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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Santa Cruz Electronic Theses and Dissertations (2024)

Recent outbreaks of infectious diseases underscore the urgent need for ultrasensitive, high-throughput, and versatile sensors to diagnose and contain them at their early stage, thereby mitigating their immense threat to public health and preventing disruption to social and economic growth worldwide. Optofluidic biosensors can offer highly sensitive single molecule detection and precise particle manipulation by combining optics with microfluidics in a single platform. A specially designed anti-resonant reflecting optical waveguide (ARROW) based optofluidic platform offers compatibility for enabling optical interaction with biomarkers and small molecules in their native low-refractive index fluidic environment. Further integration with highly selective bioassays, electronics, and signal processing techniques can improve the performance of these platforms. The goal of this thesis is to develop integrated optofluidic platforms for low complexity, remote-controllable, high-throughput, and ultrasensitive biosensing at clinically relevant biomarker concentrations. First, we explore the potential of integrating programmable fast electronics such as field programmable gate array (FPGA) for enhancing detection throughput of the MMI waveguide-based ARROW optofluidic platform. With this framework, salient experimental parameters such as target concentration and detection rate are extracted in real-time, demonstrating a highly accurate (99%) detection scheme with fluorescent nanobeads covering the entire clinically relevant range (femto to attomolar) of particle concentrations. Subsequent validation with real-time fluorescence detection of single bacterial plasmid DNA at attomolar concentrations indicates the platform's potential as a point-of-care diagnostic tool. Next, a cloud-based, Internet of Things (IoT)-enabled polydimethylsiloxane (PDMS) optofluidic platform is demonstrated for the remote operation of automated bio sample preparation and detection. This platform offers a user-friendly and intuitive workflow for on-chip liquid handling and serves as a valuable collaboration and training tool for remote access from anywhere across the globe. The rest of my thesis will discuss solid-state nanopores, essentially nanoscopic holes in thin insulated membranes, as label-free single-molecule analysis tools integrated with the optofluidic platform. When combined with a modified solid-phase extraction (SPE) bioassay, the platform offers specificity and amplification-free rapid biomarker quantification at ultra-low concentrations. The optofluidic platform allows optical trapping of target-enriched microbeads near the nanopore detector, followed by a thermal release assisting the electrophoretic target capture process. This optical trapping enhanced nanopore capture rate enhancement (TACRE) process demonstrates ~2000x enhancement factor compared to the diffusion-limited nanopore capture process. Also, this high-throughput method enabled the successful detection of SARS-CoV-2 RNAs from human nasopharyngeal swabs covering entire clinically relevant concentrations. Next, this method was modified to detect Zika and SARS-CoV-2 RNAs from non-human primate biofluids in a longitudinal infection study. This direct detection method demonstrates qRT-PCR-like performance without requiring any intermediate complex bioreactions. The versatility of the TACRE assay in analyzing six different types of biofluids and the practicality of this platform as a sensitive molecular diagnostic tool are also manifested. Finally, the integrated nanopore-optofluidic platform was utilized to characterize organoid-derived exosomes, an important biomarker for monitoring intercellular communication. This thesis concludes with a report on another application of the high-throughput and sensitive TACRE platform in monitoring the ENO-1 gene marker, a key regulatory enzyme in glycolysis, from organoid-derived exosome cargo, showing promise for further application in the clinical evaluation of cell growth and health in cell culture.

    Cover page: Integrated Optofluidic Platform for High Throughput Biomolecule Detection and Manipulation
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2021)
    The advent of single-molecule probing techniques has revolutionized the biomedical and life science fields and has spurred the development of a new class of labs-on-chip based on powerful biosensors. Nanopores represent one of the most recent and most promising single molecule sensing paradigms that is seeing increased chip-scale integration for improved convenience and performance. Due to their physical structure, nanopores are highly sensitive, require low sample volume, and offer label-free, amplification-free, high-throughput real-time detection and identification of biomolecules. Over the last 25 years, nanopores have been extensively employed to detect a variety of biomolecules with a growing range of applicatons ranging from nucleic acid sequencing to ultrasensitive diagnostics to single-molecule biophysics. Nanopores, in particular those in solid-state membranes, also have the potential for integration with other technologies such as optics, plasmonics, microfluidics, and optofluidics to perform more complex tasks for an ever-expanding demand. A number of breakthrough results using integrated nanopore platforms have already been reported, and more can be expected as nanopores remain the focus of innovative research and are finding their way into commercial instruments. This review provides an overview of different aspects and challenges of nanopore technology with a focus on chip-scale integration of solid-state nanopores for biosensing and bioanalytical applications.
      Cover page: Recent advances in integrated solid-state nanopore sensors.
