Development Of DNA nanosensors as aptasensor for lysozyme and DNAzyme sensor for zinc ions

DNA nanotechnology based on DNA aptamers and DNAzymes is highly beneficial because they provide more stable, programmable and manageable alternatives to their native counterparts (i.e. antibodies and enzymes) on top of being cost-effective. One of the widely reported features of DNA-aptamer based biosensors is by coupling it with gold nanoparticles (AuNPs), due to the unique opto-electronic characteristics of the latter. While the positively charged cysteamine-coated gold nanoparticles (cysAuNPs) present an ideal candidate to directly interact with negatively charged DNA, the fundamental understanding and precise modulation on their interaction for biosensing purposes are still largely unexplored. By carefully studying the interaction between cysAuNPs and lysozyme-binding aptamer (LBA), a colorimetric aptasensor has been developed for lysozyme detection. Despite inducing aggregation initially, further increasing the amount of LBA above critical redispersion concentration (CRC) was found to redisperse cysAuNPs, as proven by UV-visible spectroscopy and zeta-sizer study. The changing dispersion states of cysAuNPs was further exploited to construct a bimodal colorimetric aptasensor, with modes A (aggregation) and R (redispersion) utilising LBA at concentrations below and above CRC, respectively. Both modes were found to be complementarily: mode A having a linear range of 37.5 - 180 nM of lysozyme with a limit of detection (LOD) of 2.29 nM; whereas mode R can detect lysozyme linearly in the range of 500 - 4000 nM with a LOD of 375 nM. Synergistic screening via both modes also display a remarkable selectivity towards lysozyme against other control proteins. Besides, detection of lysozyme via mode R also demonstrated a novel analytical concept of inverse sensitivity, with exceptional signal-to-noise ratio (SNR) of 72.56. From another perspective, DNAzymes are frequently coupled with fluorescence resonance energy transfer (FRET) in the development of metal ion sensors. Despite allowing analysis being performed ratiometrically, FRET is hindered by several pivotal limitations, preventing such DNAzymes-driven metal ion sensors to be translated into practical scenarios. Bioluminescence resonance energy transfer (BRET) provides a good solution in line with that premise as it does not require an excitation stimulus. An 8-17 DNAzyme and its cofactor, Zn2+ ions, have been chosen for proof-of-concept study, with the BRET pair consists of Lucia luciferase and Cy3 fluorophore. The implementation of BRET was verified by observing a second luminescence peak from co-excited Cy3. Further structural and experimental optimisations have resulted in an improved BRET ratio at 0.43. The DNAzyme-driven BRET-based biosensor also showed great selectivity towards Zn2+ ions by inducing a noticeable BRET percentage changes and was able to quantify Zn2+ from 100 to 3200 nM in buffered solution. Compared to other reported Zn2+ sensors, the DNAzyme biosensor described here was able to quantify > 300 equivalent concentration of Zn2+ ions, yet still having a low LOD estimated at 62.58 nM. The study here represents a milestone that for the first time, a previously unexplored technique of BRET has been successfully implemented in DNAzyme system for metal ion sensing. In summary, the works detailed here have provided an insight on the further development of DNA nanotechnology with regards to biosensing purposes. These successful applications of both DNA aptamers and DNAzymes, when coupled with cysAuNPs and BRET, respectively, have not only addressed their currently associated limitations, but also opens up more possibility of utilising them as biosensing platforms to reliably detect biomarkers and metal ions in the near future