Selective optical sensing of biothiols with Ellman's reagent: 5,5′-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB²⁻) anion as a result of reaction with biothiols was measured at 410 nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57 μM and acceptable linearity ranging from 0.4 to 29.0 μM (r = 0.998).     

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.     

A Phenothiazine-HPQ Based Fluorescent Probe with a Large Stokes Shift for Sensing Biothiols in Living Systems

Due to the redox properties closely related to numerous physiological and pathological processes, biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), have received considerable attention in biological science. On account of the important physiological roles of these biothiols, it is of profound significance to develop sensitive and selective detection of biothiols to understand their biological profiles. In this work, we reported an efficient fluorescent probe, PHPQ-SH, for detecting biothiols in vitro and vivo, based on the phenothiazine-HPQ skeleton, with DNBS (2,4-dinitrobenzenesulfonate) as the response unit. Probe PHPQ-SH exhibited brilliant sensing performances toward thiols, including a large Stokes shift (138 nm), excellent sensitivity (for GSH, LOD = 18.3 nM), remarkable fluorescence enhancement (163-fold), low cytotoxicity, rapid response (8 min), and extraordinary selectivity. Finally, the probe PHPQ-SH illustrated herein was capable of responding and visualizing biothiols in MCF-7 cells and zebrafish.     

Hg2+-mediated aggregation of gold nanoparticles for colorimetric screening of biothiols

In this work, we report a colorimetric assay for the screening of biothiols including glutathione (GSH), cysteine (Cys), and homocysteine (Hcys) based on Hg(2+)-mediated aggregation of gold nanoparticles (AuNPs). Hg(2+) can induce aggregation of thiol-containing naphthalimide (1) capped AuNPs due to the cross-linking interactions from the resulting "thymine-Hg(2+)-thymine" (T-Hg(2+)-T) analogous structure. When Hg(2+) is firstly treated with biothiols, followed by mixing with 1-capped AuNPs suspension, AuNPs undergo a transformation from an aggregation to a dispersion state depending on the concentration of biothiols. This anti-aggregation or re-dispersion of AuNPs is due to the higher affinity of Hg(2+) for biothiols relative to compound 1. The corresponding color variation in the process of anti-aggregation of AuNPs can be used for the quantitative screening of biothiols through UV-vis spectroscopy or by the naked eye. Under optimized conditions, a good linear relationship in the range of 0.025-2.28 μM is obtained for GSH, 0.035-1.53 μM for Cys, and 0.040-2.20 μM for Hcys. The detection limits of this assay for GSH, Cys, and Hcys are 17, 9, and 18 nM, respectively. This colorimetric assay exhibits a high selectivity and sensitivity with tunable dynamic range. The proposed method has been successfully used in the determination of total biothiol content in human urine samples.     

基于碳点荧光恢复法测定生物巯基化合物

碳点(CDots)是一种新型荧光纳米材料,Cu2+可以有效猝灭其荧光;而当有生物巯基化合物存在时,碳点-Cu2+体系的荧光可以恢复.基于此原理,我们成功地构建了检测生物体内总巯基化合物的新方法.该方法具有很好的选择性,常见氨基酸和金属离子对谷胱甘肽(GSH)、半胱氨酸(Cys)和高半胱氨酸(Hcy)的检测无影响.最佳实验条件下,谷胱甘肽、半胱氨酸、高半胱氨酸的浓度在6.0×10-6mol/L～1.0×10-4mol/L与相对荧光强度呈线性,R>0.996,检出限为2.0×10-6mol/L.该体系成功用于血清样品中总巯基化合物的检测.     

