UCLA

My thesis work built on over a decade’s worth of research in the Andrews and Weiss groups aimed at discovering high-affinity oligonucleotide-based recognition elements called aptamers. Previous work focused on designing and developing solid-phase substrates with surface-tethered small-molecule targets that retained their biological functionality to enable recognition by receptors. I mastered techniques such as chemical lift-off lithography and microfluidics to pattern small-molecules in specific locations to facilitate quantification of specific binding relative to background molecules. I demonstrated recognition of surface-tethered dopamine by a previously isolated dopamine aptamer. Specific binding was validated using competitive displacement experiments, which verified that surface-tethered dopamine, despite its reduced degree of freedom, could compete with free dopamine in solution. I addressed one of the shortcomings of conventional in vitro aptamer selection by enabling on-chip determination of equilibrium dissociation constants (Kd). Using a novel patterning method to create aptamer concentration gradients on multiplexed substrates, I resolved multiple Kd values simultaneously. I demonstrated that optimized small-molecule-functionalized substrates were ready to screen for novel neurotransmitter-specific aptamers.

In parallel, however, our collaborators at Columbia University isolated high-affinity (nanomolar Kd) aptamers targeting serotonin and dopamine through the use of a solution-phase method. Thus, I advanced our research to the next step by integrating these aptamers onto the semiconducting channels of thin-film field-effect transistors (FETs). Serotonin- and dopamine-functionalized FETs were able to sense target molecules in high ionic-strength, undiluted physiological buffers, as well as in complex environments such as brain tissue. Traditionally, biological FETs have suffered from Debye length limitations under physiological conditions where the effective sensing distance is <1 nm from the surface of the semiconducting channels. We hypothesized that the mechanism that enabled sensitive detection of neurotransmitter targets even in complex fluids was driven by aptamer conformational changes. I read and synthesized every aptamer-FET paper I could find in the literature to understand the current status of the field. I found that mechanistically, there were two emerging lines of thought. The first asserts that electronic signals arise mainly from target-associated charge being brought into close proximity of FETS upon aptamer binding. The second postulates that rearrangement of charged aptamer backbones contributes to aptamer-FET target detection.

I investigated the mechanism of the serotonin and dopamine aptamer-FETs by exploring the influence of divalent cations on aptamer binding. I showed that serotonin and dopamine signal responses behaved differently based on the presence/absence of divalent cations. This meant there were aptamer-specific differences in secondary structure rearrangements upon target capture. I conducted circular dichroism and surface-enhanced Raman spectroscopy to compare alterations in aptamer secondary structures upon target capture, empirically. I demonstrated that these two techniques can be used to track aptamer conformational changes and together, they enabled prediction of sensing capabilities prior to FET incorporation of aptamers. Sensing of a neutral target (glucose) and a zwitterionic species (sphingosine-1-phosphate) further implicated target-induced rearrangement of aptamer charge at the surface of FETs as a key mechanism for small molecule sensing. This mechanism is advantageous as it is generalizable for any target of interest regardless of size or charge.

Finally, inspired by recent literature on polydopamine nanoparticles, I fabricated and characterized analogous serotonin-based nanomaterials. I demonstrated that polyserotonin nanoparticles had comparable therapeutic properties to polydopamine nanoparticles such as drug loading efficiency and photothermal capabilities. However, compared to polydopamine, polyserotonin nanoparticles showed reduced protein corona formation on the surface and improved biocompatibilities with three stem cell lines, suggesting their potential for future clinical applications.