Nucleic Acid Diagnostics using Isotachophoresis and Isothermal Amplification
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Nucleic acid amplification tests (NAAT) have become the gold standard detection technique for many infectious disease diagnoses. NAAT offer high diagnostic sensitivity and specificity that lead to confident diagnoses, as well as relatively fast results and diverse functionality (e.g. quantification, multiplexing). Despite the advantages of nucleic acid amplification tests, they currently have limited point-of-care (POC) utility due to the need for multi-step procedures and complex instruments that require skilled personnel and well-equipped laboratories. In this thesis, we aim to create integrated nucleic acid amplification technologies that are better suited for POC applications by developing devices that integrate unique sample preparation and amplification techniques. Specifically, we use a powerful electrokinetic method called isotachophoresis (ITP) to rapidly extract and concentrate nucleic acids using two buffers and an electric field. We also replace traditional polymerase chain reaction (PCR) amplification with isothermal amplification methods that do not require thermocycling equipment. We present two devices that use isotachophoresis and isothermal amplification to extract and amplify nucleic acids from complex samples. The first technology is named the NAIL device—Nucleic acid Amplification using Isotachophoresis and Loop-mediated isothermal amplification. NAIL uses ITP and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. Fluid actuation is performed by passive capillary barriers and heated air chambers that act as valves and pumps respectively. LAMP amplifies nucleic acids at constant temperature and produces a bright fluorescent signal that can be detected using a mobile phone camera equipped with fluorescent filters. We show the limit of detection (LoD) of pathogenic E. coli O157:H7 cells from whole milk samples to be 1000 CFU/mL, which is a two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples. The second device is called ITP-RPA, which combines isotachophoresis with the isothermal amplification strategy, recombinase polymerase amplification (RPA). This device offers improvements over NAIL because it simultaneously incorporates sample extraction with amplification, removing the need for any pumps, valves, etc. ITP-RPA offers detection of nucleic acids in less than 15–20 minutes, and uses low-cost glass fiber substrates that make it amenable to POC applications. We show the detection of nucleic acids spiked into serum and whole blood as we work towards creating a POC viral load monitoring diagnostic for HIV-1 antiretroviral therapy treatments. We discuss the design of ITP chemistry that can incorporate the reagents required for RPA, as well as how to adapt this chemistry for separating the reaction from sample inhibitors. We show the current LOD of the device to be 10,000 copies/mL in serum, which is one order of magnitude higher than the clinical threshold. Future work will aim to improve this value below the relevant threshold. The introduction to these thesis discusses diagnostic devices with a focus on nucleic acid diagnostics. It also presents the descriptions and relevant principles for ITP and isothermal amplification. Chapter 2 reviews and summarizes relevant metrics for developing diagnostic devices in the laboratory, as well as transitioning these devices to the clinic. Appreciating and using these metrics will benefit test developers by providing consistent measures to evaluate analytical and clinical assay performance, as well as guide the design of tests that will most benefit clinicians and patients. Discussion topics include analytical laboratory statistics, diagnostic sensitivity/specificity and clinical thresholds, clinical metrics, and case studies from relevant commercially available tests.
- Chemical engineering