###### Abstract  
Digestive health is critically dependent on the secretion of enzymes from the exocrine pancreas to the duodenum via the pancreatic duct. Specifically, pancreatic trypsin is a major protease responsible for breaking down proteins for absorption in the small intestine. Gelatin-based hydrogels, deposited in the form of thin films, have been studied as potential sensor substrates that hydrolyze in the presence of trypsin. In this work, we (1) investigate gelatin as a sensing material; (2) develop a fabrication strategy for coating sensor surfaces; and (3) implement a miniaturized impedance platform for measuring activity levels of pancreatic trypsin. Using impedance spectroscopy, we evaluate gelatin's specificity and rate of degradation when exposed to a combination of pancreatic enzymes in neutral solution representative of the macromolecular heterogeneity present in the duodenal environment. Our findings suggest gelatin's preferential degradation to trypsin compared to enzymes such as lipase and amylase. We further observe their interference with trypsin behavior in equivalent concentrations, reducing film digestion by as much as 83% and 77%, respectively. We achieve film patterns in thicknesses ranging from 300-700 nm, which we coat over interdigitated finger electrode sensors. Finally, we test our sensors over several concentrations to emulate the range of pancreatic secretions. Ultimately, our microsensor will serve as the foundation for developing in situ sensors toward diagnosing pancreatic pathologies. Gelatin; pancreas; impedance sensing; biomaterial films; biosensors; diagnostics 2018 20  
as trypsin hydrolyzes specific peptide linkages that are formed from the amino acids lysine and arginine. Gelatin is a hydrolytic product of collagen, and the above amino acids consist of around 10-20% of most final gelatin forms [10]. Gelatin is also biocompatible, and therefore an attractive substrate material. As a hydrogel that begins as a low viscosity liquid, it can be formed to a variety of structures that are limited only by the tools used until it cools and transitions from sol to gel state. One such structure is a thin film, common for various electronic sensors made from standard methods in monolithic fabrication. Deposition of hydrogel thin films has been achieved, while in the sol state, through strategies such as spin-coating, drop-casting, dip-coating, etc. [11]. Furthermore, crosslinking is utilized often to enhance the stability of the material, and is limited by the reactive functional groups in the structure's chemistry [12]. Glutaraldehyde, for example, has been used to allow for gelatin films to maintain structural integrity at higher temperatures [13; 14]. Though it can be dangerous to cells, low concentrations do not significantly enhance toxicity. Most biochemical sensors for biomedical applications are not concerned with toxicity as they are often utilized in vitro, where the samples are either in buffer or fluids that have been removed from the body. Few sensing strategies are designed to operate in vivo, as there are significant risks associated when implementing foreign objects in the physiological environment, as well as sample complexity due to molecular heterogeneity in physiological fluids. When targeting trypsin, which, as discussed above, originates near the duodenum, regional sample heterogeneity, such as potential interfering species must be considered when performing sensor testing. In this study, we developed and tested a sensing strategy for measuring trypsin activity in solutions containing several types of interfering enzymes characteristic of duodenal fluid. Crosslinked gelatin films were formed over interdigitated finger electrode (IDE) sensors, which were fabricated similarly to those described previously [15; 16; 17]. These sensors were inserted into fluidic chambers and exposed to various pancreatic enzymes while changes in the mass and impedance responses were recorded.