Project Summary Cardiovascular disease (CVD) is the leading cause of death for people in most racial and ethnic groups in the United States. Acute Myocardial Infarction (AMI) (also called heart attack) is the top cause of death in CVD. Early detection of AMI translates to earlier intervention and improves survival rates. When cardiac myocytes are damaged, cardiac troponin (cTn), a component of the heart muscle, is released into circulation. Dynamic and incremental elevation of blood cTn levels is indicative of an on-going event of heart injury with acute cardiomyocyte damage. Thus, blood cTn level is routinely measured with commercial cTn immunoassays in patients suspected of having AMI. However, current cTn immunoassays are limited in sensitivity and speed. Since two antibodies are used in the current cTn immunoassays (one as the capture and another as the detection agents), current cTn immunoassays require multiple step reactions with limited sensitivity. Additionally, the assays are not performed in real time and typically take about 30 mins to get results that could miss the early rises in cTn levels or its rate of change thus delaying diagnosis. EZ-Lab, in collaborations with investigators at Oakland Univ., Wayne State Univ., and Univ. of Missouri-Columbia, proposes to develop a novel cTn biosensor that allows highly sensitive (pg/ml) and real-time detection of cTn based on our years of research and innovations in 1) uniquely designed peptide mimotope biosensing interface for label-free affinity based electrochemical biosensor; 2) miniaturized, low cost and real-time electrochemical sensor platform. In sharp contrast to current cTn immunoassays using two antibodies, we successfully demonstrated that peptide mimotopes, in lieu of antibodies, when immobilized on the surface via self-assemble monolayer (SAM) can significantly reduce the structural variability, retain biological activity, and minimize or eliminate non-specific adsorption from interfering proteins and provide real-time, highly sensitive and selective detection of protein antigens in human serum samples. To advance this novel peptide mimotope biosensing technology for early detection and monitoring of AMI for clinical applications, we will rationally design peptides to form robust cTn biosensing interface for electrochemical cTn sensors allowing one step and real-time detection of cTn in human serum and blood samples with three research Aims: 1. Develop a highly sensitive peptide sensing interface for detection of cTn; 2. Validate peptide SAM based cTn biosensor analytical performance using human serum and blood samples; 3. Real-time cTn sensing in blood sample. The proposed peptide mimotope cTn biosensor is expected to be highly sensitive and quantitative, faster than current cTn immunoassays used in the clinics, allowing for early diagnosis of AMI at significantly lower cost for quantifying cTn biomarkers in real world clinical samples. It is expected that our easy-to-use cTn bi...