Immobilization techniques to avoid enzyme loss from oxidase-based biosensors: a one-year study.

BACKGROUND: Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. METHODS: A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. RESULTS: The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. CONCLUSIONS: After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased.

A wire-based dual-analyte sensor for glucose and lactate: in vitro and in vivo evaluation.

Continuous measurement of lactate is potentially useful for detecting physical exhaustion and for monitoring critical care conditions characterized by hypoperfusion, such as heart failure. In some conditions, it may be desirable to monitor more than one metabolic parameter concurrently. For this reason, we designed and fabricated twisted wire-based microelectrodes that can measure both lactate and glucose. These dual-analyte sensors were characterized in vitro by measuring their response to the analyte of interest and to assess whether they were susceptible to interference from the other analyte. When measured in stirred aqueous buffer, lactate sensors detected a very small amount of crosstalk from glucose in vitro, although this signal was less than 3% of the response to lactate. Glucose sensors did not detect crosstalk from lactate. Sensors were implanted subcutaneously in rats and tested during infusions of lactate and glucose. Each sensing electrode responded rapidly to changes in its analyte concentration, and there was no evidence of in vivo crosstalk. This study constitutes proof of the concept that oxidase-based, amperometric wire microsensors can detect changes in glucose and lactate during subcutaneous implantation in rats.