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

Abstract

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.

Automated microfluidic droplet sampling with integrated, mix-and-read immunoassays to resolve endocrine tissue secretion dynamics

“…However, one disadvantage of the system is the complex flow control and electrophoresis setup including external pumps, a high-voltage power supply and electrodes, and well-controlled surface chemistry for electroosmotic fluid control. Additionally, the intermittent sampling nature of those systems may cause undersampling, and the effective temporal resolution remains at the minute scale due to dispersion effects during sampling 19 . Nonetheless, both the Kennedy 16–18 and Roper 20–22 groups have proven the utility of on-chip capillary electrophoresis immunoassays for islet secretions, confirming such microfluidic systems to be uniquely qualified for temporal studies of cells.…”

“…Homogeneous immunoassays such as the molecular pincer assay 27 and related proximity assays 28–30 allow a simple mix-and-read procedure without the necessity of on-chip separation or washing, which is ideal for microfluidic secretion sampling 19 , but particularly within droplets. Compared to heterogeneous assays like the capillary electrophoresis immunoassay, application of homogeneous assays within droplets would largely simplify the microfluidic chip design.…”

Fluorescence Polarization-Based Bioassays: New Horizons

“…The developed test system allowed for the determination of half-maximal inhibitory concentration of ethylenediaminetetraacetic acid for the inhibition of DNase 1 activity to be 1.56 ± 0.91 mM. Schrell et al [ 142 ] developed a real-time system for insulin monitoring based on a microfluidic online FA immunoassay. It has a LoD of 4 nM and is suitable for use by non-specialized laboratories.…”

Design and Application of Sensors for Chemical Cytometry

“…By contrast, the temporal resolution of chemical cytometry is excellent, ranging from microseconds to seconds depending upon the cell-sampling method employed. 13, 14…”

“…83 This competitive detection achieved a temporal resolution of 10 s to follow glucose-responsive insulin secretion over time and should be applicable to a multitude of other hormones. 14…”