Amperometric Biosensing Studies Based On Poly(L-DOPA)-immobilized Enzymes

The amperometric enzyme sensors use enzymes as their molecular recognition elements to output amperometric signals from redox chemistry of the enzyme systems, which possess many advantages such as high sensitivity and selectivity. The effective immobilization of enzyme molecules is one of the key steps in constructing a biosensor.Innovation and optimization of immobilization matrices and approaches are the current frontier and focus in the amperometric biosensing area. In this thesis, on the basis of literature survey, poly(L-DOPA)(PD) is introduced as a novel enzyme-immobilization material for the first time,and several amperometric enzyme electrodes are developed for high-performance biosensing of glucose, phenols and uric acid. The main contents are as follows:1. Electrochemical/chemical oxidative synthesis and biosensing/biofuel cell applications of PD are studied versus polydopamine(PDA) as a recent hotspot biomaterial. The enzyme electrode developed by coelectrodeposition of PD and glucose oxidase(GOx), uricase(UOx) or tyrosinase(Tyr) shows biosensing performance superior to the corresponding PDA-based enzyme electrode. The chemical oxidative polymerization of L-DOPA(PDC) by NaAuCl4 in GOx-containing neutral aqueous solution is used to immobilize GOx andgold nanoparticles(Au NPs). The thus-prepared chitosan(CS)/GOx-PDC-AuNPs/Auplate/Au electrode working in the first-generation biosensing mode responds linearly to glucose concentration with a sensitivity of 152 μA mM-1 cm-2 that is larger than those of the CS/GOx-PDAC-AuNPs/Auplate/Au electrode and most reported GOx-based enzyme electrodes. This enzyme electrode also works well in the second-generation biosensing mode and as an excellent bioanode in biofuel cell construction, probably because PD as a protein-mimic ?-amino acid polymer has the higher biocompatibility and the more favorable affinity to the enzyme than PDA. The PD material of great convenience in synthesis, outstanding biocompatibility for preparing high-performance bionanocomposites, and strong capability of multifunctional coatings on many surfaces may find wide applications in diversified fields including biotechnology and surface-coating.2. Learning to nature emerges as one of the most promising ways to develop advanced functional materials and biodevices. Here, inspired by melanin formation, we report the Tyr-catalyzed polymerization of L-DOPA(versus L-tyrosine and dopamine(DA)) to immobilize enzymes for amperometric biosensing. The enzymatic polymerization is examined by UV-Vis spectrophotometry, scanning electron microscopy and electrochemical methods. A PD-Tyr/glassy carbon electrode(GCE)prepared by casting an aqueous mixture of L-DOPA and Tyr on a GCE exhibits a linear cathodic amperometric response to catechol concentration from 0.4 to 57 ?M(R2=0.997) with a sensitivity of 4.29 mA mM-1 cm-2 and a limit of detection(LOD) of 70 nM(S/N=3). A PD-GOx-Tyr/Pt electrode prepared by casting an aqueous mixture of L-DOPA, GOx and Tyr on a Pt electrode exhibits a linear anodic amperometric response to glucose concentration from 2 to 5700 ? M(R2=0.998) with a sensitivity of 78.6 ?A mM-1 cm-2 and a LOD of 0.1?M(S/N=3). The PD-based enzyme electrode shows better biosensing performance and higher bioactivity of the immobilized Tyr than those based on similarly biosynthesized poly(L-tyrosine) and PDA as well as well-established CS and Nafion systems, implying that the biosynthesized PD as a melanin-biomimetic material has promising application potential for biomacromolecular immobilization and biosensing.3. Horseradish peroxidase(HRP)-catalyzed polymerization of L-DOPA(versus DA) in the presence of H2O2(and UOx) was exploited to immobilize mono-/bi-enzymes for hydroquinone-mediated amperometric biosensing of H2O2 and uric acid(UA). The relevant polymeric biocomposites(PBCs) were prepared in phosphate buffer solution containing HRP and L-DOPA(or plus UOx) by the added H2O2.The mono-/bi-enzyme amperometric biosensors were prepared simplyby casting some of the PBCs on Auplate/Au electrodes, followed by coating with an outer-layer CS film for each. UV-vis spetrophotometry,scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were used for film characterization and/or process monitoring. The HRP immobilized by enzyme catalysis well preserved its bioactivity, as confirmed by UV-vis spectrophotometric tests. Under optimized conditions, the monoenzyme CS/HRP-PD/Auplate/Au electrode potentiostated at-0.1 V responded linearly to H2O2 concentration from 0.001 to 1.25 mM with a sensitivity of 700 ? A mM-1 cm-2 and a LOD of 0.1 ? M, and the bienzyme CS/UOx-HRP-PD/Auplate/Au electrode at-0.1 V responded linearly to UA concentration from 0.001 to 0.4 mM with a sensitivity of 349 ?A mM-1 cm-2 and a LOD of 0.1 ? M. The mono-/bi-enzyme biosensors based on biosynthesized PD performed better than many reported analogues and those based on similarly biosynthesized polydopamine.