Toward Translational Impact of Low-Glucose Strategies on Red Blood Cell Storage Optimization.

Transfusion of stored red blood cells (RBCs) to patients is a critical component of human healthcare. Following purification from whole blood, RBCs are stored in one of many media known as additive solutions for up to 42 days. However, during the storage period, the RBCs undergo adverse chemical and physical changes that are often collectively known as the RBC storage lesion. Storage of RBCs in additive solutions modified to contain physiological levels of glucose, as opposed to hyperglycemic levels currently used in most cases, reduces certain markers of the storage lesion, although intermittent doses of glucose are required to maintain normoglycemic conditions. Here, we describe an electrically actuated valving system to dispense small volumes of glucose into 100 mL PVC storage bags containing packed RBCs from human donors. The RBCs were stored in a conventional additive solution (AS-1) or a normoglycemic version of AS-1 (AS-1N) and common markers of stored RBC health were measured at multiple time points throughout storage. The automated feeding device delivered precise and predictable volumes of concentrated glucose to maintain physiological glucose levels for up to 37 days. Hemolysis, lactate accumulation, and pH values of RBCs stored in AS-1N were statistically equivalent to values measured in AS-1, while significant reductions in osmotic fragility and intracellular sorbitol levels were measured in AS-1N. The reduction of osmotic fragility and oxidative stress markers in a closed system may lead to improved transfusion outcomes for an important procedure affecting millions of people each year.

A 3D-printed multi-compartment device that enables dynamic PK/PD profiles of antibiotics.

Pathogens develop resistance to various drugs while under the selective pressure of antibiotics resulting in the emergence of bacterial strains that are resistant to multiple treatment options. Unfortunately, the resistance to antibiotics has also been accompanied by a reduction in the development of novel antibiotics to combat various pathogens. Current diagnostic tools, which are used in parts of the early developmental process of antibiotics, primarily consist of static susceptibility tests that do not resemble the pharmacokinetics of the therapy in vivo. Here, we designed and 3D-printed cubical inserts with membranes on two of the cube faces that allow diffusion of a molecule across two planes. These inserts are used with a 3D-printed device to create a two-compartment model to mimic the pharmacokinetics of a molecule in humans from multiple types of administration. Fluorescein was used to characterize the device and the diffusion of molecules from a flowing channel, through a membrane in the first plane (representing the primary compartment in vivo, or plasma), followed by measurement in the second compartment (that represents the interstitial fluid). The dynamic, two-compartment model was tested using both gram-positive and gram-negative bacterial strains in the secondary compartment. The ATP/OD600 (a measure of antibiotic activity) of a kanamycin-resistant E. coli strain challenged with the antibiotic levofloxacin increased after reaching an effective concentration of the antibiotic at 2 h, equating to a secondary compartment concentration of 3.5 ± 1.3 µM levofloxacin. The ATP/OD600 of a chloramphenicol-resistant B. subtilis strain challenged with the antibiotic levofloxacin remained steady or increased slightly after reaching an effective concentration of the antibiotic. The earliest statistical difference was detected 3 h after the start of the PK curve, which corresponds with a secondary compartment concentration of 4.8 ± 1.8 µM levofloxacin. Our results demonstrate that a fabricated 2-compartment model (1) provides realistic PK values to those published from in vivo studies and (2) can be used to determine antibiotic pharmacodynamics.

A 3D-printed, multi-modal microfluidic device for measuring nitric oxide and ATP release from flowing red blood cells.

In this paper, a 3D-printed multi-modal device was designed and fabricated to simultaneously detect nitric oxide (NO) and adenosine triphosphate (ATP) in red blood cell suspensions prepared from whole blood. Once a sample was injected into the device, NO was first detected (via amperometry) using a three-electrode, dual-opposed, electrode configuration with a platinum-black/Nafion coated gold working electrode. After in-line amperometric detection of NO, ATP was detected via a chemiluminescence reaction, with a luciferin/luciferase solution continuously pumped into an integrated mixing T and the resulting light being measured with a PMT underneath the channel. The device was optimized for mixing/reaction conditions, limits of detection (40 nM for NO and 30 nM for ATP), and sensitivity. This device was used to determine the basal (normoxic) levels of NO and ATP in red blood cells, as well as an increase in concentration of both analytes under hypoxic conditions. Finally, the effect of storing red blood cells in a commonly used storage solution was also investigated by monitoring the production of NO and ATP over a three-week storage time.

Albumin Glycation Affects the Delivery of C-Peptide to the Red Blood Cells.

Serum albumin is a prominent plasma protein that becomes modified in hyperglycemic conditions. In a process known as glycation, these modifications can change the structure and function of proteins, which decrease ligand binding capabilities and alter the bioavailability of ligands. C-peptide is a molecule that binds to the red blood cell (RBC) and stimulates the release of adenosine triphosphate (ATP), which is known to participate in the regulation of blood flow. C-peptide binding to the RBC only occurs in the presence of albumin, and downstream signaling cascades only occur when the albumin and C-peptide complex contains Zn2+. Here, we measure the binding of glycated bovine serum albumin (gBSA) to the RBC in conditions with or without C-peptide and Zn2+. Key to these studies is the analytical sample preparation involving separation of BSA fractions with boronate affinity chromatography and characterization of the varying glycation levels with mass spectrometry. Results from this study show an increase in binding for higher % glycation of gBSA to the RBCs, but a decrease in ability to deliver C-peptide (0.75 ± 0.11 nM for 22% gBSA) compared to samples with less glycation (1.22 ± 0.16 nM for 13% gBSA). A similar trend was measured for Zn2+ delivery to the RBC as a function of glycation percentage. When 15% gBSA or 18% gBSA was combined with C-peptide/Zn2+, the derived ATP release from the RBCs significantly increased to 113% or 36%, respectively. However, 26% gBSA with C-peptide/Zn2+ had no significant increase in ATP release from RBCs. These results indicate that glycation of BSA interferes in C-peptide and Zn2+ binding to the RBC and subsequent RBC ATP release, which may have implications in C-peptide therapy for people with type 1 diabetes.

A 3D-printed transfusion platform reveals beneficial effects of normoglycemic erythrocyte storage solutions and a novel rejuvenating solution.

A set of 3D-printed analytical devices were developed to investigate erythrocytes (ERYs) processed in conventional and modified storage solutions used in transfusion medicine. During storage, prior to transfusion into a patient recipient, ERYs undergo many chemical and physical changes that are not completely understood. However, these changes are thought to contribute to an increase in post-transfusion complications, and even an increase in mortality rates. Here, a reusable fluidic device (fabricated with additive manufacturing technologies) enabled the evaluation of ERYs prior to, and after, introduction into a stream of flowing fresh ERYs, thus representing components of an in vivo ERY transfusion on an in vitro platform. Specifically, ERYs stored in conventional and glucose-modified solutions were assayed by chemiluminescence for their ability to release flow-induced ATP. The ERY's deformability was also determined throughout the storage duration using a novel membrane transport approach housed in a 3D-printed scaffold. Results show that hyperglycemic conditions permanently alter ERY deformability, which may explain the reduced ATP release, as this phenomenon is related to cell deformability. Importantly, the reduced deformability and ATP release were reversible in an in vitro model of transfusion; specifically, when stored cells were introduced into a flowing stream of healthy cells, the ERY-derived release of ATP and cell deformability both returned to states similar to that of non-stored cells. However, after 1-2 weeks of storage, the deleterious effects of the storage were permanent. These results suggest that currently approved hyperglycemic storage solutions are having adverse effects on stored ERYs used in transfusion medicine and that normoglycemic storage may reduce the storage lesion, especially for cells stored for longer than 14 days.

Specific Binding of Leptin to Red Blood Cells Delivers a Pancreatic Hormone and Stimulates ATP Release.

Since its discovery in 1994, leptin continues to have new potential physiological roles uncovered, including a role in the regulation of blood flow. Leptin's role in regulating blood flow is not completely understood. Red blood cell (RBC)-derived ATP is a recognized stimulus of blood flow, and multiple studies suggest that C-peptide, a hormone secreted in equimolar amounts with insulin from the pancreatic ß-cells, can stimulate that release when delivered by albumin and in combination with Zn2+. Here, we report leptin delivers C-peptide and Zn2+ to RBCs in a saturable and specific manner. We labeled leptin with technetium-99 m (99mTc) to perform binding studies while using albumin to block the specific binding of 99mTc-leptin in the presence or absence of C-peptide. Our results suggest that leptin has a saturable and specific binding site on the RBC ((Kd = 1.79 ± 0.46) × 10-7 M) that is statistically equal to the binding affinity in the presence of 20 nM C-peptide ((Kd = 2.05 ± 0.20) × 10-7 M). While the binding affinity between leptin and the RBC did not change with C-peptide, the moles of bound leptin did increase with C-peptide, suggesting a separate binding site on the cell for a leptin/C-peptide complex. The RBC-derived ATP increased in the presence of a leptin/C-peptide/Zn2+ addition, in a concentration-dependent manner. Control RBCs ATP release increased (71 ± 5.6%) in the presence of C-peptide and Zn2+, which increased further to (94 ± 5.6%) in the presence of Zn2+, C-peptide, and leptin.

A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.

Plasma proteins are covalently modified in vivo by the high-glucose conditions in the bloodstreams of people with diabetes, resulting in changes to both structure and function. Human Serum Albumin (HSA) functions as a carrier-protein in the bloodstream, binding various ligands and tightly regulating their bioavailability. HSA is known to react with glucose via the Maillard reaction, causing adverse effects on its ability to bind and deliver certain ligands, such as metals. Here, the binding between in vivo glycated HSA and zinc (Zn2+) was determined using a novel centrifugal ultrafiltration method that was developed using a 3D-printed device. This method is rapid (90 minutes), capable of high-throughput measurements (24 samples), low-cost (<$1.00 USD per device) and requires lower sample volumes (200 µL) compared to other binding techniques. This device was used to determine an equilibrium dissociation constant between Zn2+ and a commercially obtained normal HSA (nHSA) with a glycation level of 11.5% (Kd = 2.1 (±0.5) × 10-7 M). A glycated fraction of the nHSA sample was enriched (gHSA, 65.5%) and isolated using boronate-affinity chromatography, and found to have a 2.3-fold decrease in Zn2+ binding-affinity (Kd = 4.8 (±0.8) × 10-7 M) when compared to the nHSA sample. The level of glycation of HSA in control plasma (13.0% ± 0.8, n = 3 donors) and plasma from people with diabetes (26.9% ± 6.6, n = 5 donors) was assessed using mass spectrometry. Furthermore, HSA was isolated from plasma obtained in-house from a person with type 1 diabetes and found to have a glycation level of 24.1% and Kd = 3.3 (± 0.5) × 10-7 M for Zn2+, revealing a 1.5-fold decrease in binding affinity compared to nHSA. These findings suggest that increased levels of glycated HSA result in reduced binding to Zn2+, which may have implications in complications associated with diabetes.

PolyJet 3D-Printed Enclosed Microfluidic Channels without Photocurable Supports.

Microfluidic devices have historically been prepared using fabrication techniques that often include photolithography and/or etching. Recently, additive manufacturing technologies, commonly known as 3D-printing, have emerged as fabrication tools for microfluidic devices. Unfortunately, PolyJet 3D-printing, which utilizes a photocurable resin that can be accurately printed, requires the use of support material for any designed void space internal to the model. Removing the support material from the printed channels is difficult in small channels with single dimensions of less than ∼200 µm and nearly impossible to remove from designs that contain turns or serpentines. Here, we describe techniques for printing channels ranging in cross sections from 0.6 cm × 1.5 cm to 125 µm × 54 µm utilizing commercially available PolyJet printers that require minimal to no postprocessing to form sealed channels. Specifically, printer software manipulation allows printing of one model with an open channel or void that is sealed with either a viscous liquid or a polycarbonate membrane (no commercially available support material). The printer stage is then adjusted and a second model is printed directly on top of the first model with the selected support system. Both the liquid-fill and the membrane method have enough structural integrity to support the printing resin while it is being cured. Importantly, such complex channel geometries as serpentine and Y-mixers can be designed, printed, and in use in under 2 h. We demonstrate device utility by measuring ATP release from flowing red blood cells using a luciferin/luciferase chemiluminescent assay that involves on-chip mixing and optical detection.

A rapid method for post-antibiotic bacterial susceptibility testing.

Antibiotic susceptibility testing is often performed to determine the most effective antibiotic treatment for a bacterial infection, or perhaps to determine if a particular strain of bacteria is becoming drug resistant. Such tests, and others used to determine efficacy of candidate antibiotics during the drug discovery process, have resulted in a demand for more rapid susceptibility testing methods. Here, we have developed a susceptibility test that utilizes chemiluminescent determination of ATP release from bacteria and the overall optical density (OD600) of the bacterial solution. Bacteria release ATP during a growth phase or when they are lysed in the presence of an effective antibiotic. Because optical density increases during growth phase, but does not change during bacterial lysing, an increase in the ATP:optical density ratio after the bacteria have reached the log phase of growth (which is steady) would indicate antibiotic efficacy. Specifically, after allowing a kanamycin-resistant strain of Escherichia coli (E.coli) to pass through the growth phase and reach steady state, the addition of levofloxacin, an antibiotic to which E. coli is susceptible, resulted in a significant increase in the ATP:OD600 ratio in comparison to the use of kanamycin alone (1.80 +/- 0.50 vs. 1.12 +/- 0.28). This difference could be measured 20 minutes after the addition of the antibiotic, to which the bacteria are susceptible, to the bacterial sample. Furthermore, this method also proved useful with gram positive bacteria, as the addition of kanamycin to a chloramphenicol-resistant strain of Bacillus subtilis (B. subtilis) resulted in an ATP:OD600 ratio of 2.14 +/- 0.26 in comparison to 0.62 +/- 0.05 for bacteria not subjected to the antibiotic to which the bacteria are susceptible. Collectively, these results suggest that measurement of the ATP:OD600 ratio may provide a susceptibility test for antibiotic efficacy that is more rapid and quantitative than currently accepted techniques.

Review of 3D Cell Culture with Analysis in Microfluidic Systems.

A review with 105 references that analyzes the emerging research area of 3D cell culture in microfluidic platforms with integrated detection schemes. Over the last several decades a central focus of cell culture has been the development of better in vivo mimics. This has led to the evolution from planar cell culture to cell culture on 3D scaffolds, and the incorporation of cell scaffolds into microfluidic devices. Specifically, this review explores the incorporation of suspension culture, hydrogels scaffolds, paper-based scaffolds, and fiber-based scaffolds into microfluidic platforms. In order to decrease analysis time, simplify sample preparation, monitor key signaling pathways involved in cell-to-cell communication or cell growth, and combat the limitations of sample volume/ dilution seen in traditional assays, researchers have also started to focus on integrating detection schemes into the cell culture devices. This review will highlight the work that has been performed towards combining these techniques and will discuss potential future directions. It is clear that microfluidic-based 3D cell culture coupled with quantitative analysis can greatly improve our ability to mimic and understand in vivo systems.

Ultrafiltration binding analyses of glycated albumin with a 3D-printed syringe attachment.

Protein-ligand binding assays facilitate the understanding of biomolecular interactions. Classical equilibrium dialysis methods are often used for accurate determination of binding properties. While accurate, the long equilibration times associated with the technique (> 6 h) hinder throughput. Here, in an attempt to gather high-accuracy results while reducing total analysis time, a low pressure ultrafiltration method that relies on a simple membrane-containing syringe attachment was developed. A minimal portion (1-2%) of the solution containing the binding analytes of interest is driven through the membrane pores and collected for analysis. Specifically, the device was used to investigate the binding affinity between Zn2+ and either normal human serum albumin (nHSA) or a commercially purchased glycated human serum albumin (gHSA). Both of these ligand/protein-binding systems have implications in type 1 diabetes. The device was then used to investigate the binding between the various albumin types and C-peptide, the 31 amino acid peptide that is co-secreted with insulin from pancreatic ß cells. Results for nHSA/Zn2+ binding obtained using the ultrafiltration method (Kd = 5.77 ± 0.19 × 10-7 M) were statistically equivalent with results reported using other methods. Importantly, the amount of Zn2+ bound to the nHSA was significantly different from the gHSA (97 ± 2% protein bound vs. 91 ± 3%, respectively p < 0.05). The binding affinity of C-peptide to nHSA (Kd = 2.4 ± 0.3 × 10-6 M) agreed with values reported in the literature using standard techniques. Unlike Zn2+ binding, the binding of C-peptide to nHSA was statistically equal to its binding to gHSA (77.7 ± 6.2 and 78.8 ± 7.4%, respectively), suggesting that C-peptide replacement therapy in people with T1D may be strongly dependent upon the characteristics of Zn2+ binding to human serum albumin. Graphical abstract ᅟ.

A Printed Equilibrium Dialysis Device with Integrated Membranes for Improved Binding Affinity Measurements.

Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ∼3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system's requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn2+ and human serum albumin (Kd = (5.62 ± 0.93) × 10-7 M) under physiological conditions that is statistically equal to the constants reported in the literature.

3D-printed Microfluidic Devices: Fabrication, Advantages and Limitations-a Mini Review.

A mini-review with 79 references. In this review, the most recent trends in 3D-printed microfluidic devices are discussed. In addition, a focus is given to the fabrication aspects of these devices, with the supplemental information containing detailed instructions for designing a variety of structures including: a microfluidic channel, threads to accommodate commercial fluidic fittings, a flow splitter; a well plate, a mold for PDMS channel casting; and how to combine multiple designs into a single device. The advantages and limitations of 3D-printed microfluidic devices are thoroughly discussed, as are some future directions for the field.

An In Vitro Diagnostic for Multiple Sclerosis Based on C-peptide Binding to Erythrocytes.

OBJECTIVE: To investigate the utility of a blood-based lab test as an aid in identifying patients with Multiple Sclerosis (MS). METHODS: Whole blood from subjects with MS, non-MS neurologic diseases, and healthy controls was centrifuged to isolate erythrocytes. Following the addition of exogenous C-peptide, the supernatant was assayed for remaining C-peptide using an enzyme linked immunosorbent assay (ELISA). RESULTS: The cohort included subjects with MS (n=86), other non-MS neurologic diseases (OND n=75), and healthy controls (n=39). The average C-peptide bound to erythrocytes in MS samples (3.51±0.59pmol) was significantly higher than non-MS subjects (2.23±0.51pmol; p<0.001) and healthy controls (1.99±0.32pmol; p<0.001). Using a cutoff of 3.04pmol of C-peptide uptake, the test exhibited a sensitivity of 98.3% and specificity of 89.5%. A receiver-operator characteristic (ROC) curve generated from the ratio of the sensitivity to 1-selectivity resulted in an area under the curve of 0.97. CONCLUSIONS: Exogenous C-peptide binding to erythrocytes has potential value in distinguishing MS subjects from non-MS neurologic diseases and healthy controls.

Drug penetration and metabolism in 3D cell cultures treated in a 3D printed fluidic device: assessment of irinotecan via MALDI imaging mass spectrometry.

Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with MALDI imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in 3D cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput.

A Diffusion-Based and Dynamic 3D-Printed Device That Enables Parallel in Vitro Pharmacokinetic Profiling of Molecules.

The process of bringing a drug to market involves many steps, including the preclinical stage, where various properties of the drug candidate molecule are determined. These properties, which include drug absorption, distribution, metabolism, and excretion, are often displayed in a pharmacokinetic (PK) profile. While PK profiles are determined in animal models, in vitro systems that model in vivo processes are available, although each possesses shortcomings. Here, we present a 3D-printed, diffusion-based, and dynamic in vitro PK device. The device contains six flow channels, each with integrated porous membrane-based insert wells. The pores of these membranes enable drugs to freely diffuse back and forth between the flow channels and the inserts, thus enabling both loading and clearance portions of a standard PK curve to be generated. The device is designed to work with 96-well plate technology and consumes single-digit milliliter volumes to generate multiple PK profiles, simultaneously. Generation of PK profiles by use of the device was initially performed with fluorescein as a test molecule. Effects of such parameters as flow rate, loading time, volume in the insert well, and initial concentration of the test molecule were investigated. A prediction model was generated from this data, enabling the user to predict the concentration of the test molecule at any point along the PK profile within a coefficient of variation of ∼ 5%. Depletion of the analyte from the well was characterized and was determined to follow first-order rate kinetics, indicated by statistically equivalent (p > 0.05) depletion half-lives that were independent of the starting concentration. A PK curve for an approved antibiotic, levofloxacin, was generated to show utility beyond the fluorescein test molecule.

Polymer Coatings in 3D-Printed Fluidic Device Channels for Improved Cellular Adherence Prior to Electrical Lysis.

This paper describes the design and fabrication of a polyjet-based three-dimensional (3D)-printed fluidic device where poly(dimethylsiloxane) (PDMS) or polystyrene (PS) were used to coat the sides of a fluidic channel within the device to promote adhesion of an immobilized cell layer. The device was designed using computer-aided design software and converted into an .STL file prior to printing. The rigid, transparent material used in the printing process provides an optically transparent path to visualize endothelial cell adherence and supports integration of removable electrodes for electrical cell lysis in a specified portion of the channel (1 mm width × 0.8 mm height × 2 mm length). Through manipulation of channel geometry, a low-voltage power source (500 V max) was used to selectively lyse adhered endothelial cells in a tapered region of the channel. Cell viability was maintained on the device over a 5 day period (98% viable), though cell coverage decreased after day 4 with static media delivery. Optimal lysis potentials were obtained for the two fabricated device geometries, and selective cell clearance was achieved with cell lysis efficiencies of 94 and 96%. The bottleneck of unknown surface properties from proprietary resin use in fabricating 3D-printed materials is overcome through techniques to incorporate PDMS and PS.

C-peptide and zinc delivery to erythrocytes requires the presence of albumin: implications in diabetes explored with a 3D-printed fluidic device.

People with type 1 diabetes (T1D) must administer insulin exogenously due to the destruction of their pancreatic ß-cells. Endogenous insulin is stored in ß-cell granules along with C-peptide, a 31 amino acid peptide that is secreted from these granules in amounts equal to insulin. Exogenous co-administration of C-peptide with insulin has proven to reduce diabetes-associated complications in animals and humans. The exact mechanism of C-peptide's beneficial effects after secretion from the ß-cell granules is not completely understood, thus hindering its development as an exogenously administered hormone. Monitoring tissue-to-tissue communication using a 3D-printed microfluidic device revealed that zinc and C-peptide are being delivered to erythrocytes by albumin. Upon delivery, erythrocyte-derived ATP increased by >50%, as did endothelium-derived NO, which was measured downstream in the 3D-printed device. Our results suggest that hormone replacement therapy in diabetes may be improved by exogenous administration of a C-peptide ensemble that includes zinc and albumin.

Microfluidic device with tunable post arrays and integrated electrodes for studying cellular release.

In this paper, we describe the development of a planar, pillar array device that can be used to image either side of a tunable membrane, as well as sample and detect small molecules in a cell-free region of the microchip. The pores are created by sealing two parallel PDMS microchannels (a cell channel and a collector channel) over a gold pillar array (5 or 10 µm in height), with the device being characterized and optimized for small molecule cross-over while excluding a flowing cell line (here, red blood cells, RBCs). The device was characterized in terms of the flow rate dependence of analyte cross-over and cell exclusion as well as the ability to perform amperometric detection of catechol and nitric oxide (NO) as they cross-over into the collector channel. Using catechol as the test analyte, the limits of detection (LOD) of the cross-over for the 10 µm and 5 µm pillar array heights were shown to be 50 nM and 105 nM, respectively. Detection of NO was made possible with a glassy carbon detection electrode (housed in the collector channel) modified with Pt-black and Nafion, to enhance sensitivity and selectivity, respectively. Reproducible cross-over of NO as a function of concentration resulted in a linear correlation (r(2) = 0.995, 7.6-190 µM), with an LOD for NO of 230 nM on the glassy carbon/Pt-black/0.05% Nafion electrode. The applicability of the device was demonstrated by measuring the NO released from hypoxic RBCs, with the device allowing the released NO to cross-over into a cell free channel where it was detected in close to real-time. This type of device is an attractive alternative to the use of 3-dimensional devices with polycarbonate membranes, as either side of the membrane can be imaged and facile integration of electrochemical detection is possible.