Development of a Pyruvate Sensor and Instrumentation

University Of California-San Diego, La Jolla CA

Investigators

Abstract

NSF Proposal 9987273 Development of a Pyruvate Sensor and Instrumentation Dale A. Baker, Ph.D. Project Abstract Pyruvate is a chemical, which is involved in many biological pathways and their regulation. The knowledge of its concentration is important not only in studies of human and animal physiology, but also in the control of industrial processes, such as fermentations. Currently, pyruvate is most often measured by a chemical procedure that takes several minutes to complete and requires pretreatment of turbid samples, e.g. blood, microbial growth media or food samples, in order to remove particulate matter for accurate measurement. Furthermore, the chemicals used in this method are washed away and cannot be re-used after this single measurement. Continuous measurements are not possible with this technique. To overcome these disadvantages, the pyruvate sensor in this project will use a membrane that embeds (immobilizes) an enzyme, and in conjunction with an electrode, measures the concentration of pyruvate. A pyruvate sensor will be developed that can quantitatively and continuously monitor pyruvate concentration by using the immobilized enzyme, pyruvate oxidase, in a membrane, which is coupled to an oxygen sensitive electrode. A mathematical model that describes the chemical processes within the sensor and subsequent simulations will be used to design the sensor and better understand its operation. Experiments will be conducted with sensors that are fabricated having the design features suggested from the modeling studies. The purpose of the initial experiments is to characterize the sensor response to changes in pyruvate concentration under well-controlled conditions. These experiments will also allow validation of the sensor performance before the sensor can be considered for use in more demanding physiology studies or in bioreactors. Additionally, the role of the two major properties of the enzyme membrane, the enzyme reaction kinetics and the substrate transport, will be investigated with an experimental apparatus for membrane characterization. An engineering analysis of the predominant mechanisms affecting the enzyme performance can be completed from the data collected with this apparatus. The pyruvate sensor will be used for basic biological research applications and has potential for studies of physiology in animals and humans. Many bacterial cultures depend on the level or control of pyruvate concentration to enhance growth or augment the production of valuable chemicals or enzyme products. In mammalian physiology, conditions of low blood flow (ischemia) or lowered oxygen levels (hypoxia) can cause significant changes in blood pyruvate concentration. The sensor will dynamically indicate any accumulation in pyruvate, which can reflect a transition from normal (aerobic) metabolism during these pathological conditions. It is anticipated that an acceptable biosensor can be developed to continuously monitor pyruvate concentration. These research studies will provide a systematic way to enhance the enzyme membrane, which is an essential component of the sensor used to detect and quantify the pyruvate. Improvements in the membrane will refine the sensitivity and stability of the complete sensor.