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Open Access Publications from the University of California

Scholarly Works (12 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2017)

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.

    Cover page: Aptamer-Functionalized Field-Effect Transistors For Serotonin and Dopamine Sensing
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2021)
    Oligonucleotide receptors (aptamers), which change conformation upon target recognition, enable electronic biosensing under high ionic-strength conditions when coupled to field-effect transistors (FETs). Because highly negatively charged aptamer backbones are influenced by ion content and concentration, biosensor performance and target sensitivities were evaluated under application conditions. For a recently identified dopamine aptamer, physiological concentrations of Mg2+ and Ca2+ in artificial cerebrospinal fluid produced marked potentiation of dopamine FET-sensor responses. By comparison, divalent cation-associated signal amplification was not observed for FET sensors functionalized with a recently identified serotonin aptamer or a previously reported dopamine aptamer. Circular dichroism spectroscopy revealed Mg2+- and Ca2+-induced changes in target-associated secondary structure for the new dopamine aptamer, but not the serotonin aptamer nor the old dopamine aptamer. Thioflavin T displacement corroborated the Mg2+ dependence of the new dopamine aptamer for target detection. These findings imply allosteric binding interactions between divalent cations and dopamine for the new dopamine aptamer. Developing and testing sensors in ionic environments that reflect intended applications are best practices for identifying aptamer candidates with favorable attributes and elucidating sensing mechanisms.
      Cover page: Divalent Cation Dependence Enhances Dopamine Aptamer Biosensing.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2020)
      We detect short oligonucleotides and distinguish between sequences that differ by a single base, using label-free, electronic field-effect transistors (FETs). Our sensing platform utilizes ultrathin-film indium oxide FETs chemically functionalized with single-stranded DNA (ssDNA). The ssDNA-functionalized semiconducting channels in FETs detect fully complementary DNA sequences and differentiate these sequences from those having different types and locations of single base-pair mismatches. Changes in charge associated with surface-bound ssDNA vs double-stranded DNA (dsDNA) alter FET channel conductance to enable detection due to differences in DNA duplex stability. We illustrate the capability of ssDNA-FETs to detect complementary RNA sequences and to distinguish from RNA sequences with single nucleotide variations. The development and implementation of electronic biosensors that rapidly and sensitively detect and differentiate oligonucleotides present new opportunities in the fields of disease diagnostics and precision medicine.
        Cover page: Detecting DNA and RNA and Differentiating Single-Nucleotide Variations via Field-Effect Transistors
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2017)
        Artificial receptors for hydrophobic molecules usually have moderate affinities and limited selectivities. We describe three new classes of high affinity hydrophobic receptors for nonaromatic steroids based on deoxyribonucleotides, obtained through five high stringency selections coupled with tailored counter-selections. The isolation of multiple classes of high affinity steroid receptors demonstrates the surprising breadth of moderately sized hydrophobic binding motifs (<40 nucleotides) available to natural nucleic acids. Studies of interactions with analogs indicate that two classes, four-way junctions and 4XGN motifs, comprise receptors with shapes that prevent binding of specific steroid conjugates used in counter-selections. Furthermore, they strongly prefer nonhydroxylated steroid cores, which is typical for hydrophobic receptors. The third new class accommodates hydroxyl groups in high-affinity, high-selectivity binding pockets, thus reversing the preferences of the first two classes. The high-affinity binding of aptamers to targets efficiently inhibits double-helix formation in the presence of the complementary oligonucleotides. The high affinity of some of these receptors and tailored elimination of binding through counter-selections ensures that these new aptamers will enable clinical chemistry applications.
          Cover page: High-Affinity Nucleic-Acid-Based Receptors for Steroids.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2018)
          Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or   l-tryptophan were selectively recognized by previously identified dopamine or l-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though, slightly greater than the previously determined solution dissociation constant. Using prefunctionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with l-3,4-dihydroxyphenylalanine, l- threo-dihydroxyphenylserine, and l-5-hydroxytryptophan enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons and future identification and characterization of novel aptamers targeting neurotransmitters or other important small molecules.
            Cover page: Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2015)
            We demonstrate straightforward fabrication of highly sensitive biosensor arrays based on field-effect transistors, using an efficient high-throughput, large-area patterning process. Chemical lift-off lithography is used to construct field-effect transistor arrays with high spatial precision suitable for the fabrication of both micrometer- and nanometer-scale devices. Sol-gel processing is used to deposit ultrathin (∼4 nm) In2O3 films as semiconducting channel layers. The aqueous sol-gel process produces uniform In2O3 coatings with thicknesses of a few nanometers over large areas through simple spin-coating, and only low-temperature thermal annealing of the coatings is required. The ultrathin In2O3 enables construction of highly sensitive and selective biosensors through immobilization of specific aptamers to the channel surface; the ability to detect subnanomolar concentrations of dopamine is demonstrated.
              Cover page: Fabrication of High-Performance Ultrathin In2O3 Film Field-Effect Transistors and Biosensors Using Chemical Lift-Off Lithography
