Diagnosing Active Hepatitis C Virus Infection by Electrical Detection of Protein Biomarkers using a Nanopore Device

DESCRIPTION provided by applicant This proposal will establish a novel and cost effective method for screening at risk populations for active Hepatitis C Virus HCV infection by electrically detecting individual protein biomarkers Our novel method blends the single molecule sensitivity of a nanopore with high affinity reagents designed to target only specific biomarkers of interest Achievable characteristics of our portable device include high assay sensitivity andlt IU ml and accuracy confidence ease of use in line automation rapid time to results andlt min sample in to results out and low cost $ test This proposal focuses on detecting the core antigen of HCV HCVcAg since it is a well studied and short lived biomarker that can be quantitated for the diagnosis of both acute and chronic infections To achieve HCV detection we use bi functional fusion molecules in which one end of the fusion captures the target HCVcAg in solution while the other end anchors the fusion biomarker complex to a unique location on a dsDNA scaffold When each scaffold fusion passes through the pore the occupancy status of the fusion is detected because the event depth and or duration values are larger for the larger molecule i e when the biomarker is bound providing a simple yes no criterion for every event Moreover our assays are designed so that all other background molecules e g from serum produce events distinguishable from the scaffold fusion complexes The software filters out background events thereby allowing for quantitation of the HCVcAg bound scaffolds This proposal has three aims Aim months Create the dsDNA fusion molecule and biochemically show that it binds the HCVcAg biomarker in solution Fusion molecules comprise a dsDNA binding Protein Nucleic Acid domain and an HCVcAg specific DNA aptamer Formation of dsDNA fusion HCVcAg will be confirmed using gel shift assays Aim months Using the DNA fusion reagent and nanopore technology electrically detect the presence of HCVcAg biomarkers in solution Existing mathematical tools will assess the statistical significance of detection Aim months Titrate HCVcAg to establish the limit of biomarker detection and perform experiments in the presence of background protein and diluted serum This aim investigates the effects of background on detection and helps assess if reagent redesign is needed due to non specific interactions with the dsDNA fusion PUBLIC HEALTH RELEVANCE Project Narrative The proposed hand held platform assays a biological fluid and achieves single molecule quantitation of protein biomarkers for Hepatitis C virus sensitive enough to detect whether a person is newly or chronically infected Current prototypes cost $ per device module and $ in consumables per test Such a device could reach at risk populations that do not have access to diagnostics thereby aiding in tracking disease progression vectors facilitating viral containment and preventing new cases from emerging