Attuning Forensic Sample Preparation and Microfluidics: A Path Towards Enhanced Extraction Workflows

Forensic DNA analysis plays a critical role in the criminal justice system, enabling the identification of individuals from biological evidence recovered from crime scenes. In cases of sexual violence, forensic samples often contain mixtures of biological material from both the victim and the perpetrator. To obtain a clear, single-source male DNA profile, forensic laboratories must manually separate these contributions – a meticulous, time-intensive process known as ‘differential extraction’ (DE) that involves multiple intervention steps and carries risks of sample loss and contamination. In addition, traditional extraction methods, though widely used in forensic laboratories, often rely on PCR-inhibiting reagents and therefore require additional purification steps to effectively isolate nucleic acids from cellular debris and amplification suppressors. The work described in this dissertation harnesses centrifugal microfluidics to automate and streamline forensic sample preparation, addressing these challenges by minimizing hands-on processing, reducing contamination risks, and improving the recovery of perpetrator DNA. Moreover, given their dependency on PCR-inhibiting reagents, traditional extraction methods are not ideally suited for seamless integration within a centrifugal microfluidic framework. Accordingly, this work also describes a DE process that generates PCR-ready extracts, eliminating the need for purification steps prior to downstream DNA analysis. Chapter 1 provides context for the research, highlighting key challenges in forensic science and the motivations behind the proposed advancements. Chapter 2 extends a microfluidic platform shown to enable intricate fraction isolation and temporal control of sequential process chains to forensic sample preparation by using conventional extraction methods. Chapter 3 explores alternative chemical methods that leverage temperature control of enzymatic cocktails to accelerate nucleic acid extraction from sexual assault evidence, moving beyond conventional lengthy workflows. Chapter 4 integrates these novel enzymatic extraction protocols with the microfluidic device to create a more efficient processing system. Chapter 5 introduces a parallel method for simultaneous body fluid identification and human DNA typing using these rapid, single-step extraction chemistries. Finally, Chapter 6 reflects on the broader implications of these innovations, discussing their potential to transform forensic practices while emphasizing the need for careful implementation in a field with inherently profound legal and ethical implications. Largely, this work aims to enhance the reliability, efficiency, and accessibility of forensic DNA analysis by automating the sample preparation steps through microfluidic-based approaches, paving the way for more streamlined and effective processing of complex forensic evidence.