Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2013)

The analyses of patient tumor biopsy samples and biopsy-based diagnostics have emerged as important tools for cancer diagnostics. However, the techniques employed currently are restricted to centralized laboratories as they are time-consuming, manual labor intensive and vary considerably in their effectiveness amongst institutions and countries. Point of Care testing (POCT) for cancer with the capacity for multiplexed detection of numerous biomarkers in biopsy samples in a rapid, precise and portable manner is emerging as an area with enormous potential to disseminate universal diagnostics to cancer patients. Additionally, POCT can be used as a screening tool, to discern malignant from benign tumors at the physician's office, and lead to reduction in the need for expensive and time-consuming laboratory tests, hence minimizing the cost and anxiety, for patients with benign tumors. A POCT platform for multiple biomarker analysis can not only improve the operational characteristics of assays but can also help ascertain drug efficacy, ushering in personalized medicine for the patients. The reduced volumes and diffusion distances, which enable multiplexed, portable and quick assays, in microfluidic devices makes such devices a promising platform to realize POCT systems. But current microfluidic devices for cancer diagnostics suffer from the lack of a generalized on-chip sample preparation module and a simplified fluid actuation technique.

The overall goal of the reported dissertation research is to develop microfluidic modules that will enable the development of integrated microfluidic diagnostic platforms for the multiplexed detection of cancer biomarkers in tumor biopsy samples. The main focus of the thesis is on the development of novel microfluidic sample preparation modules. The purpose of the sample preparation component is to pre-concentrate cancerous cells, remove background proteins in the sample and to subsequently lyse the cells to release the proteins of interest. The pre-concentration of the adherent cells, including the cancerous cells, in the sample is reported by trapping them using a novel hydrodynamic cell trap. The sample washing methods, to remove extracellular proteins that could interfere with downstream assays, is also optimized. An electrochemical lysis technique is then integrated to the cell pre-concentration module, to effectively lyse the cells without having to add external reagents. Microfluidic modules for the separation of bacterial and mammalian cells from mixed samples are also reported. The immortalized cancer cell lines used in this research include the human breast cancer cell lines BT-474, known to over express the Her-2 protein, and T47D along with cervical cancer cell line HeLa.

The development of a novel fluid actuation technique, termed Proximal Degas-driven Flow (PDF), is also reported in this thesis. PDF takes advantage of the high porosity and air solubility of PDMS to reduce the pressure inside the fluidic channels leading to fluid flow in the channel. This actuation technique enables bubble-free fluid flow, can be used to fill up dead-end chambers in contrast to traditional pressure (positive or negative) driven flows and does not require the priming of the channels. Unlike degas-driven flow, PDF alleviates the need for pre-degassed and sealed devices, enabling consistent and longer-lasting fluid flow. This portable technique also requires very simple and cheap hardware like a vacuum bulb or membrane pump (thumb pump).

In conclusion, several microfluidic modules to enable Point of Care biopsy-based cancer diagnostics are introduced. The research presented in this dissertation is an effort to transform point-of-care cancer testing and provide universal diagnostics and personalized medicine to cancer patients.

    Cover page: Microfluidic Modules for Enabling Point-of-Care Biopsy-based Cancer Diagnostics
    • Article
    • Peer Reviewed
    Berkeley Research Impact Initiative (BRII) (2011)
    Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      Rapid and precise detection of Chlamydia trachomatis, the leading global cause of sexually transmitted infections (STI), at the point of care (POC) is required for treatment decisions to prevent transmission and sequelae, including pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and preterm birth. We developed a rapid POC test (POCT), termed LH-POCT, which uses loop-mediated amplification (LAMP) of nucleic acids. We performed a head-to-head comparison with the Cepheid Xpert CT/NG assay using clinician-collected, deidentified paired vaginal samples from a parent study that consecutively enrolled symptomatic and asymptomatic females over 18 years of age from the Ministry of Health and Medical Services Health Centers in Fiji. Samples were processed by the Xpert CT/NG assay and LH-POCT, blinded to the comparator. Discrepant samples were resolved by quantitative PCR. Deidentified clinical data and tests for Trichomonas vaginalis, Candida, and bacterial vaginosis (BV) were provided. There were a total of 353 samples from 327 females. C. trachomatis positivity was 16.7% (59/353), while the prevalence was 16.82% (55/327) after discrepant resolution. Seven discrepant samples resolved to four false negatives, two false positives, and one true positive for the LH-POCT. The sensitivity of the LH-POCT was 93.65% (95% confidence interval [CI], 84.53% to 98.24%), and specificity was 99.31% (95% CI, 97.53% to 99.92%). Discrepant samples clustered among women with vaginal discharge and/or BV. The prototype LH-POCT workflow has excellent performance, meeting many World Health Organization ASSURED criteria for POC tests, including a sample-to-result time of 35 min. Our LH-POCT holds promise for improving clinical practice to prevent and control C. trachomatis STIs in diverse health care settings globally.
        Cover page: Development and Evaluation of a Point-of-Care Test in a Low-Resource Setting with High Rates of Chlamydia trachomatis Urogenital Infections in Fiji
        Top