Construction Of Portable Biosensing Platforms And Their Methodological Research On Virus Antibody Detection

Traditional testing methods require sampling on site-cold chain transport to the central laboratory-professional technicians for testing,with the limitations of low efficiency and high cost.The WHO calls for the urgent need for a simple,fast,online,and high-reliability testing technology and equipment,with great potential to use biosensors for testing.Biosensors can accurately and quickly analyze and identify the targets in the sample,and detect the target by establishing the semi-quantitative relationship with the detectable signals.Therefore,it is of great significance to improve the detection efficiency to establish field rapid detection biosensors and develop field detection equipment to form a portable biosensing detection platform and realize the high field sensitivity and rapid screening of samples.The biosensor can accurately and quickly analyze and identify the target in the sample,and establish a corresponding rule relationship between the concentration of the target and the detectable signal,so as to realize the semi-quantitative or quantitative detection of the target.Therefore,it is of great significance to improve the detection efficiency to establish field rapid detection biosensors and develop field detection equipment to form a portable biosensing detection platform and realize high field sensitivity and rapid screening of samples.Based on this,the study focus on the research and development of biosensors and field detection equipment,through the use of quantum dot microspheres fluorescent materials and portable devices such as smart phones,build the following portable biosensing detection platform,and used for livestock and poultry disease and SARS-CoV-2 antibody field detection,specific research content is as follows:(1)Establishment of a portable single-molecule fluorescence immunodetection platform and its methodological research in the detection of avian influenza AIV H9subtype antibodies.In order to achieve the goal of rapid,high sensitivity and high precision field detection,this study built a portable single-molecule fluorescence immunodetection platform based on smartphone-based single-molecule fluorescence microscopy.It mainly consists of the following sections:Design and preparation of The portable fluorescence counter,which with the compact size of 90 mm(diameter)X173 mm(height),the weight of～1 kg,and the cost effective devices reached the Fo V of 0.979 mm~2and the resolution of 0.55μm,thus it could collect a large amount of fluorescence signals in high resolution and accuracy;Design the software for supporting the portable fluorescence counter,which can recognize the fluorescence spots and automatically compute the fluorescence spot density;To prepare polystyrene microsphere signal probes coated with protein L,which not only to amplify the fluorescence signals,but also to reduce the fluorescence spot aggregation,and the Protein L coated polystyrene microspheres also had good bioactivity and stability;The PEG coated sample chambers were verified not only to specifically bind the targets in the liquid sample,but also to significantly reduce the non-specific absorption.Avian influenza is a serious zoonotic disease caused by influenza A virus and poses a serious threat to global public health security,especially in many less developed countries and rural areas,which raises a need for methods suitable for sophisticated field testing.Therefore,the feasibility of the portable single-molecule fluorescence immunodetection platform was verified by the AIV H9antibodies.The results show that the complete portable fluorescence immunodetection platform has the advantages of high sensitivity,high specificity,simple operation,and portable instrument,and is a potential tool for rapid field detection of AIV H9N2 antibodies(2)The Establishment of the portable quantum dot microsphere immuno-chromatography platform and the methodological study in the SARS-CoV-2 antibody detectionOn the basis of the portable single-molecule fluorescence immunodetection platform,the optical path was simplified,and a portable quantum dot microsphere immunochromatography platform was built.The COVID-19 pandemic caused by SARS-CoV-2 infection causes huge global economic and health losses,and serological antibody detection is an important tool to monitor human dynamic humoral response to SARS-CoV-2vaccination and viral infection.Therefore,the feasibility of the portable single-molecule fluorescence immunodetection platform was verified by the SARS-CoV-2 antibodies.This study mainly includes the design of a fluorescence immunochromatography test strip based on the quantum dot microspheres,and combined with a hand-held fluorescence reader to achieve the field semi-quantitative detection of IgM and IgG of SARS-CoV-2 in the tested samples within 15 min.When samples to be tested contain IgM or/and IgG,the quantum dot microsphere-nucleocapsid protein probe binds to IgM or/and IgG and can be captured by mouse anti-human IgM monoclonal antibody and mouse anti-human IgG monoclonal antibody,yielding a strong fluorescent signal.By comparing with the commercial colloidal gold strip,IgM detection is 8 times more sensitive than the traditional commercial colloidal gold strip,and IgG detection is at least 4 times more sensitive than the traditional commercial colloidal gold strip;And the specificity results of HCV,HBV and HIV serum showed that there was little nonspecific binding by fluorescence immunochromatography test strip.Given its advantages of good specificity,high sensitivity,simple operation,low cost and less time consuming,this portable quantum dot microsphere immune tomography platform is a feasible diagnostic method in emergency situations requiring timely detection of a large number of samples,and has broad prospects for the diagnosis,monitoring and control of SARS-CoV-2 worldwide.(3)Establishment of portable multimode field detection platform and its methodology in SARS-CoV-2 antibody detectionIt mainly includes the establishment of multimodal immunodetection platforms for different detection purposes,quantum dot microspheres and horseradish peroxidase coupled with goat anti-human IgG were employed as the multi-mode reporter probes,and recombinant nucleocapsid protein fixed on 96-well plate works as the capture probes.In the condition with target under detection,both reporter and capture probes can be bound by such target.When illuminated by excitation light,fluorescence signals from QD microspheres can be collected for target quantification often in fast speed.Additionally,when pursuing simple detection without using any sensing devices,HRP catalyzed TMB colorimetry immunoassay is employed;and when pursuing high sensitive detection,HRP catalyzed luminol chemiluminescent immunoassay is established.All of these fluorescence,colorimetry and chemiluminescent immunoassay can be implemented on the same multi-mode immunoassay platform.Verified by the anti-SARS-CoV-2 N humanized antibody,the sensitivities of colorimetry,fluorescence and chemiluminescent immunoassay are respectively 20,80 and640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip.Additionally,all these detecting operations can be implemented on site within 50 min relying on field-operable processing and field-portable devices.Such multi-mode immunoassay platform can provide a new solution for rapid on-site diagnostics of COVID-19.