Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N‐acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7‐tetramethyl‐8‐phenyl‐(2‐maleimide)‐difluoroboradiaza‐s‐indacene (TMPAB‐o‐M), as the labeling reagent. TMPAB‐o‐M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2–3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE‐based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.     

A Selective Near‐Infrared Fluorescent Probe for In Vivo Imaging of Thiophenols from a Focused Library

Thiophenols are highly toxic industrial materials that, once released, will accumulate in the environment, and ultimately in human bodies, thereby causing serious health problems. To achieve their selective and sensitive detection, a novel near‐infrared (NIR) fluorescent probe ( CCP‐1 ) from a focused library was developed for thiophenol species. Our studies show that CCP‐1 displays a thiophenol‐triggered 28‐fold fluorescence intensity enhancement at 706 nm, with a detection limit of 34 nm observed. It is also able to differentiate thiophenols from various other thiol‐containing analytes including hydrogen sulfide, hydrogen persulfide, and aliphatic thiols. In total, the desirable properties (e.g., excitation/emission in the NIR region, good cell‐membrane permeability, intracellular stability, and low cytotoxicity) make CCP‐1 a potential candidate for thiophenol detection both in vitro and in vivo. In addition, CCP‐1 , for the first time, successfully visualized thiophenols in mice models of thiophenol inhalation.     

Discriminative Detection of Biothiols by Electron Paramagnetic Resonance Spectroscopy using a Methanethiosulfonate Trityl Probe

Biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), coexist in biological systems with diverse biological roles. Thus, analytical techniques that can detect, quantify, and distinguish between multiple biothiols are desirable but challenging. Herein, we demonstrate the simultaneous detection and quantitation of multiple biothiols, including up to three different biothiols in a single sample, using electron paramagnetic resonance (EPR) spectroscopy and a trityl‐radical‐based probe (MTST). We term this technique EPR thiol‐trapping. MTST could trap thiols through its methanethiosulfonate group to form the corresponding disulfide conjugate with an EPR spectrum characteristic of the trapped thiol. MTST was used to investigate effects of l ‐buthionine sulfoximine (BSO) and pyrrolidine dithiocarbamate (PDTC) on the efflux of GSH and Cys from HepG2 cells.     

Ormosil Encapsulated Pyrroloquinoline Quinone‐Modified Electrochemical Sensor for Thiols

An organically modified sol‐gel glass (ORMOSIL) encapsulating pyrroloquinoline quinone (PQQ)‐modified electrode for the rapid, sensitive and simple determination of thiol‐containing compounds such as cysteine and glutathione is reported. The effect of applied potential, nature of thiol compound and pH on the response of the sensor was examined and optimum conditions were determined. The electrochemical responses and detection limits were found to be sensitive to the nature of thiols and pH. The electrochemical responses for cysteine and glutathione at an applied potential of ?0.2 V (vs. Ag/AgCl) were found to be linear with detection limits of 18 nM for cysteine and 36 nM for glutathione at pH 3.5, whereas the detection limits at pH 8.5 were 0.5 μM for cysteine and 1 μM for glutathione. The electrode retained 95% of the original response for 7 days when stored at 4 °C. The ORMOSIL‐encapsulated PQQ was also characterized by spectrophotometry. The absorbance measurement using 5,5′‐dithiobis(2‐nitrobenzoic acid) at 412 nm justify the PQQ‐mediated oxidation of glutathione whereas fluorescence measurements (excitation wavelength=380 nm; emission wavelength=480 nm) justify the successful encapsulation of PQQ in ORMOSIL matrix.     

A fluorescence turn-on probe for the detection of thiol-containing amino acids in aqueous solution and bioimaging in cells

A turn-on fluorescent probe, based on a water-soluble terphenyl derivative, for the detection of cysteine and homocysteine is reported. The aldehyde groups in the probe play crucial roles in providing reaction with thiol groups in the amino acids, leading to a formation of thiazolidine (from cysteine) or thiazinane ring (from homocysteine). As a result, the new formation of such rings alters the electronic property of the conjugated system in the probe and results in emission enhancement. The probe in aqueous solution exhibits a remarkable increase in its quantum yield upon exposure to cysteine (up to 20-fold) and to homocysteine (up to 700-fold), while slight quenching is observed in the presence of glutathione. Moreover, an investigation on time-resolved fluorescence spectra of the probe in the presence of cysteine and homocysteine reveals potential discriminatory detection of cysteine and homocysteine. Bioimaging of the thiols in live HeLa cells was successfully applied.     

