Lingyun Du

Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme Immunoassay

Abstract A simple, sensitive biotin‐avidin‐amplified electrochemical enzyme‐linked immunosorbent assay (ELISA) for the determination of estradiol (E2) was proposed in this paper. The complex of biotinylated anti‐E2 antibody and horseradish peroxidase‐labeled avidin (HRP‐avidin) were regarded as a probe in this system. The activity of labeled enzyme was measured with electrochemical methods using o‐phenylenediamine as substrate. Coupled with the plate‐coated antigen, indirect ELISA format using E2‐ovalbumin, the electrochemical detection was performed for E2 with the detection limit of 21.0 pg/ml, and the linear range of determination of 50.0–500.0 pg/ml. The proposed method has been used for the determination of E2 in river water with satisfactory results. Compared with the traditionally spectrophotometric ELISA detection, this method shows greatly heightened sensitivity. The limit of detection improved by about two orders of magnitude, which is very suitable for the conditions with extremely low concentration of analyte or very small volumes of sample present.

Electrochemical Enzyme‐Linked Immunoassay for the Determination of Estriol Using Methyl Red as Substrate

Abstract A new electrochemical substrate for horseradish peroxidase, methyl red, is reported. In this reaction system, horseradish peroxidase can catalyze the redox reaction of methyl red and H2O2. Methyl red exhibits a sensitive voltammetric peak at−0.51 V vs. Ag/AgCl reference electrode, the decrease of the peak current of methyl red is in proportion to the concentration of horseradish peroxidase (HRP). The linear range for determination of horseradish peroxidase is 5.0×10−8∼5.0×10−7 g mL−1 and the detection limit is 1.8×10−8 g mL−1. The relative standard deviation is 3.3% when 2.0×10−7 g mL−1 HRP was sequentially determined 11 times. A voltammetric enzyme‐linked immunoassay method for the determination of estriol was developed, based on this electrochemical system. The linear range for determination of estriol is 1.0∼1000.0 ng mL−1, and the detection limit is 0.33 ng mL−1. The relative standard deviation for 11 parallel determinations with 200 ng mL−1 estriol is 4.8%. Some pregnancy serum samples were analyzed with satisfactory results.

Aptamer-based fluorescent detection of ochratoxin A by quenching of gold nanoparticles

A simple, rapid, low cost and highly sensitive method for the detection of ochratoxin A (OTA) was developed based on the principle that dispersed AuNPs show a better fluorescence quenching effect than aggregated AuNPs. In the absence of OTA, the aptamer is adsorbed onto the surface of AuNPs, which helps to enhance the stability of AuNPs against salt-induced aggregation, and also enhances the fluorescence quenching of the fluorescein-labeled aptamer. With the addition of OTA, the conformation of the aptamer changed, which induced aggregation of AuNPs in the presence of high-salt conditions. The fluorescence intensity was clearly recovered. The assay showed a linear response toward OTA concentration in the range of 25 nM to 300 nM with a correlation coefficient of 0.9957. The limit of detection for OTA was experimentally determined to be 22.7 nM. This method has the advantages of simple operation, low-cost and high sensitivity compared to conventional methods and can be applied to the detection of real samples.

Determination of diethylstilbestrol based on biotin–streptavidin-amplified time-resolved fluoro-immunoassay

A rapid and sensitive time-resolved fluoroimmunoassay (TR-FIA) based on the biotin-streptavidin amplification system was developed for the determination of diethylstilbestrol (DES). Europium-labelled streptavidin derivatives combined with europium and anhydride of diethylene triamine penta-acetic acid were used to label streptavidin; biotin was coupled with goat anti-rabbit IgG to form a biotin-goat anti-rabbit IgG bridge between streptavidin-europium and the anti-DES antibody in the immunoassay. The DES assay was carried out by measuring the fluorescence of Eu(3+) -SA at 615 nm. The presented method produced a wide linear range, 0.001-1000.0 ng/mL, and a detection limit up to 0.81 pg/mL for DES. The method was applied to determine DES in serum samples, with recoveries of 97.4-107.8% and RSD 1.32-4.04%. The assay results by the present method showed that biotin-streptavidin amplified TR-FIA for DES detection; it may offer high sensitivity and promising alternative special methods in biological samples.

A competitive fluorescence quenching-based immunoassay for bisphenol A employing functionalized silica nanoparticles and nanogold

We have developed a fast and sensitive immunoassay for the determination of bisphenol A (BPA), using the fluorescence quenching effect between gold nanoparticles and fluorescein isothiocyanate (FITC). Herein, carboxyl-modified single-strand DNA (COOH-ssDNAs) and anti-BPA antibodies were simultaneously conjugated with silica nanoparticles, and FITC-labeled single-strand DNA (FITC-ssDNA, the complement of COOH-ssDNA) was hybridized with COOH-ssDNA to form a signal platform, and the BPA coated antigen-functionalized nanogold was regarded as an acceptor. In the presence of BPA, a competitive immunoreaction takes place between BPA and BPA coated antigen-functionalized nanogold for the binding sites of the anti-BPA antibodies on the signal platform. Due to the fact that the photoluminescence of FITC was strongly quenched by the AuNPs, the fluorescence emission was decreased significantly. Under optimized conditions, the fluorescence intensity had a linear signal response range with a BPA concentration from 2.0 × 10−3 ng mL−1 to 1.0 ng mL−1 with a low limit of detection of 1.44 × 10−3 ng mL−1. Furthermore, this new signal platform was successfully applied to detect real samples for low levels of BPA.