The Study Of Novel Enzyme Electrodes And Their Applications In Biosensing And Environmental Detection

Enzyme-based biosensors taking enzyme as recognition element are widely used in high sensitive and selective determination of enzyme reaction substrate or enzyme inhibitors by utilizing the biological characteristics of enzymes such as high efficiency and high specificity.With the rapid development of technology and industry,environmental pollution is more and more serious and the pollutant emissions are increasing.The development of novel enzyme biosensors to realize the high efficient detection of environmental pollutants has received much attention in biosensing areas.In this dissertation,the working principle,the fabrication and applications of enzyme biosensors,combined with the inhibition effect of heavy metal ions on the enzyme,are briefly reviewed.A series of high-performance enzyme electrodes and enzyme-inhibition biosensors are developed and applied to detect the enzyme substrate and environmental pollutants.The main contents are as follows:(1)The facile preparation of the poly(tyrosine)(PTy)-tyrosinase(Tyr)-glucose oxidase(GOx)(PTy-Tyr-GOx)composite and its application both in the Tyr substrate(bisphenol A and phenol),Tyr inhibitor(Cr(III)),GOx substrate(glucose)and GOx inhibitor(Cr(VI))sensing applications are reported for the first time.With tyrosine(Ty)as polymerization monomer and Tyr as catalyst for the catalytic oxidation of Ty polymerization,Tyr and GOx were co-immobilized in PTy,and the resultant PTy-Tyr-GOx composite was dropped on the Au electrode.The bi-enzyme biosensor was successfully prepared and used for the detection of glucose,phenolic pollutants and heavy metal ions.The results showed that the PTy-Tyr-GOx/Au electrode exhibits a sensitivity high up to 146.4?A mM-1 cm-2 in the linear range of 1.0 ?M to 2.3 × 103 ?M and a low limit of detection(LOD)of 0.22 ?M in glucose sensing;a LOD of 5 nM and a linear range of 0.01 to 0.12 ?M in Cr(VI)sensing are obtained.The PTy-Tyr-GOx/Au exhibits a linear cathodic amperometric response to bisphenol A in concentration range from 0.1 to 12.1?M with a sensitivity of 2845 ?A mM-1 cm-2 and a LOD of 16 nM;in Cr(?)sensing a LOD of 80 nM and a linear range of 0.1 to 1.0 ?M are observed.In addition,the biosensor shows high sensitivity,excellent stability and fabrication simplicity which may find wide applications in diversified fields including the detection of glucose,environmental pollutants and heavy metal ions.(2)With norepinephrine(NE)as polymer monomer,horseradish peroxidase(HRP)catalyzes the polymerization of NE in the presence of H2O2,and a novel bi-enzymes biosensor was prepared successfully.It was shown that the PNE-GOx-HRP/Pt electrode exhibits a sensitivity high up to 628.38 ?A mM-1 cm-2 in the linear range of 5.0 × 10-4?0.418 mM and a sensitivity of 208.85 ?A mM-1 cm-2 in the linear range of 0.418 mM?3.418 mM and a LOD of 0.08 ?M in glucose sensing;in Cr(VI)sensing a LOD of 0.2 nM and a linear range of 0.5?6.0 nM are obtained.The PNE-GOx-HRP-PANI/Pt electrode exhibits a linear cathodic amperometric response to H2O2 concentration from 0.05 to 30.15 mM with a sensitivity of 33.83 ?A mM-1 cm-2 and a LOD of 29 ?M;a LOD of 0.1 ?M and a linear range of 0.1 to 3.8 ?M in Cr(?)sensing are observed.Hence,an amperometric biosensor with high sensitivity,low LOD and good anti-interference ability for detection of glucose,H2O2 and heavy metal ions is developed.Compared with the conventional biosensor,the PNE-HRP-GOx/Pt electrode has been tested for a variety of substances,which reduces the cost and improves the efficiency.It is expected to be widely used in the detection of glucose,H2O2 and heavy metal ions.(3)MW CNTs-AuPtPd NPs nanocomposites were successfully synthesized by one-step hydrothermal method by taking using of the reduction and stability of cationic polyelectrolyte PDDA and the excellent conductivity and high specific surface area of MWCNTs.The GOx/MWCNTs-AuPtPd NPs/Au electrode with good performance in glucose sensing was constructed via adsorbing GOx on MWCNTs-AuPtPd NPs modified Au electrod.The prepared GOx/MWCNTs-AuPtPd NPs/Au electrode displayed a sensitivity of 74.03 ?A mM-1 cm-2 in the linear range of 10 ?M to 7 mM,with a LOD of 2.4 ?M in glucose biosensing.The resultant enzyme electrode was also utilized in Cr(?)and Ag sensing based on the inhibition effect of Cr(?)and Ag on GOx activity,with a LOD of 10 nM and a linear range from 10 nM to 110 nM obtained.It was demonstrated that the MWCNTs-AuPtPd NPs composites provide an ideal microenvironment for the immobilization of enzyme molecules,which can keep the biocatalytic activity of the enzyme and promote the electron transfer between the enzyme and the electrode.