A one-step selective fluorescence turn-on detection of cysteine and homocysteine based on a facile CdTe/CdS quantum dots–phenanthroline system

In this paper, we report a simple, selective, sensitive and low-cost turn-on photoluminescent sensor for cysteine and homocysteine based on the fluorescence recovery of the CdTe/CdS quantum dots (QDs)–phenanthroline (Phen) system. In the presence of Phen, the fluorescence of QDs could be quenched effectively due to the formation of the non-fluorescent complexes between water-soluble thioglycolic acid (TGA)-capped QDs and Phen. Subsequently, upon addition of cysteine and homocysteine, the strong affinity of cysteine and homocysteine to QDs enables Phen to be dissociated from the surface of QDs and to form stable and luminescent complexes with cysteine and homocysteine in solution. Thus, the fluorescence of CdTe/CdS QDs was recovered gradually. A good linear relationship was obtained from 1.0 to 70.0 μM for cysteine and from 1.0 to 90.0 μM for homocysteine, respectively. The detection limits of cysteine and homocysteine were 0.78 and 0.67 μM, respectively. In addition, the method exhibited a high selectivity for cysteine and homocysteine over the other substances, such as amino acids, thiols, proteins, carbohydrates, etc. More importantly, the sensing system can not only achieve quantitative detection of cysteine and homocysteine but also could be applied in semiquantitative cysteine and homocysteine determination by digital visualization. Therefore, as a proof-of-concept, the proposed method has potential application for the selective detection of cysteine and homocysteine in biological fluids.     

Determination of cysteine,homocysteine, cystine,and homocystine in biological fluids by HPLC using fluorosurfactant‐capped gold nanoparticles as postcolumn colorimetric reagents

We have demonstrated for the first time the suitability of fluorosurfactant‐capped spherical gold nanoparticles as HPLC postcolumn colorimetric reagents for the direct assay of cysteine, homocysteine, cystine, and homocystine. The success of this work was based on the use of an on‐line tris(2‐carboxyethyl)phosphine reduction column for cystine and homocystine. Several parameters affecting the separation efficiency and the postcolumn colorimetric detection were thoroughly investigated. Under the optimized conditions, cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples were determined. Detection limits for cysteine, homocysteine, cystine, and homocystine ranged from 0.16–0.49 μM. The accuracy in terms of recoveries ranged between 94.0–102.1%. This proposed method was rapid, inexpensive, and simple.     

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.     

Rapid thin-layer chromatography of homocysteine-cysteine mixtures as their thiolyte-derivatives

Summary Fluorescent labelling of cysteine and its homologue homocysteine with monobromobimane followed by TLC on silica gel allows rapid identification of both compounds through their intense yellow fluorescence when excited at 366 nm; a visual detection limit of about 2ng is reached for both thiols.     

Simultaneous Detection of Homocysteine and Cysteine in the Presence of Ascorbic Acid and Glutathione Using a Nanocarbon Modified Electrode

The simultaneous electrochemical detection of homocysteine and cysteine using an absorbed ortho‐quinone species, catechol, at the nanocarbon modified glassy carbon electrode was achieved via 1,4‐Michael addition reaction. The detection was done in the presence and the absence of each other as well as with both glutathione and ascorbic acid present in order to investigate the selectivity of homocysteine and cysteine. A determination of homocysteine sensitivity is (0.882±0.296) nA nM^?1 with a LOD of ca. 11 nM and cysteine sensitivity is (7.501±0.202) mA µM^?1 with a LOD of ca. 5.0 µM within a range of 0–0.1 mM.     

Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection

A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with a thiol-selective fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure of thiol derivatives is assessed using electrospray ionization-mass spectrometry (MS). The stability of resulting adducts in acidic medium (24 h at 10 degrees C) allows the automation of the technique and a high throughput of samples (approximately 50 per day). Separation is complete within 12 min using isocratic reversed-phase mode, and detection is operated by spectrofluorimetry (lambda ex = 385 nm and lambda em = 515 nm). Quantitation is performed by an internal standardization mode using thioglycolic acid. The LC method is fully validated, and homocysteine concentrations obtained in plasma samples are compared with values measured using either fluorescence polarization immunoassay or capillary gas chromatography-MS; a good correlation is observed between LC and both methods. The method has been applied in daily use to a large-scale study in a human healthy population, and some resulting data are discussed.     

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.     

Flow-injection determination of ascorbic acid and cysteine simultaneously with spectrofluorometric detection.

