Biosensing Studies Based On Boronic Acid Recognition

By combining the biological recognition process (e.g. enzyme-substrate, enzyme-cofactor, antibody-antigen, hormone-receptor, DNA hybridization, lectin-polysaccharide) and physical/chemical detection elements, biosensing has become one of the most attractive branches of modern analytical chemisty. The efficient immobilization of various biological recognition elements (e.g. enzyme, antibody, ssDNA, receptor, organelles, microorganisms, organization, biomimic materials) plays a key role in the fabrication of biosensors. In this dissertation, the recent progress of electrochemical enzymatic biosensors and boronic acid recognition is reviewed. A series of detailed studies on enzyme immobilization and biosensing applications based on boronic acid recognition are conducted, as summarized below.1. We propose a new protocol for efficient immobilization of a glycoprotein enzyme based on the interaction of its1,2-or1,3-diols with a boronic acid functionalized monomer. Briefly, casting a mixture of glucose oxidase (GOx) and anilineboronic acid (ABA) followed by a NaAuCl4solution to an Au-plated Au electrode surface yielded a GOx-poly(ABA)(PABA)-gold nanoparticle (Aunano) bionanocomposite, and chitosan (CS) was then cast and air-dried. In the present protocol, the small-sized Aunano or Au subnanostructures can form near/on the enzyme molecule, which greatly promotes the electron transfer of enzymatic reaction and enhances the amperometric responses. The thus-prepared CS/GOx-PABA-Aunano/Au-plated Au electrode worked well in the first-/second generation biosensing modes and as a bioanode in a monopolar biofuel cell, with analytical or cell-power performance superior to those of most analogues hitherto reported.2. A highly sensitive amperometric biosensor for uric acid (UA) was proposed. Chemical oxidation of glycoprotein-bound thiophene-3-boronic acid (TBA) monomer by chloroplatinate in neutral aqueous solution containing UOx yielded UOx-PTBA-Ptnano bionanocomposite with high specific enzymatic activity on a platinized (Ptplate) Au electrode, which was then covered by an outer-layer CS film to fabricate a CS/UOx-PTBA-Ptnano/Ptpiate/Au electrode. This electrode exhibited a linear amperometric response to UA concentration from5μM to1.2mM with a sensitivity of134μA mM-1cm-2, a limit of detection (LOD)(S/N=3) of1μM, and excellent operation/storage stability, which also worked well in serum samples.3. We propose a facile one-pot enzymatic polymerization protocol to prepare enzyme-PTBA polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Briefly, horseradish peroxidase (HRP)-catalyzed chemical oxidation/polymerization of TBA monomer was conducted in aqueous solution containing HRP (or plus GOx) by either directly added or GOx-glucose generated oxidant H2O2, and the mono-/bi-enzyme amperometric biosensors were prepared simply by casting the prepared PBCs on Au-plated Au electrode (Aupiate/Au), followed by coating with an outer-layer CS film. The PTBA is used here due to its (and TBA’s) capability of covalent bonding with enzyme at the glycosyl sites (so-called boronic acid-diol interaction) which less affects enzymatic activity, and UV-vis spectrophotometric tests confirmed that the encapsulated HRP almost possesses its pristine enzymatic specific activity. The enzyme electrodes were studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry in the presence of Fe(CN)64-mediator. The CS/HRP-PTBA/Auplate/Au electrode responded linearly to H2O2concentration from1to400μM with a sensitivity of395μA mM-1cm-2and a LOD of0.1μM. The bienzyme CS/GOx-HRP-PTBA(H2O2)/Aupiate/Au electrode responded linearly to glucose concentration from5μM to0.83mM with a sensitivity of75.1μA mM-1cm-2and a LOD of μM, and it is found that the use of Fe(CN)64-mediator here favorably avoids the "unusual amperometric responses" observed when other mediators that can also efficiently turn over GOx are used.4. A layer-by-layer (LBL) bionanocomposite assembly was fabricated based on boronic acid-glycoprotein affinity and modified multiwalled carbon nanotubes (MWCNTs) for amperometric H2O2biosensing. Boronic acid functionalized MWCNTs were prepared by chemical oxidation in one-pot manner, which combine together the electrical/mechanical properties of MWCNTs and bioaffinity of boronic acid toward glycoproteins. By using HRP of a glycosylation degree of about16.8-21wt.%as a model glycoenzyme, the LBL assembly was studied by quartz crystal microbalance, cyclic voltammetry, and electrochemical impedance spectroscopy for biosensing application. The LBL assembly provides a favorable microenvironment to retain the bioactivity of the enzyme and to prevent enzyme molecule leakage. The resulting biosensor responded linearly to H2O2concentration from1μM to0.6mM with a sensitivity of184.4μA mM-1cm-2and a LOD of0.2μM.5. Electrochemical quartz crystal microbalance was used to study the electrochemical copolymerization of ABA and aniline in a weak acidic medium, and a PABA-co-polyaniline (PANI) based molecular imprinted polymer (MIP) was prepared using N-acetylneuraminic acid (Neu5Ac) as template molecules. The Neu5Ac-eluted MIP showed good electroactivity and molecular recognition behavior in phosphate buffer solution (PBS, pH7.0), and the codeposition of PANI can notably improve the stability of the MIP films in this PBS. The specific binding of analytes to the boronic acid moieties decreased the film electroactivity, thus sensitive differential pulse voltammetric determination of Neu5Ac was performed in PBS (pH7.0) with a LOD of50μM, and high selectivity against analogue diols such as glucose was obtained.6. A sensitive colorimetric analysis was developed for tyrosinase (TR) activity using the principle of crosslinking-induced gold nanoparticles (AuNPs) aggregation. Boronic acid and tyrosine bifunctionalized AuNPs were synthesized by carbodiimide chemistry. In the presence of reducing agent ascorbic acid, TR catalyzes the oxidation of tyrosine with aid of O2, and the enzymatic product exists as the desired catechol structure. Based on the boronic acid-diols interaction, an aggregation behavior of AuNPs occurred, which is dependent on TR activity. Thus, a sensitive TR activity assay and inhibitor screening colorimetric system are achieved.