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Biosensor technology

Real time monitoring in vivo - immediate read-out, no need for HPLC analysis 
- second-by-second resolution
- "self-referencing" feature
- minimal tissue damage


In vivo electrochemistry utilizes functionalized or otherwise pre-treated microelectrodes, which are directly implanted into the mammalian CNS thereby providing a means to record electrochemical signals in the brain microenvironment. Typically, a potential is applied between the microelectrode and a reference electrode, causing the oxidation or reduction of electroactive molecules at the microelectrode surface and generation of electric current. The current generated from such Faradaic reactions is linear with respect to the concentration of the analyte, which allows continuous on-line monitoring of extracellular levels of a given analyte, e.g. a neurotransmitter glutamate. 

However, only few endogenous molecules such as hydrogen peroxide, dopamine, DOPAC or uric acid can be readily oxidized at the microelectrodes. For others, such as glutamate, lactate, choline or acetylcholine there is a need of enzymatic conversion of respective molecules, most often to easily oxidized hydrogen peroxide. This occurs via immobilization of a corresponding oxidase enzyme to the surface of the working electrode. Immobilization of the oxidase enzymes to the Pt recording sites of the electrode helps to stabilize the enzymes and keeps them active for longer periods of time.

Initially, the microelectrodes are coated with the exclusion layer in order to block or minimize undesirable electrochemically active compounds found in high concentrations in the CNS. The blockade of the undesired active molecules carrying negative net-electrical charge from reaching the Pt recording sites is conveniently achieved by coating the electrodes with negatively charged liquid polymer Nafion®. Alternatively, the electrodes are coated by electropolymerization of 1,3-phenylenediamine (mPD). Electropolymerized mPD forms a size exclusion layer that prevents larger molecules such as ascorbic acid, DA, and DOPAC from reaching the recording surface. Smaller molecules, such as hydrogen peroxide or NO, are still able to pass through the matrix. Since peroxide is a reporter molecule for oxidase enzymes, this makes mPD an ideal exclusion layer for our enzyme-based multi-site microelectrodes. A general procedure for enzyme, e.g. L-glutamate oxidase immobilization is based on cross-linking the oxidase enzyme and bovine serum albumin (BSA) via glutaraldehyde to the Pt surface of the electrode. Typically, several coatings are made by dispersing a micro-droplet of a solution containing L-glutamate oxidase, BSA and glutaraldehyde on the Pt surface. Enzyme-coated microelectrodes are cured at room temperature for 48-72 hours prior to calibration and experimentation.

The multi-site microelectrodes offer numerous advantages over the single "wire-coated" electrodes such as greater stability, reproducibility and, most importantly, a possibility of coating two adjacent pairs of electrode sites, one active site, including the oxidase enzyme and the second site, inactive - coated only with BSA protein matrix, which allows in situ "self-referencing" and subtraction of the background signals.

Adapted from
Hascup KN, Rutherford EC, Quintero JE, Day BK, Nickell JR, Pomerleau F, Huettl P, Burmeister JJ, Gerhardt GA (2007) Second-by-Second Measures of L-Glutamate and Other Neurotransmitters Using Enzyme-Based Microelectrode Arrays. In: (Eds AC Michael and LM Borland) Electrochemical Methods for Neuroscience, chapter 19. CRC Press, Boca Raton, USA, pp. 407-450.



The ceramic-based multi-site microelectrode for rapid, second-by-second measurements of extracellular glutamate levels in the CNS.
The surface of the Pt working electrode is coated with an exclusion layer (Nafion® or mPD) and the layer with immobilized Glutamate oxidase, which converts Glu to hydrogen peroxide. H2O2 can diffuse through the exclusion layer to the Pt electrode where it is oxidized and generates electric current (2 e-/molecule), which is recorded by a connected amplifier (FAST unit).

References
Bruno JP, Gash C, Martin B, Zmarowski A, Pomerleau F, Burmeister J, Huettl P, Gerhardt GA (2006) Second-by-second measurement of acetylcholine release in prefrontal cortex. Eur J Neurosci, 24:2749-2757.
Burmeister JJ, Gerhardt GA (2001) Self-Referencing Ceramic-Based Multisite Microelectrode for the detection and elimination of interferences from the measurement of L-glutamate and other analytes. Anal Chem, 73: 1037.
Burmeister JJ, Pomerleau F, Palmer M, Day BK, Huettl P, Gerhardt G.A (2001) Improved ceramic-based multisite microelectrode for rapid
measurements of L-glutamate in the CNS. J Neurosci Meth, 119:163.
Burmeister JJ,  Gerhardt GA (2003) Ceramic-based multisite microelectrode arrays for in vivo electrochemical recordings of glutamate and other neurochemicals. Trends Anal Chem, 22:498.
Gerhardt GA, Burmeister JJ (2006) Neurochemical arrays. In: Encyclopedia of Sensors (Eds. CA Grimes, EC Dickey and MV Pishko) American Scientific Publishers, USA, 525.
Hascup KN, Hascup ER, Pomerleau F, Huettl P, Gerhardt GA (2007) Second-by-second measures of L-glutamate in the prefrontal cortex and striatum of freely moving mice. J Pharmacol Exp Ther, [Epub ahead of print].
Pomerleau F, Day BK, Huettl P, Burmeister JJ, Gerhardt GA (2003) Real time in vivo measures of L-glutamate in the rat central nervous
system using ceramic-based multisite microelectrode arrays. Ann NY Acad Sci, 1003: 454.
Rutherford EC, Pomerleau F, Huettl P, Strömberg I, Gerhardt GA (2007) Chronic second-by-second measures of L-glutamate in the central nervous system of freely moving rats. J Neurochem, 102:712-722.