Study Of Microarray-based Technology For Drug Screening And Simultaneous Multi-contaminants Detection

As a highly integrated analysis and research tool,microarray chips has played a decisive role both in academic research and practical application with its advantages such as unique high-throughput,high sensity,high accuracy,good reproducibility and low time-cost.In recent years,microarray biochips has been widely used in gene expression,functional genomics,proteomics,drug screening,clinical diagnosis,and other fields.In this thesis,the microarray technology based on visual detection methods has been developed including a number of methods for simultaneous detection of a variety of harmful substances such as antibiotics,illegal additives and biotoxins in food.In addition,a fluorescence-based protein microarray chip method has also been established for dual targets drug screening.The main content includes the following aspects:1.A multiplexed screening method for agonists and antagonists of the estrogen receptor protein.The estrogen receptor(ER)is regarded as a significant drug target because of its important physical and pathological function.In this article,we describe a novel screening method to obtain agonists and antagonists of ER.ER was immobilized onto an aldehyde-modified glass slide.The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined.Two libraries,one containing 29 synthetic compounds and the other with 384 natural products that served as a model,were screened to find new ligands for ER.Finally,65 active ligands(5 synthetic compounds and 60 natural products)of ER were identified.This novel method gave identical results to a conventional fluorescence polarization assay,thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays.The presented method is sensitive,accurate,and reliable,and shows great potential for use in high-throughput drugscreening research.2.Simultaneous detection of four nitrofuran metabolites in honey by using a visualized microarray screen assayA visualized microarray sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics.Using a multiplexed approach,metabolites of four main nitrofuran antibiotics can be detected simultaneously.Individual antigens were spotted onto 96-well plates.An indirective competitive assay format,with visualized signal response,was employed.An extraction method,based on derivatization with 2-nitrobenzaldehyde and partition into ethyl acetate,was used for screening.The limits of detection were 0.10,0.04,0.04,and 0.10 ng g-1 for 3-amino-5-morpholino-2-oxazolidone(AMOZ),3-amino-2-oxazolidinone(AOZ),semicarbazide(SEM),and 1-aminohydantoin(AHD),respectively.The recovery rate ranged from 78%to 93%for the four targets.In addition,this method was easy to operate with low detection cost and fast speed.This microarray method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety monitoring.3.Simultaneous detection of four kinds of water-soluble antibiotics in honey by using a visualized microarray screen assayWe have established a microarray based method for simultaneous detection of four categories of water-soluble antibiotics in honey samples.By using this method,tetracyclines,quinolones,lincomycin and streptomycin could be detected simultaneously with single sample processing.The haptens prepared by protein conjugation method were adsorbed on 96-well plates.The assay employed an indirect competitive immunoassay to determine the content of antibiotics in honey samples.This method has high sensitivity with a detection limit of 0.98ng g-1(tetracycline),1.24 ng g-1(norfloxacin),0.65 ng g-1 and 1.76 ng g-1 for tetracyclines,quinolones,lincomycin and streptomycin respectively.The recoveries for tetracyclines,quinolones,lincomycin and streptomycin were 86%-114%,93%-117%,99%-115%and 95%-112%.The microarray method were validated by detecting 214 real samples.The results showed that the microarray method has high consistency with LC-MS/MS methods.Compared to LC-MS/MS,the microarray method has advantages such as low-cost in both time and economic,rapid analysis speed and high analysis throughput.4.Simultaneous detection of various harmful substances in milk based on visualized microarrayMelamine,cephalexin and Aflatoxin M1 are three typical harmful compounds in dairy products,representing illegal additives,residual antibiotics and internal biological toxins respectively.In order to ensure the safety of dairy products,a rapid method is needed to screen and detect these various harmful substances.This work proposed a multiplexed detection technology for testing melamine,cephalexin and Alfatoxin M1 in milk simultaneously.The method involves the visualized microarray technology using 96-well microtitre plate.Compared to detection methods based on fluorescence or chemoluminescence,the colorimetric technology provides more direct result that can be detected using a commercial scanner.The limit of detection(LoD)was defined at 16.31 ng mL-1,8.21 ng mL-1 and 0.21 ng mL-1 for melamine,cephalexin and Aflatoxin M1 respectively,which all fit the national standards for maximum residue limits.The accuracy of method was adequate for these three substances.Experiments were also conducted using actual samples;the recovery rates found in the repeatability conditions were 103.1%?106.2%for melamine,96.6%?98.3%for cephalexin and 105.4%?109.3%for Aflatoxin M1.This technology could achieve the simultaneous detection of various harmful substances and will have broad applications in the food safety analysis field.