Date of Award
Doctor of Philosophy
XiuJun (James) Li
Infectious diseases and cancer have been two main causes of global death and disability, leading to a significant impact on public health and economies globally. Early diagnosis of these diseases can improve prevention, treatment, and prognosis in clinical practice. However, current laboratory diagnostic approaches require expensive and bulky equipment, well-trained personnel, and time-consuming processes, which puts a great challenge to conventional methods to address, especially in resource-limited settings. Microfluidic lab-on-a-chip presents a unique opportunity for various biomedical applications due to multiple advantages such as low reagent consumption, integration, miniaturization, portability, and automation. Since different microfluidic platform substrates have their advantages and limitations, hybrid devices can draw more benefits from various substrates. Herein, we developed multiple low-cost paper/polymer hybrid microfluidic biochips integrated with isothermal amplification methods for high-sensitivity diagnosis of infectious diseases and cancer.At first, we developed a paper/polydimethylsiloxane (PDMS) hybrid microfluidic platform integrated with loop-mediated isothermal amplification (LAMP) for a rapid, sensitive, and specific diagnosis of the main tuberculosis-causing bacteria, Mycobacterium tuberculosis (M.tb). A battery-powered heater and a DarkBox were devised for heating and visualization steps for low-resource settings, respectively. Results could be observed by the naked eye or imaged by a smartphone camera under a portable blue light pen within 30 minutes. The limits of detection (LODs) of 5 and 15 DNA copies per LAMP zone for M.tb were achieved using two LAMP primer sets for ESAT-6 and 16s-rRNA genes of M.tb, respectively. This low-cost hybrid microfluidic device provides a simple and highly sensitive method for rapid point-of-care diagnosis of tuberculosis (TB) in low-resource settings. Based on the aforementioned singleplexed pathogen detection, by performing multiple singleplexed LAMP reactions in multiple different compartments in parallel, we developed another low-cost paper/PDMS hybrid microfluidic biochip combined with LAMP for highly sensitive and specific instrument-free multiplexed detection of the three different types of Bordetella species, B. pertussis, B. holmesii, and B. parapertussis. Results were observable by the naked eye or imaged by a smartphone camera under the portable blue light pen in the DarkBox within 45 minutes. The LODs of 5, 10, and 15 DNA copies per LAMP zone were achieved for B. pertussis, B. holmesii, and B. parapertussis, respectively. In addition to purified DNA, microorganisms of these three types of Bordetella species were directly and simultaneously detected from nasopharyngeal samples by applying our optimized bacterial lysis protocol without any laborious sample preparation procedures. In addition to infectious diseases, we also developed low-cost paper/polymer hybrid microfluidic devices to detect miRNAs as cancer biomarkers. A paper/polymethyl methacrylate (PMMA) hybrid microfluidic biochip integrated with padlock probe-based exponential rolling circle amplification (P-eRCA) was developed for quantitative point-of-care (POC) detection of miRNA-21, miRNA-155, and miRNA-141. The battery-powered heater was used for the heating steps of the P-eRCA reaction on the chip. Results were visualized under the portable blue light pen in the DarkBox and imaged by a smartphone camera. The images were analyzed using the NIH ImageJ software for quantitative analysis of miRNAs. It was found that the LODs were a few miRNA copies per P-eRCA zone for each miRNA target. In addition, the microfluidic platform was successfully applied to detect miRNAs in human serum and breast cancer cell samples. At last, to enhance the efficiency of detection of miRNAs as cancer biomarkers, we developed an origami paper/polymer microfluidic device integrated with P-eRCA for sensitive and specific multiplexed POC detection of miRNAs, including miRNA-21, miRNA-155, and miRNA-141. These three types of miRNA could be simultaneously detected in a single device. The limits of detection of 6, 5, and 8 miRNA copies per P-eRCA zone for miRNA-21, miRNA-155, and miRNA-141 were achieved, respectively. Moreover, three different miRNAs were simultaneously quantified in human serum and breast cancer cell samples using the origami hybrid microfluidic biochip. These low-cost and highly sensitive microfluidic biochips have tremendous potential for POC diagnosis of various infectious diseases and cancer, especially in low-resource settings such as physiciansÃ¢?? offices and developing nations.
Received from ProQuest
Tavakoli, Hamed, "Integration Of Genetic Isothermal Amplification On Low-Cost Hybrid Paper/polymer Microfluidic Biochips For High Sensitivity Point-Of-Care Disease Diagnosis" (2022). Open Access Theses & Dissertations. 3554.
Available for download on Saturday, June 08, 2024