BODIPY-Based Fluorescent Probes for Biothiols

Fluorescent probes for biothiols have aroused increasing interest owing to their potential to enable better understanding of the diverse physiological and pathological processes related to the biothiol species. BODIPY fluorophores exhibit excellent optical properties, which can be readily tailored by introducing diverse functional units at various positions of the BODIPY core. In the present review, the development of fluorescent probes based on BODIPYs for the detection of biothiols are systematically summarized, with emphasis on the preferable detection of individual biothiols, as well as simultaneous discrimination among cysteine (Cys), homocysteine (Hcy), reduced glutathione (GSH). In addition, organelle-targeting probes for biothiols are also highlighted. The general design principles, various recognition mechanisms, and biological applications are elaboratively discussed, which could provide a useful reference to researchers worldwide interested in this area.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

The development of a fluorescence turn-on sensor for cysteine, glutathione and other biothiols. A kinetic study

Two fluorescence probes for the detection of cysteine (Cys), glutathione (GSH) and other biothiols, such as homocysteine (Hcy) and cysteinyl-glycine (Cys-Gly), were developed. These molecular probes are coumarin-based derivatives containing a chalcone-like moiety that reacts with biothiols through a Michael addition reaction, leading to strong fluorescence enhancements. The reactivity of the tested biothiols toward both probes (ChC1 and ChC2) follows the order Cys > GSH > Hcy > Cys-Gly, ChC1 being less reactive than ChC2. Possible interference with other amino acids was assessed. ChC1 and ChC2 display a highly selective fluorescence enhancement with thiols, allowing these probes to be used for fluorimetric thiol determination in SH-SY5Y cells.     

生物硫醇荧光探针的研究进展

生物硫醇(包含半胱氨酸、高半胱氨酸和谷胱甘肽)在生命活动中扮演了重要的角色,其浓度的异常变化与某些疾病息息相关,因此对硫醇的检测具有重要意义.荧光探针因具有灵敏度高、时空分辨率好、无损伤、可视化等优势,在生物硫醇的检测方面得到了高度重视.利用硫醇在分子结构上的共同点(含巯基的氨基酸)和差异(分子大小、亲核性、空间位阻、细胞内含量),可通过迈克尔加成、亲核芳基取代、加成环化等反应实现对硫醇的选择性检测.综述了近3年来硫醇荧光探针领域的研究进展.首先介绍了对硫醇有选择性识别的荧光探针,随后分类讨论了对半胱氨酸、高半胱氨酸和谷胱甘肽各具有特异性检测的荧光探针,并重点介绍了分子设计、识别机理、荧光性质和成像应用,初步探讨了部分探针在监测细胞生命活动中的作用,同时还对本领域的发展提出了展望.     

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ_ext/λ_em = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L⁻¹ CH₃COONH₄ (pH = 2.5)/CH₃CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L⁻¹ and the LOD was 19 nmol L⁻¹. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.     

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.     

Development of a Two-photon Ratiometric Fluorescent Probe for Glutathione and Its Applications in Living Cells

Glutathione(GSH), as the most abundant intracellular biothiol, plays an important role in the redox homeostasis of the organism. Abnormal concentrations of GSH in cells may lead to many malignant diseases, such as cancer, liver damage and neurodegenerative diseases. It is urgent to develop effective methods to detect GSH in living organisms. In this work, a new two-photon ratiometric fluorescent probe Co-GSH based on the coumarin-chalcone dye platform was judiciously developed. Based on the Michael-addition reaction, Co-GSH was able to identify GSH with high selectivity and sensitivity. Furthermore, assisted by laser-scanning confocal microscopy, Co-GSH could specifically response GSH over the other biothiols, including Cys and Hcy, in living HeLa cells by using one-and two-photon modes.     

