samples.To verify the feasibility of probe 1 for Cys detection, the spectral properties of probe 1 towards Cys were firstly investigated in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS). As shown in Fig. 1, the free probe 1 showed absorption bands at 360 nm and 420 nm. Upon excitation at 420 nm, strong fluorescent signal was observed at 495 nm. After the addition of Cys (20 equiv.), the absorption at 360 nm increased with the decreased absorption band at 420 nm, while the fluorescence intensity of probe 1 significantly reduced. These results indicated that probe 1 displayed fluorescence signal response towards Cys.Open in a separate windowFig. 1(a) UV-Vis absorption and (b) fluorescence spectra of 1 (20 μM) with and without the addition of Cys (20 equiv.) in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) after 100 min.To examine the sensitivity of the probe, fluorescence titration of probe 1 (20 μM) was carried out in the presence of Cys in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) at 25 °C. The fluorescence quantum yield was evaluated to be 0.43 using coumarin 153 as a reference. Addition of 0.5 equiv. of Cys resulted in a decrease in fluorescence emission at 495 nm. The emission intensity was almost completely quenched upon addition of 20 equiv. of Cys, which showed a decrease in approximately 8-fold as compared with that of free probe 1. Furthermore, probe 1 exhibited a good linear relationship between the emission intensities at 495 nm and the concentration of Cys ranging from 0 to 100 μM with a R² value of 0.9904 (Fig. 2b). The detection limit was evaluated to be 0.18 μM based on the equation LOD (Cys) = 3σ/m, where σ is the standard deviation of blank measurements and m is the slope obtained from the calibration curve of probe 1 against Cys, indicating that probe 1 was highly sensitive to Cys.Open in a separate windowFig. 2(a) Fluorescence titration of 1 (20 μM) upon the addition of Cys (0, 10, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200, 400, 600, 1000 μM). (b) Linear correlation between emission intensities at 495 nm and concentrations of Cys (0–100 μM).We next investigated the selectivity of probe 1 for Cys. Under the same reaction conditions, other amino acids including Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val caused almost no fluorescence intensity changes, which demonstrated the high selectivity of 1 to Cys over other amino acids even at high concentration (20 equiv., 400 μM). As shown in Fig. 3a, a distinct fluorescence ratio (F₀/F) induced by Cys could be observed in contrast to other amino acids, while Hcy and GSH showed only little effect to the fluorescence intensity changes. Besides, other potential biologically relevant cations and anions were investigated, including Na⁺, K⁺, Cu⁺, Zn²⁺, Cu²⁺, Ni²⁺, Mg²⁺, Ca²⁺, Fe³⁺, Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HPO₄⁻, H₂PO₄⁻ and no significant fluorescence responses was observed (Fig. 3b).Open in a separate windowFig. 3Fluorescence changes F₀/F (λ_em = 495 nm) of 1 (20 μM) upon the addition of various (a) amino acids (20 equiv.) and (b) potential biologically-relevant ions in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) after 100 min.To study the effect of pH to the fluorescence of probe 1, the change in fluorescence emission intensity of probe 1 with and without Cys was investigated in a range of pH from 1 to 14, respectively. The fluorescence emission intensity of probe 1 at 495 nm was stable in the pH range of 6–9 (Fig. 4). Decrease in the fluorescence intensity was observed under basic conditions (pH > 9), which could be attributed to the hydrolysis of acrylate. The results suggested that probe 1 was capable of detecting Cys under physiological conditions.Open in a separate windowFig. 4Fluorescence intensity of 1 (20 μM) with and without the addition of Cys (20 equiv.) at different pH values.The response time was examined based on the change in fluorescence emission intensity of probe 1 upon reaction with 20 equiv. of Cys, Hcy, and GSH, respectively. As shown in Fig. 5, Cys caused a rapid fluorescence quenching than Hcy and GSH, and the fluorescence intensity remained stable after 100 min. However, the reaction rates of Hcy and GSH with probe 1 were significantly lower than that of Cys. This result indicated that probe 1 could selectively distinguish Cys from Hcy and GSH.Open in a separate windowFig. 5Time-dependent fluorescence changes of 1 (20 μM) upon the addition of Cys, Hcy, and GSH (20 equiv.).Align with literature reports,^54–59 we proposed the reaction mechanism of probe 1 with Cys was based on the nucleophilic addition reaction of Cys with C C bond of acrylate, followed by the cyclization–cleavage reaction and resulting in the formation of 2 with a hydroxyl group (Scheme 2). HRMS analysis of the crude reaction mixture showed the presence of peak with m/z 394.16, which revealed the formation of 2 after the reaction (Fig. S2^†). The high selectivity of probe 1 towards Cys over Hcy and GSH could be attributed to the difference in reaction rates of the intramolecular cyclization reaction. The intramolecular cyclization reaction for the formation of the seven-membered amide promoted by Cys was more kinetically favored than the formation of a strained eight or twelve-membered ring in the case of Hcy or GSH, respectively. As shown in the MS spectra (Fig. S3 and S4^†), the presence of peaks corresponding to the reaction intermediates, m/z 583.21 for Hcy and m/z 755.26 for GSH, respectively, was observed. These results indicated that Hcy and GSH exhibited slower reaction rates with probe 1.Open in a separate windowScheme 2Proposed reaction mechanism of 1 with Cys.NMR analysis of the crude reaction mixture of probe 1 with Cys (3 equiv.) was performed to provide further evidence on this reaction mechanism. As shown in Fig. 6, the hydrogen atoms on the acrylate group were located at 6.12 ppm (1H), 6.40 ppm (1H) and 6.60 ppm (1H). After reaction with Cys, the peaks corresponding to the hydrogen atoms on the acrylate group disappeared, while the shift of two peaks at 7.28 ppm (2H) and 7.51 ppm (2H) to 6.90 ppm (2H) and 7.27 ppm (2H), respectively, which corresponding to the hydrogen atoms on the phenyl ring, was observed. By comparing the NMR spectrum of isolated 2 with that of the crude reaction mixture, the result indicated that Cys reacted with the acrylate group on probe 1, resulting in the formation of 2 with the hydroxyl group.Open in a separate windowFig. 6Study of reaction mechanism using ¹H NMR analysis. (a) ¹H NMR spectrum of isolated 1; (b) ¹H NMR spectrum of isolated 2; (c) ¹H NMR spectrum of crude reaction mixture of 1 with Cys.The fluorescence was proposed to be quenched by the presence of hydroxyl substituent on the phenyl ring (i.e. phenol moiety) of the quinolizinium via intramolecular photo-induced electron transfer (PET). According to our previous study on the structure–photophysical property relationship (SPPR) studies of the quinolizinium compounds, the HOMO is composed of a π orbital of the quinolizinium and phenyl ring while the LUMO is composed of a π* orbital of the quinolizinium ring. The O atom of the phenol moiety served as an electron-donating group that donated an electron from its HOMO to the half-filled HOMO of the quinolizinium upon excitation by light, resulting in the quenching of fluorescence.To demonstrate the practical applicability of probe 1 in biological systems, cytotoxicity test and cell imaging experiments were carried out. HeLa cell lines (American Type Culture Collection) were cultured with Dulbecco''s Modified Eagle''s Medium (DMEM) (Gibco) supplemented with 44 mM sodium bicarbonate (Sigma-Aldrich), 10% v/v fetal bovine serum (Gibco), and 100 U mL⁻¹ penicillin (Gibco), 100 μg mL⁻¹ streptomycin (Gibco) at 37 °C with 5% CO₂. The cells had over 50% cell viability for concentrations of probe 1 up to 20.51 μM, revealing that probe 1 is of low toxicity and good biocompatibility. The colocalization images of HeLa cells were observed after treating with probe 1 and MitoTracker™ Red FM. As shown in Fig. 7c, the green fluorescence from probe 1 overlaid well with the red fluorescence from MitoTracker™ Red FM, indicating that probe 1 could specifically localized in the mitochondria.Open in a separate windowFig. 7Confocal fluorescence microscopic images of HeLa cells. (a) Subcellular localization of 1. (b) Subcellular localization of MitoTracker™ Red FM. (c) Merged images of (a) and (b). (d) Control experiment of 1-treated cells; (e) 1-treated cells incubated with Cys (100 μM). (f) Relative fluorescence of cells measured by ImageJ.For Cys detection in living cells, HeLa cells were first treated with 100 μM of l-cysteine for 30 min, followed by incubation with probe 1 for 2 h. l-Cysteine was replaced by PBS as the control experiment. The fluorescence imaging was conducted with a confocal microscope Leica TCS SP8 MP (Fig. 7d and e). Green fluorescence emission was observed for the control experiment, which possibly revealed that the interfering effects of other intracellular thiol-containing molecules, including Hcy, GSH and H₂S, should be negligible. The fluorescence emission was quenched by the presence of Cys in cells. These results demonstrated that probe 1 could detect Cys in living cells with mitochondrial targeting capability.We further explored the application of probe 1 in quantitative analysis of biological samples. Probe 1 was applied to the detection of Cys in mouse serum samples with literature references.^60–62 The serum samples were obtained from C57BL/6 mouse (source from The Chinese University of Hong Kong). Whole blood collected was allowed to clot by leaving it undisturbed for an hour at room temperature. The clotted blood was centrifuged at 1000 g for 10 min to remove the clot. Sera were separated and stored at −80 °C prior to the assay. The standard addition method was used to detect Cys in mouse serum. Mouse serum samples were diluted 1000-fold with PBS and Cys at different concentrations were added to the samples, respectively. After the reaction was incubated with probe 1 at 25 °C for 100 min, the fluorescence signals of samples were measured. The Cys concentration of each spiked sample was calculated from the linear calibration curve (Fig. S8^†). As shown in

Retracted Article: Facile preparation of bithiazole-based material for inkjet printed light-emitting electrochemical cell

Light-emitting electrochemical cell of bithiazole-based material was fabricated by solution processing rendered high external quantum efficiency over 12.8% and luminance of 1.8 10⁴ cd m⁻².

Light-emitting electrochemical cell of bithiazole-based material was fabricated by solution processing rendered high external quantum efficiency over 12.8% and luminance of 1.8 10⁴ cd m^−2.

