A fluorescence turn-on probe for cysteine and homocysteine based on thiol-triggered benzothiazolidine ring formation

We synthesized a new coumarin-based probe TP, containing a disulfide moiety, to detect biothiols in cells. A fluorescence turn-on response is induced by the thiol–disulfide exchange of the probe, with subsequent intramolecular benzothiazolidine ring formation giving rise to a fluorescent product. The probe exhibits an excellent selectivity for cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) and other amino acids. The fluorescent probe also exhibits a highly sensitive fluorescence turn-on response to Cys and Hcy with detection limits of 0.8 μM for Cys and 0.5 μM for Hcy. In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the probe TP could be an efficient fluorescent detector for thiols in living cells.     

Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au₈-cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au₈-cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au₈-cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au₈-cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au₈-cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot.     

A colorimetric,ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine

A chlorinated coumarin-aldehyde was developed as a colorimetric and ratiometric fluorescent probe for distinguishing glutathione (GSH), cystenine (Cys) and homocysteine (Hcy). The GSH-induced substitution-cyclization and Cys/Hcy-induced substitution-rearrangement cascades lead to the corresponding thiol-coumarin-iminium cation and amino-coumarin-aldehyde with distinct photophysical properties. The probe can be used to simultaneously detect GSH and Cys/Hcy by visual determination based on distinct different colors – red and pale-yellow in PBS buffer solution by two reaction sites. From the linear relationship of fluorescence intensity and biothiols concentrations, it was determined that the limits of detection for GSH, Hcy and Cys are 0.08, 0.09 and 0.18 μM, respectively. Furthermore, the probe was successfully used in living cell imaging with low cell toxicity.     

Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-(m-methylphenyl) benzoxazole

A new thiol fluorescence probe, 5-maleimidyl-2-(m-methylphenyl)benzoxazole (MMPB) has been developed for the direct determination of reduced glutathione (GSH) in real samples. Compared to the reported N-substituted maleimide type of thiol reagents, the main advantage of MMPB is its rather high selectivity for GSH to cysteine (Cys), which often coexists with GSH in biological samples. Under mild conditions similar to the physiological environment, MMPB reacted with GSH to give a highly fluorescent derivative with the excitation and emission wavelengths of 299.2 and 355.8 nm, respectively. In the presence of 0.40-fold (molar ratio) of Cys, a linear relationship was found in the range of 0-1.62×10⁻⁷ mol l⁻¹ with the detection limit (3σ) of 3.23×10⁻¹⁰ mol l⁻¹ for GSH determination. Many other amino acids (100-fold) did not interfere with the determination. Since the molar ratio of Cys to GSH in mammalian tissues and blood does not exceed the value of 0.40:1, the proposed method has been used in the direct determination of GSH in these kinds of biological samples, such as human blood, pig’s liver and heart with the recoveries of 94.3-104.5%     

Sensing for intracellular thiols by water-insoluble two-photon fluorescent probe incorporating nanogel

A novel “turn-on” two-photon fluorescent probe containing a π-conjugated triarylboron luminogen and a maleimide moiety DMDP-M based on the photo-induced electron transfer (PET) mechanism for biothiol detection was designed and synthesized. By simply loading the hydrophobic DMDP-M on a cross-linked Pluronic^® F127 nanogel (CL-F127), a probing system DMDP-M/CL-F127 was established, which shows quick response, high selectivity and sensitivity to cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous phase. The DMDP-M/CL-F127 system presented the fastest response to Cys with a rate constant of 0.56 min⁻¹, and the detection limit to Cys was calculated to be as low as 0.18 μM. The DMDP-M/CL-F127 system has been successfully applied to the fluorescence imaging of biothiols in NIH/3T3 fibroblasts either with single-photon or two-photon excitation because of its high biocompatibility and cell-membrane permeability. The present work provides a general, simple and efficient strategy for the application of hydrophobic molecules to sensing biothiols in aqueous phase, and a novel sensing system for intracellular biothiols fitted for both single-photon and two-photon fluorescence imaging.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

A highly selective turn-on fluorescent probe for hypochlorous acid based on hypochlorous acid-induced oxidative intramolecular cyclization of boron dipyrromethene-hydrazone

A BODIPY-based fluorescent probe, HBP, was developed for the detection of hypochlorous acid based on the specific hypochlorous acid-promoted oxidative intramolecular cyclization of heterocyclic hydrazone in response to the amount of HOCl. The reaction is accompanied by a 41-fold increase in the fluorescent quantum yield (from 0.004 to 0.164). The fluorescence intensity of the reaction between HOCl and HBP is linear in the HOCl concentration range of 1–8 μM with a detection limit of 2.4 nM (S/N = 3). Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HBP could be used as an effective fluorescent probe for detecting HOCl in living cells.     

