Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets.

Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in ß-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal's ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet ß-cells drives pulsatile insulin secretion.

Measurement of the entrainment window of islets of Langerhans by microfluidic delivery of a chirped glucose waveform.

Within single islets of Langerhans, the endocrine portion of the pancreas, intracellular metabolites, as well as insulin secretion, oscillate with a period of ∼5 min. In vivo, pulsatile insulin oscillations are also observed with periods ranging from 5-15 minutes. In order for oscillations of insulin to be observed in vivo, the majority of islets in the pancreas must synchronize their output. It is known that populations of islets can be synchronized via entrainment of the individual islets to low amplitude glucose oscillations that have periods close to islets' natural period. However, the range of glucose periods and amplitudes that can entrain islets has not been rigorously examined. To find the range of glucose periods that can entrain islets, a microfluidic system was utilized to produce and deliver a chirped glucose waveform to populations of islets while their individual intracellular [Ca(2+)] ([Ca(2+)]i) oscillations were imaged. Waveforms with amplitudes of 0.5, 1, and 1.5 mM above a median value of 11 mM were applied while the period was swept from 20-2 min. Oscillations of [Ca(2+)]i resonated the strongest when the period of the glucose wave was within 2 min of the natural period of the islets, typically close to 5 min. Some examples of 1 : 2 and 2 : 1 entrainment were observed during exposure to long and short glucose periods, respectively. These results shed light on the dynamic nature of islet behavior and may help to understand dynamics observed in vivo.

Integrated perfusion and separation systems for entrainment of insulin secretion from islets of Langerhans.

A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Automated manipulation of the height of the perfusion syringes allowed precise control of the ratio of two perfusion solutions into a chamber containing 1-10 islets. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2-8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3-6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6-10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion and can be easily modified for investigating the dynamic nature of other hormones released from different cell types.

Microfluidics-to-mass spectrometry: a review of coupling methods and applications.

Microfluidic devices offer great advantages in integrating sample processes, minimizing sample and reagent volumes, and increasing analysis speed, while mass spectrometry detection provides high information content, is sensitive, and can be used in quantitative analyses. The coupling of microfluidic devices to mass spectrometers is becoming more common with the strengths of both systems being combined to analyze precious and complex samples. This review summarizes select achievements published between 2010 and July 2014 in novel coupling between microfluidic devices and mass spectrometers. The review is subdivided by the types of ionization sources employed, and the different microfluidic systems used.

Negative feedback synchronizes islets of Langerhans.

Insulin is released from the pancreas in pulses with a period of ~ 5 min. These oscillatory insulin levels are essential for proper liver utilization and perturbed pulsatility is observed in type 2 diabetes. What coordinates the many islets of Langerhans throughout the pancreas to produce unified oscillations of insulin secretion? One hypothesis is that coordination is achieved through an insulin-dependent negative feedback action of the liver onto the glucose level. This hypothesis was tested in an in vitro setting using a microfluidic system where the population response from a group of islets was input to a model of hepatic glucose uptake, which provided a negative feedback to the glucose level. This modified glucose level was then delivered back to the islet chamber where the population response was again monitored and used to update the glucose concentration delivered to the islets. We found that, with appropriate parameters for the model, oscillations in islet activity were synchronized. This approach demonstrates that rhythmic activity of a population of physically uncoupled islets can be coordinated by a downstream system that senses islet activity and supplies negative feedback. In the intact animal, the liver can play this role of the coordinator of islet activity.

Maintaining Stimulant Waveforms in Large Volume Microfluidic Cell Chambers.

Stimulation of cells with temporal waveforms can be used to observe the frequency-dependent nature of cellular responses. The ability to produce and maintain the temporal waveforms in spite of the broadening processes that occur as the wave travels through the microfluidic system is critical for observing dynamic behaviors. Broadening of waves in microfluidic channels has been examined, but the effect that large-volume cell chambers have on the waves has not. In this report, a sinusoidal glucose wave delivered to a 1 mm diameter cell chamber using various microfluidic channel structures was simulated by finite element analysis with the goal of minimizing the broadening of the waveform in the chamber and maximizing the homogeneity of the concentration in the chamber at any given time. Simulation results indicated that increasing the flow rate was the most effective means to achieve these goals, but at a given volumetric flow rate, geometries that deliver the waveform to multiple regions in the chamber while maintaining a high linear velocity produced sufficient results. A 4-inlet geometry with a 220 µm channel width gave the best result in the simulation and was used to deliver glucose waveforms to a population of pancreatic islets of Langerhans. The result was a stronger and more robust synchronization of the islet population as compared to when a non-optimized chamber was used. This general strategy will be useful in other microfluidic systems examining the frequency-dependence nature of cellular behavior.

Temporal gradients in microfluidic systems to probe cellular dynamics: a review.