It is well known that inkjet printing works as precise and versatile patterning method for printed electronics.^1,2 As for its advantages, it is already being exploited widespread for printing electronics.^3,4 Those merits are easily processed from solutions and conveniently used for air-stable electrodes.^5,6Light-emitting electrochemical cells (LECs) have emerged as an active layer,^7,8 arousing tremendous attention over the years.^9–11 Nevertheless, LECs are simpler than organic light-emitting diodes (OLEDs).^12,13 Due to its single layer architecture, low fabrication price and operating voltages, LECs are considered as a promising, next-generation, emissive thin-film technology.^14–16The most efficient device used to date for LECs are biscyclometalated iridium(iii) (Ir) complexes, because they have highly efficient and stable devices spanning the whole visible range.^17–19 However, avoiding the use of Ir is strongly desired because of its high cost and limited supply.^20–22 Till now, thiazole has worked as active component in a LECs system with a long-lived charge-separation molecule, without additional ions in its active layer.^23–26 As for its advantages, it can reduce recombination of charge carriers and facilitate carrier transfer.^27–29 More and more importance has been attached to thiazole compounds, especially bithiazole-based ones.^30–33 Therefore, these have aroused interest in the syntheses of bithiazoles.^34–36Based on our previous research,^37–41 the use of bithiazole ligands already explored by our group for photocatalytic technology has the advantage of an easy transformation of neutral complexes into charged ones by substitution on the N-atom of the thiazole moiety.^42,43 Furthermore, we demonstrate the fabrication of LEC devices by a combination of inkjet printing and spin coating, on A4 paper substrates for low cost, disposable and flexible conductive pattern.^44,45 Their corresponding material characteristics were closely investigated, a detailed report of the material properties of the resulting coatings, and an envisaged proof of concept application are disclosed in this paper. These fully printed devices demonstrate the potential upscaling of the fabrication of optoelectronic devices.Herein we report the design for a range of bithiazole derivatives 1a–1c (Fig. 1, their synthesis is shown in Scheme S1^†), and explore their properties. First of all, the UV-visible diffuse-reflectance (UV-vis DRS) spectra of those specimens at room temperature are displayed in Fig. 2 and the data are collected in Table S1.^† This enhancement was ascribed to the increase of aromatic rings, which has an intense absorption in the visible-light region. And their bandgap energies (E_g) are between 2.97–3.01 eV (Table S1^†), and are estimated from these absorption spectra according to the Kubelka–Munk method. Expanding the conjugated system and electron density with these donor groups, it can lead to a larger bathochromic shift of the absorption maximum.^46,47Open in a separate windowFig. 1Chemical structures of compounds 1a–1c described in this work.Open in a separate windowFig. 2UV-vis DRS spectra of 1a–1c.All of the three compounds are photoluminescent at room temperature, the relevant fluorescence peak maximum ranges from 360 to 398 nm (Fig. 3). They emit blue light when they are being excited. In agreement with Fig. 3, the presence of electron-releasing groups would shift the emission maximum. Among them, the most electron-donating carbazole unit would give the most red-shifted peak at 398 nm for 1c (Table S1^†). The PL quantum yield (ϕ_PL) studies show that both 1a and 1b are as high as 62 and 78%, while another one is 85% (Table S1^†). The data for their fluorescent quantum yields largely depends on interaction effect with molecules 1a–1c in the crystal packing. In this context, π–π intermolecular interactions can inhibit the fluorescence.⁴⁸Open in a separate windowFig. 3Typical PL spectra of 1a–1c.It may indicate that enlarging the conjugation length and electron density with these donor groups plays an important role in increasing the ϕ_PL.⁴⁹ Moreover, those with high ϕ_PL may be suitable for application as efficient light-emitting material in LECs.⁵⁰In addition, time-resolved measurements of the donor lifetime (τ) in 1a–1c were carried out, and their corresponding values are given in Table S1.^† The bithiazole derivatives 1a–1c showed long-lived singlet fluorescence lifetimes (τ_F), ranging between 1.04 and 9.54 ns (Table S1^†). This was on average more than double the fluorescence lifetime of the bithiazole starting material.⁵¹ These three samples possess relatively high decay time, as a result of their inhomogeneity.⁵² Compound 1c exhibited the longest fluorescence lifetime decay τ_F = 9.54 ns. The sensitivity of the emission to the polarity of the solvent is beneficial for an intramolecular charge transfer (ICT)-like emission.⁵³ This difference probably accounts for the high concentration of donor units in the bithiazole backbone, low rates of intersystem crossing to reactive triplet states.^48,54Consequently, the composite 1c was the best performing LEC and chosen for the below test. The EL spectra obtained for both devices were almost identical, with a main peak at 498 nm and other peak at 504 nm wavelength as illustrated in Fig. 4a. It may account for trapping, cavity, and self-absorption effects from within the LEC device 1c multilayer-structure. The spin-coated emission is quantified by the commission Internationale de L’Eclairage (CIE) coordinates of (0.28, 0.42), and a colour rendering index (CRI) of 65 (Table S2^†). While inkjet-printed light emission with CIE coordinates of (0.34, 0.43) and a CRI value of 83 was achieved for device 1c (Table S2^†). It exhibits good colour rendering indices (CRI > 70), this device exhibited a warm-white appearance, and the colour rendering is considered sufficient for indoor lighting applications.Open in a separate windowFig. 4Comparison of device characteristics with spin-cast and inkjet printer emitting layer for 1c: (a) electroluminescent spectra, (b) luminance vs. voltage, (c) current density vs. voltage, (d) efficiency vs. luminance behaviour.The time-dependent brightness and current density under constant biases of 2.9–3.3 V for device 1c are shown in Fig. 4b and c. Interestingly, the device with the inkjet-printed emitting layer (EML) produced a light output of around 3600 cd m⁻², whereas 4600 cd m⁻² was achieved with the spin-coated EML. In other words, the device with the inkjet printed EML obtained about 78.3% of brightness compared with the reference device. The differences between the two devices regarding current efficiency (J) and power efficiency (PE) were rather closed, as listed in the Table S2.^† A luminous efficiency much larger than 10 cd A⁻¹ was achieved for the inkjet-printed EML, which tended to be 73.9%, and is similar to the spin-coated emitting layer.The performance gap between the devices (Fig. 4d) can be attributed to differences in the height and surface roughness of the emitting layer.⁵⁵ Lateral sizing histograms (Fig. S11^†) show that the spin-coated EML possessed a thickness of 35 nm and a very smooth surface. The inferior performance of the inkjet-printed EML is caused by a less homogeneous surface morphology and an overall fatter layer.⁵⁶ The depth of the inkjet-printed EML ranged from 25 nm (pixel centre) to 35 nm (pixel edges). As a consequence, it is an inkjet deposition process and the related evaporation dynamics reduces the light output and efficiency, but also postpones the stability of the device in the system.^10,57We then shifted our attention to the turn-on kinetics, efficiency and long-term stability of the LEC device for 1c; the typical evolution of current density and light emission is presented in Fig. 5a and b, showing a slow turn-on followed by a progressive decay over time.Open in a separate windowFig. 5(a) Current density (closed symbols) and brightness (open symbols) versus time at 3 V for a device with the representative sample 1c; (b) EQE versus time at an applied voltage of 3 V.The build-up of the light output is synchronous with that of the current density. This time-delayed response is one of the striking features of the operation of an electrochemical cell and reflects the mechanism of device operation.²² The champion device in this set exhibited a peak power efficiency (PE) of 17.4 lm W⁻¹ at a luminance of 18 000 cd m⁻². Meanwhile, the current density began to decrease, possibly due to the electrochemical oxidation or electro corrosion that occurred on component 1c through accumulated electroinduced holes, and then it kept up a durative datum.⁵⁸ Apparently it exhibited outstanding long-term operation stability beyond 40 h (Fig. 5b).The time-dependent external quantum efficiency (EQE) of LEC for the representative substrate 1c are shown in Fig. 5b. It also exhibited similar temporal tendency in device efficiency. When a bias was applied on the LECs, the EQE quickly increased since balanced carrier injection was achieved by the formation of the doped layers.⁵⁹ After attaining the crest value, the device current was still rising while the EQE reduced little by little. It implied that both growing the doped layers and weakening of the EQE was resulting from exciton quenching near the bithiazlole core in persistently extended doped layers.⁶⁰ Doping-induced self-absorption was rather adaptable to the temporal roll-off in device efficiency. This configuration shows the best performance (EQE = 12.8%) compared to those devices prepared with the single compounds, which is given in Table S3.^† The reason may be that the steady-state recombination zone in 1c device was close to the central active layer.⁶¹ Besides it contains fluorenes on its side chains, constructing plane structure, benefiting from good electron injection abilities, leading to enhancing the EQE itself.⁶²The fabrication of inkjet-printed bithiazole interdigitated electrode (IDE) is illustrated in Fig. 6. All the prepared varieties of bithiazole-based ink have been found to be highly stable with nil or miniscule precipitation in over 180 days being stored on the shelf (Fig. 6a). After HCl treatment of the paper substrate, the prepared inks have filled within ink cartridges of a low-cost desktop printer Canon iP1188 (Fig. 6b). Fig. 6c shows the photograph of inkjet-printed conductive patterns.Open in a separate windowFig. 6(a) The representative ink based on sample 1c was stable for 180 days; (b) electrode printing using Canon iP1188 printer; (c) scale showing size of printed electrodes; (d) interdigitated electrode (inset shows small size printed pattern).In addition, the most attractive prospect of the bilayer device structure at this stage is the possibility for patterned emission for the creation of a static display. Fig. 7 presents a photograph of a small portion of a larger static display, with a resolution of 170 PPI, which repeatedly exhibits a message in the form of the word “LEC”. As shown in Fig. 7, the pixel array produced are fairly luminous. The EL spectra were collected from each pixel on the substrate, the emission peak wavelength was about 510 nm and the full width at half maximum was about 170 nm. The low variation in emission intensity in the different pixels implies a small variation in thickness of the solution-coated layers. More specifically, if we assume a minimum diameter of 20 μm for an inkjetted electrolyte droplet, and a smallest inter-droplet distance of 10 μm, we attain a pitch of 30 μm, which corresponds to a high display resolution of 850 PPI. Finally, we draw attention to the herein presented static-display LEC that comprises solely air-stabile materials, and that we routinely fabricate the bilayer stack under ambient atmosphere.Open in a separate windowFig. 7(a) The patterned light emission from a bilayer LEC, with the emission pattern defined by the selected positions of the ink jetted electrolyte droplets. (b) The droplet diameter and pitch were 50 and 150 μm, respectively, and the device was driven at V = 3 V. The scale bar measures 300 μm.     

Aggregation-induced emission enhancement (AIEE)-active tetraphenylethene (TPE)-based chemosensor for Hg2+ with solvatochromism and cell imaging characteristics

An aggregation-induced emission enhancement (AIEE)-active fluorescent sensor based on a tetraphenylethene (TPE) unit has been successfully designed and synthesized. Interestingly, the luminogen could detect Hg²⁺ with high selectivity in an acetonitrile solution without interference from other competitive metal ions, and the detection limit was 7.46 × 10⁻⁶ mol L⁻¹. Furthermore, the luminogen also showed interesting solvatochromic behavior and superior cell imaging performance.

A TPE-based AIEE-active fluorescent sensor for Hg²⁺ was synthesized. Furthermore, it showed solvatochromism and cell imaging characteristics.