Selective labeling of histidine by a designed fluorescein-based probe

The synthesis of a novel fluorescent probe, 3-epoxypropoxy fluorescein (EPF), and its properties for labeling of histidine are described. The probe contained a fluorescein fluorophore with long-wavelength response and an active epoxy labeling group. In alkaline media EPF reacted selectively with histidine, rather than with other amino acids, causing a large increase in fluorescence intensity and thereby allowing a selective detection of histidine. This fluorescence increase resembled that of the fluorescein diaion with the increase of the media basicity, suggesting that the addition reaction of histidine with the epoxy group provides the fluorophore moiety with a basic molecular environment. As an application of this probe, fluorescent labeling of histidine in human serum was attempted and the obtained results were in agreement with those given by using histidine-nickel complex adsorptive voltammetry. Further, the relative S.D. of the method was 1.8% for 10 replicate determinations of 0.55 μM histidine. When 10 μM of EPF was used, the linear range for histidine was 0.007-10 μM with a detection limit (S/N=3) of 0.001 μM.     

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.     

Determination of non-protein cysteine in human serum by a designed BODIPY-based fluorescent probe

A novel fluorescent probe is designed and synthesized for the determination of cysteine in biological samples by incorporating 2,4-dinitrobenzenesulfonyl (DBS) group as a quencher into the BODIPY skeleton. The BODIPY-based probe itself shows weak fluorescence due to the strong intramolecular charge transfer process. Upon reaction with cysteine, however, the probe produces a rapid and large fluorescence enhancement through the removal of the DBS unit by nucleophilic aromatic substitution. This valuable property leads to the development of a new and simple method for cysteine assay. Under the optimized conditions, the fluorescence enhancement value is directly proportional to the concentration of cysteine in the range 2-12 μM, with a detection limit of 30 nM (S/N = 3). The applicability of the developed method has been successfully demonstrated on the determination of non-protein cysteine in human serum. Compared to most of the existing fluorescent probes proposed for cysteine, the BODIPY-based one exhibits an excellent overall performance in terms of selectivity, sensitivity and simplicity.     

Sensitive detection of biothiols and histidine based on the recovered fluorescence of the carbon quantum dots–Hg(II) system

In this paper, we presented a novel, rapid and highly sensitive sensor for glutathione (GSH), cysteine (Cys) and histidine (His) based on the recovered fluorescence of the carbon quantum dots (CQDs)–Hg(II) system. The CQDs were synthesized by microwave-assisted approach in one pot according to our previous report. The fluorescence of CQDs could be quenched in the presence of Hg(II) due to the coordination occurring between Hg(II) and functional groups on the surface of CQDs. Subsequently, the fluorescence of the CQDs–Hg(II) system was recovered gradually with the addition of GSH, Cys or His due to their stronger affinity with Hg(II). A good linear relationship was obtained from 0.10 to 20 μmol L⁻¹ for GSH, from 0.20 to 45 μmol L⁻¹ for Cys and from 0.50 to 60 μmol L⁻¹ for His, respectively. This method has been successfully applied to the trace detection of GSH, Cys or His in human serum samples with satisfactory results. The proposed method was simple in design and fast in operation, which demonstrated great potential in bio-sensing fields.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.     

Development of a fluorescence-based microtiter plate method for the measurement of glutathione in yeast

The present work was aimed to the development of a fluorescence assay using the universal 96-well microplate format, for the measurement of reduced glutathione (GSH) in yeast cells. The method relies upon the reaction between GSH and a highly selective fluorogenic probe, i.e. naphthalene-2,3-dicarboxaldehyde (NDA). The optimization of the method included the extraction step of GSH from cultured yeast cells in a cold perchloric acid solution, derivatization conditions (10-min reaction at pH 8.6 and at 20 ± 2 °C in darkness) and stability studies of the resulting fluorescent adduct. Full selectivity was observed versus other endogenous thiols (except for γ-glutamylcysteine), glutathione disulfide (GSSG) and enzymatic reducing reagents of GSSG. Linearity was verified in the range 0.3-6.5 μM (R² > 0.98) and limits of quantification and detection were 0.3 and 0.05 μM, respectively. Relative standard deviation corresponding to repeatability (n = 3) and inter-day precision (n = 5) were 2.8 and 6.1%, respectively. Mean GSH recovery from cell extracts was 95%. The method appeared highly correlated (R² = 0.96) with a previously reported HPLC method.The method was then applied to the monitoring of GSH in the yeast strain Kluyveromyces lactis during its growth period and in the presence of an inhibitor of GSH biosynthesis. The method presents the main advantage of a high throughput for the measurement of biological samples. The extent of the method to the study of the redox couple GSSG/GSH by including an enzymatic reduction step and the enhancement of the fluorescence signal using cyclodextrins were discussed.     

