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

A dual responsive fluorescent probe for selective detection of cysteine and bisulfite and its application in bioimaging

A coumarin-based dual responsive fluorescent probe with a simple structure was developed for the detection of Cys and HSO₃⁻. Under simulated physiological conditions, Cou-F displayed an on–off fluorescence response to Cys at 521 nm and an off–on fluorescence response to HSO₃⁻ at 500 nm. Furthermore, Cou-F had the advantages of high sensitivity, strong specificity and rapid response. The detection limits of Cou-F toward Cys and HSO₃⁻ were 0.54 μM and 0.65 μM, respectively. Cou-F enabled high selective responses to Cys and HSO₃⁻ over other biologically related species. The response times of Cou-F toward Cys and HSO₃⁻ were 80 s and 100 s. The fluorescence imaging of Cys and HSO₃⁻ was achieved in living RAW246.7 cells.

A coumarin-based dual responsive fluorescent probe with a simple structure was developed for the detection of Cys and HSO₃⁻.     

A molecular design towards sulfonyl aza-BODIPY based NIR fluorescent and colorimetric probe for selective cysteine detection

Cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are essential biothiols for cellular growth, metabolism, and maintenance of a biological system. Thus, the detection of biothiols is highly important for early diagnosis and evaluation of disease progression. In this article, a series of sulfonyl aza-BODIPYs was synthesized, characterized, and examined by ¹H-NMR, ¹³C-NMR, crystallization, photophysical properties and DFT calculation. Among these structures, a fluorescent probe, BDP-1, exhibited selective detection of Cys among various biothiols via nucleophilic aromatic substitution and typical size of Cys molecules. BDP-1 showed color change and near-infrared (NIR) fluorescence enhancement after reaction with Cys to generate BDP-OH, confirmed by HRMS. The red shift of absorption wavelength showed a similar tendency resulting in time-dependent density functional theory (TD-DFT). Furthermore, the calculated detection limit of BDP-1 toward Cys was 5.23 μM. This probe facilitates the colorimetric and fluorescent detection of Cys over other biothiols.

A new fluorescent and colorimetric probe based-on sulfonyl aza-BODIPY (BDP-1–3) are designed and synthesized for selective cysteine detection.     

A novel and fast responsive turn-on fluorescent probe for the highly selective detection of Cd2+ based on photo-induced electron transfer

A novel, highly sensitive and fast responsive turn-on fluorescence probe, 2,2′-((1E,1′E)-((1,10-phenanthroline-2,9-diyl)bis(methanylylidene)) bis(azanylylidene)) diphenol (ADMPA), for Cd²⁺ was successfully developed based on 2,9-dimethyl-1,10-phenanthroline and o-aminophenol. ADMPA showed a remarkable fluorescence enhancement toward Cd²⁺ against other competing cations, owing to the suppression of the photo-induced electron transfer (PET) and CH N isomerization. A good linear relationship (R² = 0.9960) was obtained between the emission intensity of ADMPA and the concentration of Cd²⁺ (0.25–2.5 μM) with a detection limit of 29.3 nM, which was much lower than that reported in literature. The binding stoichiometry between ADMPA and Cd²⁺ was 2 : 1 as confirmed by the Job''s Plot method, which was further confirmed by a ¹H NMR titration experiment. Moreover, the ADMPA probe was successfully applied to detect Cd²⁺ in real water samples with a quick response time of only 6.6 s, which was about 3–40 times faster than the reported cadmium ion probe.

A novel, highly sensitive and fast responsive turn-on fluorescence probe ADMPA for Cd²⁺ was successfully developed based on 2,9-dimethyl-1,10-phenanthroline and o-aminophenol.     

A ratiometric fluorescent probe for detection of exogenous mitochondrial SO2 based on a FRET mechanism

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO₂ was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO₃²⁻ with a fast response time (3 min) and detection limit of 0.13 μM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO₂ in cells.

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO₂ was designed and synthesized.     

A selective hybrid fluorescent sensor for fructose detection based on a phenylboronic acid and BODIPY-based hydrophobicity probe

Fructose is widely used in the food industry. However, it may be involved in diseases by generating harmful advanced glycation end-products. We have designed and synthesized a novel fluorescent probe for fructose detection by combining a phenylboronic acid group with a BODIPY-based hydrophobicity probe. This probe showed a linear fluorescence response to d-fructose concentration in the range of 100–1000 μM, with a detection limit of 32 μM, which is advantageous for the simple and sensitive determination of fructose.

