Perfusion and chemical monitoring of living cells on a microfluidic chip.
A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 microL min(-1). The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 degrees C. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1). The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.
Shackman
Jonathan G
JG
Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
Dahlgren
Gabriella M
GM
Peters
Jennifer L
JL
Kennedy
Robert T
RT
D000818:Animals; D003470:Culture Media; D004867:Equipment Design; D007118:Immunoassay; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods
D003470:Culture Media; D007328:Insulin