The Study Of Electrochemical Biosensor Based On Multiple Signal Amplification To Determinate Tau-441 Protein In Human Serum

Objective Tau protein is a class of microtubule-associated proteins.It can promote tubulin polymerization and participate in the formation of neural cytoskeleton.There are six isomers of tau protein,and their structural differences mainly lie in different amino-terminal region?N-terminal?and carboxy-terminal region?C-terminal?.Tau protein containing N-terminal can induce cognitive disorder.Cognitive disorder is a typical clinical manifestation of dementia and dementia as a progressive syndrome severely affects the living ability of patients.Tau-441,the longest isoform of tau protein,contains two N-terminal fragments.The level of tau-441 protein is closely related to cognitive disorder.Tau-441 protein is expected to assess cognitive disorder and thus become a biomarker for early diagnosis of dementia.Tau-441 protein can exist in cerebro-spinal fluid and blood.Compared with cerebro-spinal fluid,blood samples are more convenient and less invasive.However,due to the blood-brain barrier,a small amount of tau-441 protein is present in the blood.At present,the commonly used enzyme linked immunosorbent assay cannot meet the detection requirements.Electrochemical biosensors have great advantages of improving sensitivity and the application of nanomaterials can further improve the sensitivity of determination.The purpose of the research was to construct an electrochemical biosensor based on multiple signal amplification using carbon nanomaterials and gold nanoparticles to sensitively determine tau-441 protein.And the constructed method was applied in actual serum samples of normal persons,mild cognitive impairment pateints and dementia patients.It provides a new idea for early diagnosis of dementia.Methods In this study,the multi-walled carbon nanotubes-reduced graphene oxide-chitosan nanocomposite was synthesized and the nanocomposite was modified on the gold electrode surface to improve electrode conductivity.Gold nanoparticles were used to modify tau-441 protein for further signal amplification.The specific antibody was fixed on the modified electrode surface to specifically recognize the analyte.Signal amplification was verified by cyclic voltammetry?CV?,electrochemical impedance spectroscopy?EIS?,and differential pulse voltammetry?DPV?.Based on DPV,the tau-441 protein was determined.The potential value was qualitative,and the current value was quantified.Thus,a multiple signal amplification electrochemical biosensor was constructed for the determination of tau-441 protein.The volume of the multi-walled carbon nanotubes-reduced graphene oxide-chitosan nanocomposite,the volume ratio of tau-441 protein and gold nanoparticles,the incubation time of the antibody and the analyte were optimized in order to obtain a satisfactory signal value.The detection limit,specificity,accuracy,precision,stability,and reproducibility of the experiment were verified.Finally,the constructed method was applied in actual serum samples of normal persons,mild cognitive impairment pateints and dementia patients to verify its practicability.Results Both the carbon nanomaterials and gold nanoparticles in the study amplified the signal.Optimization results:the volume of the multi-walled carbon nanotubes-reduced graphene oxide-chitosan nanocomposite was 6?L,the volume ratio of tau-441 protein and gold nanoparticles was 2:1,the incubation time of the antibody and the analyte was 30 min.Under optimal parameters,DPV was used to assay tau-441protein.The sensing response of tau-441 protein with concentration range of 0.5 fmol/L to 80 fmol/L was excellent.As the concentration increased,the current value decreased.The liner regression equation was y(?35?I=I₀-I_[tau-441],?A)=1.7692x(lgc_[tau-441],fmol/L)+11.622,and the correlation coefficient was greater than 0.99.The detection limit was calculated to 0.46 fmol/L based on International Union of Pure and Applied Chemistry.Glucose,ascorbic acid,L-cysteine,?-synuclein,and human serum albumin were selected as interfering substances.The interference rates were all less than 5%.The precision?RSD?of the method were all less than 10%,the recovery was between 75%and 105%.Based on the constructed method,the current value of the electrode reserved92.86%of its initial response after 11 days storage at 4?.Under the same experimental conditions,the signal responses of different electrodes had no obvious difference.The method was applied in the analysis of actual serum samples,including 14 serum samples of normal persons,14 serum samples of mild cognitive impairment patients,14 serum samples of dementia patients,?I[normal person group]<?I[mild cognitive impairment group]<?I[dementia group].Conclusion In this study,a multiple signal amplification electrochemical biosensor was constructed for the determination of tau-441 protein.Multi-walled carbon nanotubes-reduced graphene oxide-chitosan nanocomposite was used to improve the conductivity of the electrode and the gold nanoparticle was used to modify tau-441protein for further signal amplification.Using DPV to determinate tau-441 protein,the linear range was 0.5 fmol/L?80 fmol/L,and the detection limit was 0.46 fmol/L.It had good specificity,accuracy,precision,stability and reproducibility.And the method was successfully applied in the measurement of serum samples?normal persons,patients with mild cognitive impairment,and patients with dementia?.It provided a new direction for the early diagnosis of dementia.