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2019)
              Determination of the amino acid phenylalanine is important for lifelong disease management in patients with phenylketonuria, a genetic disorder in which phenylalanine accumulates and persists at levels that alter brain development and cause permanent neurological damage and cognitive dysfunction. Recent approaches for treating phenylketonuria focus on injectable medications that efficiently break down phenylalanine but sometimes result in detrimentally low phenylalanine levels. We have identified new DNA aptamers for phenylalanine in two formats, initially as fluorescent sensors and then, incorporated with field-effect transistors (FETs). Aptamer-FET sensors detected phenylalanine over a wide range of concentrations (fM to mM). para-Chlorophenylalanine, which inhibits the enzyme that converts phenylalanine to tyrosine, was used to induce hyperphenylalaninemia during brain development in mice. Aptamer-FET sensors were specific for phenylalanine versus para-chlorophenylalanine and differentiated changes in mouse serum phenylalanine at levels expected in patients. Aptamer-FETs can be used to investigate models of hyperphenylalanemia in the presence of structurally related enzyme inhibitors, as well as naturally occurring amino acids. Nucleic acid-based receptors that discriminate phenylalanine analogs, some that differ by a single substituent, indicate a refined ability to identify aptamers with binding pockets tailored for high affinity and specificity. Aptamers of this type integrated into FETs enable rapid, electronic, label-free phenylalanine sensing.
                Cover page: Phenylalanine Monitoring via Aptamer-Field-Effect Transistor Sensors
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)
                Serotonin-based nanoparticles represent a class of previously unexplored multifunctional nanoplatforms with potential biomedical applications. Serotonin, under basic conditions, self-assembles into monodisperse nanoparticles via autoxidation of serotonin monomers. To demonstrate potential applications of polyserotonin nanoparticles for cancer therapeutics, we show that these particles are biocompatible, exhibit photothermal effects when exposed to near-infrared radiation, and load the chemotherapeutic drug doxorubicin, releasing it contextually and responsively in specific microenvironments. Quantum mechanical and molecular dynamics simulations were performed to interrogate the interactions between surface-adsorbed drug molecules and polyserotonin nanoparticles. To investigate the potential of polyserotonin nanoparticles for in vivo targeting, we explored their nano-bio interfaces by conducting protein corona experiments. Polyserotonin nanoparticles had reduced surface-protein interactions under biological conditions compared to polydopamine nanoparticles, a similar polymer material widely investigated for related applications. These findings suggest that serotonin-based nanoparticles have advantages as drug-delivery platforms for synergistic chemo- and photothermal therapy associated with limited nonspecific interactions.
                  Cover page: Polyserotonin Nanoparticles as Multifunctional Materials for Biomedical Applications
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2021)
                  While tools for monitoring in vivo electrophysiology have been extensively developed, neurochemical recording technologies remain limited. Nevertheless, chemical communication via neurotransmitters plays central roles in brain information processing. We developed implantable aptamer–field-effect transistor (FET) neuroprobes for monitoring neurotransmitters. Neuroprobes were fabricated using high-throughput microelectromechanical system (MEMS) technologies, where 150 probes with shanks of either 150- or 50-μm widths and thicknesses were fabricated on 4-inch Si wafers. Nanoscale FETs with ultrathin (~3 to 4 nm) In2O3 semiconductor films were prepared using sol-gel processing. The In2O3 surfaces were coupled with synthetic oligonucleotide receptors (aptamers) to recognize and to detect the neurotransmitter serotonin. Aptamer-FET neuroprobes enabled femtomolar serotonin detection limits in brain tissue with minimal biofouling. Stimulated serotonin release was detected in vivo. This study opens opportunities for integrated neural activity recordings at high spatiotemporal resolution by combining these aptamer-FET sensors with other types of Si-based implantable probes to advance our understanding of brain function.
                    Cover page: Implantable aptamer–field-effect transistor neuroprobes for in vivo neurotransmitter monitoring
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2018)
                    Interactions between small molecules and biomolecules are important physiologically and for biosensing, diagnostic, and therapeutic applications. To investigate these interactions, small molecules can be tethered to substrates through standard coupling chemistries. While convenient, these approaches co-opt one or more of the few small-molecule functional groups needed for biorecognition. Moreover, for multiplexing, individual probes require different surface functionalization chemistries, conditions, and/or protection/deprotection strategies. Thus, when placing multiple small-molecules on surfaces, orthogonal chemistries are needed that preserve all functional groups and are sequentially compatible. Here, we approach high-fidelity small-molecule patterning by coupling small-molecule neurotransmitter precursors, as examples, to monodisperse asymmetric oligo(ethylene glycol)alkanethiols during synthesis and prior to self-assembly on Au substrates. We use chemical lift-off lithography to singly and doubly pattern substrates. Selective antibody recognition of pre-functionalized thiols was comparable to or better than recognition of small molecules functionalized to alkanethiols after surface assembly. These findings demonstrate that synthesis and patterning approaches that circumvent sequential surface conjugation chemistries enable biomolecule recognition and afford gateways to multiplexed small-molecule functionalized substrates.
                      Cover page: Small-Molecule Patterning via Prefunctionalized Alkanethiols
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