Building a Nucleic Acid Nanostructure with DNA-Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection.

The recent surge of effort in nucleic-acid-based electrochemical (EC) sensors has been fruitful, yet there remains a need for more generalizable EC platforms for sensing multiple classes of clinically relevant targets. We recently reported a nucleic acid nanostructure for simple, economical, and more generalizable EC readout of a range of analytes, including small molecules, peptides, proteins, and antibodies. The nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the generalizable detection of larger proteins remains a challenge. Here, we adapted the sensor to quantify larger proteins in a more generic manner through conjugating the protein's minimized antibody-binding epitope to the central DNA strand. This concept was verified using creatine kinase (CK-MM), a biomarker of muscle damage and several disorders for which rapid clinical sensing is important. DNA-epitope conjugates permitted a competitive immunoassay for the CK protein at the electrode via square-wave voltammetry (SWV). Sensing through a signal-off mechanism, the anti-CK antibody limit of detection (LOD) was 5 nM with a response time as low as 3 min. Antibody displacement by native protein analytes gave a signal-on response with the CK sensing range from the LOD of 14 nM up to 100 nM, overlapping with the normal (nonelevated) human clinical range (3-37 nM), and the sensor was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall, this approach allows the quantification of a generic protein- or peptide-binding antibody and should facilitate future quantitative EC readouts of clinically relevant proteins that were previously inaccessible to EC techniques.

Thermofluorimetric Analysis (TFA) using Probes with Flexible Spacers: Application to Direct Antibody Sensing and to Antibody-Oligonucleotide (AbO) Conjugate Valency Monitoring.

Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production in low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes, such as antibody-oligonucleotide (AbO) conjugates. Previously, we demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogeneous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from the background and interferences. Expanding the applicability of TFA further, herein we demonstrate a unique proximity based TFA assay for antibody quantification that is functional in 90% human plasma. We show that the conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of poly(ethylene glycol) (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probe valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold.

Droplet-based µChopper device with a 3D-printed pneumatic valving layer and a simple photometer for absorbance based fructosamine quantification in human serum.

The development of microfluidic systems for biological assays presents challenges, particularly in adapting traditional optical absorbance assays to smaller volumes or to microfluidic formats. This often requires assay modification or translation to a fluorescence version, which can be impractical. To address this issue, our group has developed the µChopper device, which uses microfluidic droplet formation as a surrogate for an optical beam chopper, allowing for lock-in analysis and improved limits of detection with both absorbance and fluorescence optics without modifying the optical path length. Here, we have adapted the µChopper to low-cost optics using a light-emitting diode (LED) source and photodiode detector, and we have fabricated the pnuematically valved devices entirely by 3D printing instead of traditional photolithography. Using a hybrid device structure, fluidic channels were made in polydimethylsiloxane (PDMS) by moulding onto a 3D-printed master then bonding to a prefabricated thin layer, and the pneumatic layer was directly made of 3D-printed resin. This hybrid structure allowed an optical slit to be fabricated directly under fluidic channels, with the LED interfaced closely above the channel. Vacuum-operated, normally closed valves provided precise temporal control of droplet formation from 0.6 to 2.0 Hz. The system was validated against the standard plate reader format using a colorimetric fructosamine assay and by quantifying fructosamine in human serum from normal and diabetic patients, where strong correlation was shown. Showing a standard benefit of microfluidics in analysis, the device required 6.4-fold less serum volume for each assay. This µChopper device and lower cost optical system should be applicable to various absorbance based assays in low volumes, and the reliance on inexpensive 3D printers makes it more accessible to users without cleanroom facilities.

A Nucleic Acid Nanostructure Built through On-Electrode Ligation for Electrochemical Detection of a Broad Range of Analytes.

For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.

Nonfaradaic Current Suppression in DNA-Based Electrochemical Assays with a Differential Potentiostat.

One of the key factors limiting sensitivity in many electrochemical assays is the nonfaradaic or capacitive current. This is particularly true in modern assay systems based on DNA monolayers at gold electrode surfaces, which have shown great promise for bioanalysis in complex milieu such as whole blood or serum. While various changes in analytical parameters, redox reporter molecules, DNA structures, probe coverage, and electrode surface area have been shown useful, background reduction by hardware subtraction has not yet been explored for these assays. Here, we introduce new electrochemistry hardware that considerably suppresses nonfaradaic currents through real-time analog subtraction during current-to-voltage conversion in the potentiostat. This differential potentiostat (DiffStat) configuration is shown to suppress or remove capacitance currents in chronoamperometry, cyclic voltammetry, and square-wave voltammetry measurements applied to nucleic acid hybridization assays at the electrode surface. The DiffStat makes larger electrodes and higher sensitivity settings accessible to the user, providing order-of-magnitude improvements in sensitivity, and it also significantly simplifies data processing to extract faradaic currents in square-wave voltammetry (SWV). Because two working electrodes are used for differential measurements, unique arrangements are introduced such as converting signal-OFF assays to signal-ON assays or background drift correction in 50% human serum. Overall, this new potentiostat design should be helpful not only in improving the sensitivity of most electrochemical assays, but it should also better support adaptation of assays to the point-of-care by circumventing complex data processing.

