Glucose oxidase immobilization studies on boron doped CVD diamond coatings

INTRODUCTIONBoron-doped diamond (BDD) coatings obtained by chemical vapor deposition (CVD) are known to be intrinsically biocompatible [1], supporting cell adhesion and proliferation [2]. In this work we investigate the... [ view full abstract ]

INTRODUCTION

Boron-doped diamond (BDD) coatings obtained by chemical vapor deposition (CVD) are known to be intrinsically biocompatible [1], supporting cell adhesion and proliferation [2]. In this work we investigate the immobilization and activity of glucose oxidase (GOx) enzyme on different BDD coatings aiming their use as bio-platforms in devices for biossensing applications

METHODS

BDD coatings were grown on sintered Si₃N₄ disks, using an hot-filament chemical vapor deposition (HFCVD) equipment. Hydrogen and methane were used as the precursor gases for diamond growth and boron oxide diluted in ethanol and dragged by argon as the dopant. The coatings morphology (from nanocrystalline to microcrystalline) and p-type conductivity were varied by changing the CH₄/H₂ ratio and system pressure (e.g. sample 25/2 means grown at 25mbar for a Ch4 flow of 2sccm).

The covalent immobilization of glucose oxidase (GOx) to the diamond coatings was made through the carboxyl-to-amine crosslinking using carbodiimine. The enzymatic activity of the free and covalent bond GOx was performed by spectrophotometric evaluation the amount of H₂O₂ produced.

RESULTS

Data presented in Table 1 and surface morphologies depicted in Figure 1 show that the surface density of immobilized enzyme is not the same in all samples. Plots in Figure 2 illustrate the enzyme activity of GOx over the course of four weeks as a function of the main CVD growth parameters: total pressure and CH4 flow.

DISCUSSION

These preliminar results show that there is no clear correlation between surface morphology and GOx immobilization efficiency. However, the coatings with the longest GOx activity (30% still active after week four) were obtained at 50mbar with the highest methane flow (6sccm and 8sccm, respectively for samples 50/6 and 50/8). This indicates more favorable and stable chemical environment for the attachment of GOx on these coatings.

ACKNOWLEDGMENTS

This work was developed within the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID /CTM /50011/2013), financed through the FCT/MEC and co-financed by FEDER under the PT2020 Partnership Agreement.

REFERENCES

[1] A. Kromka, L. Grausova, L. Bacakova, et al (2010) Diam. Relat. Mater. 19:190-95. 

[2] M. A. Neto, C.M.F. Gomes, E.L. Silva, et al (2012) Procedia Chemistry 6:117-124.