Detection of biomarkers using DNA biosensors in a fluorescent platform to improve current one-step point-of-care methodologies

Current methodologies for patient diagnosis require the use of skilled workers, expensive reagents, and instrumentation, and most importantly, time. As these methodologies encourage inconsequential follow-up between patients and their doctors, new methods must be developed. These methods should follow the WHO guidelines of ASSURED (Accessible to those in need, Sensitive to the clinical range of the target of interest, Specific to the target of interest with no false positives or negatives, User-friendly, Rapid, under one hour for results, Equipment free, and Delivered to those in need) to create point of care technologies available for all. To this end, my studies focus on the development of convenient calibration methods for use with fluorescent, aptamer-based biosensors (aptamer beacons). Specifically, I describe here the creation, characterization, and application of a one-tube method for the calibration of aptamer beacons using sensors for the detection of DNA, silver ions, and thrombin as my test bed.A significant hurdle in the point of care testing of biomarkers in complex solutions is that there is sample variation, requiring large volumes of the target of interest and complex and cumbersome calibration. In response, I have created a simple, three-step, one-tube method for the calibration of aptamer beacons supporting the direct quantification of specific target molecules within complex sample matrices. Specifically, I have shown that the sequential addition of two inexpensive and stable oligonucleotide-based reagents allows for the determination of the absolute minimum and maximum fluorescence produced by the sensor in each solution, providing the information to calibrate in the face of sample-to-sample variations in a simple, single-tube format. Using the three associated measurements together with the pre-determined disassociation constant of the aptamer I demonstrate the single-tube calibrated quantification of multiple targets directly in complex samples