Understanding Signal and Background in a Thermally Resolved, Single-Branched DNA Assay Using Square Wave Voltammetry.

Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

Microfluidic systems for studying dynamic function of adipocytes and adipose tissue.

Recent breakthroughs in organ-on-a-chip and related technologies have highlighted the extraordinary potential for microfluidics to not only make lasting impacts in the understanding of biological systems but also to create new and important in vitro culture platforms. Adipose tissue (fat), in particular, is one that should be amenable to microfluidic mimics of its microenvironment. While the tissue was traditionally considered important only for energy storage, it is now understood to be an integral part of the endocrine system that secretes hormones and responds to various stimuli. As such, adipocyte function is central to the understanding of pathological conditions such as obesity, diabetes, and metabolic syndrome. Despite the importance of the tissue, only recently have significant strides been made in studying dynamic function of adipocytes or adipose tissues on microfluidic devices. In this critical review, we highlight new developments in the special class of microfluidic systems aimed at culture and interrogation of adipose tissue, a sub-field of microfluidics that we contend is only in its infancy. We close by reflecting on these studies as we forecast a promising future, where microfluidic technologies should be capable of mimicking the adipose tissue microenvironment and provide novel insights into its physiological roles in the normal and diseased states. Graphical abstract This critical review focuses on recent developments and challenges in applying microfluidic systems to the culture and analysis of adipocytes and adipose tissue.

Homogeneous Assays of Second Messenger Signaling and Hormone Secretion Using Thermofluorimetric Methods That Minimize Calibration Burden.

Homogeneous assays are characterized by rapidity, low cost, and simple workflows. However, relatively few specialized homogeneous platforms have garnered significant use in biological studies. Inconsistencies in matrix interferences, limited multiplexability, and the requirement for specialized instrumentation are among the various reasons for delayed acceptance. Recently, we have shown that DNA-driven protein assays using thermofluorimetric analysis (TFA) can limit matrix interference and promote multiplexing, all while requiring only a standard qPCR instrument for readout. Here, we show that homogeneous, one step (mix-and-read) TFA methods can be extended to the analysis of both a small molecule second messenger, cyclic adenosine monophosphate (cAMP), and a downstream cell-secreted hormone, insulin. Differential thermal analysis of DNA melting in these assays allowed analytical discrimination of background and signal without physical separation. The direct-readout, differential nature of TFA also promoted assay consistency and minimized calibration burden; analyte response curves were shown to be highly repeatable for up to 7 months. TFA protocols were validated by homogeneous quantification of both cAMP and insulin from single pancreatic islets undergoing a variety of treatments (glucose, KCl, glucose-responsive insulinotropic peptide (GIP), forskolin) that act upon glucose transporters, potassium and calcium channels, and G-protein-coupled receptors to modulate exocytosis. The results of this study suggest that TFA should be applicable to homogeneous quantification of a variety of small molecule messengers and protein analytes with standard instrumentation, thereby simplifying workflows in studies of cell-signaling cascades.

Automated Microfluidic Droplet-Based Sample Chopper for Detection of Small Fluorescence Differences Using Lock-In Analysis.

Fluorescence is widely used for small-volume analysis and is a primary tool for on-chip detection in microfluidic devices, yet additional expertise, more elaborate optics, and phase-locked detectors are needed for ultrasensitive measurements. Recently, we designed a microfluidic analog to an optical beam chopper (µChopper) that alternated formation of picoliter volume sample and reference droplets. Without complex optics, the device negated large signal drifts (1/f noise), allowing absorbance detection in a mere 27 µm optical path. Here, we extend the µChopper concept to fluorescence detection with standard wide-field microscope optics. Precision of droplet control in the µChopper was improved by automation with pneumatic valves, allowing fluorescence measurements to be strictly phase locked at 0.04 Hz bandwidth to droplets generated at 3.50 Hz. A detection limit of 12 pM fluorescein was achieved when sampling 20 droplets, and as few as 310 zeptomoles (3.1 × 10-19 mol) were detectable in single droplets (8.8 nL). When applied to free fatty acid (FFA) uptake in 3T3-L1 adipocytes, this µChopper permitted single-cell FFA uptake rates to be quantified at 3.5 ± 0.2 × 10-15 mol cell-1 for the first time. Additionally, homogeneous immunoassays in droplets exhibited insulin detection limits of 9.3 nM or 190 amol (1.9 × 10-16 mol). The combination of this novel, automated µChopper with lock-in detection provides a high-performance platform for detecting small differences with standard fluorescence optics, particularly in situations where sample volume is limited. The technique should be simple to implement into a variety of other droplet fluidics devices.