We have designed and synthesized a novel fluorescent probe for fructose detection through hydrophobic interactions by combining a phenylboronic acid group and a BODIPY-based hydrophobicity probe with a detection limit of 32 μM.     

Remarkably selective biocompatible turn-on fluorescent probe for detection of Fe3+ in human blood samples and cells

The robust nature of a biocompatible fluorescent probe is demonstrated, by its detection of Fe³⁺ even after repeated rounds of quenching (reversibility) by acetate in real human blood samples and cells in vitro. Significantly trace levels of Fe³⁺ ions up to 8.2 nM could be detected, remaining unaffected by the existence of various other metal ions. The obtained results are validated by AAS and ICP-OES methods. A portable test strip is also fabricated for quick on field detection of Fe³⁺. As iron is a ubiquitous metal in cells and plays a prominent role in biological processes, the use of this probe to image Fe³⁺ in cells is a substantial development towards biosensing. Cytotoxicity studies also proved the nontoxic nature of this probe.

The robust nature of a biocompatible fluorescent probe is demonstrated, by its detection of Fe³⁺ even after repeated rounds of quenching (reversibility) by acetate in real human blood samples and cells in vitro.     

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 turn-on fluorescent probe with a dansyl fluorophore for hydrogen sulfide sensing

Hydrogen sulfide (H₂S) is a biologically relevant molecule that has been newly identified as a gasotransmitter and is also a toxic gaseous pollutant. In this study, we report on a metal complex fluorescent probe to achieve the sensitive detection of H₂S in a fluorescent “turn-on” mode. The probe bears a dansyl fluorophore with multidentate ligands for coordination with copper ions. The fluorescent “turn-on” mode is facilitated by the strong bonding between H₂S and the Cu(ii) ions to form insoluble copper sulfide, which leads to the release of a strongly fluorescent product. The H₂S limit of detection (LOD) for the proposed probe is estimated to be 11 nM in the aqueous solution, and the utilization of the probe is demonstrated for detecting H₂S in actual lake and mineral water samples with good reproducibility. Furthermore, we designed detector vials and presented their successful application for the visual detection of gaseous H₂S.

H₂S turn on the fluorescence of DNS–Cu complex probe.     

An aptasensor for the label-free detection of thrombin based on turn-on fluorescent DNA-templated Cu/Ag nanoclusters

A highly sensitive thrombin aptasensor was constructed based on the alteration of the aptamer conformation induced by the target recognition and the turn-on fluorescence due to the proximity of two darkish DNA-templated copper/silver nanoclusters (DNA-Cu/Ag NCs). Two DNA templates were designed as the functional structures consisting of the Cu/Ag NC-nucleation segment located at two termini or one terminus and the aptamer segment in the middle of a DNA template. Two darkish DNA-Cu/Ag NCs came close to each other when the aptamer combined with the target due to the conformational alteration of the aptamer structure, resulting in an increased fluorescence signal readout. Thrombin was sensitively determined as low as 1.6 nM in the range of 1.6–8.0 nM with a high selectivity. Finally, this sensor succeeded in detecting thrombin in a real fetal bovine serum.

A highly sensitive thrombin aptasensor was constructed based on the alteration of the aptamer conformation induced by the target recognition and the turn-on fluorescence due to the proximity of two darkish DNA-templated copper/silver nanoclusters (DNA-Cu/Ag NCs).     

Near-infrared turn-on fluorescent probe for discriminative detection of Cys and application in in vivo imaging

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio. Herein, a highly water-soluble NIR probe (NIRHA) based on a hemicyanine skeleton and bearing an acrylate moiety was synthesized. The probe showed high selectivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The probe also had low cytotoxicity and was successfully applied in HeLa cells and mouse experiments. Results of bioimaging experiments indicated that the probe was effective for visualizing endogenous Cys in vitro and in vivo.

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio.     

A highly selective TPE-based AIE fluorescent probe is developed for the detection of Ag+

The detection of Ag⁺ in the environment is very important to determine the level of pollution from silver complexes, which have caused various human health problems. Herein, an aggregation-induced emission (AIE) chromophore (tetraphenylethane, TPE) attached to a benzimidazole group (tetra-benzimidazole, TBI–TPE) is synthesized and utilized to detect Ag⁺ in the environment. The strong chelating effect between the benzimidazole group and Ag⁺ leads to the formation of aggregates, and strong yellow fluorescence signals were observed after adding Ag⁺ into a TBI–TPE solution. The stoichiometry of the complex of TBI–TPE and Ag⁺ was established to be 1 : 2 using photochemical and mass spectra measurements. The detection limit of the Ag⁺ assay is 90 nM with a linear range from 100 nM to 6 μM. This study provides a facile method to determine Ag⁺ in real environmental samples with satisfactory results.

