Quantitative Measurement of Zinc Secretion from Pancreatic Islets with High Temporal Resolution Using Droplet-Based Microfluidics

Abstract

We assayed glucose-stimulated insulin secretion (GSIS) from live, murine islets of Langerhans in microfluidic devices by the downstream formation of aqueous droplets. Zinc ions, which are cosecreted with insulin from β-cells, were quantitatively measured from single islets with high temporal resolution using a fluorescent indicator, FluoZin-3. Real-time storage of secretions into droplets (volume of 0.470 ± 0.009 nL) effectively preserves the temporal chemical information, allowing reconstruction of the secretory time record. The use of passive flow control within the device removes the need for syringe pumps, requiring only a single handheld syringe. Under stimulatory glucose levels (11 mM), bursts of zinc as high as ~800 fg islet −1 min −1 were measured. Treatment with diazoxide effectively blocked zinc secretion, as expected. High temporal resolution reveals two major classes of oscillations in secreted zinc, with predominate periods at ~20-40 s and ~ 5-10 min. The more rapid oscillation periods match closely with those of intraislet calcium oscillations, while the slower oscillations are consistent with insulin pulses typically measured in bulk islet experiments or in the bloodstream. This droplet sampling technique should be widely applicable to time-resolved cellular secretion measurements, either in real-time or for post-processing.

Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

“…Although mature and specific, these techniques lack dynamic assessment, are costly and time-consuming, and they involve multistep protocols that often result in error. 10 To remedy these limitations, many microfluidic platforms have been developed to assess islet functionality by coupling with techniques including zinc sampling, 11 insulin ELISA, 12 Ca 2þ , 13,14 and NADPH imaging. 15 Many of the challenges with dynamic islet functionality analysis using insulin ELISA still remain when coupling with microfluidic systems.…”

Microfluidics for monitoring and imaging pancreatic islet and β -cells for human transplant

“…Additionally, droplets have also been used in β-cell study using syringe or vacuum (Chen et al ., 2008;Zhang et al ., 2009). As demonstrated by the Piston group using vacuum driven fl ow, they were able to generate a passive method for microfl uidic droplet sampling method that was used to quantify β-cell zinc secretion (zinc and insulin are co-secreted) with signifi cantly improved spatiotemporal resolution (average droplet volume was 470 ± 9 pL under a fl ow rate of 0.100 μL/min) (Easley et al ., 2009). Another microfl uidic device employing droplet-based fl uid fl ow was the chemistrode developed by the Ismagilov group (Chen et al ., 2008).…”

“…When insulin is secreted, along with c-peptide, zinc is also co-secreted into plasma. The Piston group has exploited this feature of β-cells and has developed a droplet-based device with a sampling drop volume of approximately 0.47 nL (Easley et al ., 2009). Their single islet studies demonstrated a high temporal resolution that allowed the detection of two zinc oscillatory frequencies: fast (~20-40 s) and slow (~5-10 m).…”

Micro/nanofabricated environments for synthetic biology

“…The creation of, mixing in, and fission and fusion of aqueous droplets or plugs can be controlled very precisely in both space and time in diverse ways in microchannels for initiation or quenching of specific reaction steps at key points, enabling fundamental discoveries of the nonlinear biochemical reaction mechanisms underlying complex signaling pathways [6 ,38,39]. In addition to controlled coalescence of two or more droplets within a channel, real-time stimulation and capture of secreted dilute biomarkers from cells is possible either on chip [40], or with external hydrophilic wetting layers on surfaces from microfluidic devices that function as micropipettes [41][42][43].…”