Biosensor for Neurotransmitter L-Glutamic Acid Designed for Efficient Use of L-Glutamate Oxidase and Effective Rejection of Interference

Abstract

An amperometric biosensor for l-glutamic acid (Glu) was constructed by the adsorption and dip coating of l-glutamate oxidase (GluOx, 200 U ml 21 phosphate buffer, pH 7.4) onto 60-mm radius Teflon-coated Pt wire (1 mm exposed length). The enzyme was then trapped on the surface by electropolymerisation of o-phenylenediamine that also served to block electroactive interference. This procedure afforded electrodes with similar substrate sensitivity compared with the classical approach of immobilising enzyme from a solution of monomer, and represents an approximately 10 000-fold increase in the yield of biosensors from a batch of enzyme. A number of strategies were examined to enhance the sensitivity and selectivity of the Pt/PPD/GluOx sensors operating at 0.7 V versus SCE. Pre-coating the Pt with lipid and incorporation of the protein bovine serum albumin into the polymer matrix were found to improve the performance of the electrode. The sensors had a fast response time, high sensitivity to Glu, with an LOD of about 0.3 mmol l 21 , and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

“…1 compared with previous reports for PPD-based cylinder designs 12,15,16 ). However, much of the additional enzyme activity (J max ) does not translate down to boosting the response at lower Glu concentrations due to an increased K M value, possibly caused by an anionic electrostatic barrier associated with enzyme crowding.…”

“…The results presented here also demonstrate some limitations of using GOx as a model enzyme for developing biosensors for other enzyme systems, and provide an explanation of why multiple dip-evaporation steps increased the LR slope for biosensors incorporating GOx, but not GluOx, in a previous study. 15 …”

“…Glucose oxidase (GOx, EC 1.1.3.4, Sigma, 200 U mL 21 ) or GluOx (EC 1.4.3.11, Yamasa Corp., Japan, 200 U mL 21 ) was deposited onto the metal surface by dipevaporation (1-4 dips), and then immobilised by amperometric electropolymerisation (+700 mV vs. SCE) in phosphatebuffered saline (PBS, pH 7.4) containing 300 mM o-phenylenediamine, as described previously, to form Pt/GOx/PPD and Pt/GluOx/PPD biosensors, respectively. 15 Additional sets of biosensors were prepared by pre-coating the Pt surface with either the zwitterionic lipid phosphatidylethanolamine (PEA, type II-S, Sigma, 5 mg mL 21 in chloroform) or the polycation polyethyleneimine (PEI, Aldrich, MW r y750 kDa, 1% aqueous solution), also by dip evaporation, before enzyme deposition.…”

Contributions by a Novel Edge Effect to the Permselectivity of an Electrosynthesized Polymer for Microbiosensor Applications

“…Because of the self-sealing nature of this process, longer amperometric polymerization times do not improve interference rejection of the coating. 45 The relevant electrochemical properties of PPD-coated sensors based on the Pt-Ir alloy used in this study are indistinguishable from those based on pure Pt wire, 41 and so the representation Pt is used in preference to Pt-Ir for simplicity in modified electrode nomenclature: Pt C /PPD, and so on, as discussed previously. 2 Glutamate Biosensor Fabrication.…”

Electroanalytical Flow Measurements–Recent Advances

“…This was shown, for instance, in design of a lactate biosensor with multilayer of electrodeposited polymers [80], a biosensor for l-glutamic acid with electropolymerized ophenylenediamine [81], and a urea biosensor with urease immobilized in a composite electropolymerized polypyrrole-polyion complex film [82]. Conducting polymers can be also electrodeposited in situ in flowing conditions which was shown for producing a glucose biosensor [83] and a cholesterol biosensor [84] using different supporting working electrodes.…”