Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes

Abstract

Microfluidics have enabled new cell biology experiments. Incorporating chemical monitoring of cellular secretion into chips offers the potential to increase information content and utility of such systems. In this work, an integrated, multilayer polydimethylsiloxane microfluidic chip was developed to simultaneously measure fatty acids and glycerol secreted from cultured adipocytes on-chip in near real-time. Approximately 48,000 adipocytes were loaded into a cell chamber in a reversibly-sealed chip. Cells were perfused at 0.75 μL/min. Cell perfusate was split and directed to separate, continuously operating fluorescent enzyme assay channel networks. The fluorescent assay products were detected simultaneously near the outlet of the chip. The fatty acid and glycerol assays had linear dynamic ranges to 150 μM and 110 μM, and LOD of 6 μM and 5 μM, respectively. Surface modifications including pretreatment with sodium dodecyl sulfate were utilized to prevent adsorption of fatty acids to the chip surface. Using the chip, basal fatty acid and glycerol concentrations ranged from 0.18–0.7 nmol 106 cell−1 min−1 and 0.23–0.85 nmol 106 cell−1 min−1, respectively. Using valves built into the chip, the perfusion solution was switched to add 20 μM isoproterenol, a β-adrenergic agonist, which stimulates the release of glycerol and fatty acids in adipocytes. This manipulation resulted in a rapid and stable 1.5- to 6.0-fold increase of NEFA and glycerol. The ratio of NEFA to glycerol released increased with adipocyte age. These experiments illustrate the potential for performing multiple real-time assays on cells in culture using microfluidic devices.

Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry

“…This work builds upon a previous chip that was reported to measure NEFA secretion from on-chip adipocyte perfusion [28]. In that work, cell perfusate was mixed on-chip with enzyme assay reagents in a continuous flow system to allow real-time monitoring of NEFA release; however, that method did not allow identification of individual NEFAs.…”

“…Chip fabrication used procedures similar to those described before [28]. The injection loop chip was composed of two separate pieces and all layers were formed from 10:1 base:curing agent PDMS (RTV 615, Curbell Plastics, Livonia, MI).…”

“…Murine 3T3-L1 adipocytes were cultured to day 11–13 post-induction as previously described [28]. Briefly, glass coverslips (No.…”

“…Immunoassay [23–25] or enzyme assay [26–28] with fluorescent detection, capillary electrophoresis with electrochemical detection [29], and enzyme assay with chemiluminescent detection [30] have all been used. These methods give temporal resolution and selectivity in the measurement providing excellent insight into cell function.…”

“…Adipocytes represent a particularly challenging cell type because they are fragile and buoyant, making them susceptible to changes in pressure or shear. In previous reports, NEFAs secreted from perfused adipocytes have been measured on PDMS chips with enzyme assays and laser-induced fluorescence [26, 28]. These enzyme assays only allow total NEFA measurement; MS is a more versatile detection technique, allowing detection of specific NEFAs.…”