Microfluidic Chip for Low-Flow Push-Pull Perfusion Sampling in Vivo with On-Line Analysis of Amino Acids

摘要

Multilayer soft lithography was used to prepare a poly-(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cere-brospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 (mu)m wide by 15 (mu)m high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100 000 theoretical plates were acquired at approx20-s intervals. Relative standard deviations of peak heights were 4percent in vitro, and detection limits for the excitatory amino acids were approx60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10percent in vivo. Basal concentrations of glutamate were 1.9 +- 0.6 (mu)M (n velence 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K~(+) through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of approx45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.