Comparison between capillary electrophoresis and fluorescence anisotropy competitive immunoassay for glucagon.

Glucagon plays a crucial role in regulating glucose homeostasis; unfortunately, the mechanisms controlling its release are still unclear. Capillary electrophoresis (CE)- and fluorescence anisotropy (FA)-immunoassays (IA) have been used for online measurements of hormone secretion on microfluidic platforms, although their use in glucagon assays is less common. We set out to compare a glucagon-competitive IA using these two techniques. Theoretical calibration curves were generated for both CE- and FA-IA and results indicated that CE-IA provided higher sensitivity than FA-IA. These results were confirmed in an experiment where both assays showed limits of detection (LOD) of 30 nM, but the CE-IA had ∼300-fold larger sensitivity from 0 to 200 nM glucagon. However, in online experiments where reagents were mixed within the device, the sensitivity of the CE-IA was reduced ∼3-fold resulting in a higher LOD of 70 nM, whereas the FA-IA remained essentially unchanged. This lowered sensitivity in the online CE-IA was likely due to poor sampling by electroosmotic flow from the high salt solution necessary in online experiments, whereas pressure-based sampling used in FA-IA was not affected. We conclude that FA-IA, despite lowered sensitivity, is more suitable for online mixing scenarios due to the ability to use pressure-driven flow and other practical advantages such as the use of larger channels.

Automated cellular stimulation with integrated pneumatic valves and fluidic capacitors.

Microfluidic technologies have proven to be a reliable tool in profiling dynamic insulin secretion from islets of Langerhans. Most of these systems rely on external pressure sources to induce flow, leading to difficulties moving to more elaborate systems. To reduce complexity, a microfluidic system was developed that used a single vacuum source at the outlet to drive fluidic transport of immunoassay reagents and stimulation solutions throughout the device. A downside to this approach is the lack of flow control over the reagents delivered to the islet chamber. To address this challenge, 4-layer pneumatic valves were integrated into the perfusion lines to automate and control the delivery of stimulants; however, it was found that as the valves closed, spikes in the flow would lead to abnormal insulin secretion profiles. Fluidic capacitors were then incorporated after the valves and found to remove the spikes. The combination of the valves and capacitors resulted in automated collection of insulin secretion profiles from single murine islets that were similar to those previously reported in the literature. In the future, these integrated fluidic components may enable more complex channel designs to be used with a relatively simple flow control solution.

Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with < 15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye.

Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

Online Measurement of Glucose Consumption from HepG2 Cells Using an Integrated Bioreactor and Enzymatic Assay.

Hepatocytes help to maintain glucose homeostasis in response to a variety of signals, including pancreatic hormones such as insulin. Insulin is released from the pancreas with variable dynamics, yet the role that these play in regulating glucose metabolism in the liver is still unclear. In this study, a modular microfluidic system was developed to quantitatively measure the effect of insulin dynamics on glucose consumption by a human hepatocarcinoma cell line, HepG2. A microfluidic bioreactor that contained 106 HepG2 cells was cultured for up to 10 days in an incubator. For glucose consumption experiments, the bioreactor was removed from the incubator and connected with reagents for an enzymatic glucose assay. The mixed components were then delivered into a droplet-based microfluidic system where the intensity of the fluorescent product of the enzyme assay was used to quantify the glucose concentration. By optimizing the mixing time of the reagents, the dynamic range of the enzymatic assay was adjusted to 0-12 mM glucose and had a time resolution of 96 ± 12 s. The system was used to observe rapid changes in insulin-induced glucose consumption from HepG2 cells. This assay format is versatile and can be expanded to measure a variety of hepatic metabolites, such as lactate, pyruvate, or ketone bodies, which will enable the correlation of pancreatic hormone dynamics to liver metabolism.

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing.

A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed.

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.

Multiplexing Fluorescence Anisotropy Using Frequency Encoding.

In this report, a method to multiplex fluorescence anisotropy measurements is described using frequency encoding. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and FITC-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with limits of detection of 0.6 and 5 nM for insulin and glucagon, respectively. This methodology could readily be expanded to other biological systems and further multiplexed to monitor increased numbers of analytes.

