SERS Lateral Flow Assay For Multiplex Detection Of Alzheimer’s Disease Biomarkers

Alzheimer’s disease（AD）is one of the top public health crises at 21st century.Early diagnosis,prevention and intervention can significantly reduce the risk of suffering AD.The detection of AD biomarkers in blood is expected to provide early diagnosis of AD in asymptomatic populations.Currently,AD biomarkers in blood mainly include amyloid-beta protein（Aβ）,tau protein（tau）and neurofilament light chain protein（NFL）.However,the levels of AD biomarkers in blood are extremely low,and comprehensive analysis of multiple AD biomarkers for the accurate diagnosis of AD is very necessary.Therefore,the detection of AD biomarkers is still challenging.Lateral flow assay（LFA）is a widely used low-cost rapid blood analysis platform,which can read the results in less than half an hour without professional operation and is the most widely used point-of-care testing（POCT）technology.Surface enhanced Raman scattering（SERS）nanotags can achieve multi-target detection and single-molecule level detection.Therefore,in this paper,the SERS nanotags and LFA were combined to achieve high-sensitivity detection of multiple AD biomarkers.What’s more,an ordered-structured nitrocellulose membrane with nanoscale regular porous structure was successfully prepared.And it was combined with SERS nanotags and LFA to further improve the sensitivity of AD biomarker detection.The main research contents and conclusions of this paper are as follows:（1）The gold core silica shell（Au@Si O₂）SERS nanotags were successfully prepared.It can not only obtain a very high Raman signal,but also has excellent stability and good dispersion at room temperature and 37°C.In addition,it is simple to conjugate to antibodies,and conjugation to antibodies does not reduce the Raman signal.These all lay the foundation for subsequent detection applications.（2）The Au@Si O₂ SERS nanotag based on LFA was constructed.By selecting two kinds of Raman dye-encoded SERS nanotags and setting two detection lines on the NC membrane,the simultaneous detection of four AD biomarkers(Ab_42,Ab₄₀,tau and NFL)on a single test strip was achieved.The limit of detection for Ab_42,Ab₄₀,tau and NFL was 138.1,191.2,257.1and 309.1 fg/m L,respectively,which were about two orders of magnitude lower than the concentrations of relevant AD biomarkers in blood.And the linear dynamic ranges are all 1pg/m L-1mg/m L,covering 6 orders of magnitude.As far as we know,this is the first time that SERS-LFA is proposed to simultaneously detect four AD biomarkers.（3）The SERS-LFA based on ordered-structured nitrocellulose membrane was constructed.The concentration of nitrocellulose solution and the etching time of hydrofluoric acid were optimized during the preparation of ordered-structured nitrocellulose membranes.It was concluded that the optimum concentration of NC solution is 4%,and the optimum time for hydrofluoric acid etching is 12 h.Furthermore,the ordered-structured nitrocellulose membranes with different pore sizes were successfully prepared by selecting silica particles with different particle sizes,and it was concluded that the ordered-structured nitrocellulose membranes with a pore size of 350 nm could obtain the best detection results.Finally,through the detection of four AD biomarkers(Ab_42,Ab₄₀,tau and NFL),it was concluded that the detection limit of SERS-LFA based on the ordered-structured nitrocellulose membrane was 10lower than that of SERS-LFA.As far as we know,this is the first time to propose the combination of ordered-structured nitrocellulose membranes with LFA,which provides a new idea and theoretical basis for improving the sensitivity of LFA.In conclusion,compared with the existing AD biomarker detection technology,the detection method proposed in this thesis has the advantages of high specificity,high sensitivity,simple operation,low cost,multiple detection and rapid detection,and has great application potential in the early diagnosis and monitoring of AD.