Engineering mediatorless glucose oxidase microbioreactor

Abstract

Event Abstract Back to Event Engineering mediatorless glucose oxidase microbioreactor Geraldine Merle1*, Joao H. Lopes1*, Francois-Xavier Colson1* and Jake E. Barralet1* 1 McGill University, Dentistry, Canada Introduction: Accomplishing a transfer direct of electrons (DET) between the enzyme and the electrodes has become a great challenge in the development of biosensor and biopile[7]. The electron-tunneling distance can be diminished with structured conducting nanomaterials to enable DET. Glucose monitoring has an important significance in medicine with the routine analysis of glucose in blood or in environment and food industry. Glucose oxidase (GOx) technology has been already commercialised through the disposable glucose sensor strips however, the main hurdle of this system is the limited detection (10-3-10-6 M). Here, we successfully enhanced the conductivity while immobilizing GOx by self-assemblying GOx with TiO2 and carbon nanotubes. This has led to the development of an ultrasensitive, selective and inexpensive biosensor with a detection limit in the micro-molar range, which can be further miniaturised. Experimental: Briefly, GOx, TiO2, Na3PO4 and CNT solution were mixed together with a final weight ratio of TiO2/CNT of 0.6 wt% and dried. The mixture was self-assembled into nanoporous microparticles following Bassett et al. [1] Finally, the suspension was filtered through a 75 µm sieve and the particles ≥75 μm diameter were collected and dried. Beside the determination of the physico-chemical properties of the microparticle such as specific surface area and morphology, enzymatic and electrochemical activity measurements were performed. Results and Discussion: The morphology of microparticles was investigated with SEM. It can be clearly seen at higher magnification the carbon nanotubes entangled with the TiO2 nanoparticles compared to the pristine TiO2. The biocatalytic activities of free GOx and immobilized GOx were investigated by monitoring the oxidation of glucose in the presence of horseradish peroxidase. Despite a lower Vmax for the immobilised GOx (0.063 µM/min) compared to the free enzyme (0.65 µM/min), a KM value of approximately 17.5 mM and 23.5mM was found for free and immobilised GOx respectively, confirming that the self-assembly process and the nano-environment do not alter the affinity of the enzyme for the substrate[2][3]. The residual activity for the microbioparticle remained high with a gradual decrease up to 40% of its initial activity after 30 days storage at 4°C, probably as a consequence of the desorption and leaching of enzyme. Cyclic voltammetry experiments show in the absence of GOx, no significant redox peaks whereas the microbioparticle exhibits a of well-defined peaks at -0.2 V vs SCE reference electrode, ascribed only to GOx and more specifically to the redox active center (FAD/FADH2)[4]. This confirms the key role of the CNTs assembled with TiO2 in promoting successfully the direct electron transfer between GOD and the electrode surface. The detection of glucose by chronoamperometric method shows a fast (less than 3s) response time of the biosensor to the added glucose and a current response stable in 2min. The glucose biosensor exhibits a sensitivity of 17.16 µA.mM-1cm-2 and a high selectivity for glucose.[5][6] Conclusion: Mediating DET by engineering the structures at the nanometer scale has been performed by assembling GOx with CNT and TiO2 in one step synthesis. While greatly stabilizing the GOX, this nanostructures allow the specific detection of glucose by wiring the electrons from the active site of the GOX to the electrode and opens new avenues for others biosensors. This work was carried out with the support of the São Paulo Research Foundation – FAPESP (Grant 2013/12376-5). Natural Sciences and Engineering Research Council of Canada (NSERC)