      • Article
      • Peer Reviewed
      UC Santa Cruz Previously Published Works (2022)
      Integrated optofluidic biosensors can fill the need for sensitive, amplification-free, multiplex single molecule detection which is relevant for containing the spread of infectious diseases such as COVID-19. Here, we demonstrate a rapid sample-to-answer scheme that uses a field programmable gate array (FPGA) to enable live monitoring of single particle fluorescence analysis on an optofluidic chip. Fluorescent nanobeads flowing through a micro channel are detected with 99% accuracy and particle concentrations in clinically relevant ranges from 3.4×104 to 3.4 × 106/ml are determined within seconds to a few minutes without the need for post-experiment data extraction and analysis. In addition, other extract salient experimental parameters such as dynamic flow rate changes can be monitored in real time. The sensor is validated with real-time fluorescence detection of single bacterial plasmid DNA at attomolar concentrations, showing excellent promise for implementation as a point of care (POC) diagnostic tool.
        Cover page: FPGA Integrated Optofluidic Biosensor for Real-Time Single Biomarker Analysis.
        • Article
        • Peer Reviewed
        UC Santa Cruz Previously Published Works (2020)
        We present a method to create robust, nanoscale solid-state membranes using the natural shape of a liquid meniscus as a template. A narrow, open channel is etched into a silicon substrate and then a photoresist polymer is introduced into the channel through spontaneous capillary action. The natural concave meniscus formed by the polymer is then covered by a thin chemical vapor deposited membrane. The polymer is removed by sacrificial etching, leaving behind a suspended membrane. Membranes as large as 20 μm by 9 mm can be fabricated with a thickness as low as 50 nm.
          Cover page: Solid-state membranes formed on natural menisci.
          • Article
          • Peer Reviewed
          UC Santa Cruz Previously Published Works (2021)
          Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.
            Cover page: Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor.
            • Article
            • Peer Reviewed
            UC Santa Cruz Previously Published Works (2022)
            We demonstrate an optofluidic device which utilizes the optical scattering and gradient forces for particle trapping in microchannels featuring 300 nm thick membranes. On-chip waveguides are used to direct light into microfluidic trapping channels. Radiation pressure is used to push particles into a protrusion cavity, isolating the particles from liquid flow. Two different designs are presented: the first exclusively uses the optical scattering force for particle manipulation, and the second uses both scattering and gradient forces. Trapping performance is modeled for both cases. The first design, referred to as the orthogonal force design, is shown to have a 80% capture efficiency under typical operating conditions. The second design, referred to as the gradient force design, is shown to have 98% efficiency under the same conditions.
              Cover page: Optofluidic Particle Manipulation: Optical Trapping in a Thin-Membrane Microchannel.
              • Article
              • Peer Reviewed
              UC Santa Cruz Previously Published Works (2022)
              We demonstrate a method for fabricating and utilizing an optofluidic particle manipulator on a silicon chip that features a 300 nm thick silicon dioxide membrane as part of a microfluidic channel. The fabrication method is based on etching silicon channels and converting the walls to silicon dioxide through thermal oxidation. Channels are encapsulated by a sacrificial polymer which fills the length of the fluid channel by way of spontaneous capillary action. The sacrificial material is then used as a mold for the formation of a nanoscale, solid-state, silicon dioxide membrane. The hollow channel is primarily used for fluid and particle transport but is capable of transmitting light over short distances and utilizes radiation pressure for particle trapping applications. The optofluidic platform features solid-core ridge waveguides which can direct light on and off of the silicon chip and intersect liquid channels. Optical loss values are characterized for liquid and solid-core structures and at interfaces. Estimates are provided for the optical power needed to trap particles of various sizes.
                Cover page: Optofluidic Particle Manipulation Platform with Nanomembrane.
                • Article
                • Peer Reviewed
                UC Santa Cruz Previously Published Works (2024)
                Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.
                  Cover page: Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.
                  • Article
                  • Peer Reviewed
                  UC Santa Cruz Previously Published Works (2024)
                  Despite many interventions, science education remains highly inequitable throughout the world. Internet-enabled experimental learning has the potential to reach underserved communities and increase the diversity of the scientific workforce. Here, we demonstrate the use of lab-on-a-chip (LoC) technologies to expose Latinx life science undergraduate students to introductory concepts of computer programming by taking advantage of open-loop cloud-integrated LoCs. We developed a context-aware curriculum to train students at over 8000 km from the experimental site. Through this curriculum, the students completed an assignment testing bacteria contamination in water using LoCs. We showed that this approach was sufficient to reduce the students' fear of programming and increase their interest in continuing careers with a computer science component. Altogether, we conclude that LoC-based internet-enabled learning can become a powerful tool to train Latinx students and increase the diversity in STEM.
                    Cover page: Internet-enabled lab-on-a-chip technology for education
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