Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology

Lomasney, Anna R.; Guillo, Christelle; Sidebottom, Ashley M.; Roper, Michael G.

doi:10.1007/s00216-009-2622-2

Cited by 25 publications

(19 citation statements)

References 24 publications

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“…33,34 However, since anisotropy measurements are performed under equilibrium conditions, elevated temperatures typically increase the amount of the non-covalent complex, which is beneficial for assay sensitivity and a high dynamic range. 35 Overall, this temperature system simplifies the assay conditions compared to the electrophoretic systems.…”

Section: Resultsmentioning

confidence: 99%

Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans

Schrell

Mukhitov

et al. 2017

Anal. Methods

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Insulin secretion from islets of Langerhans is a dynamic process that is essential for maintaining glucose homeostasis. The ability to measure dynamic changes in insulin levels upon glucose stimulation from single islets will allow testing of therapeutics and investigating mechanisms of defective secretion observed in metabolic diseases. Most approaches to date for measurement of rapid changes in insulin levels rely on separations, making the assays difficult to translate to non-specialist laboratories. To enable rapid measurements of secretion dynamics from a single islet in a manner that will be more suitable for transfer to non-specialized laboratories, a microfluidic online fluorescence anisotropy immunoassay was developed. A single islet was housed inside a microfluidic chamber and stimulated with varying glucose levels from a gravity-based perfusion system. The total effluent of the islet chamber containing the islet secretions was mixed with gravity-driven solutions of insulin antibody and Cy5-labeled insulin. After mixing was complete, a linearly polarized 635 nm laser was used to excite the immunoassay mixture and the emission was split into parallel and perpendicular components for determination of anisotropy. Key factors for reproducible anisotropy measurements, including temperature homogeneity and flow rate stability were optimized, which resulted in a 4 nM limit of detection for insulin with <1% RSD of anisotropy values. The capability of this system for measuring insulin secretion from single islets was shown by stimulating an islet with varying glucose levels. As the entire analysis is performed optically, this system should be readily transferable to other laboratories.

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Section: Resultsmentioning

confidence: 99%

Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans

Schrell

Mukhitov

et al. 2017

Anal. Methods

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“…The sensitivity of the IAPP assay can be increased with longer injection times, but at the expense of resolution and assay quantitation. 39 …”

Section: Resultsmentioning

confidence: 99%

Simultaneous Monitoring of Insulin and Islet Amyloid Polypeptide Secretion from Islets of Langerhans on a Microfluidic Device

Lomasney

Roper

2013

Anal. Chem.

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A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature changes in the perfusion media within 5-s allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15-cm reaction channel maintained at 37 °C. The immunoassay mixture was injected for 0.3-s onto a 1.5-cm separation channel at 11.75-s intervals and immunoassay reagents detected using 488- and 635-nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2–7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4–10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 mM to 11 mM or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

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“…The highest B/F and the variance of this point are critical as it sets the constraints for the sensitivity of the assay. We have used the B/F value at this initial point as a marker of whole assay sensitivity in the past [14]. …”

Section: Resultsmentioning

confidence: 99%

“…Peak start and finish times were approximated by events that exceeded three times the standard deviation of the baseline. To obtain a calibration curve, the average bound-to-free ratio (B/F) from 5 consecutive electropherograms was evaluated as a function of [Ins] and was fit using a four parameter logistic model [10,14]. The LOD was determined as the concentration of Ins required to induce a change in the B/F greater than three times the standard deviation of the blank.…”

Section: Methodsmentioning

confidence: 99%

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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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Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology

Cited by 25 publications

References 24 publications

Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans

Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans

Simultaneous Monitoring of Insulin and Islet Amyloid Polypeptide Secretion from Islets of Langerhans on a Microfluidic Device

Optimization of a microfluidic electrophoretic immunoassay using a Peltier cooler

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