Tunneling of redox enzymes to design nano-probes for monitoring NAD+ dependent bio-catalytic activity

Abstract

Monitoring of bio-catalytic events by using nano-probes is of immense interest due to unique optical properties of metal nanoparticles. In the present study, tunneling of enzyme activity was achieved using redox cofactors namely oxidized cytochrome-c (Cyt-c) and Co-enzyme-Q (Co-Q) immobilized on Quantum dots (QDs) which acted as a bio-probe for NAD+ dependent dehydrogenase catalyzed reaction. We studied how electron transfer from substrate to non-native electron acceptors can differentially modify photoluminescence properties of CdTe QDs. Two probes were designed, QD-Ox-Cyt-c and QD-Ox-Co-Q, which were found to quench the fluorescence of QDs. However, formaldehyde dehydrogenase (FDH) catalyzed reduction of Cyt-c and Co-Q on the surface of QDs lead to fluorescence turn-on of CdTe QDs. This phenomenon was successfully used for the detection of HCHO in the range of 0.01–100,000ng/mL (LOD of 0.01ng/mL) using both QD-Ox-Cyt-c (R2=0.93) and QD-Ox-Co-Q (R2=0.96). Further probe performance and stability in samples like milk, wine and fruit juice matrix were studied and we could detect HCHO in range of 0.001−100,000ng/mL (LOD of 0.001ng/mL) with good stability and sensitivity of probe in real samples (R2=0.97). Appreciable recovery and detection sensitivity in the presence of metal ions suggests that the developed nano-probes can be used successfully for monitoring dehydrogenase based bio-catalytic events even in the absence of NAD+. Proposed method is advantageous over classical methods as clean up/ derivatization of samples is not required for formaldehyde detection.