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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Santa Barbara Electronic Theses and Dissertations (2019)

Each moment of each day countless millions of proteins circulate through the body, performing their tasks, recognizing and binding to their targets, all with minimal crosstalk between the countless other proteins circulating alongside them. It is no wonder then, that, when faced with problems such as detecting rare molecular targets within complex backgrounds, technology developers often employ biological molecules as the foundation with which to build sensors (“biosensors”) and develop processes (“bioassays”). But that is not to say that sensors and assays based on biorecognition are perfect. Bioassays, for example, are generally complex, time-consuming processes and thus they provide actionable information only after a significant time lag. Biosensors (such as blood glucose meters), in contrast, can provide real-time information, but those few that work in realistically complex sample matrices invariably rely on the specific chemical reactivity of their targets, greatly limiting the range of molecules they can detect. Motivated by these concerns, I have been exploring biosensors and bioassays that, unlike existing approaches, are simultaneously general and capable of rapidly returning answers.

The first strategy I have explored utilizes microfluidics to adapt existing, multi-step bioassays into a rapid-and-convenient point-of-care device. Specifically, taking advantage of the inherent automation potential of microfluidics I have shown it possible to automate and speed up an established, bench-top bioassay. Once fully automated, the assay can even be used to perform continuous measurements, tracking changes in the concentration of a target protein in a clinical sample stream in real-time.

The second strategy that I explored was the development of a single-step, reagentless biosensor platform termed electrochemical DNA-based (E-DNA) sensors. This broad class of electrochemical sensors utilizes binding-induced changes in the structure or dynamics of an electrode-bound biomolecule to reagenltessly and continuously report on the concentration of its target. Specifically, this class of sensors uses DNA either as a scaffold upon which to display a recognition element (scaffold-sensors), such as an antigen, or as the recognition element itself (aptamer-based sensors). Using Monte-Carlo simulations I have examined how changing the molecular weight of a recognition element affects the performance of scaffold sensors and used this information to develop a point-of-care serological assay to help rapidly diagnose syphilis. Using a similar Monte-Carlo model I likewise optimized the performance of aptamer-based sensors by examining how modifications around the aptamer’s binding pocket affect sensor performance. Together these studies are helping to bridge the gap between complex (but generalizable) bioassays and simple to use biosensors (such as blood glucose meters) which cannot readily be adapted to other targets.

    Cover page: Miniaturizing Medicine – Strategies for Developing and Improving Point-Of-Care Biosensors
    • Article
    • Peer Reviewed
    UC Santa Barbara Previously Published Works (2019)
    The faster a disease can be diagnosed, the sooner effective treatment can be initiated, motivating a drive to replace standard laboratory techniques with point-of-care technologies that return answers in minutes rather than hours. Thus motivated, we describe the development of an E-DNA scaffold sensor for the rapid and convenient measurement of antibodies diagnostic of syphilis. To achieve this (and in contrast to previous sensors of this class, which relied on single, linear epitopes for detection), we utilized a near full-length antigen as the sensor's recognition element, allowing us to simultaneously display multiple epitopes. The resultant sensor is able to detect antibodies against Treponema pallidum pallidum, the causative agent of syphilis, at clinically relevant concentrations in samples in less than 10 min. Preliminary results obtained using sero-positive and sero-negative human samples suggest the clinical sensitivity and specificity of the approach compare well to current gold-standard tests, while being simple and rapid enough to deploy at the point of care.
      Cover page: An electrochemical scaffold sensor for rapid syphilis diagnosis
      • Article
      • Peer Reviewed
      UC Santa Barbara Previously Published Works (2018)
      The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element". The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low molecular weight haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform's signaling mechanism to aid its design and optimization; and (3) demonstrate the analytical performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of molecular targets amenable to this rapid and convenient sensing platform.
        Cover page: Expanding the Scope of Protein-Detecting Electrochemical DNA “Scaffold” Sensors
        • Article
        • Peer Reviewed
        UC Santa Barbara Previously Published Works (2020)
        The multiplexed, point-of-care measurement of specific antibodies could improve the speed with which diseases are diagnosed and their treatment initiated. To this end, we are developing E-DNA scaffold sensors, which consist of a rigid, nucleic acid "scaffold" attached on one end to an electrode and presenting both a redox reporter and an epitope on the other. In the absence of antibody, the reporter efficiently transfers electrons when interrogated electrochemically. Binding-induced steric hindrance limits movement, reducing electron transfer in a manner that is both easily measured and quantitatively related to target concentration. Previously we have used monoclonal antibodies to explore the analytical performance of E-DNA sensors, showing that they support the rapid, single-step, quantitative detection of multiple antibodies in small volume samples. Here, in contrast, we employ authentic human samples to better explore the platform's clinical potential. Specifically, we developed E-DNA sensors targeting three HIV-specific antibodies and then compared the analytical and clinical performance of these against those of gold standard serological techniques. Doing so we find that, although the multistep amplification of an ELISA leads to a lower detection limits, the clinical sensitivity of ELISAs, E-DNA sensors and lateral-flow dipsticks are indistinguishable across our test set. It thus appears that, by merging the quantitation and multiplexing of ELISAs with the convenience and speed of dipsticks, E-DNA scaffold sensors could significantly improve on current serological practice.
          Cover page: E-DNA scaffold sensors and the reagentless, single-step, measurement of HIV-diagnostic antibodies in human serum
          • Article
          • Peer Reviewed
          UC Merced Previously Published Works (2020)
          The specific detection in clinical samples of analytes with dimensions in the tens to hundreds of nanometers, such as viruses and large proteins, would improve disease diagnosis. Detection of these "mesoscale" analytes (as opposed to their nanoscale components), however, is challenging as it requires the simultaneous binding of multiple recognition sites often spaced over tens of nanometers. In response, we have adapted DNA origami, with its unparalleled customizability to precisely display multiple target-binding sites over the relevant length scale, to an electrochemical biosensor platform. Our proof-of-concept employs triangular origami covalently attached to a gold electrode and functionalized with redox reporters. Electrochemical interrogation of this platform successfully monitors mesoscale, target-binding-induced changes in electron transfer in a manner consistent with coarse-grained molecular dynamics simulations. Our approach enables the specific detection of analytes displaying recognition sites that are separated by ∼40 nm, a spacing significantly greater than that achieved in similar sensor architectures employing either antibodies or aptamers.
            Cover page: An electrochemical biosensor exploiting binding-induced changes in electron transfer of electrode-attached DNA origami to detect hundred nanometer-scale targets.
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