| Objective Three neurotransmitters,dopamine(DA),5-hydroxytryptamine(5-HT)and acetylcholine(ACh),were selected as targets in this study.Currently,the electrochemical biosensors of neurotransmitters have the following shortcomings:the lack of load and stability of the enzyme biosensor,the low conductivity and catalytic activity of nano enzyme biosensor and limit of simultaneously multiple targets detection.In this thesis,we focus on optimizing the load and stability of enzyme,and improving the conductivity and catalytic activity of the nano enzyme to establish a series of novel methods which have high activity,conductivity and stability and could detect various neurotransmitters at the same time.This article provides an efficient,accurate and convenient method for quantitative detection of multiple neurotransmitters.Methods In this study,for the construction of enzyme biosensor,graphene and chitosan co-spun nanofiber membrane with gold nanoparticles(AuNPs@GCS)prepared by electrostatic spinning and electrodeposition technique was used to immobilized acetylcholinesterase(AChE)and cholinesterase(ChOx).Simultaneously,multi-metal composite magnetic nano enzymes(FM@AuNPs,FM-Pt@AuNPs)were prepared by hydrothermal method to simulate horse radish peroxidase(HRP)activity and to construct nano enzyme biosensors.Field emission scanning electron microscopy(FESEM),Energy dispersive imaging(EDS),X-ray diffraction(XRD)and Fourier transform infrared spectroscopy(FTIR)were used to study the composition characteristics of GCS,FM@AuNPs and FM-Pt@AuNPs,including morphology characterization,element analysis,lattice type and active group.The gradual characterization of the modified electrode was detected by Electrochemical impedance spectroscopy(EIS)and Cyclic voltammetry(CV).The actual area of the modified electrode calculated by Randles-Sevcik equation,and the Michaelis constant(K_m)calculated by Michaelis equation were applied to characterize the affinity of FM@AuNPs and FM-Pt@AuNPs to the substrate.CV and Differential pulse voltammetry(DPV)were used for sensitivity analysis,electrochemical detection and optimization of main experimental parameters,such as stability,reproducibility,specificity,recovery and so on.Results Under the optimal conditions,the linear range of the enzymes based electrochemical biosensor used for ACh detection was 0.2～200 nM,and the detection limit(LOD)was 0.067 nM.The linear range of the nano enzyme biosensors for DA and5-HT was 25～8000 nM and 50～8000 nM,and the LOD was 10 nM and 16 nM,respectively.The linear ranges of the nano enzyme biosensors for simultaneous detection of DA,5-HT and ACh were 25～6000,50～6000 and 5～2000 nM,and LOD were 10,15 and 2 nM,respectively.After systematic methodological evaluation and feasibility verification,these biosensors have good stability,specificity and reproducibility.In human serum samples,the results detected by those biosensors were compared with results tested by Enzyme-linked immunosorbent assay(ELISA).T test was performed for statistical comparisons and p value more than 0.05 was considered no statistical significance.The recoveries of human serum samples ranged from 96.1%to 104.0%with RSD less than 4.74%.Conclusions In this paper,two types of enzyme biosensors including biological enzyme biosensor and nano enzyme biosensor were constructed.These enzyme biosensors can detect multiple neurotransmitters simultaneously,and the sensitivity,stability,number of targets,detection efficiency and practicability of the biosensors were effectively improved.The constructed enzyme biosensors were applied to the detection of serum samples from healthy people,and the results were consistent with ELISA,showing a good practical application prospect.It provides an efficient,accurate and convenient methodological basis for quantitative detection of complex biological sample,and also provides a new idea for diagnosis and treatment of diseases and the research and development of drugs. |
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