Microfluidic Device for the Measurement of Amino Acid Secretion Dynamics from Murine and Human Islets of Langerhans.

Islets of Langerhans are the regulators of in vivo blood glucose levels through the secretion of endocrine hormones. Amino acids, released from various cells within islets or from intrapancreatic neurons, are hypothesized to further adjust hormone secretions. In contrast to the well-accepted mechanism of glucose-stimulated insulin secretion, several questions remain as to the function of amino acids in the regulation of hormone release from islets. To understand the autocrine and paracrine roles that amino acids play in islet physiology, a microfluidic system was developed to perform online monitoring of the secretion profiles of amino acids from 2-5 islets. The device contained an islet chamber with the ability to perfuse stimulants and an amino acid measurement system with derivatization and electrophoretic separation integrated on a single microchip. The setup was optimized to allow -15 kV to be applied to the device for high efficiency and rapid separations of derivatized amino acids. The compositions of the derivatization and separation buffers were optimized to prevent precipitations in the channels, which allowed continuous monitoring of secretion for over 2 h. With this method, 10 amino acids were resolved with limits of detection ranging from 1 to 20 nM. When murine islets were perfused with 3 mM glucose, the secretion rates of 9 amino acids were measured and ranged from 30 to 400 fmol islet(-1) min(-1). As the glucose concentration was increased to 20 mM, the dynamic changes of amino acids were monitored. The biological relevance of the amino acid secretions was verified using 2,4-dinitrophenol as an inhibitor of the proton motive force. The microfluidic system was also used to measure dynamic changes of amino acid release from human islets, which showed different release profiles compared to their murine counterparts.

Interfacing Microfluidics with Negative Stain Transmission Electron Microscopy.

A microfluidic platform is presented for preparing negatively stained grids for use in transmission electron microscopy (EM). The microfluidic device is composed of glass etched with readily fabricated features that facilitate the extraction of the grid poststaining and maintains the integrity of the sample. Utilization of this device simultaneously reduced environmental contamination on the grids and improved the homogeneity of the heavy metal stain needed to enhance visualization of biological specimens as compared to conventionally prepared EM grids. This easy-to-use EM grid preparation device provides the basis for future developments of systems with more integrated features, which will allow for high-throughput and dynamic structural biology studies.

Dual Detection System for Simultaneous Measurement of Intracellular Fluorescent Markers and Cellular Secretion.

Glucose-stimulated insulin secretion from pancreatic ß-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca2+] ([Ca2+]i) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca2+]i and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca2+]i or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca2+]i and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.

Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

Micellar electrokinetic chromatography method for measuring amino acid secretions from islets of Langerhans.

Islets of Langerhans are responsible for maintaining glucose homeostasis through regulated secretion of hormones and other factors. It is hypothesized that amino acids secreted from islets play a critical role in cell functionality and viability. For example, glutamate and gamma-aminobutyric acid have been proposed to work as paracrine signaling molecules within islets to coordinate the release of hormone secretion; other amino acids, such as glutamine, leucine, alanine, and arginine, have been shown to stimulate or potentiate glucose-stimulated insulin secretion. To characterize the potential roles that these small molecules may play in islet physiology, derivatization of amino acids in high-salt buffers commonly used in islet experiments with naphthalene-2,3-dicarboxaldehyde and MEKC separation conditions were optimized. The optimized conditions used d-norvaline as the internal standard and allowed quantification of 14 amino acids with LODs ranging from 0.2 to 7 nM. The RSDs of the migration times were 0.04-0.54% and the RSDs of the peak areas were 0.2-5.8% for the various amino acids. The effects of glucose and 2,4-dinitrophenol on amino acid secretions from islets were tested and a suppressive effect of glucose on gamma-aminobutyric acid release was observed, likely acting through adenosine triphosphate inactivation of glutamate decarboxylase.

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.

Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR.

