Single Molecule Detection of Blood Biomarkers for Disease Diagnostics

Abstract

Biomarker measurements in blood are promising for various clinical applications including early detection of disease and monitoring response to treatment. However, a major challenge with methods for measuring biomarkers in blood is lack of analytical sensitivity. While biomarker levels are typically elevated in the affected tissue, once they enter the bloodstream, they become diluted and decrease in concentration. Many clinically-relevant biomarkers are present at sub pg/mL concentrations in the blood, rendering them undetectable using conventional techniques. To overcome this limitation, ultra-sensitive digital detection of protein biomarkers using Single Molecule Arrays (Simoa) was previously developed. Simoa is 1000X more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA) and has detection limits in the femtomolar range. To further improve the sensitivity of Simoa and enable detection of new protein biomarkers, we developed a method for single molecule protein detection with attomolar sensitivity using droplet digital ELISA (Cohen et al., ACS Nano 2020). We also evaluated how different antibody biotinylation approaches can improve the sensitivity of Simoa immunoassays (Cohen et al., Bioconjugate Chemistry, 2018). To expand the capabilities of Simoa for measuring different types of biomarkers, we developed a new method for microRNA detection (Cohen et al., Nucleic Acids Research 2017) and rat cytokine detection in blood (Cohen et al., Journal of Immunological Methods, 2017). We also applied Simoa assays to various clinical applications. We assessed the impact of clinical sample handling and processing on ultra-low level measurements of plasma cytokines (Cohen et al., Clinical Biochemistry, 2019). We also developed a blood biomarker panel for detecting early-stage breast cancer and discovered biomarkers for monitoring response to immunotherapy in lung cancer patients (Keegan et al., Journal for ImmunoTherapy of Cancer, 2020). Taken together, this thesis work has shown that single-molecule detection of biomarkers in blood is promising for various clinical applications.