We develop a highly selective TPE-based AIE fluorescent probe containing a benzimidazole group for the detection of Ag⁺.     

A novel fluorescent off–on probe for the sensitive and selective detection of fluoride ions

A highly sensitive and selective fluorescent probe for fluoride ions has been developed by incorporating the dimethylphosphinothionyl group as a recognition moiety into the fluorophore of coumarin. The detection mechanism is based on the fluoride ion-triggered cleavage of the dimethylphosphinothionyl group, followed by the release of coumarin, which leads to a large fluorescence enhancement at 455 nm (λ_ex = 385 nm). Under the optimized conditions, the fluorescence enhancement of the probe is directly proportional to the concentration of fluoride ions in the range of 0–30 μM with a detection limit of 0.29 μM, which is much lower than the maximum content of fluoride ions guided by WHO. Notably, satisfying results have been obtained by utilizing the probe to determine fluoride ions in real-water samples and commercially available toothpaste samples. The proposed probe is rather simple and may be useful in the detection of fluoride ions in more real samples.

A sensitive and selective fluorescent off–on probe is developed for fluoride ion detection, and its applicability has been demonstrated by determining fluoride ions in real-water samples and toothpaste samples.     

A novel ratiometric fluorescent probe for the selective determination of HClO based on the ESIPT mechanism and its application in real samples

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO. The reaction site of diaminomaleonitrile at the ortho-position of the phenolic hydroxyl group made the probe exhibit a ratiometric fluorescence response towards hypochlorous acid (HClO). The specific sensing mechanism was verified via MS, HPLC and ¹H NMR spectroscopy. Moreover, the probe showed excellent performance with high sensitivity and good selectivity towards HClO in the presence of other reactive oxygen species. In addition, the probe was successfully applied to detect HClO spiked in tap water, river water and diluted human serum with good recoveries.

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO.     

Highly selective and sensitive fluorescent probe for the rapid detection of mercury ions

Mercury (Hg) is one of the major toxic heavy metals, harmful to the environment and human health. Thus, it is significantly important to find an easy and quick method to detect Hg²⁺. In this study, we designed and synthesized a simple fluorescent probe with excellent properties, such as high sensitivity and selectivity, rapid response, and outstanding water solubility. When Hg²⁺ (5 μM) was added to the probe solution, it exhibited a very large fluorescent enhancement (about 350-fold stronger than the free probe) with the help of hydrogen peroxide (H₂O₂). Probe HCDC could quantitatively detect Hg²⁺ in the range of 0–10 μM using the fluorescence spectroscopy method and the detection limit was measured to be about 0.3 nM (based on a 3σ/slope). Analytical application was also studied, and the probe HCDC exhibited excellent response to Hg²⁺ with the addition of H₂O₂ in real water samples. So, our proposed probe HCDC provided a practical and promising method for determining Hg²⁺ in the environment.

A novel water-soluble, highly selective and sensitive rapid-response fluorescent probe was developed to monitor mercury ions in real water samples.     

A FRET based ratiometric fluorescent probe for detection of sulfite in food

A new fluorophore pyrido[1,2-a]benzimidazole based ratiometric fluorescent probe for the selective detection of sulfite ions in water was investigated. It shows large (pseudo) Stokes shifts (260 nm), high FRET efficiency, high selectivity and sensitivity. A distinct color change from red to colorless was observed and importantly, it proves to be a convenient and efficient tool to detect the sulfite levels in sugar samples.

A new fluorophore pyrido[1,2-a]benzimidazole based ratiometric fluorescent probe for the selective detection of sulfite ions in water was investigated.     

A selective and sensitive near-infrared fluorescent probe for real-time detection of Cu(i)

The disruption of copper homeostasis (Cu⁺/Cu²⁺) may cause neurodegenerative disorders. Thus, the need for understanding the role of Cu⁺ in physiological and pathological processes prompted the development of improved methods of Cu⁺ analysis. Herein, a new near-infrared (NIR) fluorescent turn-on probe (NPCu) for the detection of Cu⁺ was developed based on a Cu⁺-mediated benzylic ether bond cleavage mechanism. The probe showed high selectivity and sensitivity toward Cu⁺, and was successfully applied for bioimaging of Cu⁺ in living cells.