The design and synthesis of molecular sensors for the detection of metal ions, especially transition-metal ions, has attracted much attention.^1–3 Among all transition-metal ions, mercury (Hg²⁺) is identified as one of the most dangerous and ubiquitous heavy metals. Indeed, it is not biodegradable, and can cause extreme injury to the environment as well as human health.^4–8 Additionally, it can be accumulated through the food chain in the human body, consequently giving rise to several deleterious effects such as central nervous system defects, kidney damage, endocrine system disease and so on.^9–12 Although many governments around the world have adopted strict regulations to limit Hg²⁺ emission, the global Hg²⁺ pollution caused by human activities is still serious.^13,14 Therefore, it is highly desirable to develop a new method for the detection of Hg²⁺ with high selectivity and sensitivity.^15–17Most traditional fluorescent sensors suffer from a detrimental phenomenon called aggregation-caused quenching (ACQ), which usually results in a poor solid-state emission efficiency. Fortunately, in 2001, Tang et al. reported a fluorescent molecule named 1-methyl-1,2,3,4,5-pentaphenylsilole. Interestingly, the fluorescence emission of this luminogen was induced by aggregation, a phenomenon referred to as aggregation-induced emission (AIE).¹⁸ Subsequently, in 2002, Park et al. reported an interesting phenomenon named aggregation-induced emission enhancement (AIEE).¹⁹ In fact, both AIE and AIEE can achieve highly efficient emission in the solid state or aggregated state.^20–27 In the past years, AIE (or AIEE) phenomenon has attracted considerable research interest owing to the potential applications in a lot of fields, including bioimaging, fluorescence sensors, organic lighting emitting diode (OLED) devices and organic lasers.^28–33 Meanwhile, many stimuli-responsive materials have been reported, including photochromism, mechanochromism, and solvatochromism.^34–46 At present, the solvatochromism materials have been generally used in chemical and biological systems.⁴⁷ To date, many fluorescent chemosensors for Hg²⁺ have been reported. In contrast, the corresponding chemosensors with AIE or AIEE effect are rare, not to mention solvatochromic AIE or AIEE-active fluorescent sensors for Hg²⁺ with good cell imaging behavior. Indeed, preparing such multifunctional sensors is challenging and significative. In this paper, we reported an AIEE-active tetraphenylethene (TPE)-based fluorescent sensor (Scheme 1) for the detection of Hg²⁺. Furthermore, the luminogen also showed remarkable solvatochromism and good cell imaging characteristics.Open in a separate windowScheme 1The synthetic route of luminogen 1.To investigate the AIEE phenomenon of luminogen 1, we initially studied the UV-visible absorption spectra and photoluminescence (PL) spectra in acetonitrile–water mixtures with different water fractions (f_w). The results indicated that the absorption spectra exhibited level-off tails in the long wavelength region as the water content increased (Fig. S1^†). It is well-known that such tails are usually associated with the Mie scattering effect, which is the key signal of nano-aggregate formation.^48,49 As presented in Fig. 1, luminogen 1 showed weak fluorescence and the luminescence quantum yield (Φ) was as low as 1.35%. Interestingly, when the f_w in the acetonitrile solution was increased to 80%, an obvious emission band was formed, and a yellow-green fluorescence was observed. As the water content was increased to 90%, the emission intensity was further increased, and a bright yellow-green luminescence (Φ = 27.81%) with a λ_max at 545 nm could be observed.Open in a separate windowFig. 1(a) Fluorescence spectra of the dilute solutions of 1 (2.0 × 10⁻⁵ mol L⁻¹) in acetonitrile–water mixtures with different water content (0–90%). Excitation wavelength = 380 nm. (b) Changes the emission intensity of 1 at 545 nm in acetonitrile–water mixtures with different volume fractions of water (0–90%). (c) PL images of 1 (2.0 × 10⁻⁵ mol L⁻¹) in acetonitrile–water mixtures with different f_w values under 365 nm UV illumination.Clearly, water is a poor solvent of luminogen 1. As a result, the generation of the yellow-green emission can be attributed to the aggregate formation.^50–52 Moreover, as shown in Fig. 2, the nano-aggregates obtained were verified by dynamic light scatterings (DLS). Therefore, 1 is a typical AIEE-active fluorescent molecule, and its AIEE behavior is caused by the restricted intramolecular rotation. As shown in Fig. S2,^† solid-state compound 1 showed a strong green emission (Φ = 14.50%) with a λ_max at 497 nm, and the corresponding fluorescence lifetime is 2.66 ns (Fig. S3^†).Open in a separate windowFig. 2Size distribution curve of luminogen 1 (2.0 × 10⁻⁵ mol L⁻¹) in acetonitrile–water mixtures with f_w = 90%.On the other hand, the luminogen 1 also displayed interesting solvatochromism effect. As presented in Fig. 3, the absorption spectra and fluorescence spectra of luminogen 1 in different solvents were investigated, respectively. Obviously, the absorption spectra was barely affected by the polarity of solvents. However, the photoluminescence peaks were gradually red-shifted from 515 nm to 625 nm, and thus 1 exhibited remarkable solvatochromism behavior. Obviously, the molecular structure of luminogen 1 is distorted due to the presence of TPE unit, and the conjugation degree of molecule 1 is different in various solvents, and the intramolecular charge transfer (ICT) effect is possibly responsible for the solvatochromism behavior of 1.Open in a separate windowFig. 3(a) UV-Vis absorption spectra and (b) normalized fluorescence spectra (Excitation wavelength = 380 nm) of luminogen 1 in different solvents (2.0 × 10⁻⁵ mol L⁻¹). (c) Photographs of 1 under 365 nm UV illumination in different solvents: tol (toluene); THF (tetrahydrofuran); EA (ethyl acetate); BT (acetone); MeCN (acetonitrile); DMSO (dimethyl sulfoxide).Subsequently, the changes in the fluorescence of luminogen 1 induced by Hg²⁺ were investigated in acetonitrile (2.0 × 10⁻⁵ mol L⁻¹) at room temperature. In the fluorometric titration experiments, as shown in Fig. 4, the emission intensity significantly decreased when the Hg²⁺ concentration increased from 0 to 7.0 equivalents in acetonitrile. Meanwhile, the fluorescent color changed from orange-red to colorless, and followed by a plateau upon further titration (Fig. 5). Remarkably, the emission intensity of luminogen 1 was almost quenched completely. Furthermore, based on the titration experiments, the detection limit of luminogen 1 for Hg²⁺ on the basis of LOD = 3 × σ/B (where σ is the standard deviation of blank sample and B is the slope between the fluorescence intensity versus Hg²⁺ concentration) was 7.46 × 10⁻⁶ M (Fig. S4^†). Moreover, a good linear relationship could be obtained (R = −0.9933) and the quenching constant of luminogen 1 with Hg²⁺ was 1.9 × 10⁴ M⁻¹ (Fig. S5^†). On the other hand, the binding ratio of luminogen 1 for Hg²⁺ was established through Job''s plot and the results showed 1 : 1 stoichiometric complexation (Fig. S6^†). Next, the binding interactions between 1 and Hg²⁺ were further investigated by NMR in acetonitrile-d₃. As presented in Fig. 6, the signal of Ha from 9.42 ppm shifted to 9.62 ppm and the Hb changed from 8.43 ppm to 9.24 ppm. Besides, the Hc or Hd was slightly shifted for 0.04 ppm or 0.06 ppm, respectively. These consequences revealed that the N on the pyridine and the N on the pyrazine (near the N on the pyridine) are the most probable binding with Hg²⁺. Mass spectra were utilized to further demonstrate the binding mode of luminogen 1 toward Hg²⁺. The peak located at m/z = 820.0 was coincided well with the ensemble [1 + Hg²⁺ +2NO₃⁻ + Cl⁻]⁻, confirming the binding ratio of luminogen 1 for Hg²⁺ with 1 : 1 stoichiometry (Fig. S7^†).Open in a separate windowFig. 4Fluorescence titration spectra of luminogen 1 (2.0 × 10⁻⁵ mol L⁻¹) induced by Hg²⁺ (0–7.0 equiv.) in an acetonitrile solution. Excitation wavelength = 380 nm.Open in a separate windowFig. 5The emission intensity changes of luminogen 1 at 625 nm with different equivalents of Hg²⁺.Open in a separate windowFig. 6 ¹H NMR (acetonitrile-d₃, 400 MHz) spectra changes of luminogen 1 in the presence of Hg²⁺.Next, in order to study the selectivity behavior of luminogen 1 as a fluorescent sensor for Hg²⁺, other metal ions, such as Zn²⁺, Cd²⁺, Ba²⁺, Sr²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Pb²⁺, Ni²⁺, Co²⁺, Cu²⁺, Al³⁺, Fe³⁺, Cr³⁺, Ag⁺ and K⁺ were also measured in acetonitrile under the same experimental conditions. The corresponding UV-Vis absorption spectra were shown in Fig. S8.^† Furthermore, as showed in Fig. 7, when these cations were added separately into the solution containing luminogen 1, no obvious fluorescence changes were observed. Indeed, as noticed in Fig. S9,^† no obvious interference was observed when Hg²⁺ (7.0 equiv.) was added with other ions (7.0 equiv.). These results indicated that luminogen 1 could be served as a highly selective fluorescent sensor for detection of Hg²⁺.Open in a separate windowFig. 7(a) Fluorescence spectra of luminogen 1 (2.0 × 10⁻⁵ mol L⁻¹) towards various cations including Zn²⁺, Cd²⁺, Hg²⁺, Ba²⁺, Sr²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Pb²⁺, Ni²⁺, Co²⁺, Cu²⁺, Al³⁺, Fe³⁺, Cr³⁺, Ag⁺ and K⁺. Excitation wavelength = 380 nm. (b) Fluorescence photographs of luminogen 1 after addition of various metal ions (7.0 equiv.) under 365 nm light.Fluorescent probe is a powerful tool for optical imaging, which allows direct visualization of biological analytes.⁵³ Luminogen 1 was AIEE-active due to the restriction of intramolecular rotation in the aggregate state, which is beneficial for cell imaging. Indeed, the viability of HeLa cells incubated with luminogen 1 was evaluated by the standard MTT method (Fig. 8), and the result indicated that compound 1 exhibited low cytotoxicity. Next, cell imaging behavior of luminogen 1 was investigated using a confocal laser scanning microscopy (CLSM). HeLa cells were incubated with luminogen 1 (20 μM) for 30 min at 37 °C and the fluorescence images were obtained by CLSM.Open in a separate windowFig. 8The MTT assay of compound 1 for measuring cell viability.As presented in Fig. 9, an intense yellow-green fluorescence, which was consistent with the fluorescence of luminogen 1 in acetonitrile–water mixture with high water content (80% or 90%), was displayed inside the cells. This result indicated that luminogen 1 tended to aggregate inside the cells, and thus the bright yellow-green luminescence was clearly observed. Furthermore, the merged picture c demonstrated that picture a and picture b overlapped very well. These results indicated that luminogen 1 showed superior cell imaging performance.Open in a separate windowFig. 9Fluorescence images of HeLa cells incubated with luminogen 1 (20 μM) for 30 min at 37 °C: (a) bright field image; (b) fluorescence image; (c) merge image (a and b).In summary, a TPE-based fluorescent molecule was designed and synthesized. The luminogen exhibited obvious AIEE phenomenon. Moreover, luminogen 1 could be used as a highly selective fluorescence turn-off chemosensor for Hg²⁺, and the detection limit was 7.46 × 10⁻⁶ mol L⁻¹. Furthermore, 1 also displayed interesting solvatochromic behavior and superior cell imaging performance. Further studies on the design of new AIE or AIEE-active fluorescent chemosensors are in progress.     

A julolidine-fused anthracene derivative: synthesis,photophysical properties,and oxidative dimerization

We describe the synthesis and characterization of a julolidine-fused anthracene derivative J-A, which exhibits a maximum absorption of 450 nm and a maximum emission of 518 nm. The fluorescent quantum yield was determined to be 0.55 in toluene. J-A dimerizes in solution via oxidative coupling. Structure of the dimer was characterized using single crystal X-ray diffraction.

A julolidine fused anthracene derivative with unique photophysical and redox properties was presented.