Macro-to-micro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue.

Employing 3D-printed templates for macro-to-micro interfacing, a passively operated polydimethysiloxane (PDMS) microfluidic device was designed for time-resolved secretion sampling from primary murine islets and epidiymal white adipose tissue explants. Interfacing in similar devices is typically accomplished through manually punched or drilled fluidic reservoirs. We previously introduced the concept of using hand fabricated polymer inserts to template cell culture and sampling reservoirs into PDMS devices, allowing rapid stimulation and sampling of endocrine tissue. However, fabrication of the fluidic reservoirs was time consuming, tedious, and was prone to errors during device curing. Here, we have implemented computer-aided design and 3D printing to circumvent these fabrication obstacles. In addition to rapid prototyping and design iteration advantages, the ability to match these 3D-printed interface templates with channel patterns is highly beneficial. By digitizing the template fabrication process, more robust components can be produced with reduced fabrication variability. Herein, 3D-printed templates were used for sculpting millimetre-scale reservoirs into the above-channel, bulk PDMS in passively-operated, eight-channel devices designed for time-resolved secretion sampling of murine tissue. Devices were proven functional by temporally assaying glucose-stimulated insulin secretion from <10 pancreatic islets and glycerol secretion from 2 mm adipose tissue explants, suggesting that 3D-printed interface templates could be applicable to a variety of cells and tissue types. More generally, this work validates desktop 3D printers as versatile interfacing tools in microfluidic laboratories.

Protein quantification using controlled DNA melting transitions in bivalent probe assemblies.

Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.

A microfluidic interface for the culture and sampling of adiponectin from primary adipocytes.

Secreted from adipose tissue, adiponectin is a vital endocrine hormone that acts in glucose metabolism, thereby establishing its crucial role in diabetes, obesity, and other metabolic disease states. Insulin exposure to primary adipocytes cultured in static conditions has been shown to stimulate adiponectin secretion. However, conventional, static methodology for culturing and stimulating adipocytes falls short of truly mimicking physiological environments. Along with decreases in experimental costs and sample volume, and increased temporal resolution, microfluidic platforms permit small-volume flowing cell culture systems, which more accurately represent the constant flow conditions through vasculature in vivo. Here, we have integrated a customized primary tissue culture reservoir into a passively operated microfluidic device made of polydimethylsiloxane (PDMS). Fabrication of the reservoir was accomplished through unique PDMS "landscaping" above sampling channels, with a design strategy targeted to primary adipocytes to overcome issues of positive cell buoyancy. This reservoir allowed three-dimensional culture of primary murine adipocytes, accurate control over stimulants via constant perfusion, and sampling of adipokine secretion during various treatments. As the first report of primary adipocyte culture and sampling within microfluidic systems, this work sets the stage for future studies in adipokine secretion dynamics.

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Creating biocompatible oil-water interfaces without synthesis: direct interactions between primary amines and carboxylated perfluorocarbon surfactants.

Currently, one of the most prominent methods used to impart biocompatibility to aqueous-in-oil droplets is to synthesize a triblock copolymer surfactant composed of perfluoropolyether and polyether blocks. The resulting surfactants (EA surfactant, KryJeffa, etc.) allow generation of highly biocompatible droplet surfaces while maintaining the heat stability of the starting material. However, production of these surfactants requires expertise in synthetic organic chemistry, creating a barrier to widespread adoption in the field. Herein, we describe a simple alternative to synthetic modification of surfactants to impart biocompatibility. We have observed that aqueous-in-oil droplet surfaces can be made biocompatible and heat stable by merely exploiting binding interactions between polyetherdiamine additives in the aqueous phase and carboxylated perfluorocarbon surfactants in the oil phase. Droplets formed under these conditions are shown to possess biocompatible surfaces capable of supporting picoliter-scale protein assays, droplet polymerase chain reaction (PCR), and droplet DNA amplification with isothermal recombinase polymerase amplification (RPA). Droplets formed with polyetherdiamine aqueous additives are stable enough to withstand temperature cycling during PCR (30-40 cycles at 60-94 °C) while maintaining biocompatibility, and the reaction efficiency of RPA is shown to be similar to that with a covalently modified surfactant (KryJeffa). The binding interaction was confirmed with various methods, including FT-IR spectroscopy, NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and fluorescence microscopy. Overall, our results suggest that, by simply introducing a commercially-available, polyetherdiamine additive (Jeffamine ED-900) to the aqueous phase, researchers can avoid synthetic methods in generating biocompatible droplet surfaces capable of supporting DNA and protein analysis at the subnanoliter scale.

Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors.

A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution's ionic strength (0.125-1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.

Quantitation of femtomolar protein levels via direct readout with the electrochemical proximity assay.

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. 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.

Self-regulated, droplet-based sample chopper for microfluidic absorbance detection.

Akin to optical beam chopping, we demonstrate that formation and routing of aqueous droplets in oil can chop a fluidic sample to permit phase sensitive detection. This hand-operated microfluidic sample chopper (µChopper) greatly reduces the detection limit of molecular absorbance in a 27 µm optical path. With direct dependence on path length, absorbance is fundamentally incompatible with microfluidics. While other microfluidic absorbance approaches use complex additions to fabrication, such as fiber coupling and increased optical paths, this self-regulated µChopper uses opposing droplet generators to passively alternate sample and reference droplets at ~10 Hz each. Each droplet's identity is automatically locked-in to its generator, allowing downstream lock-in analysis to nearly eliminate large signal drift or 1/f noise. With a lock-in time constant of 1.9 s and total interrogated volume of 59 nL (122 droplets), a detection limit of 3.0 × 10(-4) absorbance units or 500 nM bromophenol blue (BPB) (29 fmol) was achieved using only an optical microscope and a standard, single-depth (27 µm) microfluidic device. The system was further applied to nanoliter pH sensing and validated with a spectrophotometer. The µChopper represents a fluidic analog to an optical beam chopper, and the self-regulated sample/reference droplet alternation promotes ease of use.

Electrochemical Sensing of the Peptide Drug Exendin-4 Using a Versatile Nucleic Acid Nanostructure.

Although endogenous peptides and peptide-based therapeutics are both highly relevant to human health, there are few approaches for sensitive biosensing of this class of molecules with minimized workflow. In this work, we have further expanded on the generalizability of our recently developed DNA nanostructure architecture by applying it to electrochemical (EC) peptide quantification. While DNA-small molecule conjugates were used in a prior work to make sensors for small molecule and protein analytes, here DNA-peptide conjugates were incorporated into the nanostructure at the electrode surfaces, and antibody displacement permitted rapid peptide sensing. Interestingly, multivalent DNA-peptide conjugates were found to be detrimental to the assay readout, yet these effects could be minimized by solution-phase bioconjugation. The final biosensor was validated for quantifying exendin-4 (4.2 kDa)─a human glucagon-like peptide-1 receptor agonist important in diabetes therapy─for the first time using EC methods with minimal workflow. The sensor was functional in 98% human serum, and the low nanomolar assay range lies between the injected dose concentration and the therapeutic range, boding well for future applications in therapeutic drug monitoring.

Passively operated microfluidic device for stimulation and secretion sampling of single pancreatic islets.

A passively operated polydimethylsiloxane (PDMS) microfluidic device was designed for sampling of hormone secretions from eight individual murine pancreatic islets in parallel. Flow control was achieved using a single hand-held syringe and by exploiting inherent fluidic resistances of the microchannels (R(sampling) = 700 ± 20 kPa s mm(-3) at 37 °C). Basal (3 mM) or stimulatory (11 mM) glucose levels were applied to islets, with stimulation timing (t(stim)) minimized to 15 ± 2 s using modified reservoirs. Using enzyme-linked immunosorbent assays (ELISA) for postsampling analyses, we measured statistically equal levels of 1 h insulin secretion (1.26 ± 0.26 and 6.55 ± 1.00 pg islet(-1) min(-1), basal and stimulated; 62 islets) compared to standard, bulk sampling methods (1.01 ± 0.224 and 6.04 ± 1.53 pg islet(-1) min(-1), basal and stimulated; 200 islets). Importantly, the microfluidic platform revealed novel information on single-islet variability. Islet volume measurements with confocal reflectance microscopy revealed that insulin secretion had only limited correlation to islet volume, suggesting a more significant role for cellular architecture and paracrine signaling within the tissue. Compared to other methods using syringe pumps or electroosmotic flow control, this approach provides significant advantages in ease-of-use and device disposability, easing the burden on nonexperts.