This study explores a microfluidic transcriptomic method for body fluid identification.
This project resulted in the optimization of chemistry for microfluidic enzymatic extraction for saliva and seminal fluid, RT-PCR amplification, and electrophoresis. Work was completed on the development of cost-effective microfluidic discs for an integrated sample preparation disc consisting of the architecture from the enzymatic extraction disc and the RT-PCR microdisc, as well as microchip electrophoresis. For the enzymatic extraction, an initial optimization was tested in order to convert the three different buffers required for each body fluid (red for blood, blue for saliva, and orange for vaginal fluid and seminal fluid) into a singular universal buffer for the implementation on-disc. This testing resulted in varied results for which body fluids were amenable with the extraction chemistry. Saliva and seminal fluid have consistently shown better results for the enzymatic extraction but fluids such as venous blood, menstrual blood, and vaginal fluid have required additional work. These experiments were then followed by various DNase testing, incubation studies, enzymatic volume titrations, inhibition studies, buffer titrations, cotton and FLOQ swab testing, and column purifications that did not result in a universal extraction method for all body fluids. Specifically, the bloods and vaginal fluid proved to be incompatible with the prepGEM enzyme, even in combination with the gold standard Qiagen RNA extraction methods with prepGEM used over the conventional Pro K. However, these results are not entirely surprising as many researchers who conduct forensic body fluid research often have trouble with blood and vaginal fluid. The enzymatic extraction was able to be used with saliva and seminal fluid resulting in successful extraction of these body fluids on the microfluidic device developed within ten minutes. This work was done alongside the ESR, as they tested their standard CellTyper II method alongside the prepGEM protocol for concordance testings along with mock samples.