Julolidine¹ is a popular structural subunit in various fluorescent dyes (Chart 1).² The restricted motion and strong electron-donating capability of the fused julolidine moiety are quite effective for improving the photophysical properties. For instance, julolidine-fused fluorophores normally display desirable photophysical characteristics, such as high quantum yield, red-shifted absorption and emission, and good photostability. Recently, julolidine derivatives have been widely exploited in various applications such as sensing,³ imaging,⁴ and nonlinear optical materials.⁵ Several julolidine dyes have been used in dye-sensitized solar cells due to their large π-conjugated system and the promising electron donating property.⁶Open in a separate windowChart 1The structure of julolidine, J-A and DAA.In this paper, we report a julolidine-fused anthracene derivative J-A, which exhibits attractive photophysical properties not observed in DAA, a dimethyl-amino substituted analogue. Both the absorption and emission of J-A show a dramatic red-shift (ca. 74 and 131 nm, respectively), compared with the unmodified anthracene (Fig. 2). The fluorescence quantum yield of J-A was determined to be 0.55 in toluene, while the emission of DAA was completely quenched. The observed spectral properties were rationalized by DFT calculations. In addition, we found that J-A was stable in the solid state, but reactive in solution. J-A dimer was formed through oxidative coupling at the para-position of the N-atom in a dichloromethane solution under air atmosphere. The structure of the dimerized product was characterized using single crystal X-ray diffraction, which unambiguously reveals the structural feature of the julolidine-fused anthracene compound. Preparation of J-A is shown in Scheme 1. Detailed synthesis and characterizations are provided in the ESI.^†Open in a separate windowScheme 1Synthetic route of J-A.Open in a separate windowFig. 2(A) Absorption spectra of J-A, AN, and DAA (1 × 10⁻⁴ mol L⁻¹ in dichloromethane); (B) emission spectra of J-A, AN, and DAA (1 × 10⁻⁵ mol L⁻¹ in dichloromethane). Excitation wavelength: 350 nm. 1 cm cuvette was used in both of the experiments. Inset: visualized fluorescence in solution was shown. ¹H-NMR signals of J-A shift to the high-field significantly, compared with DAA (Fig. 1), which indicates that the fused structure of J-A facilitates electron delocalization from the nitrogen atom to the anthracene moiety, and thus resulting in a stronger shielding effect. In the case of DAA, however, electron delocalization from the dimethyl amino group to the anthracene core is essentially inhibited due to steric hindrance, which will explain the fact that it displays a spectral feature similar to that of the unmodified anthracene.Open in a separate windowFig. 1Comparison of the ¹H-NMR spectrum between J-A and DAA in CDCl₃. Partial resonance signals in aromatic region are shown.Fusing with julolidine will exert significant effects on the photophysical properties of anthracene. The absorption and fluorescence spectra of J-A, DAA, and anthracene are shown in Fig. 2. The maximum absorption of J-A is 450 nm, which displays a red-shift of about 70 nm compared with the unmodified anthracene. J-A emits green light (^maxλ_em = 518 nm, Φ = 0.55), while anthracene emits blue light (^maxλ_em = 380 nm, Φ = 0.22). In contrast, the absorption of DAA essentially overlaps with that of anthracene, with only a minor red shift of ca. 10 nm, but its fluorescence is quenched significantly (Fig. 2). This spectral feature indicates that the dimethyl amino group is electronically separated from the anthracene moiety in the ground state, a result in good accordance with the ¹H-NMR data shown above. The quenched fluorescence of DAA may result from the photo-induced electron transfer⁷ from the lone pair of the nitrogen atom to the anthracene moiety in the excited state.The observed photophysical properties of J-A were reproduced by DFT calculations. The HOMO and LUMO orbitals are evenly distributed over the anthracene moiety and the N-atom in the julolidine, indicating the existence of a conjugated structure. The HOMO–LUMO transition (f = 0.10) corresponds to the absorption band at 450 nm. The sharper absorption at 390 nm can be assigned to the HOMO to LUMO + 1 transition. In contrast, the HOMO and LUMO orbitals of DAA resemble those of anthracene, because the dimethyl-amino group is orthogonal to the conjugated π-system (Fig. 3).Open in a separate windowFig. 3Molecular orbitals of J-A and DAA calculated at the B3LYP/6-31G(d) level of theory (iso value = 0.02). Orbital energies were given in parentheses. Excitation energies were computed by TD-DFT at the same level. Values in parentheses represent the oscillator strengths (f).J-A is stable in the solid state, but reactive in solution. The cyclic voltammetry (CV) diagram of J-A shows an irreversible oxidation potential at 0.007 V (vs. Fc/Fc⁺), indicating that J-A is easy to be oxidized (Fig. S3^†). Single crystals suitable for X-ray diffraction study were obtained by slow evaporation of a dichloromethane solution of J-A under air atmosphere. To our surprise, instead of J-A, X-ray data discloses the formation of a dimeric product (Scheme 2) of J-A. We hypothesized that the dimeric compound 5 formed via oxidative coupling reaction, a mechanism well-documented for the dimerization of the dimethylaniline compounds.⁸ The ¹H-NMR spectrum of 5 is distinct from that of J-A. All protons of the anthracene part (b′–d′) appear as a group of multiplet resonance signals (6.93–7.04 ppm) (Scheme 2). In addition, mass spectrometric analysis indicates that two hydrogen atoms were removed after the dimerization of J-A. Compound 5 exhibits a maximum absorption at 460 nm and a very weak emission (^maxλ_em = 530 nm, Φ = 0.03, Fig. S1^†). Two quasi-reversible oxidation waves were identified in the CV diagram of 5 at −0.010 V and 0.135 V (vs. Fc/Fc⁺), respectively (Fig. S5^†).Open in a separate windowScheme 2Oxidative dimerization of J-A. Inset: partial ¹H-NMR of 5 is shown.X-ray structure of 5 is shown in Fig. 4. The two connected anthracene planes are found to be orthogonal to each other with a dihedral angle of 90.17°, as a result of steric repulsion. Specifically, the bond length of N–C3 is 1.389 Å, which is similar to those of the other reported julolidine compounds (1.359–1.393 Å), while significantly shorter than that of the dimethyl-amino anthracene (1.433 Å).⁹ This result testifies the presence of electron delocalization between the fused julolidine nitrogen and the anthracene π-plane, which is in good agreement with the DFT calculations (Fig. 3). However, the two anthracene π-planes connected by the single bond (C4–C5, 1.489 Å) might not exhibit electron delocalization because of the orthogonal conformation. The fused julolidine ring-i and -ii are symmetric to each other, and both of them adopt an “envelope” conformation. The fused julolidine is nonplanar (bond angle, C3–N–C2, 115.73°, C3–N–C1, 115.92°, C1–N–C2, 113.81°). 5 is closely packed in the crystal (Fig. 4B), and no intercalated solvent molecules were observed. The closest distance between two adjacent anthracene planes is 3.922 Å, indicating a weak π–π stacking. Detailed crystal data are summarized in Table S5.^†Open in a separate windowFig. 4(A) Single crystal X-ray structure of 5. (B) View along a-axis.In summary, we report the synthesis and characterizations of a julolidine-fused anthracene derivative J-A, which demonstrates significantly red-shifted absorption (^maxλ_ab = 450 nm) and emission (^maxλ_em = 518 nm, Φ = 0.55), compared with the unmodified anthracene. The photophysical properties of J-A also contrast dramatically with a dimethyl-amino analogue DAA, which were rationalized by DFT calculations. In addition, J-A could be transformed into 5, a dimeric product, whose single crystal X-ray structure unambiguously confirmed the structural feature of the julolidine-fused anthracene.     

Highly dispersed Pt nanoclusters supported on zeolite-templated carbon for the oxygen reduction reaction

The formation of highly dispersed Pt nanoclusters supported on zeolite-templated carbon (PtNC/ZTC) by a facile electrochemical method as an electrocatalyst for the oxygen reduction reaction (ORR) is reported. The uniform micropores of ZTC serve as nanocages to stabilize the PtNCs with a sharp size distribution of 0.8–1.5 nm. The resultant PtNC/ZTC exhibits excellent catalytic activity for the ORR due to the small size of the Pt clusters and high accessibility of the active sites through the abundant micropores in ZTC.

Electrochemically synthesized highly dispersed Pt nanoclusters (PtNCs) stabilized by the nanocages of zeolite-templated carbon (ZTC) exhibit excellent electrocatalytic performance toward the oxygen reduction reaction.

