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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2020)

Circulating non-coding RNAs (ncRNAs) regulate gene expression and thereby have the ability to initiate many pathological developments, including cancer. They are also stably present in diverse body fluids of the circulatory system and, as a result, ncRNAs have great potential to serve as biomarkers for cancers and other diseases. Although ncRNAs are easily sampled non-invasively through liquid biopsies, there is still a long way to go before they can be applied reproducibly and reliably in clinical diagnosis.Effective analytical techniques have been developed in order to detect microRNAs (miRNAs), a class of ncRNAs, in biospecimens and broaden their clinical utility. Isothermal amplification of miRNAs is a more economical alternative to RT-qPCR that can be more rapid, sensitive, specific, and simple. However, it is challenging to control the former’s background signal produced in the absence of target, severely hampering detection of low-abundance targets in complex biological samples. In the present work, exponential amplification reaction (EXPAR) was improved by chemically blocking the template with hexanediol and physically as well with cobalt oxyhydroxide (CoOOH) nanoflakes and T4 gene 32 (gp32) protein to suppress nonspecific template elongation and hence background signal. Synergism among the blocking agents greatly refined the EXPAR performance, which exhibited little to no cross-reactivity with non-targets and can detect as low as 10 aM of several miRNA targets, including miR-16, miR-21, and miR-122, using SYBR™ Gold. The CoOOH nanoflakes can also selectively adsorb single-stranded nucleic acids in complex media, such as serum, and function as a vehicle to transport them into a clean detection matrix for CoOOH-assisted EXPAR. Exosomes are circulating nanovesicles that stabilize and relay miRNA during cell-cell communication. Although exosomal miRNAs are more reliable and sensitive biomarkers than circulating bare miRNAs, they are more challenging to detect due to their lower abundance and enclosure within extracellular vesicles. Herein, immunoprecipitation of exosomes and CoOOH-assisted EXPAR were utilized to fractionate raw healthy serum and screen the resulting exosome and exosome-free fractions for breast cancer miRNAs. It was found that miR-155 and miR-375 were detectable in the exosome-free fraction at aM and pM concentrations, respectively. For the purposes of this study, CoOOH-assisted EXPAR was further optimized to reach zM sensitivity, which underscores the power of CoOOH-assisted EXPAR in miRNA quantitation and its application in serum profiling of exosomal and non-exosomal miRNAs. Elucidating their relationship and diseased signatures will hone the use of two biomarker sources in diagnostic testing.

    Cover page: Development of Cobalt Oxyhydroxide Nanoflake-Assisted Exponential Amplification Reaction for the Detection of Circulating MicroRNACreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2019)
    Non-coding RNAs (ncRNAs) participate in regulation of gene expression, and are highly relevant to pathological development. They are found to be stably present in diverse body fluids, including those in the circulatory system, which can be sampled non-invasively for clinical tests. Thus, circulating ncRNAs have great potential to be disease biomarkers. However, tremendous efforts are desired to discover and utilize ncRNAs as biomarkers in clinical diagnosis, calling for technological advancement in analysis of circulating ncRNAs in biospecimens. Hence, this review summarizes the recent developments in this area, highlighting the works devoted to cancer diagnosis and prognosis. Three main directions are focused: 1) Extraction and purification of ncRNAs from body fluids; 2) Quantification of the purified circulating ncRNAs; and 3) Microfluidic platforms for integration of both steps to enable point-of-care diagnostics. These technologies have laid a solid foundation to move forward the applications of circulating ncRNAs in disease diagnosis and cure.
      Cover page: Analysis of circulating non-coding RNAs in a non-invasive and cost-effective manner
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2020)
      The detection of circulating miRNA through isothermal amplification wields many attractive advantages over traditional methods, such as reverse transcription RT-qPCR. However, it is challenging to control the background signal produced in the absence of target, which severely hampers applications of such methods for detecting low abundance targets in complex biological samples. In the present work, we employed both the cobalt oxyhydroxide (CoOOH) nanoflakes and the chemical modification of hexanediol to block non-specific template elongation in exponential amplification reaction (EXPAR). Adsorption by the CoOOH nanoflakes and the hexanediol modification at the 3' end effectively prevented no-target polymerization on the template itself and thus greatly improved the performance of EXPAR, detecting as low as 10 aM of several miRNA targets, including miR-16, miR-21, and miR-122, with the fluorescent DNA staining dye of SYBR Gold™. Little to no cross-reactivity was observed from the interfering strands present in 10-fold excess. Besides contributing to background reduction, the CoOOH nanoflakes strongly adsorbed nucleic acids and isolated them from a complex sample matrix, thus permitting successful detection of the target miRNA in the serum. We expect that simple but sensitive template-blocking EXPAR could be a valuable tool to help with the discovery and validation of miRNA markers in biospecimens. Graphical abstract.
        Cover page: Physical and chemical template-blocking strategies in the exponential amplification reaction of circulating microRNAs
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2014)

