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.     

Simultaneous Visualization of Endogenous Homocysteine,Cysteine, Glutathione,and their Transformation through Different Fluorescence Channels

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi‐signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.     

Simultaneous Visualization of Endogenous Homocysteine,Cysteine, Glutathione,and their Transformation through Different Fluorescence Channels

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi‐signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.     

Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys,Hcy, and GSH

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Minireview summarizes the most significant current challenges in the field of thiol‐reactive probes for biomedical research and diagnostics, emphasizing the needs and opportunities that have been under‐investigated by chemists in the selective probe and sensor field. Progress on multiple binding site probes to distinguish Cys, Hcy, and GSH is highlighted as a creative new direction in the field that can enable simultaneous, accurate ratiometric monitoring. New probe design strategies and researcher priorities can better help address current challenges, including the monitoring of disease states such as autism and chronic diseases involving oxidative stress that are characterized by divergent levels of GSH, Cys, and Hcy.     

A multisite-binding fluorescent probe for simultaneous monitoring of mitochondrial homocysteine,cysteine and glutathione in live cells and zebrafish

Homocysteine(Hcy), cysteine(Cys) and glutathione(GSH) play crucial roles in redox homeostasis during mitochondria functions. Simultaneous differentiation and visualization of mitochondrial biothiols dynamics are significant for understanding cell metabolism and their related diseases. Herein, a multisitebinding fluorescent probe(MCP) was developed for simultaneous sensing of mitochondrial Cys, GSH and Hcy from three fluorescence channels for the first time. This novel probe exhibited rapid fluor...     

Nonionic surfactant‐capped gold nanoparticles for selective enrichment of aminothiols prior to CE with UV absorption detection

In this study, we describe the use of Tween 20‐capped gold nanoparticles (AuNPs) as selective probes for the extraction of aminothiols from an aqueous solution. Tween 20 molecules noncovalently attached to the surface of AuNPs to form Tween 20–AuNPs were used for the selective extraction of aminothiols through the formation of Au–S bonds. After extraction and centrifugation, the aminothiols were detached from the surface of the AuNPs by adding DTT in a high concentration. We used this probe in combination with CE and UV absorption detection. On‐line concentration and separation of the released aminothiols were performed by using 1.6% v/v poly(diallyldimethylammonium chloride) as an additive in CE. Under optimal extraction and stacking conditions, the LOD at a S/N of 3 were 28, 554, and 456 nM for glutathione (GSH), cysteine (Cys), and homocysteine (HCys), respectively. In comparison with the normal injection without the extraction procedure, approximately 2280‐, 998‐, and 904‐fold improvements in the sensitivity were observed for GSH, Cys, and HCys, respectively. We have validated the application of our method on the basis of the analysis of GSH and HCys in human urine samples. It is believed that this approach has significant potential to be extended to clinical diagnosis.     

HPLC-electrospray tandem mass spectrometry for simultaneous quantitation of eight plasma aminothiols: Application to studies of diabetic nephropathy

It was reported that Hcy was related to the development of kidney disease, but it remains unknown whether Hcy is an independent biomarker for diabetic nephropathy. Analytical method for simultaneous determination of aminothiols among the Hcy metabolic cycle is desirable to discover other potential biomarkers. A high-performance liquid chromatography-electrospray tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for simultaneous quantitation of Cysteine (Cys), total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine (Cysta), methionine (Met), glutathione (GSH) and cysteinylglycine (Cys-gly) in plasma with N-(2-mercaptopropionyl)-glycine (MPG) as internal standard. The method had simple pretreatment without derivatization and the chromatograms show better separation of the eight aminothiols and the analytic time was 20 min. The results demonstrated that it provided an excellent linearity for all analytes over their respective concentration ranges and illustrated excellent precision and plasma recovery as well. Then, the method was applied in the case-control study of patients with diabetes mellitus (DM) and diabetic nephropathy (DN). In conclusion, it is an effective method to quantitate the concentrations of aminothiols in the human plasma. SAH and SAM were suggested as better potential biomarkers of DM and DN.     

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.     

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 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.     

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 Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

Distinguishable multi-substance detection based on three-channel NIR fluorescent probe in physiology and pathology of living cells and zebrafish

Mitochondria is the main organelle for the production of reactive sulfur species (RSS), such as homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and sulfur dioxide (SO₂). These compounds participate in a large number of physiological processes and play an extremely important role in maintaining the balance of life systems. Abnormal concentration and metabolism are closely related to many diseases. Due to their similarities in chemical properties, it is challenging to develop a single fluorescent probe to distinguish them simultaneously. Here, we synthesized the probe PI-CONBD with three fluorophores, NBD-Cl and benzopyranate as the reaction sites of GSH/Cys/Hcy and SO₂, respectively. Three biothiols all could cleavage ether bond to release benzopyrylium and coumarin moiety, which emitted red and blue fluorescence, but Cys/Hcy also could do intramolecular rearrangement after nucleophilic substitution, resulting in yellow fluorescence. Thus the probe can distinguish Cys/Hcy and GSH. Subsequently, only SO₂ could quench red fluorescence by adding CC of benzopyrylium. The probe also could localize well in mitochondria by oxonium ion for all kinds of cells. The probe not only could detect above sulfur-containing active substances of intracellular and extracellular but also monitor the level of them under oxidative stress and apoptosis process in living cells and zebrafish.     

Benzothiazole‐Pyimidine‐Based BF2 Complex for Selective Detection of Cysteine

Due to the similar structure and reactivity of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), the simultaneous discrimination of Cys over Hcy and GSH by a single fluorescent sensor is still a great challenge. In this work, a benzothiazole‐pyimidine‐based boron difluoride complex ( BPB ) was developed as a new fluorescent sensor for Cys. The sensor exhibits a highly selective “turn‐on” response to cysteine over Hcy, GSH and other amino acids in aqueous solution at physiological pH. The observed pseudo‐first‐order rate constant for the reaction of BPB with Cys was calculated to be about 0.062 min⁻¹. The detection limit of this sensor for Cys was determined to be 332 nm, and bioimaging of exogenous Cys by this sensor was successfully applied in living cells, thus indicating that this sensor holds great potential for biological applications.     

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.     

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.     

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.     

金纳米粒子富集-高效液相色谱同时测定人血浆中三种氨基硫醇

建立了金纳米粒子富集-高效液相色谱-紫外检测(HPLC-UVD)同时测定人血浆中3种氨基硫醇(半胱氨酸(Cys)、高半胱氨酸(Hcys)、谷胱甘肽(GSH))的新方法。以Tween 20修饰的金纳米粒子作为选择探针萃取富集氨基硫醇。经二硫苏糖醇脱附后,采用SpursilTM C18柱(250 mm×4.6 mm, 5 μm)分离氨基硫醇,以60 mmol/L磷酸盐缓冲溶液(pH 2.0)等度洗脱,检测波长为200 nm。3种氨基硫醇的浓度分别在0.025～350 μmol/L、0.02～60 μmol/L、0.01～50 μmol/L内与峰面积具有良好的线性关系,相关系数均高于0.99。方法检出限(信噪比为3)分别为5.0、6.0和2.5 nmol/L,回收率为92.8%～106.0%。该方法能显著降低血浆样品中内源性物质的干扰,提高HPLC-UVD的选择性和灵敏度。将该方法应用于心血管病人血浆中上述氨基硫醇的分离测定,结果显示: 与对照组相比,疾病组血浆中的Hcys和GSH水平存在显著性差异,Cys不存在显著性差异。     

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.     

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.