Platinum (Pt) is currently considered one of the best electrocatalysts for the oxygen reduction reaction (ORR), which occurs at the cathode of a fuel cell and is the key process determining the overall performance.^1–5 However, the high cost and scarcity of Pt limit its wide commercialization in this field. According to the US Department of Energy, the total Pt loading is required to be below 0.125 mg cm⁻², in contrast to a presently used Pt loading of 0.4 mg cm⁻² or more for fuel cell application.⁴ Therefore, reducing the Pt loading without loss or with an improvement of the cathode performance has received significant interest in electrocatalytic research for fuel cell systems.^6–10 In this regard, reducing the size of Pt particles to a nanocluster scale (size < 2 nm) and maximizing the Pt dispersion may offer an efficient way to achieve maximum utilization of the Pt electrocatalyst with appropriate consumption.^4,11–15The size of nanomaterials generally plays a critical role in controlling the physical and chemical properties for catalytic applications.^16–20 With a decrease in the particle size to the nanoscale, quantum size effects are induced, which alter the surface energy of the material due to unsaturated coordination and change in the energy level of the d orbital of metal atoms, leading to spatial localization of the electrons.^17–20 This size-induced effect on the electronic structures at the active sites modifies the capability of binding the reactant molecules in catalytic reactions, thereby altering the activity of the nanocatalyst.²⁰ When the particle contains a few to several dozens of atoms with sizes, ranging from sub-nanometer to 2 nm often termed as nanocluster that bridges nanoparticle and a single atom.²¹ However, the Pt single atom is not an appropriate electrocatalyst for the ORR in a fuel cell system as the fast four-electron (4e⁻) pathway for the reduction of O₂ to H₂O requires at least two neighboring Pt atoms.^22,23 Anderson''s group demonstrated that the ratio between the production of H₂O (product of 4e⁻ process) and H₂O₂ (2e⁻) in the ORR strongly depends on the number of atoms in the Pt cluster. Typically, it requires more than 14 atoms in a Pt cluster to produce H₂O efficiently through the 4e⁻ pathway of the ORR.²⁴ Therefore, Pt nanoclusters having more than a dozen atoms have proven to be highly efficient ORR electrocatalysts for fuel cell systems.^13–15 Upon decreasing the size of the nanoparticles to a nanocluster, the electronic state and structure are known to be changed, leading to an increase of the catalytic activity in the ORR. Therefore, it is highly desirable to synthesize a Pt nanocluster-based material as an ORR electrocatalyst with high catalytic performance. To date, several synthesis strategies, such as wet-chemical, atomic-layer deposition, and photochemical methods, have been applied for the preparation of well-dispersed Pt nanoclusters on different types of support, such as dendrimer, metal oxide, and carbon materials.^{13–15,25–31}An alternative approach to synthesize Pt nanocluster (PtNC) is the encapsulation of the cluster within nanosized pores, for example, by utilizing microporous (diameters less than 2 nm) carbon materials.³² Among the microporous carbons, zeolite-templated carbon (ZTC) has been attractive for supporting Pt clusters due to its ordered microporous structure.^33–37 ZTC is a potentially promising material as catalyst support as it offers the advantages of extremely large surface area and high electrical conductivity of graphene-like carbon frameworks constituting a three-dimensional (3D) interconnected pore structure.³⁶ Moreover, the micropores of ZTC can serve as nanocages for stabilization of the Pt nanoclusters. Coker et al. used Pt²⁺ ion-exchanged zeolite as a carbon template to synthesize Pt nanoparticles in ZTC with size in a range of 1.3 to 2.0 nm.³³ Recently, atomically dispersed Pt ionic species was synthesized via a simple wet-impregnation method on ZTC containing a large amount of sulfur (17 wt%).²³ Itoi et al. synthesized PtNC consisting of 4–5 atoms and a single Pt atom in ZTC using the organoplatinum complex.³⁷ Although these methods produced Pt nanoclusters with narrow size distribution and atomic dispersion, they required multi-step processes and/or high-temperature treatment (>300 °C). High-temperature treatment often induces the sintering of nanoclusters to aggregated clusters. Therefore, it is highly desirable to develop a simple and low-cost method for the preparation of PtNC supported on ZTC (PtNC/ZTC) for use as an efficient ORR electrocatalyst. The electrochemical reduction approach offers an alternate and efficient route for the synthesis of PtNC in the micropores of ZTC. The electrochemical method is one of the popular ways to prepare electrocatalysts because it is a simple single-step procedure and ensures electrical contact between the nanoparticles and the support.^38,39Herein, we report a facile electrochemical method for the formation of PtNC with a narrow size range of 0.8–1.5 nm supported on ZTC. The resultant PtNC/ZTC shows higher electrocatalytic activities towards ORR compared to that of commercial Pt/C. Here, ZTC plays two important roles: (i) it provides nanocages to stabilize the PtNC and (ii) it accelerates the ORR activity by enhancing the accessibility of active sites through its abundant micropores. Fig. 1a shows a schematic representation of the typical electrochemical synthesis of PtNC/ZTC. In the first step, ZTC was impregnated with a Pt-precursor dissolved in a water–ethanol mixture. As ZTC possess ordered micropores (Fig. S1a^†) with high Brunauer–Emmett–Teller (BET) surface area of 3400 m² g⁻¹ (vide infra), the uniform adsorption and anchorage of PtCl₆²⁻ ions into the micropores of ZTC was favored. After impregnating and drying, the resultant ZTC–PtCl₆²⁻ was mixed with water–ethanol and Nafion to make the ink for the preparation of the electrode. Using the prepared electrode, a potential of 0.77 V vs. reversible hydrogen electrode (RHE) (Fig. 1b) was applied followed by potential cycling between 1.12 to −0.02 V vs. RHE until the cyclic voltammogram was stabilized. The Pt content of PtNC/ZTC was determined to be ∼10 wt% (Fig. S2^†) by thermogravimetric analysis (TGA). The obtained PtNC/ZTC was electrochemically characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The cyclic voltammogram (Fig. 1c) after potential cycling in fresh KOH electrolyte shows the characteristic Pt peaks corresponding to hydrogen adsorption and desorption. The Nyquist plots (Fig. 1d) demonstrate that PtNC/ZTC has lower electrolyte resistance (42 Ω) than that of ZTC (70 Ω), implying an improvement in the conductivity of ZTC by the presence of PtNC. Due to the increase in the conductivity, PtNC/ZTC could facilitate the electron transfer more effectively than ZTC, enhancing its electrocatalytic activity.Open in a separate windowFig. 1(a) Illustration for the formation of PtNC/ZTC:Pt-precursor was impregnated into ZTC micropores, and then a potential (0.77 V vs. RHE) was exerted on the ZTC–PtCl₆²⁻ composite in a 0.1 M KOH solution to form PtNC/ZTC (b) Chronoamperometric response of ZTC–PtCl₆²⁻ at a constant potential of 0.77 V (vs. RHE) in 0.1 M KOH electrolyte. (c) Cyclic voltammogram of PtNC/ZTC in a fresh 0.1 M KOH at a scan rate of 20 mV s⁻¹. (d) Nyquist plots of ZTC and PtNC/ZTC in 0.1 M KOH. Fig. 2a and b show images from aberration-corrected scanning transmission electron microscope (STEM) with high-angle annular dark-field (HAADF). The HAADF-STEM images exhibit the typical morphology of the final product (PtNC/ZTC) after electrochemical reduction. As shown in Fig. 2a, it is very clear that isolated PtNCs are uniformly dispersed in ZTC. These PtNCs have a homogeneous distribution with a narrow size range (0.8–1.5 nm, Fig. 2b). On further magnification, the STEM image shows a cluster-like structure of Pt (Fig. 2c). The STEM image of selected PtNC (Fig. 2d) reveals that it consists of ∼20 atoms. The number of atom content in PtNC was further determined by matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry using trans-2-[3-(4-test-butylphenyl)-2-methyl-2-propenylidene]malononitrile as the matrix.^40,41 As shown in Fig. S3,^† MALDI-TOF measurement produces a mass spectra with a predominant peak centered at ∼3700 Da corresponding to the Pt₁₉ cluster. The TEM image (Fig. S4 a and b^†) validates the formation of PtNC with an average size of 0.9 nm. In addition, the energy dispersive X-ray spectrometer (EDS) mapping images clearly shows the uniform dispersion of Pt nanocluster in ZTC (Fig. S4c^†). The X-ray powder diffraction (XRD) pattern (Fig. 2e) of PtNC/ZTC showed three broad peaks associated with small size metallic Pt corresponds to (111), (200), and (311) planes (Fig. 2e, inset), along with peaks of ZTC at 2θ = 7.8° and 14.9° corresponding to the ordered microporous structure. Along with the structural analysis, the porous texture of PtNC/ZTC was examined by Ar adsorption (Fig. 2f). PtNC/ZTC had a high BET surface area of 2360 m² g_ZTC⁻¹, which is 1.4 times lower than that of pristine ZTC (3400 m² g_ZTC⁻¹). The decrease in Ar adsorption capacity after the formation of PtNC in ZTC is interpreted as a result of the filling of ZTC micropores by PtNC. This micropore filling was confirmed in the pore size distributions of the pristine ZTC and the metal-loaded carbon (inset of Fig. 2f). The X-ray photoelectron spectroscopy (XPS) results reveal the signature of Pt in ZTC (Fig. S5^†). The elemental survey (Fig. S5a^†) shows the signature of C 1s, O 1s, F 1s (Nafion), and Pt 4f. The chemical nature of Pt in PtNC/ZTC was inspected by a detailed Pt 4f XPS analysis. The deconvoluted Pt 4f XPS spectra (Fig. S5b^†) reveals the presence of both metallic and ionic Pt species. The peaks observed at 71.0 (4f_7/2) and 74.2 (4f_5/2) eV correspond to metallic Pt whereas the other peaks positioned at 72.6 (4f_7/2) and 76.0 (4f_5/2) are attributed to Pt²⁺ and the peaks at 74.9 (4f_7/2) and 77.8 (4f_5/2) eV are attributed to Pt⁴⁺ originating from the surface oxidation of metallic Pt.⁴²Open in a separate windowFig. 2(a–d) Representative spherical aberration-corrected HAADF-STEM images of PtNC/ZTC at various magnifications. (e) XRD pattern of PtNC/ZTC and (f) Ar adsorption–desorption isotherms of ZTC and PtNC/ZTC. Inset in (e) shows a 30 times magnified high-angle region of XRD of PtNC/ZTC. Inset in (f) shows the pore size distributions of the ZTC and PtNC/ZTC.The formation of narrow sized PtNC by the electrochemical method can be ascribed to the stabilization of PtNC in the ZTC micropores, which serve as cages to impose a spatial limitation on the size of the Pt clusters. For comparison, Pt supported on ZTC was also prepared by the conventional incipient wetness impregnation and subsequent H₂-reduction at high temperature (300 °C). The Pt obtained by this incipient wetness impregnation method shows the formation of Pt nanoparticles on the exterior surface of ZTC (PtNP/ZTC) (Fig. S6^†). The formation of larger Pt nanoparticles is due to the sintering at high temperature, showing that even ZTC micropores could not prevent the aggregation of PtNCs at high temperatures. Fig. 3 shows the electrochemical ORR activity of PtNC/ZTC using linear sweep voltammetry (LSV) technique on a rotating disc electrode (RDE) in a 0.1 M KOH solution saturated with O₂ at a scan rate of 5 mV s⁻¹. The ORR activity of ZTC (without PtNC) was measured for comparison as well. As shown in Fig. 3a, PtNC/ZTC exhibited higher diffusion limiting current density and higher positive onset and half-wave potential compared to ZTC alone, indicating that PtNC is the active center for the ORR. To investigate the effect of the Pt loading amount on the ORR activity, PtNC/ZTC with various Pt loadings, 2–20 wt%, was used for the measurement of LSV at 1600 rpm. With an increase in Pt content, both the onset and half-wave potential shifted towards more positive potential up to 10 wt% loading of Pt (Fig. 3a and S7^†). Upon further increase of loading of Pt on ZTC to 20 wt%, both the onset and half-wave potential of PtNC/ZTC shifted towards less positive potential along with a slight decrease in the diffusion limiting current density (Fig. 3a). The decrease in the ORR activity of PtNC/ZTC at high loading of Pt (20 wt%) was attributed to the decrease in the electrochemically active surface area (Fig. S8^†) and decrease in the specific surface area (Fig. S9^†). The STEM image clearly shows that the aggregated Pt clusters were formed on the exterior surface of ZTC at 20 wt% loading of Pt (Fig. S10c^†), blocking the accessibility of active sites. Therefore, PtNC/ZTC with the optimum loading of 10 wt% of Pt leads to superior ORR activity with a high positive onset potential of 0.99 V, which is similar to commercial Tanaka Pt/C (Pt/C-TKK) (Fig. 3b), and a half-wave potential of 0.87 V, which is ∼10 mV more positive than that of commercial Pt/C-TKK (0.86 V) (Fig. 3b). Compared to the case of PtNC/ZTC, both the onset and half-wave potential of PtNP/ZTC prepared by the conventional incipient wetness impregnation and subsequent H₂-reduction with the same loading of Pt exhibited a less positive value (Fig. S11^†). The poorer activity of PtNP/ZTC is due to the blockage of active sites by larger PtNPs formed on the exterior surface of ZTC (Fig. S6^†).Open in a separate windowFig. 3(a) RDE ORR polarization curves of PtNC/ZTC with different mass loading of Pt. (b) Comparison of PtNC/ZTC (PtNC_10%/ZTC) with commercial Pt/C-TKK at the same loading of 40 μg_Pt cm⁻². (c) RDE ORR polarization curves of PtNC/ZTC at different rotation speeds. Inset in (c) shows the corresponding K–L plots at different potentials. (d) Represents the kinetic current density values of Pt/C-TKK and PtNC/ZTC at the potential of 0.8 V vs. RHE.To investigate the kinetics of the ORR activity of PtNC/ZTC, LSV measurements were performed with RDE at different rotating rates (Fig. 3c), and the kinetics was analyzed using a Koutecký–Levich (K–L) plot (Fig. 3c, inset). From Fig. 3c, it was observed that the current density increases with the increasing speed of rotation of the electrode, which is characteristic of a diffusion-controlled reaction. The corresponding linear K–L plots (Fig. 3c, inset) with a similar slope at different potentials reveal that the number of transferred electrons was ∼4, indicating that O₂ is directly reduced to OH⁻ and the ORR is dominated by the H₂O₂-free 4e⁻ pathway. To estimate the amount of produced peroxide ion, rotating ring-disc electrode (RRDE) measurement was performed and the produce peroxide ion calculated from RRDE curve was < 4% (Fig. S12^†). The kinetic current density (J_k) obtained from K–L plot at the potential of 0.8 V (Fig. 3d) for PtNC/ZTC (J_k = 50 mA cm⁻²) is 2.2 times higher than that of commercial Pt/C-TKK (J_k = 22 mA cm⁻²).As Pt-based electrocatalysts are known to be highly active in an acidic medium, the ORR activity of PtNC/ZTC in O₂-saturated 0.1 M HClO₄ was also evaluated by comparing it with that of commercial Pt/C-TKK with the same loading of Pt on the electrode surface using RDE at a scan rate of 5 mV s⁻¹. The PtNC/ZTC-based electrode exhibited ORR activity with an onset potential of 0.96 V (Fig. 4), which is close to that of Pt/C-TKK (0.98 V), and half-wave potentials of 0.84 V, which is 20 mV more positive than that of Pt/C-TKK (0.82 V). PtNC/ZTC showed a slightly higher diffusion-limiting current density of ∼5.9 mA cm⁻² (0.4–0.7 V) compared with that of the Pt/C-TKK catalyst (∼5.6 mA cm⁻²). The kinetics of the ORR in an acidic medium was further analyzed using RDE at different rotation rates (Fig. S13^†) and it was observed that the current density increases with the increasing speed of rotation of the electrode, as in the case of the alkaline medium. The number of electron involved and the amount of produced H₂O₂ estimated by RRDE measurement were ∼4 and < 5%, respectively (Fig. S14^†). The mass activity of PtNC/ZTC obtained using the mass transport corrected kinetic current at 0.8 V is 0.15 A mg⁻¹, which is 3.2 times higher than that of Pt/C-TKK (0.046 A mg⁻¹).Open in a separate windowFig. 4(a) RDE ORR polarization curves at 1600 rpm and (b) mass activity at 0.8 V of PtNC/ZTC and Pt/C-TKK in 0.1 M HClO₄.Furthermore, the methanol tolerance of PtNC/ZTC was assessed by intentionally adding methanol to the oxygen saturated electrolyte solution (both in alkaline and acidic media). The commercial Pt/C-TKK was used for comparison as well. The peak current densities for methanol oxidation with PtNC/ZTC were ∼2.8 and ∼3 times lower than that of Pt/C-TKK in alkaline (Fig. 5a) and acidic (Fig. 5b) media, respectively. These results indicate that PtNC/ZTC has much higher tolerance towards methanol than Pt/C-TKK does. This higher methanol tolerance of PtNC/ZTC can be attributed to the small size of the Pt cluster, which may not be sufficient to catalyze the oxidation of methanol efficiently, as the oxidation of methanol requires Pt ensemble sites.⁴³Open in a separate windowFig. 5ORR polarization curves of PtNC/ZTC and Pt/C-TKK in the absence (solid line) and presence (dotted line) of 0.1 M of CH₃OH at a rotation rate of 1600 rpm in (a) alkaline and (b) acid media.The durability of PtNC/ZTC was also investigated by the amperometric technique. The test was performed at a constant voltage of the half-wave potential in an O₂-saturated alkaline medium and at 0.7 V in an O₂-saturated acidic medium at a rotation rate of 1600 rpm (Fig. S15a and b^†). The durability of the PtNC/ZTC catalyst in the alkaline medium was higher than that of Pt/C-TKK, exhibiting a 30% decrease compared to a 40% decrease of Pt/C-TKK in 5.5 h of ORR operation (Fig. S15a^†). The higher durability of PtNC/ZTC compared to Pt/C-TKK in the alkaline medium may be due to the stabilization of PtNC by pore entrapment. In the acidic medium, however, PtNC/ZTC exhibited a 54% decrease in the initial current after 5.5 h of operation while a 33% decrease was observed in the case of Pt/C-TKK (Fig. S15b^†). The decrease in ORR activity in the acidic medium may be due to the leaching out of tiny Pt nanoclusters in acid electrolyte from the ZTC micropores. To understand the decrease in the ORR durability with time, STEM measurements of PtNC/ZTC after 5.5 h of ORR operation were performed. In the alkaline medium, the STEM image of post-ORR PtNC/ZTC shows a slight change in the size of PtNC (Fig. S15c^†) while the STEM image of PtNC/ZTC after ORR in the acidic medium exhibited sintering of PtNC into large particles with an average size of 30 nm (Fig. S15d^†), resulting in a decrease of the ORR activity. In the alkaline medium, the decrease in ORR activity with time may be due to the oxidation of the ZTC support in KOH.⁴⁴We attributed the excellent ORR activity of PtNC/ZTC to the interplay between the following: (1) the structure of the Pt cluster possessing a high ratio of surface atoms that benefits the surface reactions,^45–47 (2) the microporous 3D graphene-like structure of the ZTC support that enables easy access of O₂ and electrolyte molecules to the active sites,⁴⁸ and (3) the high conductivity and large accessible surface area of ZTC that facilitates the electron transfer.^49–51     

Metallo-supramolecular polymers derived from benzothiadiazole-based platinum acetylide complexes for fluorescent security application

Metallo-supramolecular polymers with the incorporation of benzothiadiazole-substituted organoplatinum moiety have been successfully constructed. The designed monomer displays intense fluorescence signals, which are severely quenched upon the supramolecular polymerization process. On–off switching of fluorescence can be further exploited for data security materials in response to the chemical stimuli. Accordingly, the resulting supramolecular polymers can be regarded as a novel and efficient candidate toward information processing applications.

