Multiplexed protein biomarker analysis on silicon photonic microring resonators: translation towards clinical diagnostics

Protein biomarkers are valuable indicators of human physiological states. In clinical practice, they play a strong role in presymptomatic diagnosis of various diseases, as well as evaluation of disease prognosis and aid in treatment decisions making. Due to the importance of biomarkers, much efforts were made towards the discovery of good biomarker candidates, analytical methodologies for biomarker detection and quantitation, and ultimately, translation of the developed analytical platform to detect novel biomarkers in clinical practice. This dissertation places stronger emphasis on the latter two aspects of protein biomarker research: detection of biomarkers through immunoassays development, and translation of optimized assays to clinical samples analysis. For the immunoassays development aspect, assays described in this thesis were developed on a platform based on silicon photonic microring resonator technology. This sensing technology has high potential for clinical diagnostics utility, as sensor chips of this platform can be cheaply manufactured through a highly scalable process. Moreover, continuous improvements in sensor chip designs allowed rapid increase of biomarkers that can be detected simultaneously in a multiplexed panel. Multiplexed measurements are desirable due to the heterogeneity of the human population, and in many instances quantitation of multiple biomarkers are necessary to identify the disease state. Additionally, the latest generation sensing platforms have integrated fluidic systems that can be programmed for immunoassay automation, which shortens intensive training required for clinical laboratory personnel to perform assay runs. For the translational aspect of applying novel biomarker detection to the clinical laboratory, collaborations have been established with hospital physicians for access to clinical samples from diseased patients. Blood serum or plasma samples from these patients have been evaluated by the immunoassays developed on the microring resonator platform, and results from the platform’s measurements are then evaluated against other established immunoassay techniques to assess assay performance. Translational research in clinical diagnostics is a trial and error process. Good immunoassays developed for novel biomarkers might not have good diagnostics value once placed into clinical evaluations, and thus the biomarker discovery and assay development research phases repeats through again. This doctoral dissertation describes the progress of immunoassay development throughout the continuous improvements in the microring resonator platform, and eventually translates some of the developed assays to clinical samples analysis. Chapter 1 contains an introduction to protein biomarker immunoassays and their translational research value, with a more in depth description of microring resonators operation principles and the progression of the sensing platform development. Chapter 2 describes a simple detection of ricin toxin to illustrate the utility of microring resonators for protein analysis. Chapter 3 focuses on the development of an 8-plex panel to detect cancer biomarker that utilizes a protein multilayer strategy to improve assay signals. Chapter 4 explores the clinical utility of the platform by detecting monocyte chemactic protein-1 in human serum matrix. Chapter 5 describes the detection and quantitation of cardiac troponin I in serum samples from cardiac disease patients. Chapter 6 demonstrates the development of a multiplexed assay panel to detect 12 immunoregulatory markers associated with sepsis, as well as application of the panel to test plasma samples from septic patients at the intensive care unit of a local hospital. Finally, Chapter 7 outlines the future work related to the cardiac troponins project and the sepsis project described in the previous two chapters

Single Domain Antibodies for the Detection of Ricin Using Silicon Photonic Microring Resonator Arrays

Ricin is a lethal protein toxin derived from the castor bean plant. Given its notorious history as a biowarfare agent and homicidal weapon, ricin has been classified as a category B bioterrorism agent. Current ricin detection methods based on immunoassays lack the required sensitivity and specificity for many homeland security surveillance applications. Importantly, many conventional antibody-based methodologies are unable to distinguish ricin from RCA 120, a nontoxic protein also found in the castor bean plant. Single domain antibodies (sdAbs), which are recombinantly derived from immunized llamas, are known to have high affinities for ricin A or B chains and low cross-reactivity with RCA 120. Herein, we demonstrate the use of silicon photonic microring resonators for antibody affinity profiling and one-step ricin detection at concentrations down to 300 pM using a 15 min, label-free assay format. These sdAbs were also simultaneously compared with a commercial anti-RCA IgG antibody in a multicapture agent, single target immunoassay using arrays of microrings, which allowed direct comparison of sensitivity and specificity. A selected sdAb was also found to exhibit outstanding specificity against another biotoxin, saporin, which has mechanism of action similar to ricin. Given the rapidity, scalability, and multiplexing capability of this silicon-based technology, this work represents a step toward using microring resonator arrays for the sensitive and specific detection of biowarfare agents