Nano-biosensor For Detection Of Both Ebola Virus And Glutamine Release From Primary Hippocampal Neurons

In recent years,with the rapid development of science and technology,it has brought a new scientific perspective and technical theory support for the field of modern clinical laboratory diagnosis and biomedicine.Among them,graphene nanotechnology and field effect transistor(FET)biosensors have emerged in the field of clinical laboratory diagnosis because of their unique advantages.They are playing an important role in the early diagnosis of diseases and revealing disease mechanisms.The FET biosensor has become a research hotspot of many researchers because it can achieve high sensitivity,label-free and rapid detection,and integrated miniaturization of the chip.Graphene is also attracting attention among many nanomaterials due to its high electron mobility,good electrical conductivity,large specific surface area and good biocompatibility.A combined application of FET biosensors and graphene can make the two advantages as a whole so that people can achieve high sensitivity and rapid detection of target analytes in complex matrices,especially it has great potential for application in detecting serious infectious diseases caused by pathogens and studying the pathogenesis of diseases.At present,diseases caused by pathogens,such as,viruses,often occur,and even some high-risk viruses,like Ebola virus,cause major infectiousdiseases,which have a detrimental effect on human health and social stability.Therefore,early diagnosis of infectious diseases is particularly important.At the same time,for some non-infectious disease,such as,nervous system diseases,Alzheimer’s disease and Parkinson’s syndrome,it is importance to study the transduction mechanism of nervous system signaling molecules,and explore the pathogenesis of nervous system diseases by exploring the brain mystery and the corresponding treatment plan.In this thesis,this paper mainly focuses on the fabrication of reduced graphene oxide-based field effect transistor(RGO-FET)biosensors,and its application in detecting Ebola virus and glutamate release from primary hippocampal neurons in a label-free,highly sensitive and specific manner.The main research works are as follows:Part Ⅰ: Functionalized reduced graphene oxide-based FET biosensor for label-free and highly sensitive detection of Ebola virusThis part describe a field effect transistor(FET)based immunoassay for the detection of inactivated ebola virus(EBOV).An equine antibody against the EBOV glycoprotein was immobilized on the surface of the FET that was previously modified with reduced graphene oxide(RGO).The antibody against EBOV was immobilized on the modified FET,and the response to EBOV was measured as a function of the shift of Dirac voltage.The method can detect the EBOV in the phosphate buffer saline(PBS)over the concentration range from 2.4×10-12 g/m L to 1.2×10-7 g/m L and with a limit of detection as low as 2.4 pg/m L.The assay has satisfactory specificity and was applied to the quantitation of inactivated EBOV in spiked serum.Part Ⅱ: Functionalized reduced graphene oxide-based FET biosensor for real-time monitoring of glutamate released from primary hippocampal neuronsGlutamate,as one of the most important central excitatoryneurotransmitters,plays crucial roles in nerve signal transduction and is implicate in several neurological disorders.However,no effective means has been developed for specific detection of glutamate released from primary cultured neurons.This part present a reduced graphene oxide(RGO)-based field effect transistor(FET)biosensor functionalized with synthesized glutamate receptor for real time monitoring of glutamate release from primary cultured rat hippocampus neurons.Metabotropic glutamate receptors(m Glu R)was specifically synthesized and then immobilized on the RGO surface by1-Pyrenebutanoic acid succinimidyl ester(PASE)linker,after which target glutamate(p I=3.22)could specifically bind to the synthesized m Glu R in the neutral buffer,causing the charge density change.After the neurons were cultured on the sensing channel with a self-made liquid reservoir,the FET biosensor could discriminate glutamate in f M range in complete cell culture medium and generate encouraging results in real time monitoring of glutamate release from primary rat hippocampus neurons.This work is the first report of specific and direct detection of glutamate molecules released from primary culture of differentiated central neurons,which may further help understand nature of neuronal communications.Moreover,this work paves a way for the detection of electrochemically inactive small molecules released by cells.