Metallo-supramolecular polymers with the incorporation of benzothiadiazole-substituted organoplatinum moiety have been successfully constructed.

Metallo-supramolecular polymers (MSPs), which represent polymeric assemblies constructed by reversible metal–ligand coordination, are considered as an important class of organic/inorganic hybrid supramolecular materials. Owing to the incorporation of metallic complexes in the polymer chain, these macromolecular metal-containing systems not only possess the properties of traditional organic polymers (viscosity, processability, etc.), but also exhibit redox, optical, electrochromic, catalytic and magnetic properties.^1–5 In addition, due to the incorporation of reversible and weak recognition moieties on the supramolecular polymeric backbones, MSPs show unique stimuli-responsive characters, which is essential to develop environment-adaptable materials.^6–9The structure of MSPs and their physical properties can be elaborately regulated by the coordination ligand motifs.^10,11 Terpyridines and their structural analogs are the most popular chelating end-groups to form MSPs, ascribed to their capability to complex with a variety of transition metals.^10,12–16 Furthermore, the linkage on the polytopic ligand is also of crucial importance, since it dictates the structure arrangement and physicochemical properties of the targeted MSPs assemblies.¹⁰ Up to now, a variety of π-conjugated organic chromophores have been incorporated into the backbone of MSPs.^17–22 In stark contrast, the employment of π-conjugated organometallic units as the linkages has been far-less exploited.²³In this work, we sought to attain this objective, and construct a new type of MSPs with the involvement of platinum acetylide linkage. The monomeric structure is showed in Scheme 1: terpyridine moieties were incorporated on both sides of the monomer, in which the rigid benzothiadiazole-functionalized dinuclear platinum(ii) acetylide moiety serves as the linkage unit. Herein, the platinum(ii) acetylide derivatives were chosen as the linkages based on the following two considerations. First, platinum(ii) acetylide unit features the intriguing photo-physical properties such as larger stokes shifts and higher photoluminescence quantum yields, which are primarily arised from the overlapping of d-orbitals of the transition metal with p-orbitals of the alkyne ligands.^24–26 As a result, it endows the resulting supramolecular polymers with the fascinating optical and electrochromic properties.^27–31 Second, the rigid linkage incorporated in the monomer structure restrains the tendency for cyclization. The reduced critical polymerization concentration (CPC) value promotes the linear supramolecular polymerization process.^32–35 In the meantime, due to the dynamic properties of metal–ligand interactions, the resulting supramolecular polymer was anticipated to possess the stimuli-responsive properties, which display the potential applications as the smart materials.^36–39Open in a separate windowScheme 1Schematic representation for the formation of metallo-supramolecular polymer 1.The synthetic route for monomer is quite straightforward. As shown in Scheme S1,^† Sonogashira coupling reaction between terpyridine and benzothiadiazole-functionalized dinuclear platinum(ii) acetylide moieties was employed as the key step to construct the designed monomer. All of the synthetic compounds were fully characterized with NMR and ESI-MS spectra (Fig. S1–S4, ESI^†). The introduction of dinuclear platinum(ii) acetylide unit is expected to achieve low critical polymerization concentration (CPC) value for the supramolecular polymerization process. As a consequence, it facilitates to fabricate fully rigid supramolecular polymers with intriguing optical and electrochromic properties.Non-covalent complexation between the terpyridine-contained monomer and metal ion Zn²⁺ was first investigated via the spectroscopic measurements. For the monomer itself, it exhibits two absorption bands with the maximum wavelength located at 358 nm and 477 nm, respectively (Fig. 1a). The high-energy absorption at 358 nm is mainly derived from the π–π* intraligand transitions. For the low-energy absorptions at 477 nm, it could be ascribed to the interplay between the π-conjugated benzothiadiazole acetylene ligand and the transition metal Pt²⁺.²⁸ With the gradual addition of Zn²⁺ to the monomer in CHCl₃/CH₃OH (2 : 1, v/v), an obvious decrease absorbance at 370 nm was observed (Fig. 1a), indicating the transformation from free terpyridine species to the metal–terpyridine complex.³⁸ Upon excitation of monomer at 420 nm, the relative quantum yield is determined to be 3.5%, while the emission lifetime is 1.3 ns (Fig. S7^†). As shown in Fig. 1b, with the stepwise addition of the Zn²⁺, the fluorescence of monomer gradually decreases, and reaches the minimum at 602 nm when the ratio of monomer/Zn²⁺ achieves to 1.0. Also, the 1 : 1 complexation was validated by the ITC experiment (Fig. S5^†).Open in a separate windowFig. 1The intensity of changes (a) absorbance at λ = 370 nm and (b) emission intensity at λ = 602 nm upon addition of Zn(OTf)₂. Insets: arrow shows (a) UV/Vis absorption and (b) emission spectral changes of monomer (5.0 × 10⁻⁵ M) upon addition of Zn(OTf)₂.As widely documented, Zn²⁺ could complex with terpyridine to form two kinds of metallic complex species, Zn(tpy)₂²⁺ and Zn(tpy)²⁺.²² However, UV/Vis and fluorescence measurements could not distinctly distinguish such types of complex. In this regard, ¹H NMR titration measurements were employed to get further insights into the exchange kinetics of Zn²⁺–terpyridine complexes. As shown in Fig. 2, when the feed-ratio of Zn²⁺/monomer is below 1.0 (Fig. 2a–d), the terpyridine proton H₁ exhibits the remarkable upfield shift from 8.74 to 7.80 ppm due to the shielding effect, while both H₃ and H₅ exhibit obviously downfield shifts (H₃: from 7.92 to 8.17 ppm; H₅: from 8.60 to 8.95 ppm). Upon adding 1.0 equivalent Zn²⁺, the original uncomplexed terpyridine signals totally disappeared, suggesting the formation of dimeric Zn(tpy)₂²⁺. However, once Zn²⁺/monomer ratio is further increased, the newly signals evolved and gradually strengthen, suggesting the formation of Zn(tpy)²⁺.³⁸Open in a separate windowFig. 2Partial ¹H NMR spectra (300 MHz, CDCl₃: CD₃OD (2/1, v/v)): (a) monomer, (b–f) gradual titration of Zn²⁺ into monomer.After confirming the non-covalent complexation behavior between Zn²⁺and monomer in relatively low concentration as the monomeric state, the supramolecular polymerization process was further studied via concentration-dependent ¹H NMR measurement. According to Fig. S8,^† for the 1 : 1 mixture of monomer and Zn(OTf)₂ at 2.0 mM, the aromatic protons on terpyridine moiety show one set of well-defined sharp signals, implying the dominance of oligomers (Fig. S8a^†). In sharp contrast, the broadening of all signals at high concentration of monomer suggests the formation of supramolecular polymer (Fig. S8e^†). Such conclusion could be also validated by two-dimensional diffusion order spectrum experiment (DOSY), which is a convenient and efficient technique to monitor the size variation of the dynamic aggregations. When the monomer concentration increased from 0.5 to 30.0 mM, the measured diffusion coefficients of metallo-supramolecular polymer 1 decreased remarkably from 2.81 × 10⁻¹¹ to 8.91 × 10⁻¹² m² s⁻¹ (Fig. S9^†), implying the formation of large-sized supramolecular polymeric assemblies at high concentration.Next, capillary viscosity measurements were performed to study the macroscopic properties of the resulting supramolecular assemblies. All of the viscosity studies were performed in DMSO containing 0.05 M tetrabutylammonium hexafluorophosphate to exclude the polyelectrolyte effect.³⁹ As shown in Fig. 3a, the monomer shows the comparably shallow curve for the specific viscosities. In sharp contrast, the specific viscosity of 1 : 1 mixture of Zn²⁺ and monomer changes exponentially as a function of monomer concentration, revealing the formation of high-molecular-weight supramolecular polymer.Open in a separate windowFig. 3(a) Specific viscosities of metallo-supramolecular polymer 1 (red) and the corresponding monomer(blue), (b) double logarithmic plots of specific viscosity of metallo-supramolecular polymer 1versus monomer concentration.The double logarithmic plots of versus specific viscosity concentration were further obtained for 1 (Fig. 3b), which displays a clear slope change. In the low concentration range, the slope value was determined to be 1.03, which indicates the presence of oligomers with the constant size. Remarkably, a sharp rise in the viscosity was observed when the concentration exceeds the critical polymerization concentration value (around 4.0 mM), and the slope value was estimated up to 1.40. Such phenomenon is in highly consistent with the aformentioned DOSY and ¹H NMR results, suggesting the formation of large supramolecular polymeric assemblies at high concentration.Stimuli-responsive properties of the resulting supramolecular polymer were further exploited, by manipulating the dynamic property of Zn²⁺–terpyridine recognition motif. It is well known that 1,4,7,10-tetraazacyclododecane (cyclen) exhibits higher affinity toward Zn²⁺ than terpyridine unit. With the addition of cyclen, the decrease of Zn²⁺/tpy absorption bands (λ = 477 nm), together with the increase of fluorescence intensity (λ = 602 nm), suggest that cyclen competitively complexes with terpyridine to destroy the Zn²⁺/terpyridine interactions (Fig. 4a, and S10^†). Such process could also be validated by capillary viscosity experiments, which is shown in Fig. 4b. With the stepwise addition of cyclen, the specific viscosity of the metallo-supramolecular polymer decreases and gradually levels off, suggesting the disassembly of metallo-supramolecular polymer. However, with the further addition of Zn²⁺, metallo-supramolecular polymer could be recovered. The conclusion could be manifested by the reappearance of Zn²⁺/tpy absorption band, the decrease of fluorescence intensity (Fig. S10^†), and the restore of the original ¹H NMR signals (Fig. S11^†). Hence, the successive addition of the competitive ligand cyclen and Zn²⁺ provide a possible method to manipulate the reversible assembly/disassembly of the metallo-supramolecular polymer.Open in a separate windowFig. 4(a) UV/Vis absorption spectral changes of metallo-supramolecular polymer 1 upon the successive addition of cyclen and Zn(OTf)₂. (b) Viscosity changes upon adding cyclen to metallo-supramolecular polymer 1.As mentioned above, the fluorescence intensity of resulting supramolecular polymers could be regulated by stepwise addition of cyclen and Zn²⁺. By taking advantage of the “on/off” fluorescent switching properties, we sought to explore their potential applications for data security. In detail, the solution of metallo-supramolecular polymer 1 was doped into polylactic acid (PLA) and formed a bright orange film (1.2 cm × 1.0 cm). The fluorescence intensity of resulting film is relatively lower, owing to the existence of Zn²⁺/terpyridine metal–ligand interactions. The resulting film could be used as a drawing board for the applications in document security, by employing the solution of cyclen as a magic ink (Fig. 5). In detail, when a letter “Z” was written on the surface of the drawing board, there have almost no changes for the film under daylight. However, the patterned security feature of “Z” encrypted with the solution of cyclen could be readily visualized under UV light, which could be ascribed to the disassembly of Zn²⁺–terpyridine complexes. Moreover, the letter “Z” could also be wiped by immerging the film into the Zn²⁺ solution and the drawing board could be recovered.Open in a separate windowFig. 5Left was irradiated by day light, and right was irradiated by 365 nm. (a) Metallo-supramolecular polymer 1 is doping in the PLA film, (b) cyclen solution is further coated on film, (c) the film is recovered with Zn(OTf)₂ solution.In summary, we have constructed the rigid metallo-supramolecular polymer, by using metal–ligand interactions as the non-covalent connecting bonds. Benzothiadiazole-functionalized dinuclear platinum(ii) acetylide moiety was incorporated, rendering fascinating photo-physical properties to the resulting monomer and supramolecular polymeric assemblies. ¹H NMR, UV/Vis, ITC, DOSY, and viscosity measurements were employed to investigate the supramolecular polymerization process. Additionally, the metallo-supramolecular polymer 1 could be reversibly assembled and disassembled upon adding the competitive ligands. By taking advantage of the fluorescence “on/off” switch process, the resulting supramolecular polymer could be used as fluorescent security materials. Therefore, the current work provides a convenient and efficient approach to fabricate supramolecular polymer toward smart information processing materials.^40,41     

pH sensing and bioimaging using green synthesized carbon dots from black fungus

Biomass is regarded as an excellent candidate for the preparation of carbon nanomaterials. A pH sensor was established based on carbon dots synthesized from black fungus, and possesses good fluorescence response and reversibility for pH detection. Meanwhile, the CDs can also be applied to intra-cellular bioimaging, showing potential for bioimaging.

