Poster abstracts

Poster number 35 submitted by Liuhan Dai

Ultrafast, high-accuracy disease biomarker detection through fluorogenic single molecule recognition

Liuhan Dai (Department of Chemistry, University of Michigan), Pavel Banerjee, Sujay Ray (Department of Chemistry, University of Michigan), Aaron Blanchard (Department of Biomedical Engineering, Duke University), Zi Li (Pacific biosciences), Tewari Muneesh (Department of Biomedical Engineering, University of Michigan), Nils Walter (Department of Chemistry, University of Michigan)

Abstract:
Fluorogenic “light-up” probes, which yield signal almost exclusively only once bound to a target molecule, can effectively enhance the signal-to-noise ratio of molecular detection assays and enhance their sensitivity. In this study, we implemented an advanced single-molecule kinetic fingerprinting technique, termed “fluorogenic single molecule recognition by equilibrium through Poisson sampling” (FG-SiMREPS), which utilizes a fluorogenic DNA imager probe to allow identification of single cancer mutant molecules in liquid biopsies with ultrahigh accuracy through kinetic fingerprinting and digital counting, achieved within just 2 seconds. This high accuracy and data acquisition rate are facilitated by combining three advances:1) rational design of a probe sequence with mismatches and minimal self-structure; 2) utilization of an improved fluorophore-quencher pair with high fluorogenic ratio to utilize a high imager concentration of 5 µM; 3) an optimized surface passivation protocol. Based on these improvements, we were able to image more than 100 fields of view within a few minutes, yielding limits of detection for three cancer-related DNA targets, HPV, T790M, and L858R, with virtually zero background. As a proof-of-concept, we deployed FG-SiMREPS for HPV detection in human patient samples. We anticipate our approach to pave the way for single-molecule multiplexing and high-throughput detection of a multitude of biomarkers of disease.

Keywords: Fluorogenic, Single-molecule fluorescence, Biosensor