ABSTRACT
Doctoral School of Biological Sciences, Neuroscience Section
Dept. of Neuroscience, Medical School – University of Sassari, Italy
Neurochemical pathways involved in brain physiology or disease pathogenesis are mostly unknown either in physiological conditions or in neurodegenerative diseases.
Previously we have successfully monitored, in real-time, neurochemical changes in the striatum of freely-moving animals using amperometric sensors interfaced with a telemetric device [1,2]. This system was coupled with microsensors and biosensors for the detection of ascorbic acid (AA), oxygen (O2), nitric oxide (NO), glucose and lactate in the striatum of untethered, freely-moving rats.
The aim of this study is the development of carbonanotubes-based neurosensors for in vivo monitoring of brain energy metabolism.
The carbon nanotubes (CNT) are carbon compounds of nanometers in size and are built with a length to diameter ratio significantly higher than any other material. They possess strong electrical and conductive properties. For this study we have selected multiwalled carbon nanotubes (MWCNT) with an average external diameter of 150 nm.
Carbon nanotubes are mostly insoluble; even so they are soluble in some organic solvents [3]. In order to make MWCNT compatible with components of microsensors and biosensors, we derivatised them modifying a previously described method [4]. Then, the resulting MWCNT powder has been mixed with polyethilenimmine (PEI, 5%) to avoid precipitation. In this manner we obtained CNT soluble for months.
O2 microsensor construction and calibration were performed as previously described [2]. AA microsensors were manufactured in the same way as oxygen microsensors without the collodion layer or any further surface modification. The NO microsensor wasmodified with O-phenylenediamine (OPD) and nafion as previously reported [5,6]. The design of the glucose biosensors has been previously described in detail [7]. This is a classical oxidase-based biosensor with the production and the subsequent oxidation of hydrogen peroxide on MWCNT surface.
The fabrication of the lactate biosensors has been derived from a previously published procedure [8].
The determination of extracellular adenosine-5’-triphosphate (ATP) is of great importance for understanding the brain energy metabolism, for this purpose we have produced the ATP sensor. It is a dual enzyme biosensor developed by co-immobilization of the enzymes glucose oxidase (GOX) and hexokinase (HEX). The ATP detection required two calibration steps. The first step was performed in presence of glucose. The electrochemical signal is generated by the oxidation of H2O2 at the electrode surface. The second step was made in the presence of glucose, HEX, ATP and Mg2+. In presence of ATP, the enzymatic reaction catalyzed by HEX was consuming glucose and the signal decreased. The change in current responses is proportional to the ATP concentration.
All sensors were calibrated before in vivo implantation.
Male rats were used in all in vivo experiments. Stereotaxic surgery was performed under chloral hydrate anesthesia. The microsensors and biosensors were implanted in the right striatum, while the biotelemetric device was fixed to the skull.
Preliminary results showed real-time changes of the neurochemical signals in brain of freely-moving rats.
The biotelemetric approach confirmed previous experimental results obtained using classical microdialysis probes and could be used as a rapid model for studying the effects of different drugs in conscious animals.
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2) Calia G. et al (2009) – Sensors 9: 2511-2523.
3) Bahr J. L. et al (2001) – Chem. Commun. 193-194.
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5) Friedemann M. N. et al (1996) – Anal. Chem. 68: 2621-2628.
6) Zhang X. et al (2002) – Electroanalysis 14: 697-703.
7) Serra P. A. (2006) – Sens. Actuat. B 122: 118-126.
8) McMahon C. P. (2006) – Analyst. 131: 68-72.