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 real-time ratiometric fluorescent probe for imaging of SO2 derivatives in mitochondria of living cells

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer (ICT) of the hemicyanine dye platform. The mechanism of using the probe is mainly through the Michael addition that occurs between IN-CZ and sulfite with a detection limit of 2.99 × 10⁻⁵ M. IN-CZ displays a fast response (within 1 minute) and is highly selective for SO₃²⁻/HSO₃⁻ over ROS, biologically relevant ions, biological mercaptans and other reactive species. More importantly, IN-CZ was suitable for ratiometric fluorescence imaging in living cells, by real-time monitoring of SO₃²⁻/HSO₃⁻ changes in mitochondria targeted in living cells.

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer of the hemicyanine dye platform.     

A novel ratiometric fluorescent probe for rapid detection of hydrogen peroxide in living cells

In this work, we present a new ratiometric fluorescent probe JNY-1 for rapid and convenient detection of H₂O₂. The probe could selectively and sensitively respond to H₂O₂ within 10 min. In addition, this probe was successfully applied for monitoring and imaging of H₂O₂ in liver cancer HepG2 cells under physiological conditions.

A new ratiometric fluorescent probe JNY-1 for sensitive detection of H₂O₂ is presented with selectivity over other reactive oxygen species, reactive nitrogen species, and biologically relevant species. Imaging of H₂O₂ in liver cancer HepG2 cells was achieved.     

A ratiometric fluorescent probe for the detection of β-galactosidase and its application

Herein, a coumarin fluorescent probe (Probe 1) was developed for the ratiometric detection of β-galactosidase (β-gal) activity. The detection range was 0–0.1 U mL⁻¹ and 0.2–0.8 U mL⁻¹, and the limit of detection (LOD) was 0.0054 U mL⁻¹. Moreover, the luminous intensity of Probe 1 increased gradually with increase in β-gal activity. It could be observed under 254 nm UV irradiation by the naked eye. Furthermore, this method only required a small amount of sample (20 μL) and a short analytical time (30 min) for the detection of β-gal activity with a low LOD. Probe 1 was successfully used to detect β-gal activity in real fruit samples, and can be applied to the quantitative and qualitative detection of β-gal activity.

A ratiometric fluorescent probe was successfully used as a tool to determine β-galactosidase activity in fruits.     

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.     

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.     

11-Mercaptoundecanoic acid capped gold nanoclusters as a fluorescent probe for specific detection of folic acid via a ratiometric fluorescence strategy

A novel ratiometric fluorescence strategy is developed for specific detection of folic acid (FA) by using 11-mercaptoundecanoic acid protected gold nanoclusters (AuNCs@MUA). In this design, the fluorescence color of the probe can be switched among red, pink, violet and blue by varying the concentration of FA. AuNCs@MUA possesses strong fluorescence peaking at 612 nm (R-signal) and FA exhibits blue emissive auto-fluorescence at 446 nm (B-signal), showing a large emission shift of ∼170 nm. When AuNCs@MUA approaches FA through electrostatic binding, the R-signal decreases while the B-signal increases with titration of FA. Based on the above phenomenon, a radiometric analysis platform is constructed for FA target detection, with a wide linear response range from 0 to 20 μM, and an excellent detection limit of 26 nM. This new ratiometric strategy exhibits low background, and wide signal changes in a low concentration range, which presents obvious advantages over most previous FA detections based on single-responsive fluorescence methods. Furthermore, the proposed method is successfully applied to determine FA in human serum samples.

A novel ratiometric fluorescence strategy is developed for specific detection of folic acid (FA) by using 11-mercaptoundecanoic acid protected gold nanoclusters (AuNCs@MUA).     

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.     

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 novel ratiometric AIEE/ESIPT probe for palladium species detection with ultra-sensitivity

Existing fluorescent probes for palladium (Pd) species detection have revealed their vulnerabilities, such as low sensitivity, poor anti-interference ability and long reaction time. In order to develop a faster and more accurate detection method for palladium species at extremely low concentrations, in this study, we designed a novel ratiometric AIEE/ESIPT probe (HPNI-1) based on the Tsuji–Trost reaction for Pd. According to the data obtained, the probe was able to detect Pd species with an ultra-high anti-interference ability (Pd : other metals = 1 : 1000), rapid detection time (within 2 minute) and ratiometric fluorescent signal changes with a 1.34 nM detection limit. This study not only proves that existing methods can be improved but also provides future prospects for HPNI-1 as one of the greatest probes for Pd species detection.

Existing fluorescent probes for palladium (Pd) species detection have revealed their vulnerabilities, such as low sensitivity, poor anti-interference ability and long reaction time.     

