Electrochemical Biosensor Based On Single-layer Reduced Graphene Oxide For Assaying Tau-441 Protein In Human Serum

Objective Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases.In 2010,there were 36 million AD patients worldwide,by2030,the number of AD patients is expected to increase to 65.7 million.The neurofibrillary tangles formed by the abnormal aggregation of Tau protein are the main pathological features of AD,and the increase of the Tau-441 protein of the 4R isoform is related to the decline of cognitive function.Tau-441 may serve as a biomarker for early diagnosis of AD.The purpose of this study was to construct a high-sensitivity electrochemical biosensor based on a single-layer reduced graphene oxide to assay Tau-441 protein in human serum,providing technical support for the early diagnosis of AD.Methods In this study,1,3,6,8-pyridine tetrasulfonic acid tetrasodium salt(PTSA)was used to exfoliate the reduced graphene oxide(rGO)into a single-layer reduced graphene oxide with better conductivity.After the glassy carbon electrode was modified by single-layer reduced graphene oxide with a carboxyl group,an antibody with an amino group could be fixed under the action of EDC/NHS,and then the Tau-441 protein is bound to the electrode through the specific recognition of the antibody and the Tau-441 protein.Cyclic voltammetry(CV)was used for electrochemically characterization,the Square Wave Voltammetry(SWV)was used to study the interaction between antibodies and Tau-441 protein,and the potential value was qualitative and the current value was quantified.An electrochemical biosensor based on single-layer reduced graphene oxide was constructed to determine the Tau-441 protein content.In order to obtain the best detection results,this study optimized four conditions in the experiment:the volume of the composite material,the incubation time of the antibody,the volume of the antibody,and the incubation time of the antibody antigen.The detection limit,specificity,accuracy and precision of the method were verified.Finally,the method was applied to the detection of Tau-441 protein in human serum,which confirmed the feasibility of its application to actual samples.Results As a result of optimized detection conditions,the volume of the composite material was 6μL,the volume of the antibody was 8μL,the incubation time of the antibody was 3 hours,and the incubation time of the antigen antibody was30 minutes.Under optimal conditions,the measured peak current difference had a good linear correlation with the logarithm of the standard series concentration of Tau-441 protein.The linear regression equation was y=20.962x+44.726,R~2=0.9956,and the linear range is 0.08 pM～80 pM.In the SWV detection chart,the anodic peak of the oxidation of Tau-441 protein is about 0.23 V,and the oxidation peak current decreases with the increase of Tau-441 concentration.According to the International Union of Pure and Applied Chemistry(IUPAC)method,the detection limit of this method was 75 fM,the components that interfered with Tau-441 protein(glucose(Glu),α-synuclein(α-syn),bisphenol A(BPA),L-cysteine(L-cys),and ascorbic acid(AA))were performed in a 1:1000 diluted serum interference experiment,and the accuracy of the method was better.The average spike recovery of the experimental method was between 75%-105%,and the precision(RSD)is between 3.9%-4.6%.The constructed method was used for the detection of 30 human serum samples,including 15 normal elderly and 15 AD patients.The peak value of SWV detection in AD patients was lower than that in normal elderly people,and serum Tau-441 protein concentration in AD patients was higher than the concentration in normal elderly.Conclusions This study established an electrochemical biosensor based on single-layer reduced graphene oxide for the detection of Tau-441 protein in human serum.The results of CV showed that the composite material and antibody were successfully modified on the glassy carbon electrode.Under the optimal conditions,SWV technology was used to assay Tau-441 protein at different concentrations.The concentration detected by electrochemical immunosensors ranged from 0.08 pM to 80pM,and the detection limit is as low as 75 fM.The sensor showed good accuracy and high precision.These characteristics made the method successfully applied to the detection of human serum samples,which had clinical significance for the detection and diagnosis of AD.