Integrating a Simple Photometric System with Programmable Droplet Formation for Quantification of Diabetes Biomarkers and to Study Adipose Tissue Secretions

Abstract

The chapters presented within this dissertation give a comprehensive account of the development and adaptation of cost-effective optical components tailored for integration into microfluidic devices, with a primary focus on absorbance-based quantification techniques for analytes relevant to diabetes research. This innovative system was made possible through the utilization of 3D printing technology. This technology facilitated the generation of fluidic layer template molds, from which polydimethylsiloxane (PDMS) replicas housing intricately engraved microchannels were fashioned. Notably, the dynamic nature of the microfluidic device's evolution in design was enabled by the inherent flexibility of 3D printing, allowing for swift and facile modifications as necessitated by our research.

Another pivotal contribution to the viability of absorbance measurements within microfluidic devices was the implementation of droplet-based lock-in detection. This method, previously pioneered and applied within our laboratory, was shown to mitigate background noise during the analytical processes. Locking the detector into the droplet frequency enabled detection of low signals that are otherwise obscured in microfluidic systems due to their diminutive optical pathlengths, which usually renders absorbance measurements insensitive compared to other optical methods.

Although the preliminary iterations of these devices were passively operated and demonstrated functionality in droplet generation, it became evident that downstream data analysis of these droplets posed considerable challenges. The inherent irregularities in droplet velocity and morphology necessitated a reevaluation of the system's operability. Consequently, a compelling need arose to exert precise control over droplet generation and solution flow, which led to the adaptation of normally open pneumatic valves within the microfluidic framework as discussed in Chapter 2. This incorporation enabled the systematic characterization of the optimal frequency for droplet formation, ensuring alignment with the optical system's detection capabilities. Moreover, this active control of droplet generation paved the way for the seamless integration of lock-in detection, affording the ability to synchronize each droplet with its specific frequency and phase, thereby enhancing precision and accuracy in the analytical processes.

Subsequent chapters delve into several biological applications of the photometric and hybrid system, combining PDMS, fluidic layers, and 3D printed pneumatic layers. Initially, as described in Chapter 3, this system was harnessed for the quantification of fructosamine, a well-established glycemic biomarker commonly employed in conjunction with the conventional hemoglobin A1C test, particularly for individuals afflicted with hemoglobin anomalies such as anemia. The quantification of fructosamine was accomplished using sera derived from both healthy individuals and diabetic patients. The results demonstrated a congruence with those from a standard 96-well plate reader, thereby attesting to the functionality and effectiveness of our devised system in quantifying fructosamine even in complex matrices such as serum.

Finally, in Chapter 4, our investigations were extended to the quantification of glycerol secreted from murine adipose tissue explants, a vital analyte in the context of studying the mechanistic underpinnings of diabetes, obesity, and metabolic syndrome. Glycerol, as a product of lipolysis, assumes a pivotal role in elucidating the intricacies of such metabolic disorders. To this end, adipose tissue explants were subjected to treatments involving isoproterenol (ISO) as a pro-lipolytic agent and BAY 5944 as an anti-lipolytic agent, with subsequent quantification of glycerol levels in both conditions. The findings, which once again corroborated with those obtained from a conventional plate reader, underscored the effectiveness of our devised system in quantifying glycerol.

The work presented in this dissertation shows that by employing 3D printing technology, and lock-in detection, and by actively controlling droplet formation, a simple photometric system such as the one developed and described herein, absorbance-based measurements in microfluidic systems can be made feasible and accessible. In the future, these devices could be applied in low resource environments or with less trained personnel. Lastly, suggested changes and additions for making the system much better, are shared in Chapter 5.