Amperometric Biosensing Studies Based On Novel Methods For Polymeric Immobilization Of Biorecognition Molecules And For Nano Enhancement

By combining the recognition ability of bioactive species (enzyme, antibody/antigen, nucleic acid, aptamer, etc.) and the physical/chemical transducer, biosensing analysis has become one of the most important branch of modern analytical chemistry. Biosensor is well acknowledged as a device that can detect the target with high selectivity, time/cost-effectiveness, and operation facility, being competent for working in complex samples, on-line monitoring, and even in vivo analysis. Since the inception of the first biosensor in 1960s, biosensing research has gained an explosive development and received wide academic and industrial attention. The effective immobilization of biorecognition materials is regarded as one of the key steps to construct a biosensor, and the use of chemical or electrochemical polymerization to prepare biocompatible polymeric biosensing films is always one of the most important methods to immobilize biorecognition molecules. Recently, along with the explosive development of nanoscience and nanotechnology, nanobiosensing has become the frontier and focus of this field. In this dissertation, the recent advances of electrochemical biosensors as well as the biosensing applications of polymers and nanomaterials are reviewed, and a series of biosensing studies are conducted based on novel methods for polymeric immobilization of biorecognition molecules and for nano enhancement, as summarized below.1. Combining the chemical polymerization and electrochemical polymerization, we propose the one-pot chemical preoxidation and electropolymerization of monomer (CPEM) in enzyme-containing aqueous suspensions (or solutions) as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors. We investigated the monomer of 1,4-benzenedithiol (BDT),1,6-hexanedithiol (HDT), o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. The typical experimental procedures are as follows. Into an aqueous suspension of BDT and GOx, the preoxidant of K3Fe(CN)6 was added to obtain BDT oligomer-enzyme composite; then high-performance enzyme film was gained by co-electrodeposition of these composites and poly(1,4-benzenedithiol). Compared with the biosensor based on protocol of conventional electropolymerization (CEP), the CPEM-based glucose biosensor presented a 32.4-fold sensitivity. We also found that the load and activity of the immobilized enzymes was largely improved, as estimated by electrochemical quartz crystal microbalance (EQCM) and UV-vis spectrophotometry. 2. We propose the biochemical preoxidation and electropolymerization of monomer (BPEM) using enzyme generated-H2O2 (EG-H2O2) as the preoxidant to immobilize enzyme with high load and activity, thus to construct a high-performance amperometric glucose biosensor. Basic experimental procedures are as follows. In an aqueous suspension of 2,5-dimercapto-1,3,4-thiadiazole (DMcT) and GOx, glucose was added, and the EG-H2O2 biochemically oxidized DMcT to DMcT oligomer (DMcTO), which entrapped GOx and yielded DMcTO-GOx composites, followed by co-electrodeposition of these composites and DMcT polymer to form a high performance enzyme film. Compared with the CEP protocol, the BPEM-based glucose biosensor presented 119-fold sensitivity, as well as a detection limit of 2 orders lower. We found that the performance of BPEM-based biosensor was superior to that based on the CPEM protocol using externally-added H2O2 as the preoxidant, which may be ascribed to the improved proximity of biochemical preoxidation and thus an increased enzyme load. We also found that DMcT polymer could be cathodically detached from the electrode surface, favorably enabling the electrochemical regeneration of the electrode substrate, as monitored by EQCM.3. We propose the preparation of novel poly dopamine (PDA)-enzyme-metallic nanoparticles nanocomposites using the CPEM protocol, with dopamine (DA) as the monomer and HAuCl4 or H2PtCl6 as the preoxidant, to immobilize GOx (or galactose oxidase) for fabricating high performance glucose (or galactose) biosensors. The PDA exhibited excellent adsorbability and biocompatibility, being favorable for highly efficient immobilization of enzyme molecules and nano enhancement. Compared with the CEP protocol, the PBNCs-based biosensor showed obviously