The disruption of copper homeostasis (Cu⁺/Cu²⁺) may cause neurodegenerative disorders.     

A highly sensitive and selective fluorescent probe without quencher for detection of Pb2+ ions based on aggregation-caused quenching phenomenon

Lead is a highly toxic heavy metal, and various functional nucleic acid (FNA)-based biosensors have been developed for the detection of Pb²⁺ in environmental monitoring. However, most fluorescence biosensors that have been reported were designed on the basis of a double-labeled (fluorophore and quencher group) DNA sequence, which not only involved an inconvenient organic synthesis but also restricted their wider use in practical applications. Here, we utilized a G-rich DNA sequence as a recognition probe and conjugated fluorene (CF) to develop a fluorescence sensor without a quencher based on the aggregation-caused quenching (ACQ) effect. In the presence of Pb²⁺, the degree of aggregation of CF was reduced because Pb²⁺ induced the formation of a G-quadruplex structure of the CF-DNA probe, and the fluorescence signal increased with the concentration of Pb²⁺ (0–1 μM), with a limit of detection of 0.36 nM. This fluorescent probe without a quencher enables the sensitive and selective detection of Pb²⁺. On the basis of these advantages, the CF-DNA probe represents a promising analytical method for detecting Pb²⁺.

Fluorescent probe with only a fluorophore but no quencher for detecting Pb²⁺ on the basis of the aggregation-caused quenching (ACQ) phenomenon.     

A fluorescent microsensor for the selective detection of bifenthrin

Based on the fluorescence quenching phenomenon, a smart fluorescent microsensor was synthesized. The bifenthrin (BI) microsensor inherited the high selectivity of molecular imprinted polymers (MIPs) and the excellent fluorescence properties of aqueous CdTe quantum dots (QDs). Aqueous CdTe QDs are functionalized by octadecyl-4-vinylbenzyl-dimethyl-ammonium chloride (OVDAC). A type of functional monomer, 4-vinylphenylboronic acid (VPBA), was used and its boronic acid groups could covalently combine with a cis-diol compound for direct imprinting polymerization. The OVDAC-functionalized aqueous CdTe QDs were used as solid supports and auxiliary monomers. Under optimal conditions, experimentation showed that BI had a linear detection range of 10 to 300 μmol L⁻¹ with a correlation coefficient of 0.9968 and a high imprinting factor (IF) of 4.53. In addition, the prepared MIP-OVDAC/CdTe QDs were successfully used to detect BI in water samples. Therefore, this work provided a highly selective and sensitive fluorescence probe for the detection of BI. In addition, the fluorescence probe could be used to detect other targets by changing the functional monomers.

Based on the fluorescence quenching phenomenon, a smart fluorescent microsensor was synthesized.     

Development of a turn-on graphene quantum dot-based fluorescent probe for sensing of pyrene in water

Polycyclic aromatic hydrocarbons (PAHs) are potentially harmful pollutants that are emitted into the environment from a range of sources largely due to incomplete combustion. The potential toxicity and carcinogenic effects of these compounds warrants the development of rapid and cost-effective methods for their detection. This work reports on the synthesis and use of graphene quantum dots (GQDs) as rapid fluorescence sensors for detecting PAHs in water. The GQDs were prepared from two sources, i.e. graphene oxide (GO) and citric acid (CA) – denoted GO-GQDs and CA-GQDs, respectively. Structural and optical properties of the GQDs were studied using TEM, Raman, and fluorescence and UV-vis spectroscopy. The GQDs were then applied for detection of pyrene in environmental water samples based on a “turn-off-on” mechanism where ferric ions were used for turn-off and pyrene for turn-on of fluorescence emission. The fluorescence intensity of both GQDs was switched on linearly within the 2–10 × 10⁻⁶ mol L⁻¹ range and the limits of detection were found to be 0.325 × 10⁻⁶ mol L⁻¹ and 0.242 × 10⁻⁶ mol L⁻¹ for GO-GQDs and CA-GQDs, respectively. Finally, the potential application of the sensor for environmental water samples was investigated using lake water and satisfactory recoveries (97–107%) were obtained. The promising results from this work demonstrate the feasibility of pursuing cheaper and greener environmental monitoring techniques.

Graphene quantum dots provide a more environmentally friendly fluorescence sensor for pyrene.