Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample.;In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of molecules, this dissertation establishes the utility of microring resonator chips for multiparameter analysis of several challenging protein targets in cell cultures, human blood sera, and other clinical samples such as cerebrospinal fluid.;Various sandwich immunoassay formats for diverse protein analytes are described herein, but the bulk of this dissertation focuses on applying the technology to cytokine analysis. Cytokines are small signaling proteins that are present in serum and cell secretomes at concentrations in the pg/mL or ng/mL range. Cytokines are very challenging to quantitate due to their low abundance and small size, but play important roles in a variety of immune response and inflammatory pathways; cytokine quantitation is thus important in fundamental biological studies and diagnostics, and complex and overlapping cytokine roles make multiplexed measurements especially vital. In a typical experiment, microfluidics are used to spatially control chip functionalization by directing capture antibodies against a variety of protein targets to groups of microring sensors. In each case, binding of analytes to the rings causes a change in the local refractive index that is transduced into a real-time, quantitative optical signal. This photonic sensing modality is based on the interaction of the propagating evanescent field with molecules near the ring surface. Since each microring sensor in the array is monitored independently, this technology allows multiple proteins to be quantified in parallel from a single sample.;This dissertation describes the fabrication, characterization, development, and application of silicon photonic microring resonator technology to multiplexed protein measurements in a variety of biological systems. Chapter 1 introduces the field of high-Q optical sensors and places microring resonator technology within the broader context of related whispering gallery mode devices. The final stages of cleanroom device fabrication, in which 8" silicon wafers that contain hundreds of ring resonator arrays are transformed into individual functional chips, are described in Chapter 2. Chapter 3 characterizes the physical and optical properties of the microring resonator arrays, especially focusing on the evanescent field profile and mass sensitivity metrics. Chapter 4 demonstrates the ability to apply ring resonator technology to cytokine detection and T cell secretion analysis. Chapter 5 builds on the initial cytokine work to demonstrate the simultaneous detection of multiple cytokines with higher throughput to enable studies of T cell differentiation. In preparation for reaching the goal of cytokine analysis in clinical samples, Chapter 6 describes magnetic bead-based signal enhancement of sandwich immunoassays for serum analysis. Additional examples of the utility of nanoparticles and sub-micron beads for signal amplification are described in Chapter 7, also demonstrating the ability to monitor single bead binding events. Chapter 8 describes an alternative cytokine signal enhancement strategy based on enzymatic amplification for human cerebrospinal fluid (CSF) analysis. Chapter 9 adds work with other CSF protein targets that are relevant to the continuing development of a multiparameter Alzheimer's Disease diagnostic chip. Future directions for multiplexed protein analysis as it pertains to important immunological studies and in vitro diagnostic applications are defined in Chapter 10. (Abstract shortened by UMI.).