Carbon dots derived from black fungus were prepared and applied as a pH sensor for real water samples.

Carbon dots (CDs) are spherical fluorescent nanoparticles with sizes less than 10 nm.¹ Due to their attractive properties of high chemical stability, bright fluorescence, water solubility, facile synthesis and easy modification, CDs have attracted lots of attention in recent years. Many works revealed green synthetic approaches of CDs from renewable or natural materials and have advantages such as non-toxicity, economic production, cleanliness and facile accessibility.^2,3 For example, banana,^4,5 eggshell membrane,⁶ roasted chickpeas,⁷ tapioca flour,⁸ spinach,⁹ green tea leaf residue,¹⁰ pea¹¹ and betel leaves¹² have all been utilized as carbon precursors. The main synthesis approaches could be classified into two categories, top-down and bottom-up strategies. The top-down method refers to cutting down large carbon materials into fine nanoparticles by acid etching or laser cauterization.¹³ While, the bottom-up method involves chemically fusing organic molecules via carbonization and dehydration under thermal/hydrothermal/solvothermal conditions, which is effective and simple. A hydrothermal method has been proved to be facile and rapid for CD fabrication.¹⁴ CDs can be directly obtained from various green materials under high temperature and pressure conditions. The as-prepared CDs, as promising fluorescent materials, were applied widely in many practical applications including biological labeling, optoelectronic devices, biosensors, gene delivery, drug delivery, antimicrobial coating and so on, showing their potential in replacing heavy metal-doped quantum dots and organic dyes.^15–20The pH value plays an important role in both environmental and biological processes. Slight change of intracellular pH can also lead to organ damage or neurological problems. Recently, fluorescent CDs-based nanosensors have been established for the pH detection in environmental samples and vivo/vitro bioanalysis.²¹ In this communication, blue-emissive CDs with good biocompatibility and high fluorescence were fabricated by facile hydrothermal treatment of black fungus at 200 °C. The obtained CDs were utilized in pH determination and cell imaging for preliminary study.²²The CDs were synthesized via a rapid and low-cost hydrothermal treatment using black fungus as the carbon precursor. The black fungus was grounded and mixed with distilled water. The mixture was then poured into a 50 mL Teflon reaction vessel and autoclaved at 200 °C for 4 h. After cooling to room temperature, the reaction yielded a kind of brown solution with strong blue fluorescence. After purification by filter and dialysis, pure CDs were obtained. The products could also be dried to solid by freeze-drying. Full details for the preparation conditions optimization (reaction time and temperature of the hydrothermal treatment) were listed in the ESI^† (Tables S1 and S2). Under the same preparation conditions, the reaction phenomena were consistent with different batches. The overall scheme of the preparation of CDs derived from black fungus is presented in Scheme 1.Open in a separate windowScheme 1Schematic illustration of CDs synthesis.The CDs were analyzed with high resolution transmission electron microscopy (HRTEM) images to ascertain their morphology. Fig. 1a shows the as-prepared CDs are nearly spherical in shape and well distributed with average sizes of 2.23 nm. The lattice fringes spacing in HRTEM image is observed to be 0.24 nm corresponding to the (1 1 2) facet of graphitic carbon, which indicates good crystallinity of the CDs (Fig. S1^† and inset of Fig. 1a). The X-ray diffraction (XRD) pattern in Fig. 1c exhibits a broad peak at about 22.4°, associated with the graphitic structure of the CDs.Open in a separate windowFig. 1TEM image (inset: HR-TEM image) (a), particle size distribution (b), XRD pattern (c), FTIR spectrum (d), XPS spectrum (e), and Raman spectrum (f) of the CDs.Fourier transform infrared spectrum (FTIR) and X-ray photoelectron spectroscopy (XPS) were conducted to confirm the states of the elements and analyze the surface functional groups of the CDs. As shown in Fig. 1d, the characteristic peaks at 3445, 1642, 1138, 1071 cm⁻¹ corresponded to the stretching vibrations of O–H/N–H, asymmetric stretching vibrations of C N/C O, aromatic stretching vibrations of C–N and tensile vibrations of C–O. The full range XPS spectrum confirms the main peaks of CDs are C, N, O and S at 284.8, 401.0, 531.5 and 170.4 eV with atomic contents of 64.14%, 10.01%, 24.24%, and 1.61%, respectively, indicating the incorporation of N and S into the carbon matrix. The abundance of proteins in black fungus provides the doping of heteroatoms. The fitted peaks of C_1s, N_1s and O_1s were shown in high-resolution XPS to analyze the chemical bonds (Fig. S2^†). The C_1s spectrum could be deconvoluted into four peaks at 284.0 eV, 284.7 eV, 285.8 eV, 287.5 eV, corresponding to the four kinds of C-bonds, including C C, C–C, C–N/C–O, C N/C O. The O_1s spectrum was deconvoluted into two components of C O (531.5 eV) and C–OH (532.8 eV). The deconvolution of N_1s spectrum demonstrated the presence of three types of N-bands: pyridinic N at 398.7 eV, amino N at 399.5 eV and pyrrolic N at 401.1 eV, respectively. The XPS data and FTIR spectrum are in good agreement, which determined that many hydrophilic groups on the surface endow CDs with good water solubility. Raman spectrum is shown in Fig. 1f. The feature centered at 1378 cm⁻¹ (D band) implying the sp³ C–C bands or defects in graphene framework, while the feature centered at 1570 cm⁻¹ (G band) indicates the character of sp² nature for C C bands.The optical properties of the CDs from black fungus were characterized by UV-vis and fluorescence spectroscopy. The absorption spectrum of the CDs in aqueous solution was shown in Fig. 2a. The peaks at 230 nm and 282 nm are ascribed to the π–π* of C C bonds and n–π* of C C/C O bonds transitions,²³ respectively. The fluorescence spectra of as-prepared CDs excited by different wavelengths are shown in Fig. 2b. The as-prepared CDs demonstrated excitation-dependent FL behavior, which approximates to reported literatures of CDs.²⁴ Increasing the excitation wavelength from 320 nm to 420 nm, the emission peak red-shifts gradually. At 370 nm excitation, the CDs show highest intensity at emission peak of 450 nm (Fig. S3^†). The CDs solution emitted bright blue fluorescence under irradiation of 365 nm UV lamp (Fig. 2b, inset), which can be used as invisible ink for information encryption or painting patterns (Fig. S4^†). The fluorescence quantum yield of as-prepared CDs was estimated to be 11.3% by the direct measurement, which is comparable with other reported CDs derived from biomass (Table S3^†).Open in a separate windowFig. 2Absorption spectrum (a), fluorescence emission spectra of CDs: excitation wavelength starts from 320 to 420 nm; inset: photos of the CDs dispersed in water under daylight (left) and UV irradiation (right) (b), the relationship between the FL intensity and pH variation in the range of 1–13 (excited at 370 nm) (c), the fitted linear curve with pH variation around physiological condition (4.0–13.0) (d).The stability of CDs under different conditions was investigated as shown in Fig. S5.^† Although the FL intensity of CDs was slightly affected by NaCl concentrations, there was still above 90% of intensity in a NaCl solution with a high concentration of 2.0 M (Fig. S5a^†). The effect of exposure time on FL intensity was shown in Fig. S5b.^† It can be found that the FL intensity remained negligible change after continuous 365 nm UV exposure of 60 min, indicating good photobleaching resistance. Moreover, after long time storage for 20 days, the CDs solutions showed little effect, suggesting good time stability (Fig. S5c^†).The CDs exhibited an obvious pH-sensitive FL property due to the protonation–deprotonation of the surface functional groups. Fig. 2c depicts the effects of solution pH value on the FL intensity at the emission wavelength of 450 nm when excited at 370 nm. The FL intensity of as-prepared CDs has an incredibly fast response to the change of solution pH value. Increasing the pH value from 1–13, the intensity of CDs showed an increasing trend from pH 1 to 4 and then decreases gradually. There is an excellent linear relationship between the FL intensity and the pH value with a pH range from 4–13. The linear regression equation is (F − F₀)/F₀ = −0.1062 pH + 0.4452 and the linear dependence coefficient (R²) is 0.9925. After 5 times of changing pH value from 5 to 11 and then back to 5, the FL intensity does not show apparent change from the original value under the same solution pH conditions, demonstrating good pH reversible performance and response ability of the CDs (Fig. S6^†). A series of control experiments were carried out to evaluate the sensitivity and selectivity of the CDs when used as the pH detection probe. Unlike significant fluorescence changes with adjusting the pH value of CDs solution, common cations, anions and compounds showed negligible effect on the fluorescence intensity, indicating excellent selectivity to pH values (Fig. S7^†). Therefore, the CDs can be served as potential pH sensor in both environmental samples or biological systems. As illustrated in Table S4,^† different samples were taken as analytical models. The results calculated by the proposed CDs-based probe and measured by pH-meter are basically the same.In view of good fluorescence properties of the CDs, the fluorescence imaging in vitro was conducted. HeLa cells were used as the model system to investigate the cytotoxicity of CDs. The toxicity of the obtained CDs towards HeLa cells could be negligible even after 48 h. The cell viability was higher than 80% at a high concentration of 800 μg mL⁻¹. The HeLa cells were cocultured with the as-prepared CDs (100 μg mL⁻¹) at 37 °C for 4 h and then subjected to the confocal fluorescence imaging (Fig. 3). Bright blue fluorescence signal of the cells could be observed, which demonstrates that the CDs have potential applications in biological imaging. To further investigate the biocompatibility of the as-prepared CDs, we conducted the hydroponics experiments of soybeans with CDs solutions. We grew soybeans sprouts using CDs aqueous solution. After incubation for 3–4 days at room temperature, the soybeans have sprouted normally, and their sprouts displayed blue fluorescence emissions under UV light (365 nm), demonstrating that the CDs were non-toxic and easily taken into the soybeans (Fig. S8^†). Therefore, the obtained CDs have good biocompatibility.Open in a separate windowFig. 3Bright-field image (a) and the fluorescence image in vitro of HeLa cells with CDs (the excitation wavelength was 370 nm) (b).     

Electrochemical preparation system for unique mesoporous hemisphere gold nanoparticles using block copolymer micelles

Gold nanoparticles (AuNPs) are widely used in various applications, such as biological delivery, catalysis, and others. In this report, we present a novel synthetic method to prepare mesoporous hemisphere gold nanoparticles (MHAuNPs) via electrochemical reduction reaction with the aid of polymeric micelle assembly as a pore-directing agent.

Mesoporous hemisphere Au nanoparticles using self-assembled micelles, for the first time, are demonstrated by using electrochemical reduction on a Ti substrate.

