Enhancing Bioanalytical Assays with Photocleavable DNA Strands and Valve-Based Microfluidics

Abstract

The detection and quantification of biomarkers, especially in the early stages of diseases, play a crucial role in developing new therapeutics and increasing treatment effectiveness. Hence it is a vital part of advancing biomedical research and clinical diagnostics. Quantification of biomarkers in extremely low concentrations remains complex and challenging, highlighting the importance of the development of new bioanalytical assays focused on simplicity, sensitivity, and fast response times. In this dissertation, we present our contributions to the development of photo-controlled oligonucleotide-based assays for detecting and amplifying biomarker signals. We also present our contribution to the development of an integrated microfluidic droplet system with promising capabilities for tissue sampling at high resolution. Chapter 1 introduces the basic principles of oligonucleotide-based assays, focusing on assay design and optimizations, oligonucleotide modification that increase assay application flexibility, and a deep perspective on proximity binding assays and amplification techniques for biosensing. In chapters 2 and 3, we present two different oligonucleotide-based assays that take advantage of design flexibility by embedding a photocleavable (PC) linker into oligonucleotide strands for the development of a photo-controlled bioanalytical assay. Chapter 2 describes a new approach to thermofluorimetric assays (TFA) by performing a photo-controlled destabilization of double-stranded DNA (dsDNA), which simplifies the assay instrumentation by avoiding real-time thermal scanning while retaining the advantages of TFA, such as analytical signal separation from background without any physical separation technique. Additionally, this technique offers an enzyme-free, rapid, and precise cleavage of oligonucleotide probes with high temporal resolution. Chapter 3 shows our work on a new cyclic amplification method for nucleic-acid detection that incorporates photocleavable oligonucleotide strands and magnetic beads for strand trapping. The magnetic bead is designed to trap the analyte, and the release of fluorescent marker is controlled by UV light exposure and strand photo-cleavage. The fluorescence intensity, proportional to analyte concentration, can be measured after each cycle, demonstrating great potential for real-time amplification. Recognizing the importance of automation and high-throughput of sensitive techniques for biomarker detection and tissue studies, chapters 4 and 5 will focus on droplet-based microfluidic techniques. Chapter 4, introduces the fundamentals of microfluidics, describing the development and fabrication of devices, followed by droplet flow control and manipulation. It concludes with a discussion of the main applications of droplet-based microfluidics in bioanalysis. Chapter 5 presents our approach to a simplified droplet-based microfluidic device utilizing only four pneumatic valves. This device takes advantage of pulse-width flow modulation (PWFM) for flow rate control and a reusable wire electrode interface for droplet merging via high alternating current (AC) electric fields. The device achieves high temporal resolution of sampling and segmentation, demonstrating its potential for studying fast kinetic reactions and detailed tissue sampling capability. The dissertation concludes with Chapter 6, where we summarize the research performed and discuss further opportunities to improve upon the presented methods, including coupling our photocleavable oligonucleotide-based assays with microfluidic technology.