Construction Of Nanoporous Gold/Bioenzyme Electrochemical Sensor And Its Application In Amino Acids Detection

Cysteine,tyrosine,tryptophan and glutamate are not only involved in the composition of proteins,but also are precursors of many biologically active compounds,which are closely related to people’s health and even production or life.Therefore,the sensitive detection of free amino acids plays an important role in clinical diagnosis and industrial production guidance.In view of the limitations of traditional detection methods such as expensive equipment and complicated operation,this study uses NPG with unique nanostructure and function as GCE modification material,supplemented by rGO which is modified to enhance the sensing performance.NPG/GCE and rGO/NPG/GCE electrochemical sensors were constructed to investigate the enzyme-free electrochemical detection of cysteine,tyrosine and tryptophan,respectively;meanwhile,we proposed an indirect electrochemical sensing strategy for glutamate using the excellent electrocatalytic activity of NPG for NADH in synergy with the efficient catalytic properties of the bioenzyme.Firstly,NPG was prepared by dealloying method.The microstructure of NPG was observed by SEM,which showed that the prepared NPG had nanoscale pore channels and continuous gold ligaments,of which the average width of gold ligaments was about 30 nm and the pore channel diameters ranged from several nanometers to tens of nanometers.The nanoporous structure of NPG confers a large specific surface area,good structural stability,excellent electron transfer efficiency and an abundance of active catalytic sites.Subsequently,NPG was loaded on the GCE surface to construct NPG/GCE.The electrochemical characterization results showed that NPG had good electrocatalytic activity for cysteine,and the sensing performance of the modified GCE was significantly enhanced.The electrocatalytic oxidation of cysteine on the electrode surface was an adsorption-controlled irreversible reaction.The sensitive detection of cysteine was achieved in the range of 3～50μM with a sensitivity of 376.15 μA mM-1 and a detection limit as low as 0.032 μM.The sensor showed good anti-interference ability and selectivity,the recoveries of cysteine spiked in fermentation broth and urine samples were in the range of 90%～110%with the RSD values of less than 5.92%for repeated experiments.The above results showed that the constructed NPG/GCE had good accuracy and reproducibility for the detection of cysteine.Then,the rGO/NPG/GCE electrochemical sensors were constructed to investigate the electrochemical behaviors of tyrosine or tryptophan on the electrode surface,and inferred the reaction mechanism based on the number of electron and proton transfer of the reactions.Using the excellent electrocatalytic oxidation activity of the electrode,the standard curves for electrochemical detection were established,in which the electrochemical detection range of tyrosine was 20～1250 μM with the detection limit as low as 0.215 μM,while the linear range of tryptophan detection was 10～300 μM with the sensitivity and detection limit of 106.13 μA mM-1 and 0.565 μM,respectively.The sensors showed good anti-interference ability,and the spiked recoveries of fermentation broth and urine samples were in the range of 90%～110%with the RSD values of less than 5.39%for repeated experiments,which indicated that the constructed rGO/NPG/GCE electrochemical sensors could achieve rapid and sensitive detection of tyrosine or tryptophan.Finally,an electrochemical sensing strategy for the indirect detection of glutamate by NADH assay was established using glutamate dehydrogenase in synergy with NPG.Using the excellent electrocatalytic oxidation activity of NPG on NADH,a standard curve for the electrochemical detection of NADH was established with a linear range of 0.05～10 mM,a sensitivity of 4.588 μA mM-1,and a detection limit of 2.916 μM.Therefore,the indirect detection of glutamate was achieved based on the electrocatalytic oxidation of NADH by NPG using glutamate dehydrogenase to catalyze the generation of α-ketoglutarate accompanied by the generation of NADH.A standard curve for the electrochemical detection of glutamate was established with a linear range of 50～700 μM,a sensitivity of 1.950 μA mM-1 and a detection limit of 6.821 μM.In addition,the recoveries of glutamate spiked in the fermentation broth were in the range of 95%～105%,and the RSD values of the repeated experiments were less than 4.97%.The established glutamate electrochemical assay showed good selectivity and antiinterference ability.In summary,four amino acid electrochemical detection methods based on NPG and its synergistic interaction with biological enzymes were proposed in this study.All four constructed amino acid electrochemical detection methods have the advantages of low cost,simple preparation and operation,convenience,high sensitivity,and high efficiency.And they have shown good potential for application in actual sample detection.The research work in this thesis provides technical support for sensitive detection of amino acids in clinical diagnosis,food and drug testing,amino acid fermentation and industrial production.