Development of an online microdialysis-microchip electrophoresis system for near real time monitoring of amino acids

Abstract

The brain extracellular space is complex chemical environment consisting of ions, peptides, neurotransmitters, growth factors, metabolites and extracellular matrix molecules. Microdialysis is a popular and well-characterized sampling technique that makes it possible to continuously recover analytes from the extracellular space. Systems that couple microdialysis with capillary electrophoresis (CE) based separations have gained popularity due to their ability to perform online analysis with high temporal resolution. Such systems are appropriate to study in vivo neurotransmission where the fluctuations in neuroactive analytes can occur in the second to millisecond time scale. In this work, a separation based microdialysis-microchip electrophoresis system with fluorescence detection was developed for the determination of amino acid neurotransmitters from microdialysis samples collected from rat brain striatum. Amino acid standards and microdialysis samples were derivatized with naphthalene 2, 3 dicarboxaldehyde (NDA) and sodium cyanide (NaCN) to form fluorescent cyno-2-substituted-benz(f) isoidole (CBI) derivatives prior to analysis. Initial experiments employed capillary electrophoresis with laser induced fluorescence (CE-LIF) detection to evaluate the optimum buffer conditions for the separation of amino acid standards. This was followed up with the analysis of a rat brain microdialysate sample by CE-LIF in which aspartate (Asp) and glutamate (Glu) were the primary amino acids of interest. Lastly, microdialysis samples were analyzed for Glu release in vivo triggered by a high potassium infusion. The CE separation of amino acids served as the basis for optimization of the amino acid separation in the microchip electrophoresis experiments. First, offline analysis was carried out on brain microdialysate samples to optimize the method for the detection of Asp and Glu. The optimal separation conditions consisted of 200 mM borate, 80 mM SDS, 5% acetonitrile using a 21 cm long serpentine channel in a PDMS microchip. Subsequently, the chip was modified for online monitoring with precolumn derivatization by introducing three separate tubing inlets for microdialysis sample and reagents introduction followed by mixing in a serpentine reaction channel. The derivatized sample was then injected, separated and detected by microchip electrophoresis with fluorescence detection. This device was successfully employed for the continuous monitoring of Asp, Glu and fluorescein in the striatum of an anesthetized Sprague-Dawley (SD) rat. Fluorescein was also included in the separation for the possibility of monitoring blood brain barrier (BBB) integrity simultaneously with amino acid neurotransmitter release. A possible application of this device is monitoring changes in excitatory amino acids and BBB integrity due to stroke. During stroke, the concentration of Glu is increased leading to excitotoxicity and loss of integrity of the BBB. Our work established an online microdialysis microchip setup that can be further modified to perform high temporal resolution studies for studying dynamic changes of neuroactive analytes in the brain following stroke.