A curcumin-based AIEE-active fluorescent probe for Cu2+ detection in aqueous solution

Curcuminoids have been extensively investigated as metal ion probes, but the intrinsic aggregation-caused-quenching (ACQ) characteristic of curcumin would hinder their applications in aqueous solution. Fortunately, tetraphenylethylene (TPE) could endow the compounds with aggregation-induced emission (AIE)/aggregation-induced enhanced emission (AIEE) characteristics to eliminate the ACQ effect. According to this strategy, a series of TPE-modified curcumin derivatives L1–4 were prepared and studied for their AIEE properties. Among the four TPE-curcumin analogues, only L1 particles have been successfully used as an on-off fluorescence probe for detecting Cu²⁺ in aqueous solution. The fluorescence titration experiment determined its detection limit of 1.49 × 10⁻⁷ mol L⁻¹, and the binding ratio between L1 and Cu²⁺ was estimated as 2 : 1, which was in agreement with the results of high resolution mass spectrum and Job''s plot. In addition, the binding constant was evaluated as 6.77 × 10² M⁻¹ using a Benesi–Hildebrand plot. Finally, the obtained L1-based indicator paper showed significant fluorescence response to Cu²⁺ aqueous solution. This TPE-modified strategy improves the detection capability of curcumin probe in aqueous solution and provides a feasible way to obtain other probes with ACQ characteristics.

A curcumin-based AIEE-active L1 was synthesized and used to prepare an on-off fluorescent probe for Cu²⁺ detection in aqueous solution.     

A ratiometric fluorescent sensor for detection of metformin based on terbium–1,10-phenanthroline–nitrogen-doped-graphene quantum dots

Metformin (MTF), an effective biguanide and oral antihyperglycemic agent, is utilized to control blood glucose levels in patients with type II diabetes mellitus, and the determination of its concentration in biological fluids is one of the main issues in pharmacology and medicine. In this work, highly luminescent nitrogen-doped graphene quantum dots (N-GQDs) were modified using terbium (Tb³⁺)–1,10-phenanthroline (Phen) nanoparticles (NPs) to develop a dual-emission ratiometric fluorescent sensor for the determination of MTF in biological samples. The synthesized N-GQDs/Tb–Phen NPs were characterized using different techniques to confirm their physicochemical properties. The N-GQDs/Tb–Phen NPs showed two characteristic emission peaks at 450 nm and 630 nm by exciting at 340 nm that belong to N-GQDs and Tb–Phen NPs, respectively. The results indicated that the emission intensity of both N-GQDs and Tb–Phen NPs enhanced upon interaction with MTF in a concentration-dependent manner. Also, a good linear correlation between the enhanced fluorescence intensity of the system and MTF concentration was observed in the range of 1.0 nM–7.0 μM and the limit of detection (LOD) value obtained was 0.76 nM. In addition, the prepared probe was successfully used for the estimation of MTF concentration in spiked human serum samples. In conclusion, the reported dual-emission ratiometric fluorescent sensor can be used as a sensitive and simple fluorimetric method for the detection of MTF in real samples.

Shcematic representation of the MTF detection by an enhancing mechanism.     

Construction of a lysosome-targetable ratiometric fluorescent probe for H2O2 tracing and imaging in living cells and an inflamed model

Hydrogen peroxide (H₂O₂), an important reactive oxygen species (ROS) with unique destructive oxidation properties, can be produced in lysosomes to fight off pathogens. Although many fluorescent probes have been developed for the detection and imaging of H₂O₂, the development of a ratiometric fluorescent probe for H₂O₂ detection and imaging in lysosomes and an inflammation model remains rather scarce. Therefore, it is important to develop an efficient tool for monitoring H₂O₂ in inflamed tissues to evaluate the physiological and pathological relationship between inflammation and lysosomal H₂O₂. In this work, a new naphthalimide-based lysosome-targeting fluorescent probe (NPT-H₂O₂) for ratiometric detection and imaging was developed in vitro and in vivo. The probe exhibited two well-resolved emission peaks separated by 125 nm, rapid response (<40 s), and high selectivity and sensitivity toward H₂O₂, as well as low cytotoxicity in vitro. Inspired by prominent features of these results, we further successfully applied NPT-H₂O₂ for H₂O₂ imaging with a dual-channel in living cells, demonstrating that our probe NPT-H₂O₂ was targeted in the lysosomes. Finally, NPT-H₂O₂ was used for H₂O₂ detection in inflamed tissues and achieved satisfactory results. We predict that our probe can be used as a powerful tool to reveal the relationship between physiology and pathology of inflammation and lysosomal H₂O₂.

Hydrogen peroxide (H₂O₂), an important reactive oxygen species (ROS) with unique destructive oxidation properties, can be produced in lysosomes to fight off pathogens.     

Triple-FRET multi-purpose fluorescent probe for three-protease detection

A new, robust and reliable methodology for three-protease screening in a single-enzyme mode has been developed and verified, employing a multi-purpose peptide probe with three selectively cleavable sites furnished with four fluorophores. A triple-FRET-based single-excitation quadruple-emission concept for unambiguous sensing of trypsin, chymotrypsin and caspase-8 in the lowest detectable concentrations of 0.5 ng mL⁻¹, 0.2 μg mL⁻¹, and 2 U mL⁻¹, respectively, has been applied and graphically depicted. Then the developed 4-dye probe has been also studied from the perspective of simultaneous two-protease screening, which was found only partially feasible, primarily due to unselective chymotrypsin cleavage.