Intracellular Thiols and Photo‐Illumination Sequentially Activate Doubly Locked Molecular Probes for Long‐Term Cell Highlighting and Tracking with Precise Spatial Accuracy

A novel photoconvertible fluorescent probe, which can be activated by intracellular thiols, has been synthesized. Such a molecular probe comprises three parts: a 7‐aminocoumarin phototrigger, a thiol‐removable energy acceptor, and a caged fluorescein scaffold with intracellular thiols reactivity as the fluorescent reporter. Extracellularly, the energy acceptor blocks the emission of the coumarin that regulates the photocleavage and photoactivation of the fluorescein. Intracelluarly, the high concentration of thiols releases the energy acceptor, thus activating the S1 state of the phototrigger, which emits coumarin blue fluorescence for pre‐visualization and liberates the caged green‐fluorescent fluorescein to highlight the specific cell upon illumination. Compared to traditional photoactivated organic dyes, the intracellular thiols activated probe requires double activations: one by intracellular thiols and the other by light activation. The dual activations restrict fluorescence precisely inside live cells and at the particular spatial region of light activation, thus a probe with precise spatial accuracy in live cells.     

A ratiometric fluorescent probe for thiols based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold

In this work, we have designed and synthesized the compound Ratio-HPSSC, based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold, as a new ratiometric fluorescent probe for thiols. The ratiometric probe Ratio-HPSSC is highly selective and sensitive to thiols. Importantly, the novel ratiometric probe exhibited a remarkable change in emission color from red to blue. This key feature allows Ratio-HPSSC to be employed for thiol detection by simple visual inspection. Furthermore, we have demonstrated that Ratio-HPSSC is suitable for ratiometric fluorescence imaging of thiols in living cells. We believe that the new ratiometric probe will find interesting applications in chemistry, biology, and medicine.     

高效液相色谱法测定人血浆中总高半胱氨酸含量

建立了快速检测人血浆中总高半胱氨酸含量的高效液相色谱（HPLC）方法。采用反相HPLC分析前,以巯基特异性荧光试剂ABDF对血浆中巯基进行衍生．方法简便、灵敏、准确,无干扰峰影响。平均回收率为97．75％,日内和日间精密度分别为4．49％和9．79％。     

Rational Design of an Ultrasensitive and Highly Selective Chemodosimeter by a Dual Quenching Mechanism for Cysteine Based on a Facile Michael‐Transcyclization Cascade Reaction

Differentiation of biologically important thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) is still a challenging task. Herein, we present a novel fluorescent chemodosimeter capable of selectively detecting Cys over other biothiols including Hcy and GSH and other amino acids by a facile thiol‐Michael addition/transcyclization rearrangement cascade click process. The unique transcyclization step is critical for the selectivity as a result of the kinetically favorable formation of a six‐membered ring with the Cys Michael adduct. Moreover, the probe adopts a distinctive dual quenching mechanism—photoinduced electron transfer (PET) and photoinduced intramolecular charge transfer (ICT) to deliver a drastic turn‐on fluorescence response only at the Cys‐selective transcylization step. The judicious selection of strong electron‐withdrawing naphthalimide fluorophore with maleimide group enhances the electrophilicity and thus reactivity for the cascade process leading to fast detection and ultrasensitivity with a detection limit of 2.0 nm (S/N=3). The probe has demonstrated its practical utility potential in Cys imaging in live cells.     

Determination of thiols by capillary micellar electrokinetic chromatography with laser induced fluorescence detection using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as labeling reagent

A sensitive and effective micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection approach was described for the determination of low molecular‐mass thiols using 1,3,5,7‐tetramethyl‐8‐phenyl‐(4‐iodoacetamido) difluoroboradiaza‐s‐indacene as the labeling reagent. After precolumn derivatization, baseline separation of six thiol compounds including cysteine, glutathione, N‐acetylcysteine, homocysteine, 6‐mercaptopurine, and penicillamine were achieved within 18 min. The optimal running buffer was composed of mixtures involving 25 mM sodium dodecyl sulfate, 25% (v/v) acetonitrile and 15 mM sodium phosphate buffer, pH 7.5. The detection limits (S/N = 3) were found as low as 40 pM under argon ion laser‐induced fluorescence detector (λ_ex/λ_em = 488/520 nm), which were much better than the reported approaches. The accuracy and specificity of this assay for real samples were assured by a standard addition method. The proposed method has been applied to the analysis of thiols both in human plasma and plum flower samples with recoveries of 92.0–109.4%.     