A simple and sensitive spectrofluorometric method was developed for the simultaneous determination of ascorbic acid and cysteine by a flow-injection system. This method is based on the reduction of Tl(III) with ascorbic acid or cysteine in acidic media, producing fluorescence reagent, TlCl3(2-) (lambdaex = 227 nm, lambdaem = 419 nm). The injected sample solution was divided into two separate streams. The first stream was treated with Tl(III) at pH 3.0 and then passed through a 270 cm reaction coil to the flow cell of the spectrofluorometer, where the fluorescence intensity was measured. This signal is related to ascorbic acid and cysteine concentration. The second part of the injected sample solution was treated with Tl(III) in HCl solution and then passed through a 50 cm reaction coil to the flow cell and the fluorescence intensity was measured. This signal is related only to cysteine. Thus, the ascorbic acid content was determined directly by the difference according to the calibration curve. Ascorbic acid and cysteine can be determined in the range of 1 x 10(-6) to 5.0 x 10(-5) M, at a rate of 16 samples per hour. The limits of detection (S/N = 3) were 8 x 10(-7) M for ascorbic acid and 7 x 10(-7) M for cysteine. The influence of potential interfering substances was studied. The proposed method was successfully applied to the simultaneous determination of both analytes in real samples.     

水溶性半胱氨酸和高半胱氨酸探针的合成及应用

以对硝基甲苯和变色酸为原料,经硝化、重氮化反应合成了水溶性偶氮化合物(1),其结构经1H NMR和元素分析表征.利用UV-vis滴定法研究了在缓冲溶液(pH 7.4)中1对半胱氨酸(Cys)和高半胱氨酸(Hcy)的识别能力.结果表明:1与Cys或Hcy作用后引起吸收光谱蓝移～69 nm,能够用于Cys和Hcy的裸眼识别.     

Simultaneous determination of serum homocysteine,cysteine, and methionine in patients with schizophrenia by liquid chromatography–tandem mass spectrometry

Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances in the homocysteine metabolism are an important factor in the pathophysiology of schizophrenia. In this research, a novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) quantification procedure was developed to investigate three significant compounds of homocysteine metabolism: homocysteine, cysteine, and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2 min. The LC–MS/MS method was validated according to CLSI C62-A and the Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range of 0.5–6.9%, an accuracy of 90.4–101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for a schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of the transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.     

Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography

A new analytical method is proposed for simultaneous determination, by liquid chromatography, of the three main urinary thiols–cysteine, cysteinylglycine, and homocysteine. To measure the total amount of these thiols urine is reduced with sodium borohydride, to convert disulfides to thiols which are then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium thiol derivatives formed were achieved by high-performance liquid chromatography with detection at 355 nm. Validation showed the method enabled reliable simultaneous determination of these aminothiols in urine. The calibration graphs for each analyte, obtained by use of normal urine spiked with increasing amounts of cysteine, cysteinylglycine, and homocysteine, were linear (R ²≥0.997) over the range covering most practical situations. The recovery of the assay was 98–100% and sensitivity was 0.12–0.25 μmol L⁻¹. The method was applied to 91 different samples of normal urine to establish reference values for the aminothiols, normalized on creatinine.     

Indirect Simultaneous Kinetic Determination of L‐Cysteine and Homocysteine by ANNs

Abstract

Simultaneous determination of cysteine and homocysteine in binary mixtures was performed by application of neural networks on the spectral kinetic data. This method is based on the complexation of bivalent iron with 2,2′–bipyridin (bipy). Iron(III) is quantitatively reduced to iron(II) with cysteine and homocysteine in the presence of 2,2′–bipyridin producing iron(II)–bipy complex (λmax=522 nm), and it can be used as a visible spectrophotometric signal for indirect simultaneous determination of the cysteine and homocysteine concentrations. On the basis of the difference in the rate between the two reactions, these two amino acids can be determined simultaneously using principal component‐artificial neural networks (PC‐ANN). The parameters controlling behavior of the system were investigated and optimum conditions selected. Determinations were made over the concentration range 0.10–5.50 µg · mL^?1 of cysteine and 0.1–5.00 µg · mL^?1 of homocysteine. Applying this method satisfactorily to simultaneous determination of these amino acids with total relative standard error less than 5% validated the proposed method.     

Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine-cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis

An elevated level of thiol amino acid homocysteine is associated with several complex disorders. Homocysteine ability to bind proteins, thereby modulating their structure and function, is proposed to be one of the mechanisms of homocysteine induced pathogenecity. Homocysteine and homocysteine thiolactone bind to protein cysteine and lysine residues respectively. A major hurdle in studying protein homocysteinylation is the lack of suitable analytical techniques to determine simultaneously the concentrations of reduced and oxidized forms of homocysteine and cysteine (especially homocysteine-cysteine mixed disulfide) together with thiolactone formed during the reaction of homocysteine or thiolactone with proteins. Herein we report a capillary electrophoresis method to determine simultaneously the levels of these intermediates. For this 40 mmol/L Tris phosphate buffer at (pH 1.60) was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 15 kV and an overimposed pressure of 0.1 psi. A rapid separation of these intermediates in less than 6 min with a good reproducibility of both peak areas (CV < 2%) and migration time (CV < 0.2%) was obtained. The applicability of our method was validated by incubating reduced homocysteine and albumin and measuring the reaction intermediates in the solution mixture.     

Electrochemical studies of homocysteine and homocystine at mercury electrodes

The behaviour of homocysteine and cysteine at mercury electrodes is compared. The one-electron oxidation associated with thiols is shown to be the same for both compounds in acidic phosphate buffer, giving rise to an adsorbed thiol—mercury complex, (RS)₂Hg, at the electrode surface. Formation of this complex is utilized in the cathodic stripping voltammetric determination of homocysteine; the detection limit is 10^?9 M after a deposition time of 90 s at a hanging mercury drop electrode. The similar E_{$\text{1}\text{2}$} values for homocysteine and cysteine mean that prior separation is needed for their individual determination. Amperometric detection with a mercury-coated goal electrode after separation by cation-exchange liquid chromatography provides a method for the simultaneous determination of both compounds. Reduction of homocystine at the mercury electrode is also compared to that of cystine. The more negative reduction potential, and the maximum observed for homocystine on d.c. polarograms, which is not seen for cystine, is attributable to different reaction kinetics at the mercury electrode; the products of both the 2-electron reductions are the corresponding thiol-containing amino acids.     

Determination of biologically active low-molecular-mass thiols in human blood. II. High-performance capillary electrophoresis with photometric detection

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm x 50 microm I.D.) at 30 degrees C. With the electric field of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was approximately 5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.     

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).     

Electrochemically Initiated Catalytic Oxidation of 5‐Thio‐2‐Nitrobenzoic Acid (TNBA) in the Presence of Thiols at a Boron Doped Diamond Electrode: Implications for Total Thiol Detection

The electrochemical response of 5,5‐dithiobis(2‐nitrobenzoic acid) (DTNB) to increasing additions of thiol species has been examined at a boron doped diamond electrode. A reaction has been shown to occur with a range of biologically relevant thiols and proceeds via a CECC' process. A total thiol detection methodology has been developed showing that the sensitivities of the standard addition plots are independent of the individual thiol species added to the solution. The analytical utility of the reaction process has been assessed using chronoamperometry with the corresponding data producing detection limits of 5.7 μM, 4.4 μM and 5.8 μM for the detection of cysteine, homocysteine and glutathione respectively.     

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.     

Specific detection of cysteine and homocysteine: recognizing one-methylene difference using fluorosurfactant-capped gold nanoparticles

Aggregation of fluorosurfactant-capped gold nanoparticles could be induced selectively by cysteine and homocysteine and, when solution ionic strength was low, the kinetics of homocysteine-induced aggregation of large size nanoparticles (approximately 40 nm) was much faster than that induced by cysteine, leading to specific detection of homocysteine in the presence of excess cysteine.     

Miniaturized capillary electrophoresis system with a carbon nanotube microelectrode for rapid separation and detection of thiols

Multi-walled carbon nanotube (CNT) was mixed with epoxy to fabricate microdisc electrode used as a detector for a specially designed miniaturized capillary electrophoresis (CE)-amperometric detection system for the separation and detection of several bioactive thiols. The end-channel CNT amperometric detector offers favourable signal-to-noise characteristics at a relatively low potential (0.8 V) for detecting thiol compounds. Factors influencing the separation and detection processes were examined and optimized. Four thiols (homocysteine, cysteine, glutathione, and N-acetylcysteine) have been separated within 130 s at a separation voltage of 2000 V using a 20 mM phosphate running buffer (pH 7.8). Highly linear response is obtained for homocysteine, cysteine, glutathione, and N-acetylcysteine over the range of 5-50 μM with detection limits of 0.75, 0.8, 2.9, and 3.3 μM, respectively. Good stability and reproducibility (R.S.D. < 5%) are obtained reflecting the minimal adsorption of thiols at the CNT electrode surface. The new microchip protocol should find a wide range of bioanalytical applications involving assays of thiol compounds.