Expanding the toolbox for precision medicine with silicon photonic microring resonators and microfluidic technologies
The goal of precision medicine is to use molecular profiles of disease to identify a targeted treatment that results in the best available patient outcomes. Although the concept of individualizing treatment is not new to medicine, genomic technologies and therapies targeted to genetic drivers of disease have inspired an era of precision medicine. Obtaining molecular profiles of disease requires analyzing biological samples that present daunting analytical challenges with thousands of potentially interfering analytes, often at concentrations much higher than the analytes of interest. Many analytes harbor fragile chemical modifications, such as phosphorylation of signaling proteins, and therefore, require careful design of protocols for sample handling to preserve the relevant biological information. Classifying patients into subgroups that will benefit from tailored therapies demands moving beyond single biomarker diagnostics to multiplexed detection methods. The analytical toolbox for studying the molecular basis of human disease has grown tremendously in recent decades and has been motivated in part by the human genome project, with the most dramatic changes seen in sequencing technologies. The next frontier for molecular diagnostics is the development of diagnostic tools for non-genomic molecular profiles of disease, such as non-coding RNAs and proteins. This thesis details efforts to improve multiplexed protein detection for precision medicine diagnostics. Most of the following work uses microring resonator arrays as the detection platform, a versatile silicon photonic biosensing technology. Chapter 1 reviews the applications of optical resonators in analytical chemistry. Microring resonators arrays are a class of whispering gallery mode resonators featured throughout the review. A conventional focus of optical resonator development has been on designing label-free sensors for biomolecule detection. However, much of the recent work pushing limits of detection for the microring resonator platform have used immunoaffinity labels and enzymatic enhancement to perform detection in clinically relevant samples. Initially, we had anticipated needing to fractionate interfering species out of these biological samples to perform multiplex measurements on the fractions of interest using the microring resonator platform. We found solutions that avoided separations prior to sample analysis, but the microring resonators offered an interesting property uncommon among chromatography detectors: a universal detector with an enormous dynamic range. Chapter 2 details interfacing the microring resonator platform with liquid chromatography. Optical resonators are surface sensitive and most commonly used to observe binding events on a modified sensor, but they can also serve as bulk refractive index detectors. In comparison to commercial refractive index detectors, the microring resonator platform is compatible with solvent gradient chromatography because of the large dynamic range. Commonly studied small molecule pharmaceuticals were used for proof-of-concept experiments, and ongoing work seeks to extend the platform to polymer analysis, an analyte class that lacks chromogenic signatures. The next two chapters detail my contributions to protein detection on the microring resonator platform and can be summarized as the implementation of protein and phosphoprotein detection in whole cell lysates and tissue homogenates. Protein detection in cell lysate was achieved by modifying the signaling amplification schemes developed by previous lab members and altering the chemical strategy for covalent modification of proteins to the sensor surface. Chapters 3 and 4 describe the specifics of this strategy. These projects were designed in part as proof-of-principle studies to demonstrate the application of microring resonators to novel samples and biomolecules. Signaling pathways are often dysregulated in tumors, resulting in uncontrolled growth and proliferation, and these signaling pathways are driven by phosphorylation cascades. In Chapter 3, a multiplex protein and phosphoprotein panel was used to monitor the levels across multiple signaling pathways in glioblastoma, the most common and aggressive brain cancer in adults. Chapter 4 builds on the phosphoprotein panel developed in Chapter 3 to dynamically monitor signaling networks of patient derived xenografts in response to targeted therapeutics. The phosphoprotein levels in these samples indicated pathway signatures unique to treatment time and mutational status of the sample. This approach could potentially be used to provide actionable information to clinicians by determining tumor susceptibility to treatment based off its signaling state. The phosphoprotein panels described in Chapters 3 and 4 center around the PI3K/Akt/mTOR signaling network. However, these panels lack the membrane proteins that initiate the signaling cascade. To include membrane protein analysis, I developed a microfluidic platform for Nanodisc assembly and purification, referred to as the μNAP platform and detail in Chapter 5. The μNAP platform capitalizes on sample preservation and small volume processing inherent to miniaturization and microfluidics and achieves Nanodisc assembly by combining a reagent mixing chamber and packed detergent removal bed onto a microfluidic device. The platform also includes an affinity chromatography module for rapid purification on the microfluidic scale. Cytochrome P450 3A4 was used to demonstrate the capabilities of the μNAP platform. Chapter 6 details the future directions for each of the described projects. The next steps for phosphoprotein detection with microring resonator arrays is to use the targeted panel to reconstruct the aberrant signaling networks from tumor biopsy samples. Network reconstruction has been performed using global profiling methods, such as next generation sequencing and proteomics with mass spectrometry, but reconstructing key signaling networks from minimal network data could provide a less cumbersome approach to obtain actionable information with higher throughput. The future work for the μNAP platform will include generating Nanodisc libraries from valuable samples, such as tumor biopsies. Nanodisc libraries have been shown to accurately represent the membrane protein composition of the sample, and these libraries formed from patient samples could be used for functional screening of membrane proteins in response to targeted therapeutics. Finally, interfacing the microring resonator platform with on-chip electrophoresis could prove to be a remarkably useful combination. The microring resonator platform would allow for on-chip multiplexed detection of biomolecules combined with the separation efficiency of electrophoresis. The interface would substantially reduce reagent consumption and shrink the footprint of the sensor platform by eliminating the need for external pumping. By applying the previously developed electrophoretic methods for sample stacking along with the improved mass transfer properties of non-laminar flow, on-chip electrophoresis combined with microring resonator arrays could represent a significant analytical advancement.
Biosensor, Precision medicine, Silicon photonics, Microfluidics, Nanodiscs
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign