Study On LM Gene-Sensor Based On The Principle Of Surface Enlarged By Golden Nanoparticle

A rapid, accurate and convenient approach for detecting of LM was established empolying DNA sensor,which is based on the surface enlarged on the surface of gold electrode by Au nanoparticle and employing hlyA gene as a molecular recognizing component,piezoelectric quartz crystal as a mass sensitive component. The experiment included five parts and main results were as follows:1 Listeria monocytogenes probe was designed and species specificity was detectedAiming at hlyA gene, taking into account of specificity and length of DNA probe, oligonucleotide probe which suited for constructing piezoelectric DNA sensor was designed by bio-informatics technique.It was proved that the probe had favourable LM species specificity by dot hybridization method.2 Study on the self-assemble process and capability of mercaptan self-assemble piezoelectric DNA sensorProbe marked with mercaptan at the 5′end was immobilized on the surface of gold electrode by self-assemble,the effect of probe concentration and pH value of the solution on sensor were discussed and the sensor was used to detect single-strand DNA in solution.The result showed that if the probe concentration was below 1.0μmol/L, the amount of immobilization increased along with probe concentration increasing.In acidic environment,the amount of immobilization on piezoelectric quartz crystal was highest and the amount of hybridization was lowest, while the result was contrary in neutral environment. When the concentration of ssDNA in hybridation solution between 0.01～1.0μmol/L,there was linear relation between detected signals and DNA concentrations.3 Probes labelled by amino were immobilized on the surface of gold electrode Active couplet TAPD was synthesized in the study.TAPD cound self-assemble on the surface of gold electrode,probes were immobilized by the reaction of amino in probe and active esterfunction in TAPD, the sensor was used to detect ssDNA in hybridization solution.The result showed that the frequency only declined by 38 Hz when the sensor was used to detect 1μmol/L ssDNA.4 Study on LM gene-sensor based on the principle of surface enlarged by golden nanoparticle16nm gold nanoparticle and gold nanoparticle-oligonucleotide probe was synthesized in the study. The ssDNA was immobilized on the surface of gold electrode and hybridized with the gold nanoparticle-oligonucleotide complementary DNA probes,then the rest gold nanoparticle-oligonucleotide probes detected ssDNA in sample. The sensitivity of the sensor was two times that of unimmobilized Au nanoparticle.5 Listeria monocytogenes were detected by sensor in milkThe result showed that the sensor had good specificity for detection of 15 strains of listeria and 1 strain of staphylococcus aureus, and allowed detection at level of as low as 10 cfu/mL milk.The sensor could be primarily used in Listeria monocytogenes as qualitative and quantitive detection.Compared with immunoassay and PCR method which used to LM detection in food ,the sensor based on the principle of surface enlarged by gold nanoparticle detect LM in food had several advantages, rapid and simple procedure, easy sample preparation, low price and high sensitivity.The DNA was extracted by boiling and no special reagents were needed,and the whole detection process only take 2 hours. It wound bring profound influence on the evaluation of food safety, clinical diagnosis and environmental monitoring.