Anal. Chem. 2005, 77, 7702-7708
Microfluidic Electrophoresis Chip Coupled to Microdialysis for in Vivo Monitoring of Amino Acid Neurotransmitters Zechariah D. Sandlin,† Minshan Shou,† Jonathan G. Shackman,† and Robert T. Kennedy*,†,‡
Department of Chemistry and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-1055
Microfluidic electrophoresis devices were coupled on-line to microdialysis for in vivo monitoring of primary amine neurotransmitters in rat brain. The devices contained a sample introduction channel for dialysate, a precolumn reactor for derivatization with o-phthaldialdehyde, a flowgated injector, and a separation channel. Detection was performed using confocal laser-induced fluorescence. In vitro testing revealed that the initial device design had detection limits for amino acids of ∼200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30 200 theoretical plates when applying an electric field of 370 V/cm. A second device design that allowed electric fields of 1320 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156 000 theoretical plates. Flow splitting into the electrokinetic network from hydrodynamic flow in the sample introduction channel was made negligible for sampling flow rates from 0.3 to 1.2 µL/min by placing a 360-µm-diameter fluidic access hole that had flow resistance (0.15-7.2) × 108-fold lower than that of the electrokinetic network at the junction of the sample introduction channel and the electrokinetic network. Using serial injections, the device allowed the dialysate stream to be analyzed at 130-s intervals. In vivo monitoring was demonstrated by using the microdialysis/ microfluidic device to record glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain. Neurotransmitter monitoring is an essential tool in elucidating normal and pathological nervous system functions.1-5 In vivo * Corresponding author. Phone: 734-615-4363. Fax: 734-615-6462. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Pharmacology. (1) Kalra, S. P.; Dube, M. G.; Pu, S. Y.; Xu, B.; Horvath, T. L.; Kalra, P. S. Endocr. Rev. 1999, 20, 68-100. (2) Greenamyre, J. T.; Young, A. B. Neurobiol. Aging 1989, 10, 593-602. (3) Magnusson, O.; Nilsson, L. B.; Westerlund, D. J. Chromatogr., B 1992, 582, 1-5. (4) Carlsson, M.; Carlsson, A. Trends Neurosci. 1990, 13, 272-276. (5) Meldrum, B. S. Antiepileptic Drugs; Spinger: Berlin, 1995.
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chemical measurements in the brain are especially important in studying the role of neurotransmitters in pharmacological, physiological, and behavioral effects. Several techniques capable of measuring chemicals in the extracellular space of the brain in vivo have been developed including positron emission tomography, sensors, and microdialysis sampling coupled with analytical methods. Microdialysis has become a popular method for in vivo monitoring because of its potential for good temporal resolution and its compatibility with sensitive analytical techniques enabling detection of many different analytes.6-8 In microdialysis, a semipermeable membrane probe that is continuously perfused is implanted into the brain region (or other tissue) of interest. Molecules below the molecular weight cutoff of the membrane diffuse across the membrane into the probe according to their concentration gradient and are collected in the dialysate stream for analysis.7 Microdialysis is most commonly coupled with high-performance liquid chromatography (HPLC).9-12 It has been demonstrated that coupling microdialysis to miniaturized techniques with high mass sensitivity, such as capillary liquid chromatography (LC)13-16 and capillary electrophoresis (CE),17-20 can greatly improve the information content of a dialysis experiment. With (6) Ungerstedt, U.; Forster, C.; Herrera-Marschitz, M.; Hoffman, I.; Jungnelius, U.; Tossman, U.; Zetterstrom, T. Neurosci. Lett. 1982, (Suppl. 10), 493. (7) Ungerstedt, U. In Measurement of Neurotransmitter Release In Vivo; Marsden, C. A., Ed.; John Wiley & Sons: New York, 1984; pp 81-105. (8) Robinson, T. E.; Justice, J. B., Jr. Microdialysis in the Neurosciences: Techniques in the Behavioral and Neural Science; Elsevier: Amsterdam, 1991. (9) Davies, M. I. Anal. Chim. Acta 1999, 379, 227-249. (10) Torto, N.; Laurell, T.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1999, 379, 281-305. (11) Davies, M. I.; Lunte, C. E. Chem. Soc. Rev. 1997, 26, 215-222. (12) Vanstaden, J. F. Fresenius J. Anal. Chem. 1995, 352, 271-302. (13) Ruban, V. F. J. Chromatogr., B 1993, 619, 111-115. (14) Shen, H.; Lada, M. W.; Kennedy, R. T. J. Chromatogr., B 1997, 704, 4352. (15) Boyd, B. W.; Witowski, S. R.; Kennedy, R. T. Anal. Chem. 2000, 72, 865871. (16) Kennedy, R. T.; Watson, C. J.; Haskins, W. E.; Powell, D. H.; Strecker, R. E. Curr. Opin. Chem. Biol. 2002, 6, 659-665. (17) Bert, L.; Robert, F.; Denoroy, L.; Stoppini, L.; Renaud, B. J. Chromatogr., A 1996, 755, 99-111. (18) Tellez, S.; Forges, N.; Roussin, A.; Hernandez, L. J. Chromatogr., B 1992, 581, 257-266. (19) Hogan, B. L.; Lunte, S. M.; Stobaugh, J. F.; Lunte, C. E. Anal. Chem. 1994, 66, 596-602. (20) Lada, M. W.; Schaller, G.; Carriger, M. H.; Vickroy, T. W.; Kennedy, R. T. Anal. Chim. Acta 1995, 307, 217-225. 10.1021/ac051044z CCC: $30.25
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such techniques, probes may be operated at low flow rates where the recovery (i.e., the concentration in the probe divided by the concentration outside the probe) approaches 100% allowing quantitative monitoring while avoiding the difficulties of in vivo calibration.20 Furthermore, samples may be collected more frequently, allowing improved temporal resolution.19,21-29 Using CE or capillary LC, samples have been collected at 1-120-s intervals whereas with conventional HPLC columns with 2.0-4.6mm inner diameter, sampling intervals are usually 10 min or greater because of the requirement to collect sufficient sample for analysis. While it is feasible to collect samples off-line, it is also possible to obtain near real time in vivo monitoring with automated on-line analysis. In such experiments, the temporal resolution becomes limited by the separation time. The high speed of CE has allowed on-line separations of 3-120 s to be achieved.19,21,22,24,28 Such temporal resolution is valuable in tracking rapid chemical changes that occur in the brain following neuronal stimulation or behavioral changes.30-33 While microdialysis with on-line CE is a powerful tool for in vivo chemical monitoring, its adoption for routine use by many laboratories may be limited by the requirement for assembling complex fluidic connections with precise distances and volumes. For example, the flow-gated interface used in some studies requires capillaries to be aligned with gaps of
