| Objective Alzheimer's disease(AD)is a neurodegenerative chronic disease that occurs to the elderly(over 65 years of age).According to the World Alzheimer Report 2018,a new case of AD occurred every 3 seconds around the world.The worldwide population of AD in 2018 was 50 million,and the number is expected to triple to 152 million by 2050.The total estimated worldwide cost of AD in 2018 was $1 trillion and it is expected to increase to $2 trillion by 2030.A key advance in the field of AD research is the confirmation that two classical neuropathological changes,i.e.,senile plaque and neurofibrillary tangle,are the final outcome of the pathological process of AD.Among them,tau protein is the main component of neuronal fiber tangles,and tau-381 protein is one of the six subtypes of tau protein,which may be used as a biomarker for early detection of AD.Traditional enzyme-linked immunosorbent assay(ELISA)is not sufficient to detect ultra-trace(pmol/L level)tau-381 protein,and electrochemical biosensor is currently one of the most sensitive techniques.By using dual recognition of aptamers and antibodies,this study proposed a novel electrochemical aptamer-antibody sandwich analysis method capable of highly specific and sensitive detection of tau-381 protein.The analytical method constructed in this study was used to detect human serum samples,providing new insights into early diagnosis and clinical analysis of AD.Methods In this study,the gold electrode was pretreated by surface modification technology,and the antibody was immobilized on the surface of the electrode.The aptamer was assembled on gold nanoparticles by covalent binding.The specific binding of the antibody to tau-381 protein was used to bind the tau-381 protein to the surface of the electrode.Under the action of the aptamer,the specific adsorption of the tau-381 protein was enhanced,and then the interaction between aptamer-antibody sandwich structure and tau-381 was studied by the differential pulse voltammetry(DPV).A novel aptamer-antibody sandwich electrochemical biosensor was constructed to detect the content of tau-381 protein.Combined with the characteristics of tau-381 protein,the fiveconditions of tau antibody concentration,pH value of cysteamine solution,incubation time of conjugates,volume ratio of conjugates to tau-381 protein and incubation time of aptamer-antibody sandwich were optimized.The limit of detection,accuracy,precision and specificity of the method were verified.In order to verify whether the newly constructed aptamer-antibody electrochemical biosensor is superior to the traditional antigen-antibody electrochemical biosensor,an antigen-antibody method was constructed to detect tau-381 protein.The linear range,limit of detection and others of the two methods were compared.Finally,the feasibility of this method was proved by detecting tau-381 protein in human serum.Results The optimal determination conditions were as follows: tau antibody concentration was 0.05 mg/mL,the pH value of cysteamine solution was 5,the incubation time of conjugates was 3 h,the volume ratio of conjugates to tau-381 protein was 1:1,and the incubation time of aptamer-antibody sandwich was 45 min.Under the condition of this method,there was a good linear correlation between the difference of peak current and logarithm of standard series concentration,with the correlation coefficient greater than0.999.The determination range was 0.50 pmol/L~100.00 pmol/L.The linear regression equation was y=7.9674x+2.9256.The anodic peak corresponding to tau-381 protein oxidation can be observed in DPV detection figure at about 0.21 V potential,and the oxidation peak current increased with the concentration of tau-381 protein.According to IUPAC method,limit of detection was 0.42 pmol/L;the precision of the experimental method(RSD)was 4.8%~5.7%,and the average recovery was 75% ~105%.Anti-interference experiments were carried out in 1:100 diluted serum containing ascorbic acid(AA),L-cysteine(L-cys),glucose(Glu)and tau-441 protein,and the interference percentage was calculated to be less than 5%.Comparing with the adapter-antibody electrochemical biosensor,the anodic peak corresponding to tau-381 protein oxidation could be observed in DPV detection diagram at about 0.21 V potential,just like the traditional antigen-antibody electrochemical biosensor,but its oxidation peak current decreased as its concentration increased.In the range of 0.50~100.00 pmol/L,the standardsolution of tau-381 protein demonstrated a good linear correlation with the response value,the correlation coefficient was greater than 0.99,but limit of detection of this method was0.49 pmol/L.The novel method constructed in this study was applied to actual human serum samples.The serum samples collected from 10 normal elderly people(normal control group)and 10 patients with AD(experimental group)were measured by DPV technology.The average concentration of current difference in experimental group was higher than that in normal control group.The average concentration of current difference in experimental group was 298.95 pmol/L.Conclusions In this study,a novel electrochemical biosensor based on aptamerantibody sandwich was constructed to detect the content of tau-381 protein in human serum.Cyclic voltammetry(CV)and Electrochemical impedance spectroscopy(EIS)technique were used for electrochemical characterization.DPV technique was used to record the signal response values of tau-381 protein with different concentrations.The linear range was 0.50 pmol/L ~100.00 pmol/L,and the limit of detection was 0.42 pmol/L.In order to further verify the high sensitivity of this method,the selection of traditional antigen-antibody electrochemical biosensor was compared,and good results were obtained.Finally,the feasibility of this method was verified by detecting tau-381 protein in human serum.This method could quantitatively detect tau-381 protein in human serum,thus providing a new method for the early diagnosis of AD,and having applications for clinical practice. |
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