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

McMahon, Colm P.; Rocchitta, Gaia; Serra, Pier Andrea; Kirwan, Sarah M.; Lowry, John; O’Neill, Robert

doi:10.1039/b511643k

Cited by 50 publications

(83 citation statements)

References 54 publications

(98 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These data are consistent with the hypothesis that for anionic substrates such as Glu interacting with polyanionic proteins (pI for GluOx is 6.2), 19 high loading of enzyme leads to an electrostatic barrier, reducing the affinity of enzyme for its substrate. 79 The overall outcome of this interaction is that increasing the density of GluOx on the disk surface increases the K M (Glu) significantly (see Figure 3) but has little effect on the LRS; indeed, there was no correlation (R 2 ) 0.01, n ) 62) between Glu sensitivity in the linear response region (LRS) and enzyme loading (J max ) over the range of J max shown in Figure 3.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

et al. 2006

Self Cite

View full text Add to dashboard Cite

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K M (Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K M (O 2 ). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K M (O 2 ) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.L-Glutamate (Glu) is the most widespread excitatory neurotransmitter in the mammalian central nervous system, 1 plays a major role in a broad range of brain functions, and has been implicated in a number of neurological disorders. 2 Indeed, the development of devices for Glu detection has become an important research area due to the value of monitoring this key amino acid in a number of complex matrixes, including food processing, 3,4 cell cultures, 5-7 tissue slices ex vivo, 8,9 and intact brain in vivo. [10][11][12][13][14][15] A number of designs of biosensors for Glu monitoring have been reported and, although Glu receptors 16 Brain Res. Bull. 2002, 58, 401-404. (10) Burmeister, J. J.; Gerhardt, G. A. Trends Anal. Chem. 2003, 22, 498-502. (11)

show abstract

Section: Resultsmentioning

confidence: 99%

“…We are presently exploring the behavior of these biosensors in the intact brain in terms of sensitivity, selectivity, and stability in vivo. The oxygen dependence of PECbased biosensors, whose Glu sensitivity has been increased significantly by the incorporation of polyelectrolytes, 79 is also under investigation.…”

Section: Discussionmentioning

confidence: 99%

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…(1), it is evident that the selectivity coefficient depends on the sensitivity of the biosensors to glutamate. To improve the Glu sensitivity of the biosensor, a cationic polymer PEI was incorporated into the sensor matrix [22]. Disc sensors were therefore formed by dip coating PEI onto platinum discs, followed by dip evaporation of the enzyme and electropolymerization of the monomer o-PD with BSA at 0.65 V for 15 min.…”

Section: Pt D /Pei/gluox/ppd-bsamentioning

confidence: 99%

“…Some electrode configurations were dipped in a 5% solution of Nafion and dried before being cured for 7-10 min at 170-180 • C [20,21]. The electrodes were dipped in a 1% solution of a polycationic polymer PEI and dried for 15 min immediately before immobilizing GluOx [22]. Deposition of the enzyme was carried out by immersing the electrodes in a buffered solution of the enzyme GluOx for 5 min to allow the adsorption of the enzyme on the discs and letting them dry for 5 min.…”

