| In developing nations, deaths associated with infectious diseases are often due to the lack of appropriate tools for diagnosis rather than effective and economical prevention and treatment options. Compared to traditional techniques such as culturing and microscopy, nucleic acid amplification testing (NAAT) is faster and more accurate, but its adoptability is limited due the high cost and complexicity of the assay. Therefore, this work aimed at developing an affordable and easy-to-use NAAT device by leveraging advances in the fields of genomics, low-cost microfluidics, and isothermal techniques for NAAT.;The complexity of pathogen detection due to the extensive genetic variability that exists for many pathogens was evaluated to substantiate the need for multiplexed detection In this context, a virulence and marker gene (VMG) biochip was developed for parallel detection of multiple waterborne pathogens, using several VMGs per pathogen and multiple probes per VMG. While the cost of the assay was, at the time of development, considered reasonably low on a per pathogen basis, recent advances in the arena of low-cost microfluidics drastically changed this notion. Therefore, development of a disposable microfluidic chip and accompanying device for pathogen detection was undertaken as the next step in this project.;Loop-mediated isothermal amplification (LAMP) was used instead of the more common polymerase chain reaction (PCR) since the former eliminates the need for thermal cycling while achieving a level of accuracy and rapidity that is comparable or even better than that of PCR. In terms of assay format, a 64-channel polymer microfluidic chip was designed and fabricated for parallel analysis of four samples, with each sample being tested for up to 15 genetic markers. To improve usability of the chips in the hands of untrained personnel, the chip was designed to allow dispensing of the sample using a common pipettor in a single step, after which the chip is sealable using tape. For fabrication of the chips, a technique involving hot embossing of thin polymer films using inexpensive molds and a sacrificial thermoplastic counter tool was established. Furthermore, a novel application of xurography and film lamination was developed for facile integration of tens to hundreds of microvalves and air vents for flow control. Also, by exploiting the high amplification yield of LAMP in combination with an optimal fluorescent dye, the optics could be drastically simplified. An array of individually addressable LEDs was employed to read the reaction wells with a single photodiode, without the need for mechanically moving components. This optical module was demonstrated to provide identical performance characteristics, in terms of assay accuracy and speed, with a commercial real time PCR machine but was significantly cheaper.;Based on these two key components, a fully functional prototype of the device was designed and assembled, with the necessary electronics and software being developed by other researchers involved in this project. This device and the 64-channel chip were validated for multiplexed detection of six major diarrheal pathogens. Due to its robustness, low cost and compact size, it is expected that this device could play a key role in improving human health in developing nations by providing accurate and rapid diagnostics in an affordable and easy-to-use format. |
