Microchannel-assisted thermal-lens spectrometry for microchip analysis

We present a new method for homocysteine quantitation in human plasma based on in-capillary reaction of homocysteine with 2,2′-dipyridyl disulfide. Homocysteine is in this so-called thiol-exchange reaction quantitatively transformed in mixed disulfide concomitantly with formation of an equimolar amount of 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined specifically at 343 nm. The concentration of homocysteine is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay was from 0.03–3 mM (correlation coefficient 0.994) with a detection limit of 6 μM and a limit of quantitation 20 μM. The inter-day reproducibility of the peak area and the migration time were 1.37% and 0.05%, respectively. The method is simple, relatively rapid and can be easily automated. Moreover the common capillary electrophoresis apparatus with a UV detector can be used to distinguish between normal and pathological hyperhomocysteinemia plasma samples.     

Comparison of pulsed amperometric detection and integrated voltammetric detection for inorganic sulfur compounds in liquid chromatography

A Variety of potential–time waveforms are useful in pulsed electrochemical detection (PED) when applied for the amperometric detection of numerous polar organic compounds following their separation by liquid chromatography (LC). Here, we compare the waveforms for pulsed amperometric detection (PAD) and integrated voltammetric detection (IVD) applied for detection of organosulfur compounds at Au electrodes in acidic media. In PAD waveforms, electrodes response is measured at a constant detection potentials. In IVD waveforms, electrodes current is integrated throughout a fast cyclic scan of the detection potential. As a consequence of this difference in detection strategy, the background signal for IVD is significantly smaller for PAD in the detection of organosulfur compounds whose response mechanisms require the concomitant formation of surface oxides on Au electrodes. Furthermore, in comparison to Pad, IVD has a larger sensitivity and a diminished system peak from 0₂ dissolved in the sample. Use of a preadsorption step increases detection sensitivity in both PAD and IVD. The limit of detection (S/N=3)for cysteine in LC-IVD is ca. 6 nM for a 50-μl injection (i.e., 300 fmol) using a detection waveform that includes a 1000-ms preadsorption period.     

Comparison of Methionine α,γ‐Lyase and Homocysteine α,γ‐Lyase for Electrochemical Determination of Homocysteine

Recently, a novel enzymatic method was developed for determination of homocysteine. This method utilizes the electrochemical hydrogen sulfide sensor along with methionine α,γ‐lyase to accomplish the fast, accurate, sensitive and selective measurements. As a continuation of this work, another enzyme, homocysteine α,γ‐lyase, was used and the parallel experiments of using both enzymes were carried out against the effect of pH, sensitivity, linearity, and interferences, in an intended comparison between these two enzymes. The excellent linearity of amperometric currents against homocysteine concentrations, high sensitivities and low detection limits for both enzymes reconfirmed that the electrochemical method is superior over other analytical means. The high enzymatic activity of methionine α,γ‐lyase surpassing homocysteine α,γ‐lyase endowed the former higher sensitivity, lower detection limit and faster response than the latter, suggesting methionine α,γ‐lyase a better candidate for homocysteine measurement by electrochemical method. The differences between these two enzymes on the trends of response time and sensitivity at different pH environments, reactivity toward several forms of homocysteine as well as on the interference from several agents were also addressed and discussed.     

A turn on ESIPT probe for rapid and ratiometric fluorogenic detection of homocysteine and cysteine in water with live cell-imaging

5(Benzothiazol-2-yl)-4-hydroxyisophthalaldehyde (BHI), an intense ESIPT containing molecule in mixed media loses its properties due to resonance-assisted H-bond (RAHB) in absolute water. Due to resonance-assisted H-bond the o-aldehyde is more reactive than the other one. With addition of cysteine/homocysteine into this solution the o-aldehyde group gets transformed into thiazolidine/thiazine ring, respectively, and the phenolic proton becomes free enough for transfer to nitrogen of the benzothiazole ring in excited state, that is, the ESIPT of BHI is turned on. Thus we can detect cysteine/homocysteine in water as well as in live cells.     

