Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip

Shackman, Jonathan G.; Reid, Kendra R.; Dugan, Colleen E.; Kennedy, Robert T.

doi:10.1007/s00216-012-5755-7

Cited by 39 publications

(50 citation statements)

References 30 publications

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“…These results represent the first step toward 3D printed microfluidic devices for integrated analyses of nucleic acids and other molecules in which many active and passive components are incorporated in a single device. [35][36][37][38][39][40] …”

Section: Microfluidicsmentioning

confidence: 99%

3D printed microfluidic devices with integrated valves

2015

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We report the successful fabrication and testing of 3D printed microfluidic devices with integrated membrane-based valves. Fabrication is performed with a low-cost commercially available stereolithographic 3D printer. Horizontal microfluidic channels with designed rectangular cross sectional dimensions as small as 350 μm wide and 250 μm tall are printed with 100% yield, as are cylindrical vertical microfluidic channels with 350 μm designed (210 μm actual) diameters. Based on our previous work [Rogers et al., Anal. Chem. 83, 6418 (2011)], we use a custom resin formulation tailored for low non-specific protein adsorption. Valves are fabricated with a membrane consisting of a single build layer. The fluid pressure required to open a closed valve is the same as the control pressure holding the valve closed. 3D printed valves are successfully demonstrated for up to 800 actuations.

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Section: Microfluidicsmentioning

confidence: 99%

3D printed microfluidic devices with integrated valves

2015

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“…1,2 For these reasons, there is a growing interest in the development of rapid analytical methods for the determination of glucagon. Typically, glucagon has been determined by complex indirect assays that require its labeling with optical 3,4 or radioactive tags. 5,6 Electrochemical methods are potentially useful for direct determination of glucagon; however, little is known about the electrochemistry of this peptide.…”

Section: Introductionmentioning

confidence: 99%

Hormone glucagon: electrooxidation and determination at carbon nanotubes

Karra

Griffith

Kennedy

et al. 2016

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The oxidation of glucagon, which is one of the key hormones in glucose homeostasis, was studied at electrodes modified with carbon nanotubes (CNT) that were dispersed in a polysaccharide adhesive chitosan (CHIT). Such electrodes displayed improved resistance to fouling, which allowed for the investigation of both the electrolysis/mass spectrometry and electroanalysis of glucagon. The off-line electrospray ionization and tandem mass spectrometric analyses showed that the -4 Da mass change to glucagon upon electrolysis at CNT was due to the electrooxidation of its tryptophan (W25) and dityrosine (Y10, Y13) residues. The methionine residue of glucagon did not contribute to its oxidation. The amperometric determination of glucagon yielded the limit of detection equal to ~20 nM (E = 0.800 V, pH 7.40, S/N = 3), sensitivity of 0.46 A M−1 cm−2, linear dynamic range up to 2.0 μM (R2 = 0.998), response time <5 s, and good signal stability. Free tryptophan and tyrosine yielded comparable analytical figures of merit. The direct amperometric determination of unlabeled glucagon at CHIT-CNT electrodes is the first example of a rapid alternative to the complex analytical assays of this peptide.

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“…Although chemical measurements can be performed off-chip [7], integrating analytical measurements with cells on microfluidic systems eliminates the need for sample collection and off-line analyte detection, and can provide real-time recording of cellular dynamics [8,9]. Several examples of this approach have been described including on-line immunoassays [10], sensors [11], and enzyme assays [12]. Most such systems perform a single assay on cells.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip

Cited by 39 publications

References 30 publications

3D printed microfluidic devices with integrated valves

3D printed microfluidic devices with integrated valves

Hormone glucagon: electrooxidation and determination at carbon nanotubes

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

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