Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids

Multilayer soft lithography was used to prepare a poly(dimethyisiloxane) 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 cerebrospinal 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 similar to 20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were similar to 60 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 10% in vivo. Basal concentrations of glutamate were 1.9 +/- 0.6 mu M (n = 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 similar to 45 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.