EAGER: Enzymatic Perturbation Specificity Test for Self-Validating, In-Situ Regenerable Nanoengineered Molecularly Imprinted Polymer Sensors in Interstitial Fluid

Wearable and point of care biosensors can deliver earlier and more personalized health information than laboratory tests. However, many sensors cannot verify that the signals they produce truly come from a targeted molecule, especially in a complex and changing biofluid. This project will develop a built in “specificity check” for sensors operating under the skin. The Enzymatic Perturbation Specificity Test (EPST) will measure a sample and then measure it again after an enzyme briefly converts or removes the target molecule. A predictable drop in signal provides evidence that the sensor response is driven by the correct target. The project will pair EPST with reusable synthetic receptor sensors that can be electrically reset between measurements. To show that the method applies across chemistry types, the work will focus on two stress related biomarkers from different molecular classes: cortisol (a steroid hormone) and neuropeptide Y (a peptide). Outcomes will include openly shared protocols, datasets, and analysis tools, plus training for students in materials, microfluidics, biochemistry, and data analysis. By improving confidence and reducing false positives, EPST could help advance reliable, affordable biosensing that supports the national interest in health, prosperity, and welfare. This EAGER project will develop an Enzymatic Perturbation Specificity Test (EPST) to self-validate biosensors based on nanoengineered molecularly imprinted polymers (NCMIPs). EPST will collect paired native and enzyme perturbed measurements in which the free target concentration is reduced by a verified conversion/depletion fraction. It will use the differential suppression to compute a Specificity Confidence Metric. The project will fabricate restorable NCMIP recognition layers and will optimize an in situ restoration cycle for stable repeated operation in buffer and interstitial fluid mimicking matrices. Then, the project will build interchangeable immobilized enzyme microreactors with composition matched ON/OFF controls and will map conversion/depletion fraction as a function of residence time using orthogonal chromatography/mass spectrometry assays. A time multiplexed single sensor architecture will be the primary testbed. Split path sensing and norepinephrine perturbation will be conditional stretch targets. EPST performance and Specificity Confidence Metric thresholds will be calibrated to a ≤5% false positive rate using designed negative controls across independent sensor batches. Deliverables will include a transferable EPST playbook with open datasets and code for reproducible specificity auditing 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: 2615022 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Rahim Esfandyarpour | Institution: University of California-Irvine, IRVINE, CA | Award Amount: $210,000 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2615022 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2615022.html