Near real-time microfluidic monitoring of cellular secretions using fluorescent enzyme assays.

Abstract

Microfluidic devices can be used to perform sophisticated cell physiology experiments. Additionally, microfluidic devices have the ability to decrease labor, reagent, and sample required relative to off-line studies through miniaturization, automation, and integration of analytical components. An interesting application of on-chip cell physiology studies is to monitor adipocyte metabolism. Obesity is a growing epidemic worldwide, and its prevalence underscores the need to understand adipocyte physiology in order to address obesity and obesity-related disorders. An initial microfluidic platform consisting of a perfusion cell chip and an enzyme assay mixing chip was developed to couple on-line perfusion of cells with chemical analysis of effluent to study adipocyte lipolytic secretions. A continuous-flow fluorescent enzyme assay for glycerol was developed and transferred to the chip-based format enabling on-line mixing and detection resulting in a limit of detection (LOD) of 4 microM for glycerol. This dual-chip system was used to monitor glycerol release from ∼50 000 cultured adipocytes with 90 s temporal resolution and with less than 1% of reagent usage required for off-line studies. A second device improved upon the initial platform by integrating the two separate chips into one multilayer device and by extending the functionality of the device to monitor non-esterified fatty acid (NEFA) release from cultured adipocytes. Integration of the device allowed 87% fewer cells to be sampled, decreased the amount of perfusion buffer used by an order of magnitude, and resulted in a temporal resolution of 83 s. A fluorescent enzyme assay for NEFAs was developed and resulted in a LOD of 5 microM NEFA on-line, enabling collection of complementary lipolysis information relative to the previous glycerol assay. The dual-chip system was used in a collaborative study to examine the effect of Wnt3a (a secreted cysteine-rich protein) on lipolysis in near real-time. Adipocytes were perfused on-chip with and without Wnt3a treatment to assess effects on lipolysis and provided kinetic information to augment static glycerol and NEFA release data. This study suggested that Wnt signaling occurs in mature adipocyte and regulates the metabolic process.