CAREER: Expanding the dimensional and multiplexing frontiers of ultrasensitive single-molecule protein detection

The collection of active proteins in the body contains vast information that can improve disease understanding and diagnosis. However, many disease-related proteins exist at very low levels and are difficult to detect. Furthermore, many detection tools measure protein abundance but not protein function. This CAREER project will develop ultrasensitive tools to measure simultaneously low abundance proteins and their activities. The project will build on a recently developed single-molecule protein detection technology that enables protein measurements at concentrations far below the limits of conventional assays. The project will establish a design framework to detect many low abundance proteins and eliminate false signals. It will develop a complementary approach to measure both protein function and abundance. The platforms will be high-throughput, modular, and compatible with common laboratory equipment. These technologies will enable researchers and clinicians to maximize biological information from valuable samples. Results will help accelerate discovery of key proteins involved in disease processes. The project will help train the next generation of diagnostics innovators and promote diagnostics literacy. Activities will include mentored research experiences for high school and undergraduate students, hands-on K-12 diagnostics workshops with educational videos, and a new graduate course in biomolecular engineering. These efforts will promote scientific progress in protein measurement tools, strengthen the future biotechnology workforce, and enhance preparedness for public health challenges. Proteins play critical roles in biological processes. The extraordinary complexity of the human proteome presents a vast, untapped reservoir for understanding disease. Many current protein measurement tools lack the analytical sensitivity, multiplexing capacity, and broad accessibility required to probe low-abundance proteins and their activities in complex biofluids with high throughput and scalability. To overcome these challenges, this CAREER project will advance the multiplexing and dimensional frontiers of ultrasensitive single-molecule protein detection, integrating DNA barcoding and activity-based detection with a high-throughput digital immunoassay platform. The project will pursue two orthogonal research objectives: (1) establishing the foundational design principles for a cross-reactivity-free multiplex digital immunoassay platform; and (2) engineering a platform for multiparametric ultrasensitive protease activity profiling using single-molecule detection. These tools will not only maximize the biological insight from valuable clinical and research samples for biomarker discovery and disease understanding, but also advance knowledge in single-molecule assay design. Through integration with an education and outreach program focused on diagnostics literacy and early hands-on learning experiences, this project will also foster training of the next generation of biotechnology innovators in measurement science and diagnostics. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2541796 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Connie Wu | Institution: Regents of the University of Michigan - Ann Arbor, ANN ARBOR, MI | Award Amount: $549,998 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2541796 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2541796.html