Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans

Guillo, Christelle; Roper, Michael G.

doi:10.1016/j.chroma.2011.05.006

Cited by 25 publications

(35 citation statements)

References 39 publications

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“…Labeling of bovine insulin with Cy5 (Insulin*) was performed as previously described. 29 All buffers were prepared using ultrapure deionized water (NANOpure Diamond TM deionization system, Barnstead International, Dubuque, IA) and filtered using 0.2 µm nylon syringe filters (Pall Corporation, Port Washington, NY).…”

Section: Methodsmentioning

confidence: 99%

Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans

Wang

Dhumpa

et al. 2015

Lab Chip

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A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Automated manipulation of the height of the perfusion syringes allowed precise control of the ratio of two perfusion solutions into a chamber containing 1 – 10 islets. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2 – 8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3 – 6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6 – 10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion and can be easily modified for investigating the dynamic nature of other hormones released from different cell types.

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Section: Methodsmentioning

confidence: 99%

Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans

Wang

Dhumpa

et al. 2015

Lab Chip

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“…Glucagon and/or amylin assays have been developed in the competitive immunoassay format using Ab as affinity ligands 22, 23 and transferred to microfluidic devices to monitor their dynamic secretion from islets of Langerhans. 26 However, the LOD of the developed competitive immunoassays were at unsatisfactorily high levels, which as stated before, were intrinsically determined by the K d of the Ab-Ag* binding reaction.…”

Section: Resultsmentioning

confidence: 99%

“…A dual color detection scheme was used for simultaneous assays of amylin and insulin as previously described. 22, 23 All separations were performed in a 50 μm i.d. × 360 μm o.d., 30 cm long fused-silica capillary (Polymicro Technologies, Phoenix, AZ).…”

Section: Methodsmentioning

confidence: 99%

Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes

Wang

Bethge

et al. 2016

Analyst

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The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

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“…For production of Cy5-labeled insulin (Ins*) Cy5 monofunctional N-hydroxysuccinimide ester was obtained from GE Healthcare Bio-Sciences (Piscataway, NJ). The methodology for labeling bovine insulin has been described previously [7] and was performed according to the manufacturer’s guidelines.…”

Section: Methodsmentioning

confidence: 99%

Optimization of a microfluidic electrophoretic immunoassay using a Peltier cooler

Mukhitov

Schrell

et al. 2014

Journal of Chromatography A

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Successful analysis of electrophoretic affinity assays depends strongly on the preservation of the affinity complex during separations. Elevated separation temperatures due to Joule heating promotes complex dissociation leading to a reduction in sensitivity. Affinity assays performed in glass microfluidic devices may be especially prone to this problem due to poor heat dissipation due to the low thermal conductivity of glass and the large amount of bulk material surrounding separation channels. To address this limitation, a method to cool a glass microfluidic chip for performing an affinity assay for insulin was achieved by a Peltier cooler localized over the separation channel. The Peltier cooler allowed for rapid stabilization of temperatures, with 21 °C the lowest temperature that was possible to use without producing detrimental thermal gradients throughout the device. The introduction of cooling improved the preservation of the affinity complex, with even passive cooling of the separation channel improving the amount of complex observed by 2-fold. Additionally, the capability to thermostabilize the separation channel allowed for utilization of higher separation voltages than what was possible without temperature control. Kinetic CE analysis was utilized as a diagnostic of the affinity assay and indicated that optimal conditions were at the highest separation voltage, 6 kV, and the lowest separation temperature, 21 °C, leading to 3.4% dissociation of the complex peak during the separation. These optimum conditions were used to generate a calibration curve and produced 1 nM limits of detection, representing a 10-fold improvement over non-thermostated conditions. This methodology of cooling glass microfluidic devices for performing robust and high sensitivity affinity assays on microfluidic systems should be amenable in a number of applications.

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Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans

Cited by 25 publications

References 39 publications

Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans

Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans

Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes

Optimization of a microfluidic electrophoretic immunoassay using a Peltier cooler

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