Supramolecular glucose oxidase-SWNT conjugates formed by ultrasonication: effect of tube length, functionalization and processing time

BACKGROUND: Generation-3 (Gen-3) biosensors and advanced enzyme biofuel cells will benefit from direct electron transfer to oxidoreductases facilitated by single-walled carbon nanotubes (SWNTs). METHODS: Supramolecular conjugates of SWNT-glucose oxidase (GOx-SWNT) were produced via ultrasonic processing. Using a Plackett-Burman experimental design to investigate the process of tip ultrasonication (23 kHz), conjugate formation was investigated as a function of ultrasonication times (0, 5, 60 min) and functionalized SWNTs of various tube lengths (SWNT-X-L), (X = −OH or -COOH and L = 3.0 μm, 7.5 μm). RESULTS: Enzyme activity (K(M), k(cat), k(cat)/K(M), v(max) and n (the Hill parameter)) of pGOx (pristine), sGOx (sonicated) and GOx-SWNT-X-L revealed that sonication of any duration increased both K(M) and k(cat) of GOx but did not change k(cat)/K(M). Functionalized tubes had the most dramatic effect, reducing both K(M) and k(cat) and reducing k(cat)/K(M). UV–vis spectra over the range of 300 to 550 nm of native enzyme-bound FAD (λ(max) at 381 and 452 nm) or the blue-shifted solvated FAD of the denatured enzyme (λ(max) at 377 and 448 nm) revealed that ultrasonication up to 60 minutes had no influence on spectral characteristics of FAD but that the longer SWNTs caused some partial denaturation leading to egress of FAD. Circular dichroism spectral analysis of the 2° structure showed that sonication of any duration caused enrichment in the α-helical content at the sacrifice of the unordered sequences in GOx while the presence of SWNTs, regardless of length and/or functionality, reduced the β-sheet content of pristine GOx. Surface profiling by white light interferometry revealed that ultrasonication produced some aggregation of GOx and that GOx effectively debundled the SWNT. CONCLUSIONS: Supramolecular conjugates formed from shorter, -OH functionalized SWNTs using longer sonication times (60 min) gave the most favored combination for forming bioactive conjugates.