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Piezoresistive Microcantilever For The Detection Of Listeria Monocytogenes In Food

Posted on:2013-02-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:L HanFull Text:PDF
GTID:1118330374960930Subject:Nutrition and Food Hygiene
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The invention of the atomic force microscope (AFM) in1986, and its subsequentapplication in the fields of biotechnology and nanotechnology led to the creation of anew modality of sensing: the cantilever. The growing interest in using cantilevers asbiosensors is due to the high sensitivity and label free. Cantilevers have been used innumerous applications to measure physical properties such as density and viscosity,These tools have been used to analyse a wide range of analytes, including cells,pathogens, toxins, DNA hybridization, and proteins In this research, two types ofpiezoresistive micracantilevers sensors, i.e. silicion and silicon dioxidemicrocantilever sensor, have been studied. Base on the design, fabrication andcharacterization, the scheme and performance of the microcantilever sensors in genedetection have been invertigated.The paper consists of two parts: preparation for biological samples and researchon piezoresistive microcanrilever biosensors.The Primer5.0software package was used to design gene probes, and DNA wasextracted from bacteria using a DNA extraction kits. Polymerase chain reaction (PCR)technology, which allows the amplification of a specific fragment of nucleic acid wasused to amplify the DNA of the target strains. In order to identify PCR products ofLM, the recombinant plasmid pMD-18T/hlyA was also bulit.The hlyA DNA from LM was amplified, and the Prokaryotic expression vectopGEX-3X/hlyA was also bulit. LLO protein was expressed in E.coli. By means ofcutting GST fragments, highly purity of LLO has been obtained. The theroreticalmodels of the micracantilever sensors were studied. The parameters of the siliconmicrocantilever sensor have been systematically analyzed and optimized. Thecontinuous multilayered laminated plate theory was employed to improve sensitivity and resolution. A novel segment model based upon discontinuous multilayeredlaminated palte theory has been builded. Self-heat effect of silicon dioxide has beenstudied. Bridge in differential mode was designed for reading signals of silicondioxide microcantilever biosensor in order to reduce thermal noise.The gene probe was immobilized onto the microcantilevers by self-assembledmonolayers, and a functionalisation method was developed and verified using XPS.After functionalisation by coating Au, silicon dioxide the microcantilever sensorsshowed highly sensitivity to LM (LOD0.1μg/mL). The LOD of silicon piezoresistiveis1.0μg/mL. The fact was confirmed that the sensitivity of silicon dioxidepiezoresistive microcantilever biosensor was higher than silicon piezoresistivemicrocantilever biosensor. The result is accordant with theorical analysis.The mass of biosample for amplifying the signal of biosensor was increased, butin this research we found the mass can not affect the strength of the biosensor signals.The P type of silicon dioxide microcantilever biosensor has been used to detectthe immune response in the process of purifiing protein LLO with polyclonalantibodies.The LM in milk has been detected by P type of silicon dioxide microcantileverbiosensor.
Keywords/Search Tags:LM, microcantilever sensors, piezoresistive, gene detection
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