The sensor is based on the reaction:
pyruvate + phosphate + O2 → acetyl phosphate + CO2 + H2O2
This reaction is catalyzed by the enzyme pyruvate oxidase and is highly specific for pyruvate. It uses oxygen sensors to detect a difference in oxygen concentration between a reference electrode and the sensor electrode. The difference in oxygen correlates to the amount of pyruvate, the limiting reagent, consumed in the enzymatic reaction. This sensing method is similar to how the widely available glucose sensors works.
Design functions of the pyruvate sensor were determined. In order for it to be useful in the clinical setting, the pyruvate sensor must be sensitive to pyruvate over its physiological range in humans (approximately 0.04mM to 0.12mM). These levels should occur in the sensor’s linear response range for better measurement accuracy. The response time of the sensor should be low, about two minutes. Having a fast response time is important because often, instantaneous data is needed so that correct medical decisions can be made based on the data. The sensor must be stable enough for use over a period of several hours to a few days. Having a sensor operating longer periods allows for continuous pyruvate monitoring without having to implant a replacement sensor. This project will test a sensor on the bench top over the course of one week and will not attempt to engineer a sensor that will last longer than a week. Improvements in durability are difficult and may involve genetically modifying the central enzyme used (pyruvate oxidase) or by utilizing different enzyme immobilization techniques for enhanced enzyme stability. Even though this project will not perform in vivo tests, the sensor must be biocompatible and small enough that is able to be implanted. The sensor will be housed in silicone rubber, a strong, flexible material that is also found to be safe in a variety of toxicological and biocompatibility tests [1].
The central enzyme required for sensor operation, pyruvate oxidase, will be entrapped in a gel matrix and located at the tip of the silicone sensor tubing. The immobilization technique that will be used is glutaraldehyde crosslinking to albumin. Crosslinking will produce a stable enzyme entrapped in a mesh of albumin. The dimensions of the sensing region will be length = 3.0 mm and diameter = 2.0 mm. These dimensions will produce a predictable diffusion rate of oxygen that can be used in our mass transfer model to calculate sensor behavior. This configuration also ensures that there is excess oxygen present for the enzyme reaction since oxygen is able to diffuse radially through the silicon tube. The reference oxygen electrode (used to sense background oxygen levels) will be housed adjacent to the sensing electrode. The signal difference between the sensing electrode and the reference electrode is the indicator of oxygen consumption by the enzyme reaction and therefore a measurement of pyruvate levels.
Drawing
representing crosslinking
[1] Mohanan, P.V. Biocompatibility studies on silicone rubber Proceedings RC IEEE-EMBS & 14th BMESI. 1995.