A frequency-modulated fluorescence encoding method was used as a means to increase the number of fluorophores monitored during infrared-mediated polymerase chain reaction. Laser lines at 488 nm and 561 nm were modulated at 73 and 137 Hz, respectively, exciting fluorescence from the dsDNA intercalating dye, EvaGreen, and the temperature insensitive dye, ROX. Emission was collected in a color-blind manner using a single photomultiplier tube for detection and demodulated by frequency analysis. The resulting frequency domain signal resolved the contribution from the two fluorophores as well as the background from the IR lamp. The detection method was successfully used to measure amplification of DNA samples containing 10(4)-10(7) starting copies of template producing an amplification efficiency of 96%. The utility of this methodology was further demonstrated by simultaneous amplification of two genes from human genomic DNA using different color TaqMan probes. This method of multiplexing fluorescence detection with IR-qPCR is ideally suited as it allows isolation of the signals of interest from the background in the frequency domain and is expected to further reduce the complexity of multiplexed microfluidic IR-qPCR instrumentation.

Simultaneous monitoring of insulin and islet amyloid polypeptide secretion from islets of Langerhans on a microfluidic device.

A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4-10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey's reagent and reversed phase LC-MS/MS.

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h-1; D-Gln: 300 ± 100 fmol islet-1h-1) or high (D-Ser: 23 ± 1 fmol islet-1h-1; D-Gln: 120 ± 50 fmol islet-1h-1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.

Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans.

The cell-to-cell signaling role of d-amino acids (d-AAs) in the mammalian endocrine system, particularly in the islets of Langerhans, has drawn growing interest for their potential involvement in modulating glucose metabolism. Previous studies found colocalization of serine racemase [produces d-serine (d-Ser)] and d-alanine (d-Ala) within insulin-secreting beta cells and d-aspartate (d-Asp) within glucagon-secreting alpha cells. Expressed in the islets, functional N-methyl-d-aspartate receptors are involved in the modulation of glucose-stimulated insulin secretion and have binding sites for several d-AAs. However, knowledge of the regulation of d-AA levels in the islets during glucose stimulation as well as the response of islets to different levels of extracellular d-AAs is limited. In this study, we determined the intracellular and extracellular levels of d-Ser, d-Ala, and d-Asp in cultures of isolated rodent islets exposed to different levels of extracellular glucose. We found that the intracellular levels of the enantiomers demonstrated large variability and, in general, were not affected by extracellular glucose levels. However, significantly lower levels of extracellular d-Ser and d-Ala were observed in the islet media supplemented with 20 mM concentration of glucose compared to the control condition utilizing 3 mM glucose. Glucose-induced oscillations of intracellular free calcium concentration ([Ca2+]i), a proxy for insulin secretion, were modulated by the exogenous application of d-Ser and d-Ala but not by their l-stereoisomers. Our results provide new insights into the roles of d-AAs in the biochemistry and function of pancreatic islets.

Online coupling of digital microfluidic devices with mass spectrometry detection using an eductor with electrospray ionization.

MS detection coupled with digital microfluidic (DMF) devices has most commonly been demonstrated in an offline manner using matrix assisted laser desorption ionization. In this work, an eductor is demonstrated which facilitated online coupling of DMF with electrospray ionization MS detection. The eductor consisted of a transfer capillary, a standard ESI needle, and a tapered gas nozzle. As a pulse of N(2) was applied to the nozzle, a pressure differential was induced at the outlet of the ESI needle that pulled droplets from the DMF, past the ESI needle, and into the flow of gas exiting the nozzle, allowing detection by MS. Operating position, ionization potential, and N(2) pressure were optimized, with the optimum ionization potential and N(2) pressure found to be 3206 V and 80 psi, respectively. Online MS detection was demonstrated from both open and closed DMF devices using 2.5 µL and 630 nL aqueous droplets, respectively. Relative quantitation by DMF-MS was demonstrated by mixing droplets of caffeine with droplets of theophylline on an open DMF device and comparing the peak area ratio obtained to an on-chip generated calibration curve. This eductor-based method for transferring droplets has the potential for rapid, versatile, and high-throughput microfluidic analyses.