Glutamine biosensors for biotechnology applications, with suppression of the endogenous glutamate signal.

Glutamine membranes for amperometric measurements are described. The interference of the endogenous glutamate is greatly diminished by using a supplementary "anti-glutamate" layer consisting of immobilized glutamate oxidase and catalase on top of the glutamine-sensitive layer having co-immobilized glutaminase and glutamate oxidase. The use of polycarbonate membranes with different permeability characteristics for the control of the substrate's access to the enzyme layers is presented, as well as the effect of the density of the enzyme layer on the sensitivity of these membranes. The fabricated membranes have good operational stability (at least 5 days) and very good linearity (up to 10 mM glutamine). Using an appropriate choice of membranes and cross-linking conditions, membranes with good rejection of glutamate have been fabricated (less than 6% RSD for a 5 mM glutamine sample containing 5 mM glutamate as interferent). These membranes are suitable for monitoring of glutamine levels in mammalian cell cultures without the need of a separate measurement for glutamate.

An implanted peritoneal oxygen tonometer that can be calibrated in situ.

Oxygen tension in the peritoneum has been continuously measured with a Silastic tonometer having an integral oxygen electrode and inlet and outlet tubes for gasequilibrated electrolyte solution. Remote kinetic calibration of the system is periodically performed. Tonometers were implanted in six rabbits. Peritoneal oxygen tension was measured in awake and anesthetized rabbits under various oxygen breathing conditions.

Long-term stability of electroenzymatic glucose sensors implanted in mice. An update.

Protection of the enzyme layer of glutaraldehyde vapor-stabilized glucose oxidase-based glucose sensors from attack by proteolytic enzymes and peritoneal macrophages can be accomplished by covering with a regenerated cellulose (viscose) membrane, as commonly used for laboratory dialysis. These implanted sensors are not externally polarized, but continuously consume oxygen and glucose and generate gluconic acid and hydrogen peroxide. On the average (sensors in 45 mice) the activity declines with time, almost vanishing by 600 days. However, the fact that some sensors retain nearly all of their activity for over 500 days indicates that glucose sensors can be made with a life span compatible with the requirements for an artificial pancreas (glucose sensor/insulin pump). Limited observation with similar implanted lactate sensors indicates their life span to be shorter.

Long-term implantation of voltammetric oxidase/peroxide glucose sensors in the rat peritoneum.

Methods for designing, fabricating, testing in vitro and in vivo, and improving chronically implantable oxidase/peroxide-type polarographic glucose sensors are described. Voltammetric means to evaluate oxygen supply to the sensor and to measure the nearby microcirculation with hydrogen washout techniques using the implanted glucose sensor are outlined. Because some peritoneally implanted sensors have, perhaps surprisingly, remained functional for months, such devices may prove with further development to be useful as the sensing components in artificial pancreatic beta cells for the control of diabetes.

Polarographic cerebral oxygen availability, fluorocarbon blood levels and efficacy of oxygen transport by emulsions.

In order to relate blood perfluorocarbon (PFC) level to brain tissue oxygen availability (aO2) and respiratory oxygen (FIO2), twelve conscious rabbits with chronically implanted platinum cathodes were infused with six types of emulsions in 14 infusions and their response to oxygen and carbogen breathing recorded. Blood was removed for sampling and to avoid a volume overload. Blood lactate was measured as an indicator of adequacy of perfusion. Glucose, osmotic pressure, packed cell volume, PFC by combustion and volatilization were also measured in blood samples. Methyl prednisolone was administered to 5 rabbits. Blood PFC levels measured by the commonly used centrifugation method (Fluorocrit) were subject to considerable variation, depending mainly upon centrifugation time. Fluorocrit values after Oxypherol infusion decreased from an average of 43% for 5 minutes to 15% for 30 minutes centrifugation. Fluorocrit tended to slightly increase with time of centrifugation when phospholipid was used as the emulsifier, early in PFC infusion and on the next day. Blood fluorocarbon was always lower, about half that of 30 minutes of centrifugation, when determined by combustion or by volatilization, than by centrifugation. The increase in brain a O# response to oxygen and carbogen was observed at a lower PFC blood level than expected and in some animals appeared in either the right or left hemisphere, but not in both, suggesting that oxygen transport, at least near the electrode, was by other than oxygen solubility in blood PFC. Blood lactate proved to be an excellent monitor of whole body perfusion. Animals with a blood lactate above 5 mM/L at 1 hour post infusion died the following day. The fluorocrit after 5, 10, 20, and 30 minutes of centrifugation, which we have named the "fluoro-gram," can have a sharp downward, a slight upward curve, or stay level, depending upon the type of emulsifier, the amount of PFC circulating and the time it has circulated. The fluorocarbon-induced increases in aO2 persist through the third day. This enhanced cerebrocortical aO2 current can be very roughly calculated as: aO2 current equals the air aO2 current plus (the 30 minute fluorocrit times K) where K for oxygen is 0.05 and for carbogen is 0.07. Some enhancement of the aO2 current in oxygen lingers to the fifth day after the blood PFC level is vanishingly small. Methyl prednisolone has a transient effect in suppressing the PFC enhanced oxygen and carbogen aO2 responses and no effect on the Oxypherol fluorogram.(ABSTRACT TRUNCATED AT 400 WORDS)