(184a) Design of FRET-Based Reversibly Photoswitchable Quantum Dot-DNA Conjugates
AIChE Annual Meeting
Monday, November 11, 2019 - 3:30pm to 5:00pm
Super-resolution fluorescence microscopy is an important tool for biological imaging applications, as it enables live-cell imaging with high labeling specificity and resolution below the diffraction limit of light. In stochastic optical reconstruction microscopy (STORM), single molecule localization is achieved through temporal resolution of spatially overlapping images of fluorescent probes. Currently used fluorescent dyes limit the achievable resolution of STORM because of low photon emission rates and susceptibility to photobleaching. Compared to such dyes, quantum dots (QDs) possess higher photon emission rates, higher absorption cross-sections, and greater stability against photobleaching. Their broad absorption spectra and narrow, tunable emission spectra also enable color multiplexing. However, they are not inherently photoswitchable. In a previous design, our lab engineered QD-gold nanoparticle (AuNP) probes linked by azobenzene modified ssDNA to achieve stochastic photoswitching through Förster resonance energy transfer (FRET). Complementary ssDNA is conjugated to both nanoparticles, and modulation of the excitation source induces cis-trans conformational changes of the azobenzene groups to regulate DNA hybridization. Regulation of DNA hybridization alters the interparticle distance between QDs and AuNPs, which affects FRET efficiency. Although some degree of photoswitching was observed in this model, it was inconsistent and did not provide sufficient contrast between on/off states because of insufficient DNA conjugation and interparticle distance. Ideally, >95% quenching should be achieved for STORM probes. In this work, we employ the âloops-trains-tailsâ polymer adsorption model to achieve a compact QD surface coating to help minimize interparticle distance. We then conjugate ssDNA to QDs using EDC/Sulfo-NHS crosslinker chemistry and copper-free click chemistry. As an alternative, we also embed DNA in the shell of the QD core/shell structure, circumventing problems associated with QD bioconjugation. Here, we report our nanoparticle synthesis techniques and results, as well as the photoswitching kinetics and FRET efficiencies of our systems.