Amperometric Biosensing Studies Based On Several New Nanocomposite Enzyme Films

Biosensors consist of biomolecular recognition elements (e.g., enzyme, antigen/antibody, aptamer) and various physical/chemical transducers, and they have been widely used for analysis/test of a variety of life and chemical substances. Amperometric enzyme-based biosensors integrate various enzyme-immobilization techniques with electrochemical sensing techniques, which have the advantages of high sensitivity and selectivity for direct assay of target analytes in real samples. Recently, nanocomposite materials have received much attention in various areas, and developing new nanocomposite enzyme film is essential to improve the biosensing performance and has become the frontier and focus in the field. In this thesis, we briefly review the recent advances of Prussian blue (PB) in the field of amperometic enzyme electrode, and constructed several biosensors based on gold-Prussian blue ((Au-PB)REd) nanocomposite of ultrahigh electroactivity, and poly(anilineboronic acid), polydopamine and polyelectrolyte nanocomposites for high load/bioactivity immobilization of enzymes and high-performance biosensing. The main contents are as follows.1. We report a new and efficient protocol for rapid electrodeposition of (Au-PB)REd nanocomposite of ultrahigh electroactivity on Au electrode by cyclic voltammetry in0.1M aqueous K2SO4containing1mM K3Fe(CN)6,1mM HAuCl4, and0.1mM Fe2(SO4)3, and electrochemical quartz crystal microbalance and scanning electron microscopy were utilized to investigate the electrodeposition. The (Au-PB)REd/Au electrode was then cast-coated with an urate oxidase (UOx)-poly(anilineboronic acid)(PABA)-Pt nanoparticles (PtNPs) bionanocomposite and chitosan (CS) for high-performance amperometric biosensing of uric acid (UA) in the dual-potential mode. The UOx-PABA-PtNPs bionanocomposite was prepared through chemical oxidation of anilineboronic acid (ABA) by sodium chloroplatinate in the presence of UOx. The thus-fabricated CS/UOx-PABA-PtNPs/(Au-PB)REd/Au enzyme electrode worked well under optimized conditions through both oxidation and reduction determination of enzyme-generated H2O2, which responded linearly to UA concentration from0.3μM to0.65mM with a sensitivity of223μA mM-1cm-2and a limit of detection (LOD) of0.2μM (0.7V vs SCE), and from0.2μM to0.25mM with a sensitivity of247μA mM-1cm-2and a LOD of0.1μM (-0.05V vs SCE), being superior to most analogues hitherto reported for biosensing of UA.2. We studied the rapid oxidation of dopamine (DA) by K3Fe(CN)6, chloroauric acid or chloroplatinic acid in the presence of glucose oxidase (GOx), yielding chemically polymerized DA (PDA) with GOx effectively entrapped. The GOx-entrapped PDA with coexisting noble metal nanoparticles was then cast-coated on an (Au-PB)REd/Au electrode, followed by CS strengthening for dual-potential amperometric biosensing of glucose. In the enzyme-generated H2O1-reduction mode, thus-prepared CS/GOx-PDA-PtNPs/(Au-PB)REd/Au electrode displayed a high sensitivity (145.7μA mM-1cm-2), a low LOD (0.5μM), fast response (less then5s), good suppression of interferences and excellent operation/storage stability, which also worked well in biofuel cell.3. We developed a novel method for effectively immobilization of GOx by mixing sodium alginate (AlgNa) and ploy(diallyldimethylammonium)(PDDA), yielding an interpenetrating polymer composite based on electrostatic interaction. To improve the electron-conductivity and biocompatibility, we introduced AuNPs or PtNPs into the polyelectrolyte composite. The final nanocomposite and then the mixture of GOx and Fe3+was cast-coated and air-dried on the Au plated Au electrode. In the present protocol, Fe3+can react with AlgNa to form AlgFe hydrogel for further robust immobilization of enzyme. Thus-prepared GOx-PDDA-AlgFe-AuNPs/Au or GOx-PDDA-AlgFe-PtNPs/Au plated Au electrode displayed a good anti-interference ability and satisfactory sensitivity.