The Study Of Screen-Printed Electrochemical Biosensor(array) For Detection Of Pathogenic Microorganisms

bjective: To establish a detection of pathogenic microorganisms, screen printingDNA biosensors, with rapid detection, high sensitivity, low cost, simple process. Thisstudy were prepared by screen printing technology sensing electrode by controllingthe potential for fixing DNA probe for typing of pathogenic microorganisms and theircontent in the sample detection. Provide a fast, sensitive and inexpensive detectiontechnique for the rapid determination of pathogenic microorganisms in the samplecontent. It also provides a preliminary basis for the establishment of new microbialmolecular typing.Methods: To establish the method of detection of DNA hybridization, theelectrochemical DNA sensors and electrochemical indicator were used. Thesingle-stranded DNA （ssDNA） was fixed to screen-printed carbon electrodeconstituting the electrochemical DNA sensors. Co（phen）₃³⁺electrochemical indicatorwas prepared by cobalt salt and the ligand phenanthroline complex. The functionalgroups and the electrochemical characteristics of the indicator were analyzed byplasma emission spectrometry （ICP-AES）, nuclear magnetic resonance method（NMR） and cyclic voltammetry Act （CV） respectively. This paper discusses the fixedmethod of direct adsorption, electrostatic adsorption and bonding of three kinds ofssDNA on the electrode surface.Results:（1） In this paper, screen-printed carbon electrode were prepared, the electrodepreparation, repeatability and reproducibility surface were well. The response of thebackground signal of the electrochemical detection was low and stable.electrochemical indicator Co（phen）₃³⁺were also prepared, which was analyzed byplasma emission spectroscopy and NMR¹H spectral and the results showed that, Coelement and1,10-phenanthroline complexation reaction was correct. The modifiedelectrode electrochemical characterization results show that the Co（phen）₃³⁺dsDNAmodified electrode surface has better adsorption effect.（2） Electrostatic adsorption and key legal fixed amount of ssDNA, using theelectrostatic adsorption electrode fixed probe hybridization target DNA, Co （phen）₃³⁺easy to embed a double-stranded DNA （dsDNA）, the CV peak current （ip）, the signalincrease with the concentration of target DNA. In this paper, the electrostatic adsorption of ssDNA electrode was used to detect DNA hybridization experiments.The results showed that when the concentration of probe fixative ssDNA reached5mg/L, the target DNA concentration ranged from6.65×10^-8-4.26×10-6mol/L,Co（phen）₃³⁺ip value and the concentration of DNA in the dsDNA modified electrodeshowed a good linear relationship, R²was0.9819.（3） The Listeria monocytogenes Bureau specificity of inl A, act A, and the hly Agene were verified by PCR and gel electrophoresis and the detection sensitivitywas9.2×10²cfu/mL; The act A gene detection DNA probe was designed as5’-AGTTAC AGA AAG AAA TAA AGA GG-3’,according to gene sequence determinationand comparison.The real-time PCR detection standard curve of Inl A, act A and hly A weredrawed in the concentration range of9.2×101cfu/mL-9.2×105cfu/mL. The threelinear correlation coefficients were0.987,0.980and0.991respectively.Electrochemical indicator MB was adsorbed and detected on the electrode afterthe DNA hybridization, the response to ip can not be formed between the DNAconcentration and ip; When Co（phen）₃³⁺was used as electrochemical indicator, thestandard curve of DNA hybridization was successfully mapped with the linearregression equation y=0.223Ln（x）+3.7843. The linear correlation coefficient was0.9871; Hybridization standard curve was used for quantitative detection calculating,then results were compared with that of real-time PCR. The detection resultsconsistent with the quantitative PCR test results, the error is less than10%.Conclution: Here established a technology of screen-printed DNA sensor fordetecting of pathogenic microorganisms. This technology improved the sensoranalytical accuracy and sensitivity, and it will be used to testing both short sequenceDNA and genomic DNA. It also provided a method for the detection andclassification of pathogenic microorganisms.