A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

Okkyoung Choi, Byung Chun Kim, Ji Hye An, Kyoungseon Min, Yong Hwan Kim, Youngsoon Um, Min Kyu Oh, Byoung In Sang

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro [1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58μAcm -1 by adding 1mM glucose at an applied potential of -0.5V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67mM (S/N=3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.

Original languageEnglish
Pages (from-to)441-445
Number of pages5
JournalEnzyme and Microbial Technology
Volume49
Issue number5
DOIs
Publication statusPublished - 2011 Oct 10

Keywords

  • Biosensor
  • Biosilicification
  • Enzyme immobilization
  • Fused enzyme
  • Glucose oxidase
  • Silaffin

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

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