Microfluidic devices have found a unique place in cellular studies due to the ease of fabrication, their ability to provide long-term culture, or the seamless integration of downstream measurements into the devices. The accurate and precise control of fluid flows also allows unique stimulant profiles to be applied to cells that have been difficult to perform with conventional devices. In this review, we describe and provide examples of microfluidic systems that have been used to generate temporal gradients of stimulants, such as waveforms or pulses, and how these profiles have been used to produce biological insights into mammalian cells that are not typically revealed under static concentration gradients. We also discuss the inherent analytical challenges associated with producing and maintaining temporal gradients in these devices.

A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR.

The endemic of Avian Influenza Virus (AIV) in Asia and epizootics in some European regions have caused serious economic losses. Multiplex reverse-transcriptase (RT) PCR has been developed to detect and subtype AIV. However, the number of targets that can be amplified in a single run is limited because of uncontrollable primer-primer interferences. In this paper, we describe a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip. A simple UV cross-linking method was used to immobilize the DNA probes on unmodified glass surface, which makes it convenient to integrate microarray with microfluidics. This solid-phase RT-PCR method combined RT amplification of extracted RNA in the liquid phase and species-specific nested PCR on the solid phase. Using the developed approach, AIV viruses and their subtypes were unambiguously identified by the distinct patterns of amplification products. The whole process was reduced to less than 1 hour and the sample volume used in the microfluidic chip was at least 10 times less than in the literature. By spatially separating the primers, highly multiplexed amplification can be performed in solid-phase PCR. Moreover, multiplex PCR and sequence detection were done in one step, which greatly simplified the assay and reduced the processing time. Furthermore, by incorporating the microarray into a microchamber-based PCR chip, the sample and the reagent consumption were greatly reduced, and the problems of bubble formation and solution evaporation were effectively prevented. This microarray-based PCR microchip can be widely employed for virus detection and effective surveillance in wild avian and in poultry productions.

Rapid detection of avian influenza virus in chicken fecal samples by immunomagnetic capture reverse transcriptase-polymerase chain reaction assay.

Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings. In this study, magnetic beads were applied for immunoseparation and purification of AIV from spiked chicken fecal sample. The beads were conjugated with monoclonal antibodies against the AIV nucleoprotein, which is conserved in all the AIV. The bead-captured virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) without RNA extraction because of effective removal of RT-PCR inhibitors. The developed bead-based assay showed a similar detection limit comparable to the RNA extraction and the classic virus isolation method. Using ready-to-use antibody-conjugated bead, the method requires less than 5 h. Furthermore, the method has potential to integrate into a Lab-on-a-chip system for rapid detection and identification of AIV.

DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7.

Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal and tracheal swab specimens was completed in less than 2 h with 94% accuracy.

Rapid sample preparation for detection and identification of avian influenza virus from chicken faecal samples using magnetic bead microsystem.

Avian influenza virus (AIV) is an infectious agent of birds and mammals. AIV is causing huge economic loss and can be a threat to human health. Reverse transcriptase polymerase chain reaction (RT-PCR) has been used as a method for the detection and identification of AIV virus. Although RT-PCR is a sensitive method for detection of AIV, it requires sample preparation including separation and purification of AIV and concentrate viral RNA. It is laborious and complex process especially for diagnosis using faecal sample. In this study, magnetic beads were used for immunoseparation of AIV in chicken faecal sample by a magnetic microsystem. Using this system, all the 16 hemagglutinin (H) and 9 neuraminidase (N) subtypes of AIV were separated and detected in spiked faecal samples using RT-PCR, without an RNA extraction step. This rapid sample preparation method can be integrated with a total analysis microsystem and used for diagnosis of AIV.

Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR.

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.

Particle flow assays for fluorescent protein microarray applications.

Microarray technology has brought a paradigmatic change in bioanalytics. However, highly sensitive and accurate assays are still needed for a real breakthrough. We present a simple and generic approach for fluorescent signal amplification with fluorescent microparticle labels. The assay principle was demonstrated using a reverse array model consisting of spots of bovine serum albumin with a small fraction of the proteins biotinylated. Specific binding of streptavidin coated fluorescent microparticles to the spots was promoted by applying a controlled continuous microparticle flow. The surface bound beads were visualized and quantified with confocal microscopy images. Comparison with standard fluorescent and flow discrimination assays has revealed several advantages of our approach. First, non-specific particle binding could be reduced to less than 1 particle/spot making therefore the visualization of single biomolecular bonds possible. Second, the amplification scheme presented here is generic and can be applied to any fluorescent microarray. Furthermore, this assay makes use of a biotin-streptavidin linkage and can therefore be applied to all kind of assays. Finally, single fluorescent microbeads can be easily visualized with standard optical equipments, so that no high performance equipment is required.