RESUMO
Insulin levels in the blood oscillate with a variety of periods, including rapid (5-10 min), ultradian (50-120 min), and circadian (24 h). Oscillations of insulin are beneficial for lowering blood glucose and disrupted rhythms are found in people with type 2 diabetes and their close relatives. These in vivo secretion dynamics imply that the oscillatory activity of individual islets of Langerhans are synchronized, although the mechanism for this is not known. One mechanism by which islets may synchronize is negative feedback of insulin on whole-body glucose levels. In previous work, we demonstrated that a negative feedback loop with a small time delay, to account for the time required for islets to be exposed to a new glucose concentration in vivo, results in small 3-6 islet populations synchronizing to produce fast closed-loop oscillations. However, these same islet populations could also produce slow closed-loop oscillations with periods longer than the natural islet oscillation periods. Here, we investigate the origin of the slow oscillations and the bistability with the fast oscillations using larger islet populations (20-50 islets). In contrast to what was observed earlier, larger islet populations mainly synchronize to longer-period oscillations that are approximately twice the delay time used in the feedback loop. A mean-field model was also used as a proxy for a large islet population to uncover the underlying mechanism for the slow rhythm. The heterogeneous intrinsic oscillation periods of the islets interferes with this rhythm mechanism when islet populations are small, and is similar to adding noise to the mean-field model. Thus, the effect of a time delay in the glucose feedback mechanism is similar to other examples of time-delayed systems in biology and may be a viable mechanism for ultradian oscillations.
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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 â¼300-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 â¼3-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.
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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.
Assuntos
Técnicas Analíticas Microfluídicas , Animais , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Insulina , Microfluídica , Imunoensaio , PerfusãoRESUMO
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.
Assuntos
Ilhotas Pancreáticas , Humanos , Insulina/análise , Glucagon , Peptídeos/análise , Espectrometria de Massas , Glucose/análiseRESUMO
Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest. LAY SUMMARY: To understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.
Assuntos
Doenças do Cão , Micetoma , Scedosporium , Animais , Cães , Hospedeiro Imunocomprometido , Micetoma/microbiologia , Micetoma/veterinária , Scedosporium/genética , Especificidade da EspécieRESUMO
The European XFEL delivers up to 27000 intense (>1012 photons) pulses per second, of ultrashort (≤50â fs) and transversely coherent X-ray radiation, at a maximum repetition rate of 4.5â MHz. Its unique X-ray beam parameters enable groundbreaking experiments in matter at extreme conditions at the High Energy Density (HED) scientific instrument. The performance of the HED instrument during its first two years of operation, its scientific remit, as well as ongoing installations towards full operation are presented. Scientific goals of HED include the investigation of extreme states of matter created by intense laser pulses, diamond anvil cells, or pulsed magnets, and ultrafast X-ray methods that allow their diagnosis using self-amplified spontaneous emission between 5 and 25â keV, coupled with X-ray monochromators and optional seeded beam operation. The HED instrument provides two target chambers, X-ray spectrometers for emission and scattering, X-ray detectors, and a timing tool to correct for residual timing jitter between laser and X-ray pulses.
RESUMO
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.
Assuntos
Corantes Fluorescentes/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptor Muscarínico M3/metabolismo , Células Cultivadas , Corantes Fluorescentes/química , Glucose/química , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Dispositivos Lab-On-A-Chip , TemperaturaRESUMO
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.
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Reatores Biológicos , Ensaios Enzimáticos , Glucose Oxidase/metabolismo , Glucose , Peroxidase do Rábano Silvestre/metabolismo , Técnicas Analíticas Microfluídicas , Glucose/análise , Glucose/metabolismo , Células Hep G2 , Humanos , Tamanho da Partícula , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
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.
RESUMO
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.
Assuntos
Relógios Biológicos/fisiologia , Sinalização do Cálcio/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Animais , Células Cultivadas , Simulação por Computador , Retroalimentação Fisiológica/fisiologia , Glicólise/fisiologia , Humanos , Secreção de InsulinaRESUMO
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.
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Aminoácidos/metabolismo , Ilhotas Pancreáticas/metabolismo , Microfluídica/métodos , Aminoácidos/análise , Animais , Humanos , CamundongosRESUMO
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.
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Polarização de Fluorescência , Glucagon/análise , Imunoensaio , Insulina/análise , Corantes Fluorescentes/química , LasersRESUMO
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.
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Técnicas Analíticas Microfluídicas , Microscopia Eletrônica de Transmissão/métodos , Animais , Linhagem Celular , Metais Pesados/química , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia Eletrônica de Transmissão/instrumentação , Modelos Moleculares , Tamanho da Partícula , Canais de Potássio de Abertura Dependente da Tensão da Membrana/isolamento & purificação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/ultraestrutura , Ratos , Coloração e Rotulagem/métodosRESUMO
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.
Assuntos
Cálcio/análise , Insulina/análise , Técnicas Analíticas Microfluídicas/instrumentação , Imagem Óptica/instrumentação , Animais , Cálcio/metabolismo , Desenho de Equipamento , Corantes Fluorescentes/análise , Fura-2/análise , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Glucagon/metabolismo , Imunoensaio/métodos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Humanos , Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , CamundongosRESUMO
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.
Assuntos
Aminoácidos/análise , Aminoácidos/metabolismo , Ilhotas Pancreáticas/metabolismo , 2,4-Dinitrofenol/farmacologia , Aminoácidos/química , Animais , Cromatografia Capilar Eletrocinética Micelar , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Limite de Detecção , Modelos Lineares , Masculino , Camundongos , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Retroalimentação Fisiológica , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Animais , Transporte Biológico , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , CamundongosRESUMO
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.
Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Fourier , Lasers , Espectrometria de FluorescênciaRESUMO
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.