Xiurong Yang

Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein-streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 microg/ml.

Impedance study of (PEO)10LiClO4–Al2O3 composite polymer electrolyte with blocking electrodes

A composite solid polymer electrolyte (SPE) of (PEO)(10)LiClO4-Al2O3 was prepared and Pt and stainless steel(SS) blocking electrodes were used for an impedance study. It was found that the semicircle in the high frequency range and the straight line in the low frequency range depend upon different blocking electrodes and polarization potentials applied in the experiments. In the equivalent circuit. two constant phase elements (CPE) have been used instead of the pure geometrical and double layer capacitances. respectively. A theoretical line calculated from their estimated values has a good correlation with the experiment data. Moreover. the equivalent circuit also can be used to explain the impedance properties of Pt and stainless steel (SS) blocking electrodes both in the high and the low frequency ranges

Capacitive detection of glucose using molecularly imprinted polymers

A novel glucose biosensor based on capacitive detection has been developed using molecularly imprinted polymers. The sensitive layer was prepared by electropolymerization of o-phenylenediamine on a gold electrode in the presence of the template (glucose). Cyclic voltammetry and capacitive measurements monitored the process of electropolymerization. Surface uncovered areas were plugged with 1-dodecanethiol to make the layer dense, and the insulating properties of the layer were studied in the presence of redox couples. The template molecules and the nonbound thiol were removed from the modified electrode surface by washing with distilled water. A capacitance decrease could be obtained after injection of glucose. The electrode constructed similarly but with ascorbic acid or fructose only showed a small response compared with glucose. The stability and reproducibility of the biosensor were also investigated.

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Determination of Surface pKa of SAM Using an Electrochemical Titration Method

A self-assembled monolayer (SAM) of 3-mercaptopropionic acid (HSCH2CH2COOH) was formed on a gold electrode. The effect of the charge of the end group on the electrochemical response of Fe(CN)(6)(3-) at the SAM modified electrode was studied by using cyclic voltammetry. At high pH, when the -COOH groups are dissociated, Fe(CN)(6)(3-) current is suppressed; as the solution pH is lowered, Fe(CN)(6)(3-) current increases. The electrochemical titration curve was obtained by correlating the currents to the different electrolyte pH values, from which the surface pK(a) was obtained to be 5.2+/-0.1. Furthermore, a calculation equation was presented to simulate the electrochemical titration. As comparison, the surface pK(a) was also measured by contact angle titration as 5.6+/-0.1. The surface pK(a) values determined by the two methods in our work are consistent and accurate.

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 microM.

Aptamer-DNAzyme hairpins for biosensing of Ochratoxin A

We report an aptasensor for biosensing of Ochratoxin A (OTA) using aptamer-DNAzyme hairpin as biorecognition element. The structure of this engineered nucleic acid includes the horseradish peroxidase (HRP)-mimicking DNAzyme and the OTA specific aptamer sequences. A blocking tail captures a part of these sequences in the stem region of the hairpin. In the presence of OTA, the hairpin is opened due to the formation of the aptamer-analyte complex. As a result, self-assembly of the active HRP-mimicking DNAzyme occurs. The activity of this DNAzyme is linearly correlated with OTA concentration up to 10 nM, showing a limit of detection of 2.5 nM.

New technique for capillary electrophoresis directly coupled with end‐column electrochemiluminescence detection

A new end‐column electrochemiluminescence (ECL) detection technique coupling to capillary electrophoresis (CE) is characterized. A 300 νm diameter Pt working electrode was used to directly couple with a 75 νm inner diameter separation capillary without an electric field decoupler. The hydrodynamic cyclic voltammogram (CV) of Ru(bpy)32+ showed that electrophoretic current did not affect the ECL reaction. The presence of high‐voltage (HV) field only resulted in the shift of the ECL detection potential. The distance of capillary to electrode was an important parameter for optimizing detection performance as it determined the characteristics of mass transport toward the electrode and the actual concentration of Ru(bpy)32+ in the detection region. The optimum distance of capillary to electrode was decided by the inner diameter of the capillary, too. For a 75 νm capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 220–260 νm. The effects of pH value of ECL solution and molecular structure of analytes on peak height and theoretical plate numbers were discussed. Using the 75 νm capillary, under the optimum conditions, the method provided a linear range for tripropylamine (TPA) between 1×10−10 and 1×10−5 mol/L with correlation coefficient of 0.998. The detection limit (signal‐to‐noise ratio S/N = 3) was 5.0×10−11 mol/L. The relative standard deviation in peak height for eight consecutive injections was 5.6%. By this new technique lidocaine spiked in a urine sample was determined. The method exhibited the linear range for lidocaine from 5.0×10−8 to 1.0×10−5 mol/L with correlation efficient of 0.998. The limit of detection (S/N = 3) was 2.0×10−8 mol/L.

Determination of biogenic amines by capillary electrophoresis with pulsed amperometric detection

The biogenic amines, putrescine, cadaverine, spermidine and spermine were separated and quantified by capillary electrophoresis with pulsed amperometric detection. Detection potential of the pulsed amperometric detection was optimized as 0.6 V. Optimal separation of the biogenic amines was achieved using a separation buffer of 30 mM citrate at pH 3.5, while keeping the buffer in the detection cell as 20 mM NaOH. Using these conditions, the four biogenic amines were baseline separated. Extrapolated limits of detection for putrescine, cadaverine, spermidine and spermine were 400, 200, 100 and 400 nM for the standard mixture (polyamines dissolved in running buffer), respectively. These are lower than ultraviolet detection and comparable or even lower than laser-induced fluorescence detection results as reported in the literature. The number of theoretical plates was maintained at the 10(5) level, which is absolutely higher than any reported method. When applying capillary electrophoresis-pulsed amperometric detection to milk analysis, only spermidine was found in amounts varying between 0.1 and 0.5 mg/kg.

Plasticizer effect on the ionic conductivity of PEO-based polymer electrolyte

The effects of plasticizer ethylene carbonate (EC) on the AC impedance spectra and the ionic conductivity are reported. With increasing of EC concentration the semicircle in high frequency disappears, and the slope of the straight line in low frequency decreases. The data obtained from impedance experiments can be explained using an equivalent circuit proposed. On the other hand, the room temperature conductivity increases with EC concentration because of the increase of the segmental flexibility of PEO. For lower EC concentration samples, the temperature dependence of conductivity in low temperature range follows Arrhenius type, but when EC concentration is larger than 20%, the temperature dependence of conductivity obeys the Vogel-Tamman-Fulcher (VTF) equation in all temperature ranges.