improved sensitivity of 99μA cm-2 mmol-1 L and 129μA cm-2 mmol-1 L for glucose at bare Au and platinized Au electrode, respectively. Also, the prepared PBNCs electrode is able to work well in the second generation biosensing mode.4. We propose a novel protocol of chemical polymerization with magnetic separation/immobilization for high-sensitivity amperometric glucose biosensing. We prepared Fe3O4@Au core-shell hybrid magnetic nanoparticles (MNPs) using the method of chemical co-precipitation, then synthesized poly(1,6-hexanedithiol) (PHDT)-GOx-Fe3O4@Au nanocomposites using chemical polymerization, which were magnetically separated and immobilized on the magnetism electrode. This protocol was simple, time-saving, and highly efficient. The glucose biosensor showed a high detection sensitivity of 110μA cm-2 mmol-1L, detection limit of 0.3μmol L-1, S/N=3).5. We propose electrosynthesized PHDT as a new matrix to immobilize AuNPs and anti-body for performance-enhanced piezoelectric immunosensing. Using the EQCM, we investigated the process and mechanism for HDT electropolymerzation, and estimated the coverage of AuNPs and the nano enhancement effect theoretically and experimentally. We comparatively examined the adsorption of anti-human immunoglobulin G (anti-hIgG) and the subsequent immunoreaction with immunoglobulin G (hIgG). Compared with the electrode modified with HDT self-assembled monolayer (SAM), the PHDT modified electrode showed better stability and can be constructed more time-effectively, demonstrating that the novel thiol-polymer materials may become a useful alternative to thiol-based SAM and find wide biosensing applications.6. Novel PDA-PtNPs-anti-hIgG nanocomposites were prepared using chemical synthesis, which present satisfactory immuno-recognition efficiency and strong catalysis ability to electrochemical reduction of H2O2 for high performance sandwich-type amperometric immunosensing. The immunosensor has a limit of detection of 0.018 ng mL-1, as well as good reproducibility, stability, regeneration ability, specificity, and satisfactory feasibility for assay in clinical human serum samples.7. Chemical oxidation was used to prepare PDA-PtNPs-GOx nanocomposites, on the surface of which AuNPs were biochemically prepared from reduction of HAuCl4 by the entrapped GOx-generated H2O2 to effectively immobilize antibody and GOx, yielding GOxads/antibody/AuNPs/PDA-PtNPs-GOx PBNCs for high performance sandwich-type amperometric immunosensing. The thus-prepared PBNCs feature high load and activity of the enzyme label immobilized both in the interior and on the surface for ultrasensitive signal readout. Using p-benzoquinone/hydroquinone as the mediator, the proposed immunosensor could detect the target antigen of hIgG down to a concentration of 2 pg mL-8. The AuNPs/PDA-PtNPs-GOx nanocomposites were cast onto an Au electrode to covalently bind a thiolated aptamer for thrombin, and a "signal-off'electrochemical aptasensor was thus constructed based on signal output of the GOx label. The binding of thrombin with the captured aptamer obviously blocked the mass-transfer inside the film on the electrode, leading to the suppression of the enzymatic reaction on the electrode and the decrease of the oxidation current of EG-H2O2. The thus-prepared aptasensor could detect thrombin down to a concentration of 0.1 nmol L/-1.9. We propose a novel sandwich-type amperometric aptasensor using aptamer-wrapped single-walled carbon nanotubes (SWCNTs) as an amplification platform and the magnetic separation/immobilization technique to effectively improve the sensitivity. In a suspension containing Fe3O4 MNPs modified with thrombin aptamerⅠ(aptamer@MNPs) and SWCNTs modified with thrombin aptamerⅡ(aptamer@SWCNTs), the target thrombin was added to yield the sandwich-type composites of aptamer I@MNPs-thrombin-aptamerⅡ@SWCNTs. The composites were magnetically separated and collected onto the surface of a magnetism Au electrode, methylene blue was added to detach the aptamerⅡfrom SWCNTs via its stronger interaction with SWCNTs than aptamerⅡdoes, and the methylene blue-adsorbed SWCNTs modified electrode was obtained after magnetically removing the aptamer I@MNPs-thrombin-aptamerⅡcomposites. Based on the differential pulse voltammetric detection of methylene blue, the thus-prepared aptasensor could detect thrombin down to a concentration of 3 pmol L-1.