Authors: Colleen E Dugan James P Grinias Sebastian D Parlee Mahmoud ElAzzouny Charles R Evans Robert T Kennedy
Publish Date: 2016/10/19
Volume: 409, Issue: 1, Pages: 169-178
Abstract
Microfluidics is an enabling technology for both cell biology and chemical analysis We combine these attributes with a microfluidic device for online solidphase extraction SPE and mass spectrometry MS analysis of secreted metabolites from living cells in culture on the chip The device was constructed with polydimethylsiloxane PDMS and contains a reversibly sealed chamber for perfusing cells A multilayer design allowed a series of valves to control an onchip 75 μL injection loop downstream of the cell chamber with operation similar to a sixport valve The valve collects sample and then diverts it to a packed SPE bed that was connected inline to treat samples prior to MS analysis The valve allows samples to be collected and injected onto the SPE bed while preventing exposure of cells to added back pressure from the SPE bed and organic solvents needed to elute collected chemicals Here cultured murine 3T3L1 adipocytes were loaded into the cell chamber and nonesterified fatty acids NEFAs that were secreted by the cells were monitored by SPEMS at 30 min intervals The limit of detection for a palmitoleic acid standard was 14 μM Due to the multiplexed detection capabilities of MS a variety of NEFAs were detected Upon stimulation with isoproterenol and forskolin secretion of select NEFAs was elevated an average of 15fold compared to basal levels Despite the 30min delay between sample injections this device is a step towards a miniaturized system that allows automated monitoring and identification of a variety of molecules in the extracellular environmentThis research was funded by NIH R37 DK046960 to RTK JPG was supported by NIH F32 EB019800 Spray tips and PicoClear unions were generously provided by New Objective Inc TOFMS experiments utilized Core Services of the Michigan Regional Comprehensive Metabolomics Resource Core MRC2 supported by grant DK097153 of NIH to the University of Michigan
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