Zhixian Gao

Simultaneous and rapid detection of six different mycotoxins using an immunochip

Mycotoxins are highly toxic contaminants in food, animal feed, and commodities. The study has developed an immunochip for quantifying the concentrations of six mycotoxins: aflatoxin B1, aflatoxin M1, deoxynivalenol, ochratoxin A, T-2 toxin, and zearalenone, which were added to drinking water. The complete antigens (Ags) of the mycotoxins were contact printed and immobilized onto agarose-modified glass slides with 12 physically isolated subarrays, based on the reaction of both diffusion and covalent bond. The optimal concentration of each antigen and antibody (Ab) was obtained using an Ag-Ab immunoassay. Based on the indirect competitive immunoassay for the simultaneous detection of six mycotoxins in one single chip, six standard curves with good logistic correlation (R(2)>0.97) were respectively plotted. The working ranges (0.04-1.69, 0.45-3.90, 20.20-69.23, 35.68-363.18, 0.11-1.81, and 0.08-7.47 ng/mL, respectively) were calculated, as well as the median inhibitory concentrations (0.31±0.04, 1.49±0.21, 34.54±1.30, 134.06±11.75, 0.49±0.05, and 1.54±0.22 ng/mL, respectively), when six mycotoxins were detected simultaneously. Finally, the recovery rates in drinking water generally ranged from 80% to 120% on the same chip, with an intra-assay coefficient of variation lower than 15%. We successfully established an immunochip for simultaneous detection of six mycotoxins within 4h, with advantages of using minimal samples and being visually semiquantitative with our naked eyes. In summary, the method could be developed on one single chip for detecting multiple contaminants in actual samples.

Flow injection chemiluminescence sensor using molecularly imprinted polymers as recognition element for determination of maleic hydrazide

A novel flow injection chemiluminescence (CL) sensor for the determination of maleic hydrazide (MH) using molecularly imprinted polymer (MIP) as recognition element is reported. The MH-MIP was synthesized by thermal initiated polymerization in methanol, using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the maleic hydrazide template molecule. Molecular modeling was employed to simulate the possible recognition process of the MIP, and to achieve high selectivity. Then the synthesized MH-MIP was employed as recognition element by packing into flow cell to establish a novel flow injection CL sensor. The CL intensity responded linearly to the concentration of MH in the range 3.5x10(-4)-5.0x10(-2) mg/mL with a detection limit of 6.0x10(-5) mg/mL (3sigma), which is lower than that of conventional methods. The relative standard deviation (RSD) for the determination of 1.0 microg/mL of MH was 2.7% (n=11). The sensor is reusable and has a great improvement in sensitivity and selectivity for CL analysis. As a result, the new MIP-CL sensor had been successfully applied to the determination of MH in vegetable samples.

Identification of differential hepatic proteins in rare minnow (Gobiocypris rarus) exposed to pentachlorophenol (PCP) by proteomic analysis

Pentachlorophenol (PCP) is a ubiquitous contaminant that has been shown to lead to hepatoxicity and is implicated in the incidence of liver tumors in human. A number of previous studies have described the toxic effects of PCP based on conventional toxicological indices. However, little evidence on protein levels is available at present. For further understanding of mechanisms of action and identifying the potential protein biomarkers for PCP exposure, two-dimensional electrophoresis coupled with mass spectrometry has been used to identify proteins differentially expressed in the livers of rare minnow (Gobiocypris rarus) following PCP exposure of 0.5, 5, 50 μg/L. After comparison of the protein profiles from treated and control groups, 39 protein spots were found altered in abundance (>2-fold) from male and female PCP-treated groups. Matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/MS) analysis allowed the unambiguous identification, and 18 protein spots were identified successfully, 12 proteins in females and 6 proteins in males, respectively. These proteins were involved in transport, metabolism, response to oxidative stress and other biological processes. Of these proteins, four differentially expressed mRNA encoding proteins underwent quantitative analysis by quantitative real-time PCR (QRT-PCR). The consistent and discrepant results between mRNA and protein levels suggested that complicated regulatory mechanisms of gene expression were implicated in the response to PCP exposure. In addition, marked gender differences in response to PCP have been described from the comparison of the male and female liver protein profiles.

Simultaneous and combined detection of multiple tumor biomarkers for prostate cancer in human serum by suspension array technology

Tumor markers (TMs) play an important role in clinical rapid screening and diagnosis for prostate cancer (PCa). In this study, we describe a competitive method to establish the multiplex suspension array by tumor biomarkers coated on distinguishable microbeads which competing with free biomarkers for their complementary antibodies (Ab) in one single reaction system for simultaneous and combined detection of prostate TMs in human serum. The volumes of the targets coupled onto the beads and their complementary Abs were optimized. The suspension array standard curves correlated well with PCa biomarkers (R(2)>0.9968). PCa biomarker levels were quantified using median fluorescent intensities. The working ranges of prostate-specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP) were 0.47-502.94, 1.00-923.35, 1.00-524.79, and 1.73-176.07ngmL(-1) in serum samples, respectively. This method was compared to indirect competitive enzyme linked immunosorbent assay. It was found that high concordance between the two technologies resulted from serum samples of the eight PCa patients. The multiplex suspension array technology is specific to PCa biomarkers, displayed no significant cross-reactivity, and remains stable for 6 months. We also characterized the bead surface microstructures under different conditions employing a field emission scanning electron microscope. The suspension array is a straightforward and reliable method for analysis of multiple TMs with simple operation, high sensitivity at a low cost.

Immunochip for the detection of five kinds of chemicals: Atrazine, nonylphenol, 17-beta estradiol, paraverine and chloramphenicol.

This paper reports an immunochip for the detection of five kinds of chemicals: atrazine, nonylphenol, 17-beta estradiol, paraverine and chloramphenicol, which are harmful to human health and environment due to unconscionable use or abuse. Five antigen conjugates were synthesized, purified and then labeled with Cy3 dye. Meanwhile, the immunochip was prepared by immobilization the five kinds of specific antibodies on aldehydized slides as probes. A series of experimental conditions were optimized and selected. Standard curves were plotted and the detection ranges were 0.001-5 microg/mL with good logistic correlation and linear correlation (R(2)>0.99). All the five chemicals can be simultaneously and quantitatively determined in a chip, and the relative standard deviations were within 10%. The immunochip will become a highly sensitive, rapid, alternative analytical tool for detection of chemicals or toxicants.

Studies on detection of atrazine and papaverine by protein arrays

The high toxicity of pesticides and their wide use in modern agriculture has increased public concern. People are also worried about health threats caused by deleterious materials added to food. Current detection methods for these materials have their shortcomings. Microarray technology provides a highly sensitive and precise technique for obtaining information from biological samples, with the added advantage that it can handle a large number of samples simultaneously for rapid analysis. A direct competitive immunoassay for the screening of poisonous chemical haptens is proposed. Complete antigens of atrazine and papaverine were labeled with fluorescence. The fluorescence intensity linearly increased with decreasing concentration of analytes. The detection limits of atrazine and papaverine were 0.001 /spl mu/g/ml and 0.01 /spl mu/g/ml respectively.