Development of an Organic Dye-Conjugated β-Diketonate-Europium Complex Luminescence Probe for Discrimination and Detection of Intracellular Biothiols

Small molecular biothiols, cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play important roles in organisms, and their concentration levels are indicative of some human diseases. Herein we report an organic dye-conjugated β-diketonate-Eu³⁺ complex, [Eu(NBD-keto)₃(DPBT)] (NBD-keto: 7-nitro-2,1,3-benzoxadiazole (NBD)-conjugated to 1,1,1,2,2-pentafluoro-5-phenyl-3,5-pentanedionate through a “O” ether bond; DPBT: 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine), which acts as a unique luminescent probe for detecting and discriminating biothiols. [Eu(NBD-keto)₃(DPBT)] itself is not luminescent due to intramolecular interactions between NBD and β-diketonate-Eu³⁺ moieties. Upon reaction with biothiols, the β-diketonate-Eu³⁺ complex [Eu(keto)₃(DPBT)] is generated, which emits long-lived red emission at 610 nm. Meanwhile, three biothiol-substituted NBD derivatives that exhibit different luminescence behaviors, green emissive (short-lived) NBD-NR (R=Cys or Hcy) at 540 nm and non-luminescent NBD-SR (R=GSH), are also generated. These luminescence response behaviors allow time-gated and steady-state luminescence modes to be combined for detecting total biothiols and discriminating GSH and Cys/Hcy. Using this probe, the quantitative detection and discrimination of GSH and Cys/Hcy in lysis solutions of HeLa cells were realized, which revealed the potential of the probe for biomedical applications.     

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.     

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.     

柱前衍生高效液相色谱-荧光检测法测定水稻中7种巯基化合物

建立了水稻中半胱氨酸(Cys)、谷胱甘肽(GSH)和植物螯合肽(phytochelatin, PC:PC₂、PC₃、PC₄、PC₅、PC₆)7种巯基化合物的柱前衍生高效液相色谱-荧光检测分析方法.样品经0.1%三氟乙酸(TFA)(含6.3 mmol/L二乙烯三胺五乙酸(DTPA))超声提取,然后以单溴二胺(mBrB)为衍生剂在pH 8.0的4-羟乙基哌嗪丙磺酸(HEPPS)缓冲溶液中衍生化.采用的色谱分离柱为Agilent Eclipse plus C_l8柱,流动相为0.1%TFA(pH 2.5)和100%乙腈(ACN),梯度洗脱,流速为0.8 mL/min.荧光检测的激发波长和发射波长分别为380 nm和470 nm.结果表明,7种巯基化合物在0.7~100.0 mg/L范围内,峰面积与质量浓度之间的线性关系良好(r²≥0.9991);检出限为0.03~0.20 mg/L;加标回收率为89.26%~99.42%,相对标准偏差为2.05%~5.87%.该方法准确、灵敏度高、重现性好,为水稻中巯基化合物的研究提供了检测手段.     

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 red-emitting water-soluble fluorescent probe for biothiol detection with a large Stokes shift

A highly water soluble fluorescent probe was developed for sensitive and selective detection of biothiols with a red emission and a large Stokes shift. The probe was successfully applied to detect biothiols both in aqueous solution and 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.     

基于萘酰亚胺的生物硫醇探针的合成及对含硫醇氨基酸的检测

合成了以4-羟基萘酰亚胺为荧光团,2,4-二硝基苯磺酰氧基为特异性识别基团的生物硫醇探针4-(2,4-二硝基苯磺酰氧基)-正丁基-1,8-萘酰亚胺(DNSBN).吸收光谱和荧光光谱结果表明, DNSBN对半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)3种生物硫醇分子具有高效的检测识别能力,不受其它17种天然氨基酸的干扰.同时,通过荧光滴定实验证实了此探针是一种比率型探针,555 nm处的荧光强度与溶液中的生物硫醇分子浓度在0 ~ 20 μmol/L范围内呈良好的线性关系,对Cys、Hcy和GSH的检出限(3σ)分别为25.9、92.0和77.9 nmol/L.而吸收光谱、荧光光谱和质谱表征数据显示,生物硫醇与2,4-二硝基苯磺酸酯发生亲核取代反应并导致磺酸酯的分解.随着识别基团的解离,探针分子的d-PeT (donor-excited photoinduced electron transfer) 效应被解除,并出现非常明显的比色与荧光变化.HeLa细胞成像实验表明,探针DNSBN具有良好的生物相容性,能够对细胞外源性生物硫醇分子进行检测.