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 Novel Fluorescent Probe Based on Perylene Derivative for Hg2+ Ions and Biological Thiols and its Application in Live Cell Imaging and Theoretical Calculations

The selective and sensitive detection of biothiols; cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous solutions is of considerable importance because of their pivotal roles in maintaining the reducing environment in the cells. This study describes a strategy for the determination of biothiols based on the PDI/Met‐Hg²⁺complex platform. We designed and fabricated methionine modified perylene diimide molecule as a selective sensing probe for Hg²⁺ ions in aqueous solutions ( PDI/Met‐Hg ²⁺). The complex between perylene bisimide derivative ( PDI/Met) and Hg²⁺ was investigated and it demonstrated turn‐on fluorescence response for the detection of the biological thiols. Besides, PDI/Met displayed fluorescence quenching response in the presence of mercury ions and the emission intensity of PDI/Met‐Hg²⁺ was recovered after transferring biothiols (Cys, Hcy, and GSH). Thus, PDI/Met could be utilized as a fluorescent chemosensor for the sequential recognition of mercury ions and biological thiols.     

Cysteine‐Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn‐On

The development of sensitive and selective small molecular probes that enable real‐time detection of endogenous cysteine (Cys) has become an attractive topic because of the essential roles played by Cys in controlling the cellular nitrogen balance and in maintaining biological redox homeostasis. Herein, we report a Cys‐specific probe, 2‐cyanobenzothiazol‐6‐yl acrylate (CBTOA), that shows not only fluorescence turn‐on for sensitive detection of endogenous Cys but also enhanced probe retention inside cells for real‐time monitoring of Cys levels upon external stimulation. Cys‐mediated intracellular formation of luciferin from CBTOA was the key strategy leading to this new type of fluorogenic probe. CBTOA showed fast response to Cys in living cells and liver tissue slices with high sensitivity and selectivity. By using CBTOA as a real‐time probe, we were able to monitor the change in Cys levels in living HeLa cells under ROS‐induced oxidative stress as well as in human mesenchymal stem cells during adipogenic differentiation.     

A sensitive and selective detection method for thiol compounds using novel fluorescence probe

In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0 μM for DTT, 0.6 μM for GSH, and 0.8 μM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection.     

Synthesis of Fe3O4@phenol formaldehyde resin core-shell nanospheres loaded with Au nanoparticles as magnetic FRET nanoprobes for detection of thiols in living cells

A magnetic, sensitive, and selective fluorescence resonance energy transfer (FRET) probe for detection of thiols in living cells was designed and prepared. The FRET probe consists of an Fe(3)O(4) core, a green-luminescent phenol formaldehyde resin (PFR) shell, and Au nanoparticles (NPs) as FRET quenching agent on the surface of the PFR shell. The Fe(3)O(4) NPs were used as the core and coated with green-luminescent PFR nanoshells by a simple hydrothermal approach. Au NPs were then loaded onto the surface of the PFR shell by electric charge absorption between Fe(3)O(4)@PFR and Au NPs after modifying the Fe(3)O(4)@PFR nanocomposites with polymers to alter the charge of the PFR shell. Thus, a FRET probe can be designed on the basis of the quenching effect of Au NPs on the fluorescence of Fe(3)O(4)@PFR nanocomposites. This magnetic and sensitive FRET probe was used to detect three kinds of primary biological thiols (glutathione, homocysteine, and cysteine) in cells. Such a multifunctional fluorescent probe shows advantages of strong magnetism for sample separation, sensitive response for sample detection, and low toxicity without injury to cellular components.     

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.     

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.     

A validated HPLC‐fluorescence method with a semi‐micro column for routine determination of homocysteine,cysteine and cysteamine,and the relation between the thiol derivatives in normal human plasma

A semi‐micro column HPLC‐fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium‐7‐fluorobenzo‐2‐oxa‐1,3‐diazole‐4‐sulfonate (SBD‐F) were isocratically separated within 12 min on a semi‐micro ODS column (Daisopak‐SP‐120‐5‐ODS‐BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH₃CN as a mobile phase. The purity and similarity of SBD‐thiols by a multi‐wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal‐to‐noise ratio of 3 were 0.16, 0.47 and 0.03 µm , respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572–1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and −0.35, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

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.