PROJECT SUMMARY Early detection of cancer is amongst the most important problems to address in biomedical research because the earlier cancer is detected, the better chance of a cure or a successful treatment. In contrast to the current approach of DNA liquid biopsy, we propose to develop an ultra-sensitive immunoassay technology that enables protein liquid biopsy for early cancer detection. By leveraging our insights in improving signal-to-background ratio, we have developed a prototype immunoassay named NULISA, or NUcleic acid-Linked Immuno- Sandwich Assay, that has achieved unprecedented level of detection sensitivity of any commercially available immunoassays at ~20,000 molecules/mL (i.e. ~30 aM). In this proposal, we aim to optimize the NULISA assay to further boost lower limit of detection and limit of quantitation (LOD and LOQ) and test a few candidate markers for their potential in early cancer detection. The specific aims of this Phase 1 SBIR proposal include: 1) Optimize single plex NULISA for ultrasensitive detection of protein markers in plasma and demonstrate minimally 10- fold better detection sensitivity over the most sensitive immunoassay on market; and 2) Demonstrate initial proof of concept of NULISA for early cancer detection. At the end of this project, we expect to have established a high performance NULISA assay and the feasibility of novel markers for early cancer detection. The longer-term goal of the project is to develop noninvasive single- and/or pan-cancer screen tests that could detect cancer much earlier for more successful or curative treatment.