Wenrui Jin

Determination of bovine serum albumin by capillary zone electrophoresis with end-column amperometric detection at the carbon fiber microdisk array electrode

Abstract Capillary zone electrophoresis (CZE) was employed to the analysis of bovine serum albumin (BSA) using an end-column amperometric detector with a carbon fiber microdisk array electrode. The optimum conditions of separation and detection are 0.025 mol l−1 KH2PO4-0.02 mol l−1 NaOH for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, and 0.90 V vs. SCE for the detection potential. The limit of detection is 4 × 10−7 mol l−1 or 0.7 fmol ( S N = 2 ). The method was applied to determining BSA in bovine serum.

Electrochemiluminescence of CdTe quantum dots as labels at nanoporous gold leaf electrodes for ultrasensitive DNA analysis

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S(2)O(8)(2-) as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to -2V to record the curve of ECL intensity versus potential. The maximum ECL intensity (I(m,ECL)) on the curve is proportional to t-DNA concentration with a linear range of 5 x 10(-15) to 1 x 10(-11)mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection.

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end‐column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 μm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal‐to‐noise ratio. The limit of detection is 1 × 10—7 mol/L or 26 amol and the linear dynamic range is 2 × 10—7 to 2 × 10—5 mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser‐induced fluorescence detection.

Determination of molybdenum in soils and plants using the polarographic adsorptive complex catalytic wave of Mo(VI)-cupferron

Abstract The very sensitive adsorptive complex catalytic wave of Mo(VI)-cupferron with acetic acid-postassium acetate as supporting electrolyte in differential-pulse, normal-pulse direct current and linear-sweep polarographic techniques has been successfully applied to the determination of available molybdenum in soils and molybdenum in plants. Soil and ashed-plant samples are extracted with hot water and the extract solution can be analysed directly without preseparation. The procuedure is very simple and the method is very sensitive for the determination of Mo(VI). The mechanims of the polarographic wave was studied; it behaves as an adsorptive complex and also as an EC (electrode reaction-chemical reaction) parallel catalytic wave.

Ultrasensitive Electrochemical DNA Assay Based on Counting of Single Magnetic Nanobeads by a Combination of DNA Amplification and Enzyme Amplification

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection.

The precapillary derivatization of 20 amino acids with naphthalene‐2,3‐dicarboxaldehyde (NDA) and CN– was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7×10–7 and 1.8×10–6 mol/L or 84 and 893 amol (according to the signal‐to‐noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91–109%.

Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene‐2,3‐dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection

A method is described for the direct identification and quantification of amino acids in individual mouse peritoneal macrophages by capillary zone electrophoresis with electrochemical detection after on‐column derivatization with naphthalene‐2,3‐dicarboxaldehyde (NDA) and CN–. In this method, individual macrophages and then the lysing/derivatizing buffer are injected into the front end of the separation capillary by electromigration with the aid of an inverted microscope. The front end of the separation capillary is used as a chamber to lyse the macrophage and derivatize its contents, which minimizes dilution of amino acids of a single macrophage during derivatization. Six amino acids (serine, alanine, taurine, glycine, glutamic acid, and aspartic acid) in single mouse peritoneal macrophages have been identified. Quantitation has been accomplished through the use of calibration curves, where the concentration ratios of these standard amino acids are similar to the concentration ratios of amino acids in macrophages. Cellular levels of the amino acids in these cells range from 0.27 ± 0.20 fmol/cell for alanine to 6.4 ± 4.6 fmol/cell for taurine.

Electrochemiluminescence resonance energy transfer between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor and its biological application

Novel electrochemiluminescence resonance energy transfer (ECRET) between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor is investigated. The ECRET technique can be used to study the interactions and conformational changes of proteins.

Capillary zone electrophoresis with end-column amperometric detection for the determination of glutathione at a gold/mercury amalgam microelectrode without deoxygenation

Abstract Capillary zone electrophoresis was employed to the analysis of glutathione by using end-column amperometric detection with a gold/mercury amalgam microelectrode at a constant potential of 0.02~0.06 V vs. SCE. In this procedure deoxygenation is not necessary. The calibration plot was found to be linear over 4 orders of magnitude and the limit of detection was 6.3 × 10 −7 mol l −1 (or 126 amol). The effect of the concentration of the buffer, and the high separation voltage across the capillary on the migration time and separation efficiency, and the dependence of the injection conditions on the detection signal, the separation efficiency and the coulometric efficiency have been investigated. The method was applied to the determination of glutathione in human blood and erythrocytes.

Quantitative counting of single fluorescent molecules by combined electrochemical adsorption accumulation and total internal reflection fluorescence microscopy.

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6- CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 x 40 microm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 x 10(-15) to 5 x 10(-12) mol/L for R6G, 3 x 10(-15) to 2 x 10(-12) mol/L for IgG(H+L)-488, and 3 x 10(-15) to 2 x 10(-12) mol/L for DNA-R6G.