Project Summary/Abstract Monitoring the state of a biological system in terms of biochemical composition is of significance to many research and patient care situations, from controlling bioreactors and in vitro cell culture systems to animal subjects and human patients. Metabolite monitoring has become essential to basic understanding of biological processes and the ability to track metabolic changes over time is considered critical to precision medicine. However, current approaches to such measurements are limited in availability, flexibility, and general functionality or are simply too costly or complex for use in many situations. A versatile, expandable approach to provide specific, frequent (real-time/on-demand), and stable measurements of various metabolic biomarkers is needed to facilitate high-throughput data collection on small-molecule metabolites, as this will lead to enhanced understanding of and control over a wide variety of biological systems. We propose to address this problem with a versatile metabolite sensing platform based on a pairing of biocompatible ?smart? materials with customized optical instrumentation. Specifically, phosphorescent hydrogels and miniaturized optical readers will be developed. A novel materials approach will be applied to produce hydrogels with embedded spherical microdomains to precisely control diffusion-reaction processes, providing control over enzymatic operation at different substrate supply rates. The hydrogel provides a stable biocompatible interface to the surrounding environment (tissue/cells, etc.) and maintains the microdomains in a fixed location so they may not migrate and may be removed at a later point. The materials are moldable into form factors allowing attachment to culture substrates or needle insertion into organ cultures or living tissue. This new sensing platform requires successful integration of two core features that connect directly to the Specific Aims: 1) robust hydrogel-based phosphorescent sensors that can integrate directly into any biological system (tissue, cell culture, etc) and 2) robust optical measurement systems that can be miniaturized and directly interfaced with any relevant biological system. Stable, biocompatible hydrogels functionalized with oxygen- sensitive phosphors will be designed to integrate with in vitro and in vivo disease models (Specific Aim 1). Design rules for enzymatic hydrogels to function in different environments will be established, enabling materials that will respond in proportion to concentration of target analytes (Specific Aim 2). Reference materials will be incorporated to ensure accurate and robust analysis (Specific Aim 3). Optical instrumentation to noninvasively monitor hydrogels in various vessels and form factors will be designed, built, and tested (Specific Aim 4).