[Chemistry 304] It is understood that acute insulin secretion is altered or abolished in the disease states of obesity, insulin resistance, and diabetes. However, much less is known about the acute endocrine function of adipose tissue (fat), since current methods are insufficient to interrogate dynamics. We propose to replace large-scale sampling methods (Petri dishes or well-plates) with simple microfluidic systems that can interrogate much smaller samples of tissue. As such, we must develop compatible assays for hormone quantitation in these small volumes. In this work, I will discuss how we have broadened our capabilities in studying hormone secretion from intact tissue such as adipocytes and pancreatic islets.
We first applied microfluidic aqueous-in-oil droplet formation (~0.5 nL droplets) to sample and measure zinc secretions from single murine pancreatic islets at high temporal resolution (~1 s). To assay proteins in such small volumes, we have recently developed proximity immunoassays that employ fluorescence resonance energy transfer (pFRET). Our insulin pFRET is capable of direct detection of 300 pM insulin, and it was recently combined with microfluidic droplet sampling to monitor secretions in real time from as few as 12 pancreatic islets during glucose stimulation and pharmacological treatments. Microfluidic secretion sampling from 3D cultures of fat tissue (adipocytes) was shown functional, and we are currently working to combine this system with droplet sampling and pFRET assays for leptin and adiponectin. While we are interested in uncovering the nature of rapid adipokine secretion, this integrated system should provide a unique and generalizable platform for interrogating the dynamics of hormone secretion from various types of tissue at small scales.
In addition, we have developed an electrochemical proximity assay (ECPA) capable of direct detection of proteins at femtomolar concentrations. ECPA can directly assay insulin in 20-fold diluted mouse serum (unspiked), and our reusable ECPA approach was proven functional for real-time interrogation of insulin secretion from <20 islets at ~5-7 minute temporal resolution. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.