A triple-FRET four-dye system for detection of three proteases has been developed and verified.     

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 ratiometric fluorescence probe for the selective detection of H2S in serum using a pyrene-DPA–Cd2+ complex

A ratiometric and selective hydrogen sulfide (H₂S) detection probe was proposed based on the pyrene-DPA–Cd²⁺ complex through the metal ion displacement approach (MDA) mechanism. While most MDA-based fluorescence probes with paramagnetic Cu²⁺ have focused on the development of a simple turn-on sensor using the broad spectral range of fluorescence enhancement, this ratiometric probe exhibited unchanged monomer emission as a built-in internal reference with an increase in excimer emission with added H₂S. The demonstrated probe showed a rapid response (within 1 min) and a high sensitivity, with 70 nM as the limit of detection. The selectivity for H₂S over cysteine, homocysteine and glutathione was confirmed, and reliable fluorescence enhancement, which could be monitored by the naked eye, was observed upon irradiation with handheld UV light. In addition, this detection system was successfully applied to detect H₂S in human serum without interference from biological molecules.

The pyrene-DPA–Cd²⁺ complex is demonstrated as a ratiometric fluorescence probe for selective hydrogen sulfide detection in serum based on a metal displacement approach.     

A gold nanoparticle based colorimetric and fluorescent dual-channel probe for acetylcholinesterase detection and inhibitor screening

Based on the competitive host–guest interaction between p-sulfonatocalix[6]arene (p-SC₆A) capped AuNPs and Rhodamine B (RhB)/acetylthiocholine, a fluorescent and colorimetric dual channel probe was developed for rapid detection of AChE with high sensitivity and selectivity. The detection limit was estimated to be 0.16 mU mL⁻¹. Crucially, due to the specific host–guest interaction, the high selectivity of the bioassay permitted the discrimination of AChE from other cations and proteins including biothiols and enzymes. Furthermore, the present method was also successfully applied to determinate AChE levels and screen AChE inhibitors in real cerebrospinal fluid (CSF) samples, which suggested that our proposed method has great potential to be applied in monitoring the disease progression and drug treatment effects of Alzheimer''s disease (AD).

A novel colorimetric and fluorescent dual-channel probe was developed for acetylcholinesterase detection and inhibitor screening with high sensitivity and selectivity.     

An ink-jet printed dual-CD ratiometric fluorescent paper-based sensor for the visual detection of Cu2+

Copper ion (Cu²⁺) plays an important role in the human body because it is beneficial for metabolism. However, an excessive or slight amount of Cu²⁺ can cause various symptoms. Therefore, it is necessary for human health to realize the trace and visual detection of Cu²⁺. Referring to traditional fluorescence test papers, the qualitative and semi-quantitative detection of Cu²⁺ could be realized by a dual-carbon dots (CDs) ratiometric fluorescent paper-based sensor with the advantages of environmental protection, portability and low cost. In this paper, the inkjet-printed test paper with the best mixing ratio of the two CDs has been researched to maximize the spectral energy transfer of ion detection (maximum color gamut expansion). Among them, the preparation method of b-CDs has been improved, increasing the photoluminescence quantum yield (PLQY) to 88.9%. The sensitivity detection limit of the double emission ratio sensor was 0.15 nM in solution, and the human eye can distinguish at least 3 μmol L⁻¹ Cu²⁺ in the paper-based sensor. Compared with the traditional single-emission sensor, the human eye was more sensitive to the color change of the emission ratio sensor. The results indicate that we not only realized the micro detection of Cu²⁺ with convenient methods, but also provided a promising strategy for the visual detection of Cu²⁺.

A fluorescent test paper sensor for qualitative and semi-quantitative detection of Cu²⁺ is designed based on high photoluminescence quantum yield (PLQY) carbon dots (CDs).     

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 novel hydrophilic fluorescent probe for Cu2+ detection and imaging in HeLa cells

Copper is an essential element in living systems and plays an important role in human physiology; therefore, methods to detect the concentration of copper ions in living organisms are important. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe that can be used for the detection of Cu²⁺. The probe, BNQ, has high selectivity and sensitivity. The fluorescence intensity of the probe at 520 nm was visible to the naked eye under a UV lamp; upon the gradual addition of Cu²⁺, there was a colour change from green to nearly colourless. Furthermore, the detection limit of BNQ for Cu²⁺ was 45.5 nM. The detection mechanism was investigated using a Job''s plot and density functional theory (DFT) calculations. In addition, owing to great biocompatibility, we were able to successfully use BNQ to detect Cu²⁺ in living HeLa cells with low toxicity.

Probe BNQ was successfully used for detection of exogenous Cu²⁺ in cells using a rare ESDPT sensing mechanism.