Horseradish peroxidase-catalyzed synthesis of poly(thiophene-3-boronic acid) biocomposites for mono-/bi-enzyme immobilization and amperometric biosensing

We report here on a facile enzymatic polymerization protocol to prepare enzyme—poly(thiophene-3-boronic acid) (PTBA) polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Horseradish peroxidase (HRP)-catalyzed polymerization of thiophene-3-boronic acid (TBA) monomer was conducted in aqueous solution containing HRP (or plus glucose oxidase (GOx)) by either directly added or GO x -glucose generated oxidant H 2 O 2 . The mono-/bi-enzyme amperometric biosensors were prepared simply by casting the dialysis-isolated PBCs on Au-plated Au electrode (Au plate /Au), followed by coating with an outer-layer chitosan (CS) film. The boronic acid residues are capable of covalent bonding with enzyme at the glycosyl sites (boronic acid-diols interaction), which should less affect the enzymatic activity as compared with the common cases of covalent bonding at the peptide chains, 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) 6 4− mediator. The CS/HRP–PTBA/Au plate /Au electrode responded linearly to H 2 O 2 concentration from 1 to 300   μM with a sensitivity of 390   μA   mM −1   cm −2 and a limit of detection (LOD) of 0.1   μM. The bienzyme CS/GO x −HRP−PTBA(H 2 O 2 )/Au plate /Au electrode responded linearly to glucose concentration from 5   μM to 0.83   mM with a sensitivity of 75.1   μA   mM −1   cm −2 and a LOD of 1   μM, and it is found here that the use of Fe(CN) 6 4− that can only efficiently mediate HRP favorably avoids the “unusual amperometric responses” observed when other mediators that can efficiently turn over both HRP and GO x are used.