Section: Preparation Of the Working Electrodesmentioning

confidence: 99%

Highly selective and stable microdisc biosensors for l-glutamate monitoring

Govindarajan

McNeil

Lowry

et al. 2013

Sensors and Actuators B: Chemical

Self Cite

View full text Add to dashboard Cite

a b s t r a c tGlutamate mediates most of the excitatory synaptic transmission in the brain, and its abnormal regulation is considered a key factor underlying the appearance and progression of many neurodegenerative and psychiatric diseases. In this work, a microdisc-based amperometric biosensor for glutamate detection with highly enhanced selectivity and good stability is proposed. The biosensor utilizes the enzyme glutamate oxidase which was dip-coated onto 125 m diameter platinum discs. To improve selectivity, phosphatidylethanolamine was pre-coated prior to enzyme deposition, and electropolymerization of o-phenylenediamine was performed to entrap the enzyme within a polymer matrix. A variety of coating configurations were tested in order to optimize biosensor performance. For stability measurements, biosensors were biased continuously and calibration curves calculated each day for a period of 5-6 days. The optimized biosensors exhibited very high sensitivity (71 ± 1 mA M −1 cm −2 ), low detection limit of ∼2.5 M glutamate, selectivity (over 87% against ascorbic acid), very good temporal stability during continuous use, and a response time of <5 s. These biosensors are therefore good candidates for further development as devices for continuous monitoring during traumatic brain injury or neurosurgery.

show abstract

“…Using implantable sensors allows for stable, long-term recording of a number of common analytes in the extracellular fluid (ECF) of the brain including oxygen (Lowry et al, 1996Lowry and Fillenz, 2001;Bolger and Lowry, 2005), glucose (Hu and Wilson, 1997;Fillenz and Lowry, 1998a;Lowry et al, 1998a,b,c;Lowry and Fillenz, 2001;Dixon et al, 2002), nitric oxide (Brown et al, 2009) and glutamate (Kulagina et al, 1999;McMahon et al, 2006aMcMahon et al, , 2006bMcMahon et al, , 2007Qin et al, 2008;Tian et al, 2009). Unlike other methods implanted into the brain tissue (Clark et al, 1958;Krolicki and Leniger-Follert, 1980;Doppenberg et al, 1998;Gupta et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous recording of hippocampal oxygen and glucose in real time using constant potential amperometry in the freely-moving rat

Kealy

Bennett

Lowry

2013

Journal of Neuroscience Methods

Self Cite

View full text Add to dashboard Cite

h i g h l i g h t sWe show that average concentrations of hippocampal oxygen and glucose are 100.26 ± 5.76 M and 0.60 ± 0.06 mM respectively. We show that there are uncoupled changes in oxygen and glucose during neuronal activation. Anaesthesia and carbonic anhydrase inhibition both significantly increase hippocampal oxygen. Anaesthesia, dimethyl sulfoxide administration and carbonic anhydrase inhibition significantly increase hippocampal glucose. We show that changes in hippocampal metabolism can be detected in real time using constant potential amperometry. a r t i c l e i n f o t r a c tAmperometric sensors for oxygen and glucose allow for real time recording from the brain in freelymoving animals. These sensors have been used to detect activity-and drug-induced changes in metabolism in a number of brain regions but little attention has been given over to the hippocampus despite its importance in cognition and disease. Sensors for oxygen and glucose were co-implanted into the hippocampus and allowed to record for several days. Baseline recordings show that basal concentrations of hippocampal oxygen and glucose are 100.26 ± 5.76 M and 0.60 ± 0.06 mM respectively. Furthermore, stress-induced changes in neural activity have been shown to significantly alter concentrations of both analytes in the hippocampus. Administration of O 2 gas to the animals' snouts results in significant increases in hippocampal oxygen and glucose and administration of N 2 gas results in a significant decrease in hippocampal oxygen. Chloral hydrate-induced anaesthesia causes a significant increase in hippocampal oxygen whereas treatment with the carbonic anhydrase inhibitor acetazolamide significantly increases hippocampal oxygen and glucose. These findings provide real time electrochemical data for the hippocampus which has been previously impossible with traditional methods such as microdialysis or ex vivo analysis. As such, these sensors provide a window into hippocampal function which can be used in conjunction with behavioural and pharmacological interventions to further elucidate the functions and mechanisms of action of the hippocampus in normal and disease states.

show abstract

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

Cited by 50 publications

References 54 publications

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

Highly selective and stable microdisc biosensors for l-glutamate monitoring

Simultaneous recording of hippocampal oxygen and glucose in real time using constant potential amperometry in the freely-moving rat

Contact Info

Product

Resources

About