PROJECT SUMMARY Rapid, inexpensive detection of biomarkers at the point of care is vital for many clinical purposes. However, limitations in current detection platforms have prevented the sensitive detection of many protein and small molecule biomarkers, forcing clinicians to rely on either potentially inaccurate empirical diagnosis or expensive lab tests to make critical treatment decisions. Sensitive detection of nucleic acid targets has been readily achieved by exploiting Watson-Crick base pairing to amplify signals (e.g., PCR), but there has been a lack of innovation for detection of low concentration antigens and small molecules at the point of care. Nature, on the other hand, has evolved intricate mechanisms for rapidly amplifying protein signals in vivo via post-translational modification and protein-based signaling networks that could be adapted for biomarker detection. Towards the goal of developing novel, rapid, ultrasensitive diagnostics, the central hypothesis of this project is that in vitro, protein-based signaling networks incorporating self-amplifying motifs can detect clinical biomarkers. Specifically, we plan to incorporate three mechanisms of protein signaling networks with potential for diagnostics: split enzyme reconstitution, autocatalytic positive feedback loops, and small molecule biosensors. We have previously demonstrated the in vitro use of split adenylate cyclase for small molecule detection via the simultaneous binding of two proteins (i.e. a sandwich assay in solution), bringing two halves of adenylate cyclase together and producing cAMP. Aim 1 (K99) will incorporate binding domains for detection of clinically relevant biomarker targets and investigate detection in human sample matrices. We will test different binding domains fused to split adenylate cyclase fragments to detect Hepatitis C Core Antigen (HCVcAg). Towards the development of a point-of-care diagnostic, Aim 2 (K99) will optimize reaction components for lyophilization and long-term storage and develop a hand-held point-of-care reader to detect the shift in fluorescence produced by the FRET-based cAMP biosensor. Lab-bench validation of all components will be performed using HCVcAg spiked into commercially purchased pooled blood samples. Aim 3 (R00) will investigate the use of split adenylate cyclase and cAMP receptor protein to create an autocatalytic feedback loop in vitro. This work would result in the first known in vitro protein signaling network with bistable switch-like sensing, a significant advancement for the diagnostics field. If successful, this system would be broadly applicable for protein and small molecule detection and could be used to detect a wide range of target analytes with known binding domains. As such, future work includes the incorporation of binding domains to detect other high value protein and small molecule analytes currently unable to be rapidly detected at the point of care and the clinical translation of the described diagnostic device with pilot clinical trials in collaboration with both domestic and foreign clinical partners.

Rapid, inexpensive detection of protein biomarkers of disease at the point of care is critical for many clinical purposes but is currently not possible for many important clinical targets. Here, we propose a new detection technique that uses in vitro, protein-based signaling networks incorporating self-amplifying enzymatic pathways that results in biomarker detection platforms with unparalleled sensing capabilities in less than two minutes. While initial detection designs will be applied for the point-of-care detection of Hepatitis C Virus, the detection system can be applied to sense any protein or small molecule biomarker with known binders, and as such could be used as a platform for the detection of many analytes currently unable to be rapidly detected at the point of care.