Daily Quantification of Cancer-Associated Exosomal miRNA in Patient Blood by Photonic Crystal-Enhanced Quantum Dot Emission

点击翻译按钮获取中文摘要

ABSTRACT The primary goal of this proposal is to develop a new technology that can allow patients to repeatedly measure nucleic acid-based cancer biomarkers from a single drop of blood on a daily basis. This assay is being developed for specific clinical applications to determine drug treatment efficacies, prognosticate survival, and monitor post- treatment intervention by evaluating candidate nucleic acids shed from the tumor into blood of a cancer patient. We are focusing on detecting microRNA due to a very strong correlation with survival that our team has recently identified for patients with metastatic prostate cancer. We hypothesize that by measuring the concentrations of these markers in patients on a frequent basis during the course of therapy, we can precisely adjust therapeutic regimens for individual patients. However, accurately measuring microRNA in blood requires an extremely high limit of detection due to low concentrations, detection over a broad range of concentrations, and high sequence- specificity, attributes that are not currently possible for routine screening of fingerstick blood samples using standard methods of detection, such as PCR. Toward this end, our clinical needs have inspired a new form of assay to measure nucleic acids in blood through direct molecular counting in a microscope. This is now possible because we have developed novel ways to amplify the signals from individual molecules through a series of synergistic technologies, including light-emitting quantum dots, electric field-enhancing photonic crystals, and single-step sequence-specific enzymatic growth of microRNA. We now will combine these technologies to set the stage for measurement of microRNA using low-cost equipment that is already available in clinical diagnostic laboratories to minimize translational barriers to clinical adoption. To achieve these goals, our multi-investigator team has extensive expertise in probes for single-molecule imaging (Andrew Smith), optical detection in low- cost devices (Brian Cunningham), clinical oncology (Manish Kohli), biomarker discovery (Liang Wang), and epidemiology/biostatistics (Rebecca Smith). We will optimize our platform using a combination of synthetic and clinical blood specimens and thoroughly analyze the sequence selectivity of our assay, particularly focusing on microRNA variants, and closely compare our results with those from quantitative PCR assays. By the end of this award period, we expect to have developed the first direct-readout microRNA assay for use in human samples that is compatible with low-cost equipment, optimized the synergistic integration between quantum dots and photonic crystals, and measured, for the first time, the precise (digital) concentrations of microRNA in the blood of 100 subjects, prospectively enrolled and followed over 6 days each during the course of standard of care treatments for which no predictive or prognostic biomarkers currently exist in the treatment of metastatic prostate cancer. If successful, the outcome of this work will fill a major clinical gap in knowledge of how to match and finely tune treatments to individual molecular profiles.