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.     

Electrochemical Determination of Homocysteine at Disposable Graphite Electrodes

In the case of disruption of Hcy metabolism, the blood level of Hcy increases and it causes particularly the cardiovascular diseases, cancer, dementia and Parkinson’s disease. Thus, the sensitive analysis of Hcy levels in biological fluids is very important. Hcy analysis was performed herein using very practical and cost‐effective protocol using differential pulse voltammetry and graphite electrode. Detection limit of Hcy was found to be 1.21 µM in the linear range from 2 µM to 20 µM. The electrochemical Hcy detection in artificial urine medium was also successfully performed even in the presence of L ‐Cysteine, L ‐Methionine and Glutathione.     

Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma

Homocysteine present in human blood plasma is derivatized with thiol selective ultraviolet labelling reagent, 2-chloro-1-methylpyridinium iodide, and separated from other plasma thiol derivatives by high-performance liquid chromatography (HPLC) with detection at 312 nm. The separation is carried out isocratically on LiChrospher RP-18 column using mobile phase consisting of pH 2.5 0.04 M trichloroacetic acid buffer and methanol in the ratio 9:1 (v/v) pumped at 0.5 ml min⁻¹ at 40°C. The homocysteine S-pyridinium derivative elutes at 6.5 min. To determine total and protein-bound homocysteine it is necessary to cleave disulphide bounds by the use of tri-n-butylphosphine in order to form free sulfhydryl group. The method provides quantitative information on total and protein-bound homocysteine based on assays with derivatization after reduction of whole plasma, and derivatization after reduction of acid precipitated proteins. The calibration graph is linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol sensitivity and is reproducible; intra- and inter-day, relative standard deviation range from 1.79 to 5.09% and from 2.80 to 5.60%, respectively. The method is applied to the determination of total and protein-bound homocysteine in the plasma of healthy individuals.     

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.     

Selective determination of homocysteine levels in human plasma using a silver nanoparticle-based colorimetric assay

The first use of silver nanoparticles (AgNPs) for the rapid, simple, and selective determination of homocysteine (Hcy) levels in human plasma was studied. Hcy and five other amino acids, including cysteine (Cys), could be distinguished by their different aggregation kinetics, which caused a change in the visible color and a shift in the UV-vis absorption spectra. The difference in the cross-linking (aggregation) rate between Hcy and Cys was used as the basis for developing a selective probe for Hcy and allowed the detection of Hcy in the linear range of 2-12 μM (R² = 0.9936). The limits of detection and quantification were found to be 0.5 μM and 1.7 μM, respectively. To investigate its selectivity and potential applicability, this AgNP-based method was successfully applied for the determination of Hcy levels in actual biological (human plasma) samples, where the determined levels of Hcy were within the error range of the measured level using the traditional chemiluminescence microparticle immunoassay (CMIA). Thus, the use of AgNPs is a feasible and potentially reliable method for the determination of Hcy levels in biological samples.     

Use of capillary electrophoresis with chemiluminescence detection for sensitive determination of homocysteine

A sensitive capillary electrophoresis (CE) method with chemiluminescence (CL) detection was developed for the determination of homocysteine (HCys) in human plasma. In this work, N‐(4‐aminobutyl)‐N‐ethylisoluminol was used as tagging reagent to label the analyte for achieving high assay sensitivity. N‐(4‐Aminobutyl)‐N‐ethylisoluminol‐tagged HCys after CE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase, producing CL emission. Experimental conditions for labeling analyte, CE separation, and CL detection were studied. The CL intensity was proportional to the concentration of HCys in the range of 2.5×10^?8 to 5.0×10^?6 M. Detection limit (S/N=3) was 7.6×10^?9 M. Human plasma samples from healthy donors were analyzed by the presented method. HCys levels were found to be in the range of 9.50–15.3 μM.