        Context

        Exposure to secondhand smoke (SHS) has been reduced in the USA by banning smoking in public places. These restrictions have not had the same effect on children's exposure to SHS as adults suggesting that children are exposed to SHS in locations not covered by bans, such as private homes and cars.

        Objectives

        Assess exposure to SHS in the backseat of a stationary vehicle where a child would sit, quantify exposures to fine particulates (PM2.5), polycyclic aromatic hydrocarbons (PAH), carbon monoxide (CO) and nicotine. Estimate the impact on a child's mean daily exposure to PM2.5.

        Methods

        SHS exposures in stationary vehicles with two different window configurations were monitored. A volunteer smoked three cigarettes in a one-hour period for twenty-two experiments. PM2.5, CO, nicotine and PAH where measured in the backseat of the vehicle. 16 PAH compounds were measured for in gas and particle phases as well as real-time particle phase concentrations.

        Results

        The mean PAH concentration, 1325.1 ng/m(3), was larger than concentrations measured in bars and restaurants were smoking is banned in many countries. We estimate that a child spending only ten minutes in the car with a smoker at the mean PM2.5 concentration measured in the first window configuration--1697 mg/m(3)--will cause a 30% increase to the daily mean PM2.5 personal average of a child.

        Conclusions

        Estimates made using the measured data and previously reported PM2.5 daily mean concentrations for children in California showing that even short exposure periods are capable of creating large exposure to smoke.
          Creative Commons 'BY-NC-SA' version 4.0 license
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2017)
          The present work investigates the capability of single-stranded DNA (ssDNA) in enhancing the intrinsic peroxidase-like activity of the g-C3N4 nanosheets (NSs). We found that ssDNA adsorbed on g-C3N4 NSs could improve the catalytic activity of the nanosheets. The maximum reaction rate of the H2O2-mediated TMB oxidation catalyzed by the ssDNA-NSs hybrid was at least 4 times faster than that obtained with unmodified NSs. The activity enhancement could be attributed to the strong interaction between TMB and ssDNA mediated by electrostatic attraction and aromatic stacking and by both the length and base composition of the ssDNA. The high catalytic activity of the ssDNA-NSs hybrid permitted sensitive colorimetric detection of exosomes if the aptamer against CD63, a surface marker of exosome, was employed in hybrid construction. The sensor recognized the differential expression of CD63 between the exosomes produced by a breast cancer cell line (MCF-7) and a control cell line (MCF-10A). Moreover, a similar trend was detected in the circulating exosomes isolated from the sera samples collected from breast cancer patients and healthy controls. Our work sheds lights on the possibility of using ssDNA to enhance the peroxidase-like activity of nanomaterials and demonstrates the high potential of the ssDNA-NSs hybrid in clinical diagnosis using liquid biopsy.
            Cover page: Enhancement of the Intrinsic Peroxidase-Like Activity of Graphitic Carbon Nitride Nanosheets by ssDNAs and Its Application for Detection of Exosomes
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