A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 muL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10min. Following extraction, the 1500bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10min for 30cycles. The total assay time was 23min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 muL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.