Glutaraldehyde Cross-Linked Glutamate Oxidase Coated Microelectrode Arrays: Selectivity and Resting Levels of Glutamate in the CNS

Abstract

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.

Microelectrode Biosensors for in vivo Analysis of Brain Interstitial Fluid

“…This length may determine the flexibility of the protein matrix and the degrees of freedom that proteins retain once they are immobilized, thereby affecting substrate specificity. Interestingly, when glutaraldehyde is preincubated in bovine serum albumin (BSA) prior to enzyme immobilization, these effects on substrate specificity are attenuated . It is possible that under these conditions, enzyme immobilization is achieved with glutaraldehyde molecules that are already bound to BSA, and this would provide a longer chain of cross‐linked molecules for immobilization.…”

“…). This recent convergence of results is probably related to technical improvements in microelectrode designs and enzyme immobilization procedures . The effect of neuronal firing on resting glutamate concentrations is also debated.…”

REAL-TIME IN VIVO NEUROTRANSMITTER MEASUREMENTS USING ENZYME-BASED CERAMIC MICROELECTRODE ARRAYS: WHAT WE HAVE LEARNED ABOUT GLUTAMATE SIGNALING

“…However, one recent report suggested that the high selectivity of GluOx may be altered when glutaraldehyde is used as the cross-linking agent and thereby resulting in an increase in perceived resting glutamate levels when using wire-type microelectrodes (Vasylieva et al, 2013). However, studies using ceramic-based MEAs support that GluOx is selective over other amino acids when using our coating protocol of entrapping GluOx in a glutaraldehyde cross-linked BSA matrix (Burmeister et al, 2013). For one, we only observed minimal responses to select amino acids with glutaraldehyde cross-linked glutamate oxidase-coated MEAs, and furthermore, functional glutamate MEA biosensors employed for in vivo experiments use multiple means to minimize the potential interference from electroactive molecules (e.g., exclusion layers and self-referencing subtraction).…”

“…For example, first one uses a highly selective enzyme, like glutamate oxidase (GluOx) which is extremely selective for l-glutamate over other amino acids (Kusakabe et al, 1983). Then GluOx is entrapped in a protein matrix such as bovine serum albumin (BSA) that is cross-linked with glutaraldehyde to retain the high enzyme-selectivity and activity after immobilization onto the MEA surface ( (Burmeister et al, 2013) green area shown in Figure 5.2).…”

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

“…Glutaraldehyde is a crosslinking agent that causes changes in the properties of albumin. The color change seen in Figure 1 is attributed to the formation of the aldimine linkages [19][20][21] . The glutaraldehyde reacts predominantly with the BSA through the amino groups of lysine to form the intermolecular covalent bonds 14 .…”