Optimization of a microfluidic electrophoretic immunoassay using a Peltier cooler

Abstract

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.

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

“…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.…”

Multiplexing Fluorescence Anisotropy Using Frequency Encoding

“…The LODs were 0.6 and 5 nM for insulin and glucagon, respectively, which are comparable to what has been obtained using more conventional methods for insulin or glucagon. 3,22,23,30–32 …”

Interfacing Microfluidics with Negative Stain Transmission Electron Microscopy

“…The extraction divot was etched to a depth of 45 μm using conventional methods. 18 The channels and grid chamber were then fabricated using a second etch to a depth of 50 μm. The same process was repeated for another piece of glass, producing the mirror image of the design features to serve as the bottom complement.…”