Gold (Au) is one of the most stable and versatile elements utilized in various fields, including catalysis, optics, and industrial purposes. Consequently, various shapes and sizes of AuNPs have been intensively studied to improve the performance of Au in different applications.^1–6 Previously, nanoporous or dendritic metal nanostructures, including Au nanostructures, have been synthesized by employing different reagents and conditions such as SH-terminated amphiphilic surfactant,⁷ pH controlling,⁸ and hard-templates.^9,10 The reported porous and dendritic Au nanostructures possess high surface areas and rich active sites, which in turn lead to highly enhanced catalytic activities.Recently, a soft-template method using self-assembled micelles or lyotropic liquid crystals as pore-directing agents has allowed the successful synthesis of mesoporous nanoparticles^11–13 and films^14–17 with different metal compositions. The metals with mesoporous structures demonstrate superior catalytic activity per weight or surface area over their nonporous bulk forms. Previously, our group reported a several-fold increase in the catalytic activity of mesoporous metals in reactions such as the methanol oxidation reaction (MOR),^14,15 ethanol oxidation reaction (EOR),^13,15–17 and nitric oxide reduction¹² as compared to their bulk nanoparticles and films. Such improvement in the catalytic activity of mesoporous structures is mainly attributed to their significantly larger surface areas, more exposed catalytically active sites, and increased durability against aggregation.Interestingly, nanoporous or mesoporous Au structures had been successfully synthesized by using a dealloying method¹⁸ and a hard templating method.⁹ Such methods, however, are a little complicated, and pore-directing templates often remain within the pores, thus leading to severe contamination. Using a thiol group is an alternative way to synthesize mesoporous Au nanospheres.⁷ A significant drawback of using a thiol group, however, is its strong chemical bonding with Au, thus becoming unable to be removed. The synthesis of mesoporous structures using self-assembled polymeric micelles as soft-templates, on the other hand, is a more facile method with fewer synthetic steps, and it is also known to be free of contaminations within the pores. Although a soft-templating method using polymeric micelles has been utilized for the preparation of mesoporous Au and Au-based alloy films towards surface-enhanced Raman scattering (SERS) signals,¹⁹ glucose sensing,^20,21 and MOR,²² the obtained morphologies have been limited to only films.Despite such apparent benefits arising from mesoporous structures and their synthesis using soft-templates, the synthesis of mesoporous AuNPs using soft-templates has not been achieved yet. It is mainly due to the physical and chemical properties of Au which make it extremely hard to form mesoporous structures. Herein, we adopt an electrochemical approach and the soft-template method to synthesize MHAuNPs successfully. As discussed above, we expect MHAuNPs to be highly efficient in various applications in medical diagnosis,²³ optical sensing,²⁴etc.In this report, MHAuNPs with different shapes and sizes are for the first time reported by changing various electrochemical deposition conditions such as applied potentials between electrodes and deposition times. Scheme 1 shows the schematic illustrations of the entire process of precursor preparation (Scheme 1a) and the MHAuNPs fabrication process (Scheme 1b), including the deposition and the detachment of the nanoparticles. The characterization methods implemented in this paper are mentioned in ESI.^†Open in a separate windowScheme 1(a) The process of Au precursor solution preparation and (b) fabricating MHAuNPs by electrochemical reduction.In a typical experiment, a p-doped silicon (Si) wafer was cleaned by using acetone, isopropyl alcohol, and deionized water (DIW) with sonication for 5 minutes, followed by nitrogen (N₂) gas blowing to dry the Si wafer. After the wet cleaning process, the Si surface was treated by oxygen (O₂) plasma for 5 minutes (Oxford Instruments PlasmaPro 80 Reactive Ion Etcher) to remove residual organic impurities. Then, 10 nm of titanium (Ti) layer and 100 nm of Au layer were deposited sequentially by electron beam evaporation (Temescal FC-2000 e-beam evaporator) at 10⁻⁶ torr. Commercially available Au etchant (Sigma-Aldrich) was used to etch the Au film to expose the Ti area (the left image in Scheme 1b). During etching, about 20 percent of Au area was left to be connected to the electrochemical work station, as drawn in Scheme 1b. In preparation of the Au precursor solution, 5 mg of poly(styrene)-block-poly(ethylene oxide) (PS-b-PEO, the number of average molecular weight (M_w) for each block is 18 000 for PS and 7500 for PEO, respectively) was mixed in 1.5 ml of tetrahydrofuran (THF) followed by stirring at 300 rpm for 8 hours. Then, 0.75 ml of ethanol, 0.5 ml of HAuCl₄ aqueous solution (40 mM), and 1.25 ml of DIW were added sequentially. The solution was stirred for another 30 minutes at 200 rpm. The existing block copolymer micelles can be confirmed by TEM observation, and the average diameter is 25 nm, as shown in Fig. S1.^† For the electrochemical deposition, an electrochemical workstation (CH Instruments Inc. 660e) with three electrode system was used to deposit MHAuNPs on the Ti/Si substrate. After the deposition, the particles were carefully washed by chloroform, followed by a rinse using DIW to remove the residual micelles completely. To detach and collect MHAuNPs from the Ti/Si substrate, the substrate was soaked in ethanol and strongly sonicated for a few minutes (Scheme 1b).Fig. S2^† shows the details of the growth mechanism of MHAuNPs by different deposition times. At the initial stage (Fig. S2a^†), small nanoparticles are generated by reducing Au ions in the precursor solution throughout the substrate. Then, the seed starts growing and forming MHAuNPs as the deposition time increases (Fig. S2b–e^†). This similar growth mechanism is the same as the previous report.¹⁹ The high-angle annular detector dark-field scanning transmission electron microscopy (HAADF-STEM) image (Fig. S2f^†) shows the mesopores inside the MHAuNPs are homogeneously generated. As-obtained MHAuNPs consist of a pure Au element without any impurities, as shown in Fig. S3.^† Fig. 1 and S4^† show scanning electron microscope (SEM) images of MHAuNPs deposited at different voltages from −0.2 V to −0.9 V vs. Ag/AgCl at high magnification and low magnification, respectively. Different deposition voltages lead to significant changes in the particle sizes but slight differences in the particle shapes. The size distributions of MHAuNPs and the plots of the average diameters of MHAuNPs by different deposition voltages are described in Fig. 2. The distribution graphs show the large sizes of particles, such as more than 1 μm in diameter, when the high voltage (−0.2 V vs. Ag/AgCl) is applied (Fig. 2a). The distribution becomes narrower upon the lower applied voltage. The average diameter-applied voltage plots in Fig. 2b show that the average particle size decreases from around 1.1 μm at −0.2 V to about 300 nm at −0.9 V. Thus when the lower deposition voltages are applied (i.e., the deposition rate is higher) (Fig. 1g–h), the smaller particles with a higher degree of size uniformity are obtained. The opposite trend is observed at higher deposition voltages (i.e., the deposition rate is lower) (Fig. 1a and b), at which the particles become larger and their size uniformity decreases. This trend is because the higher voltage allows only a limited number of seed particles to be deposited on the Ti/Si substrate, and each seed individually grows with no additional seed formation. Whereas the lower voltage can allow a higher number of seeds, leading to a uniform supply of electrons from the working Ti/Si electrode (Fig. S5^†). In addition, the lower deposition voltages make the particle shape more hemispherical in Fig. 1f–h.Open in a separate windowFig. 1The SEM images of MHAuNPs electrochemically deposited at (a) −0.2 V, (b) −0.3 V, (c) −0.4 V, (d) −0.5 V, (e) −0.6 V, (f) −0.7 V, (g) −0.8 V, and (h) −0.9 V for 500 s. The scale bars indicate 200 nm.Open in a separate windowFig. 2(a) Size distributions of MHAuNPs generated by different voltages and (b) the average diameter–the applied voltage plots. Fig. 3 shows the SEM images of MHAuNPs deposited at −0.2 V and for different deposition times from 250 s to 1000 s. Although longer deposition time does not change the number of MHAuNPs, it leads to the growth of MHAuNPs in lateral and vertical directions. Although the MHAuNPs grow more than about two or three times larger at long deposition time, the mesoporous formation does not seem to be changed, as shown in insets in Fig. 3. This point indicates that the deposition time is not the main factor affecting the formation of mesoporous structures as well as the number of particles (seeds), but it affects the sizes of particles.Open in a separate windowFig. 3The SEM images of MHAuNPs deposited at −0.2 V (vs. Ag/AgCl) for (a) 250 s, (b) 500 s, and (c) 1000 s. The scale bars indicate 10 μm. The insets in each figure are magnified SEM images of each condition (The scale bars in insets indicate 500 nm).In this report, 10 nm Ti layer on Si wafer plays an important key role in the formation of MHAuNPs, as previously mentioned in the experimental procedure. The use of the Ti substrate with low conductivity (ca. 2.38 × 10⁶ S m⁻¹), which is about only 5.8% in comparison with that of Au (ca. 4.10 × 10⁷ S m⁻¹), is not common in the electrochemical plating research field.^25–30 Most of the papers on mesoporous metal structures synthesized by electrochemical deposition have utilized Au or Pt substrates due to its chemical stability and high electrical conductivity.^14–17 Fig. S6^† shows the amperometry (i–t) curves during the deposition of MHAuNPs (black dots) on a Ti/Si substrate and mesoporous gold films (red dots) on an Au substrate at the same deposition condition. As shown in Fig. S6,^† around 1/7 times less current flows on the Ti/Si substrate throughout the deposition time. This low current density on the Ti/Si substrate is one of the factors for fabricating MHAuNPs. Low current density causes the formation of a few particles (i.e., seeds) at the initial stage of the deposition and leads to seed growth in a few places, as explained in Fig. S2.^† Furthermore, the use of Ti/Si substrates affects the bottom parts of MHAuNPs to become an arch shape. Only edges of MHAuNPs attach onto the Ti/Si substrates, as shown in Fig. 4. This attachment is because the interaction between the deposited MHAuNPs and the Ti substrate surface (probably, the Ti surface can be partially oxidized, forming TiO_x) is very weak. Therefore, the deposited MHAuNPs can be easily detached from the Ti/Si substrates by sonicating the substrates in solvents (Scheme 1b). The collected MHAuNPs in a solvent are obtained as colloidal particles as shown in Fig. S7.^† Such interesting hemispherical mesoporous nanoparticles have advantages to electrocatalytic activities in comparison to spherical mesoporous metals.³¹ The method using a Ti/Si substrate as a working electrode can be repeatedly implemented with one substrate and without change of the precursor solution, thus it can be effective for mass production in the future.Open in a separate windowFig. 4The SEM image of MHAuNPs deposited at −0.6 V. The arrow shows that the bottom of the MHAuNPs is an arch.Finally, surface-enhanced Raman scattering (SERS) effects on MHAuNPs were investigated by using an adsorbate called rhodamine 6G (R6G), as shown in Fig. S8.^† The resulting MHAuNPs at all conditions (−0.3 V, −0.6 V, and −0.9 V) show substantially strong SERS intensity (Fig. S8a^†), while Ti/Si and Si substrates without MHAuNPs show noise level of intensity. To further investigate enhancement factor (EF) and limit of detection (LoD), various concentrations of R6G with MHAuNPs fabricated at −0.9 V were used for the SERS studies (Fig. S8b^†). The main peak of SERS is 1363 cm⁻¹, and it disappears from less than 10⁻⁶ M concentration, while the 1183 cm⁻¹ peak still exists at 10⁻⁸ M (Fig. S8c^†). The maximum EFs at 1363 cm⁻¹ (10⁻⁶ M) and 1183 cm⁻¹ (10⁻⁸ M) are 1.5 × 10⁴ and 3.1 × 10⁶, respectively (Fig. S8d^†). Transmission electron microscope (TEM) images in Fig. S9^† show the detailed particle structures and the electron diffraction (ED) pattern confirmed the crystal structure is the face-center cubic (FCC) structure. The sharp surface structures and the pores on MHAuNPs provide abundant hot spots that have been reported as the origin that enhances SERS intensity owing to the plasmon resonances.^19,32 Besides, the high density of small-sized MHAuNPs (Fig. S5 and S10^†) boosted higher SERS intensity.In conclusion, we have synthesized MHAuNPs by using 10 nm Ti-coated Si substrates as a working electrode on a Si wafer and electrochemical deposition using self-assembled polymeric micelles as pore-directing agents. The low current generates Au seeds at only a few places, and it acts as the points that MHAuNPs start growing. The particle shapes and sizes can be controlled by changed applied voltages and deposition times. The lower voltages make small particles and the great hemispherical AuNPs with mesoporous architecture. The long-time deposition does not affect any mesoporous formation, but the particle shape and size. Besides, the low affinity between Au and Ti (probably, oxidized layer) results in the arch on the bottom of MHAuNPs, which helps the particles detached from the substrates easily. These results indicate that different thicknesses and compositions of working electrodes can provide different metal deposition phenomena, which can bring out unique shaped particles with mesoporous architectures in the future.