Abstract
Detection and analysis of biochemical markers is essential in many disciplines. The projects described in this dissertation were focused on the development of centrifugal microfluidic approaches to address unmet needs and technology gaps in the forensic, biomedical, and National Defense and Security communities. Conventional laboratory instrumentation currently used for these purposes is often expensive, non-portable, and dictates labor-intensive and time-consuming benchtop protocols conducted by highly trained personnel. These limitations can be overcome through advancements in and lab-on-a-disc (LoaD) microfluidic technologies that allow for integration and automation of numerous unit operations within a cost-effective, compact, and portable platform amenable to use by nontechnical personnel. Among these, rotationally driven microfluidic platforms can be complete sample-to-answer, push-button micro total analysis systems (μTAS) (e.g., narcotics and explosives detection) or they may simply automate a portion of a much larger process chain (e.g., dynamic solid phase extraction (dSPE) of nucleic acids or differential extraction (DE) of forensic sexual assault evidence).
The projects outlined in this dissertation were driven by clear unmet needs or technology gaps, with focus on adapting existing laboratory processes to LoaD platforms. The centrifugal differential extraction project (CDx) project was aimed at automating the manually intensive, time-consuming sample preparation workflow associated with analyzing sexual assault evidence samples, i.e., reducing analyst hands-on time and enhancing sperm fraction enrichment. Similarly, the centrifugal vertical flow project (cVF) aimed to develop a portable, field deployable device capable of supplanting existing paper-based lateral flow immunoassay technology with more sensitive, rapid pathogen detection while maintaining independence from central laboratories. Given the complex nature of both parent projects, a large portion of research exploration was necessarily dedicated to describing, characterizing, and integrating novel or improved microfluidic unit operations that enhance on-disc functionality by expanding the ‘microfluidic toolbox.’
With regard to the CDx project, the central research goal was to design and develop a centrifugal microfluidic disc capable of performing, automating, and multiplexing a complete forensic DE sample preparation. Initial design and testing efforts towards that goal detailed in Chapter 2 demonstrate that a full forensic differential extraction (DE) can be performed on a single microfluidic disc that provides for timed reagent release, temperature control for sequential enzymatic reactions, and fluidic fractionation that yields discrete sperm (SF), non-sperm (NSF), and waste fractions from sexual assault evidence samples. Optimization of flow control and microvalving strategies were major hurdles in the development of this microfluidic architecture. As such, Chapter 3 is devoted to a detailed discussion of the development, characterization, and optimization of two microvalve closure methods, i.e., laser-based and contact heating closure strategies.
The driving motivator for the cVF project was to develop a disc-based system capable of outperforming existing lateral flow immunoassay (LFI) test strips; operational limitations of traditional LFI test strips include poor control over incubation times, reliance upon capillary flow, limited ability to process larger sample volumes, and dependance upon porous materials that rapidly saturate with fluid. During the cVF analysis, the three-dimensional flow path directs the fluid stream orthogonally through embedded nanoporous membranes that are permanently bonded to the disc substrates during the disc fabrication process. The work presented in Chapter 4 describes visualization and characterization of on-disc flow and fluid drainage patterns, achieved largely through high-speed videography. These studies highlight critical observations impacting ongoing centrifugal vertical flow (cVF) experiments and efforts, including changes in drainage profiles attributed to differing sample matrix composition and the loss of hydraulic pressure with changes in fluid column fill height during continued flow. The work presented in Chapter 5 describes crucial pilot and proof-of-principal studies for on-disc pathogen detection via cVF sandwich-type immunocapture.
In large part, the future success the CDx and cVF parent projects hinges upon key microfluidic functionality, chiefly microvalving and on-disc flow through porous media. Understanding the advantages and limitations of these approaches will influence future decision-making and research objectives. Substantial work on the CDx and cVF parent projects remains, yet the exploratory and unit operation development studies outlined here bode well for the future of both ventures. Once fully developed, these rotationally-driven CDx system is poised to significantly impact the forensic science community by ameliorating persistent evidence backlogs, improving laboratory turnaround times, and enhancing sperm fraction enrichment. Likewise, the cVF project will provide enhanced pathogen detection capabilities by offering control over sample volume and incubation times and eliminating reliance upon capillary flow and wicking materials.