Enzymatic rotating biosensor for cysteine and glutathione determination in a FIA system

The high sensitivity that can be attained using an enzymatic system and mediated by catechols has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of catechols, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV. Thus, when l-cysteine (Cys) or glutathione (GSH) was added to the solution, these thiol-containing compounds participate in Michael addition reactions with catechols to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. Cys was used as the model thiol-containing compound for the study. The highest response for Cys was obtained around pH 7. This method could be used to determine Cys concentration in the range 0.05-90 μM (r = 0.998) and GSH concentration in the range 0.04-90 μM (r = 0.999). The determination of Cys and GSH were possible with a limit of detection of 0.7 and 0.3 nM, respectively, in the processing of as many as 25 samples per hour. Current response of the HRP-rotating biosensor is not affected by the oxidized form of GSH and Cys (glutathione disulfide, GSSG, and l-cystine, respectively), by sulfur-containing and alkyl-amino compounds such as methionine and lysine, respectively. The interferences from easily oxidizable species such as ascorbic acid and uric acid are lowest.     

Hypochlorous acid turn-on boron dipyrromethene probe based on oxidation of methyl phenyl sulfide

A boron dipyrromethene (BODIPY)-based fluorometric probe, HCS, has been successfully developed for the highly sensitive and selective detection of hypochlorous acid (HOCl). The probe is based on the specific HOCl-promoted oxidation of methyl phenyl sulfide. The reaction is accompanied by a 160-fold increase in the fluorescent quantum yield (from 0.003 to 0.480). The fluorescent turn-on mechanism is accomplished by suppression of photoinduced electron transfer (PET) from the methyl phenyl sulfide group to BODIPY. The fluorescence intensity of the reaction between HOCl and HCS shows a good linearity in the HOCl concentration range 1–10 μM. The detection limit is 23.7 nM (S/N = 3). In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the HCS probe could be an efficient fluorescent detector for HOCl in living cells.     

Highly sensitive and selective detection of biothiols using graphene oxide-based “molecular beacon”-like fluorescent probe

A fluorometric method for quantity analysis of biothiols was developed using a graphene oxide (GO)-based “molecular beacon”-like probe, which consisted of FITC labeled thymine (T)-rich single-stranded DNA (ssDNA), GO and Hg²⁺ ions. The labeled ssDNA containing T–T mismatches would self-hybridize to duplex in the presence of Hg²⁺, which can avoid its adsorption on GO and the fluorescence of this GO-based probe was recovered. The fluorescence of the probe quenched after the addition of biothiols such as glutathione (GSH) and cysteine (Cys) owing to thiol groups can selectively competitive ligation of Hg²⁺ ions with T–T mismatches. In the present work, the GO-based probe was used for the determination of GSH and Cys. Under the optimal conditions, a linear correlation was established between fluorescence intensity ratio I₀/I and the concentration of GSH in the range of 2.0 × 10⁻⁹–5.0 × 10⁻⁷ mol L⁻¹ with a detection limit of 1.0 × 10⁻⁹ mol L⁻¹. The linear range for Cys is from 5.0 × 10⁻⁹ to 4.5 × 10⁻⁷ mol L⁻¹ with a detection limit of 2.0 × 10⁻⁹ mol L⁻¹. The proposed method was applied to the determination of GSH in human serum and cell extract samples with satisfactory results.     

Exceptional time response,stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform

A phenyl-selenium-substituted coumarin probe was synthesized for the purpose of achieving highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ∼100-fold fluorescent enhancement compared with the signal generated for other closely related amino acids, including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH at the 4-position of the probe, which is doubly-activated by two carbonyl groups, occurs extremely fast, showing subsecond maximum fluorescence intensity attainment; equilibrium was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.     

Simple and practical,highly sensitive and responsive recognition of cysteine: Design,synthesis and mechanism study of a novel curcumin fluorescent probe

A novel class of curcumin-derived fluorescent probes was designed. This kind of probe introduces easy leaving groups methylsulfonyl and phenylsulfonyl respectively to achieve the detection effect through the nucleophilic attack of amino acids. At the same time, BF₂ group is introduced to increase the emission wavelength of the probe. Probes 4 and 5 can respond quickly with amino acids, but can specifically recognize Cys. In UV detection, the maximum absorption wavelength of the probes can be blue-shifted by 81 nm with the addition of Cys and still show a strong fluorescence signal. The detection limits for compounds 4 and 5 were determined to be 0.40 μM and 0.87 μM, respectively, with a goodness-of-fit of 0.99. In addition, a rapid response of the probe to Cys could be observed with the naked eye within 1 min. These results provide a new method for rapid detection of Cys; And this kind of probe has the drug structure of curcumin, which can provide ideas for the design of drug-probe.