Evaluating the effects of estradiol on endothelial nitric oxide stimulated by erythrocyte-derived ATP using a microfluidic approach

Recently, estrogens have been reported to have protective effects against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Although the molecular mechanism for such a protective effect is currently incomplete, we hypothesized that estradiol may reduce the release of ATP from erythrocytes (ERYs), thereby lowering the production of nitric oxide (NO) by endothelial cells. Here, we report on the use of a microfluidic device to investigate the direct effects of the estrogen estradiol on endothelial cell nitric oxide production. In addition, the incorporation of a thin polycarbonate membrane into the device enabled the passage of ERYs through the device to determine indirect effects of estradiol on NO production that may be meditated by ERYs. When these ERYs were incubated with increasing concentrations of estradiol, the NO production from the endothelial cells was attenuated to a value that was only 59 ± 7% of ERYs in the absence of estradiol. This decrease in NO production coincides with reductions in ERY-derived ATP release in the presence of estradiol. Estradiol is typically reported to have NO-stimulating effects; however, such reports have employed in vitro experimental designs that include only a single cell type. To demonstrate the potential importance of this attenuation of ATP from ERYs, results from a small-scale study show that the ATP release obtained from healthy controls was 138 ± 21 nM (n = 18) while the release from the ERYs obtained from people with MS was 375 ± 51 nM (n = 11). The studies reported here involving multiple cells types (endothelial cells and ERYs) may lead to a reappraisal of the in vivo activities of estradiol.     

A microchip-based endothelium mimic utilizing open reservoirs for cell immobilization and integrated carbon ink microelectrodes for detection

This paper describes the fabrication and characterization of a microfluidic device that utilizes a reservoir-based approach for endothelial cell immobilization and integrated embedded carbon ink microelectrodes for the amperometric detection of extracellular nitric oxide (NO) release. The design utilizes a buffer channel to continuously introduce buffer or a plug of stimulant to the reservoir as well as a separate sampling channel that constantly withdraws buffer from the reservoir and over the microelectrode. A steel pin is used for both the fluidic connection to the sampling channel and to provide a quasi-reference electrode for the carbon ink microelectrode. Characterization of the device was performed using NO standards produced from a NONOate salt. Finally, NO release from a layer of immobilized endothelial cells was monitored and quantified using the system. This system holds promise as a means to electrochemically detect extracellular NO release from endothelial cells in either an array of reservoirs or concurrently with fluorescence-based intracellular NO measurements.     

Analysis of Nitric Oxide from Chemical Donors Using CMOS Platinum Microelectrodes

Electrochemical detection of NO generated from chemical donors is reported. Because NO is an important biological messenger, many donor sources and detection methods have been developed. Few reports have characterized NO donors using electrochemistry despite electrochemical techniques being sensitive and selective. Here, a CMOS platinum microelectrode array is interfaced with a microfluidic device for the electrochemical analysis of NO from (Z)‐1‐[N‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate (DETA/NO). The donor parent amine fouls the electrode, resulting in substantial signal loss, but an electrochemical cleaning method was developed that substantially reduces fouling and allows detection of NO between 90 nM and 1 µM.     

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

To elucidate the protein–protein interactions of hemoglobin (Hb) variants A and A₂, HbA was first shown to bind with HbA₂ in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA₂ outside the RBC was shown by cross electrophoresis. The starch–agarose gel electrophoresis of hemolysate, RBCs, freeze‐thawed RBCs and the supernatant of freeze‐thawed RBCs showed that the interaction between HbA and HbA₂ was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA₂ in RBCs (RBC HbA‐HbA₂) and hemolysate (hemolysate HbA‐HbA₂) were isolated from the starch–agarose gel and separated by 5–12% SDS‐PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA‐HbA₂ but not in hemolysate HbA‐HbA₂. Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α‐globin, δ‐globin and β‐globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.     

Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing

We immobilized adherent human embryonic kidney (HEK) cells—which are able to trace adenosine triphosphate (ATP) —inside a microfluidic free-flow electrophoresis (μFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 μM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.     

Simultaneous determination of cell aging and ATP release from erythrocytes and its implications in type 2 diabetes

Glucose-6-phosphate dehydrogenase (G6PD) is a determinant in the antioxidant status of the red blood cell (RBC) and is also used as an indicator of cell age. However, it is unknown if the relationship among antioxidant status, cell age, and RBC-derived adenosine triphosphate (ATP) occurs immediately or over a period of time. Therefore, the development of a simultaneous determination of G6PD activity (via the determination of nicotinamide adenine dinucleotide phosphate (NADPH)) in RBCs and the determination of deformation-induced RBC-derived ATP is described. The NADPH and ATP were determined while undergoing a chemically induced aging process via inhibition of G6PD with dehydroepiandroesterone (DHEA). Upon incubation with DHEA for 30 min, NADPH levels measured in a flow stream decreased to 7.96 ± 1.10 μM from an original value of 13.20 ± 1.80 μM in a 0.02% solution of RBCs. In order to demonstrate a direct relationship between G6PD activity and deformation-induced ATP release from RBCs, a simultaneous microflow determination of G6PD activity and ATP release was performed. Upon inhibition with DHEA, NADPH levels decreased to 8.62 ± 0.29 μM from its original value of 12.73 ± 0.50 μM while ATP release decreased from 0.21 ± 0.07 μM to 0.06 ± 0.02 μM. These values were validated by an examination of NADPH levels in, and ATP release from, RBC fractions containing younger and older cells (separated by cell density centrifugation). This determination provides evidence that antioxidant status in the RBC and its ability to release ATP, a known stimulus of nitric oxide production, are closely related.     

Measuring the simultaneous effects of hypoxia and deformation on ATP release from erythrocytes

It is known that adenosine triphosphate (ATP) is released from red blood cells (RBCs) due to various forms of stimulation such as deformation, pharmacological stimuli, and hypoxia. To date, these various stimuli have been investigated individually. Here, we have combined a microflow system capable of initiating deformation-induced release of ATP from the RBCs at various levels of hypoxia as measured by percent oxygen saturation in the RBC sample. When values of ATP released from deformation and hypoxia are compared to values of ATP release due to hypoxia alone, the relationship between the two stimuli can be deduced. Measurement of RBC-derived ATP with the well-known chemiluminescence assay employing luciferin/luciferase indicates that RBCs deoxygenated for 4 min released 1.84 +/- 0.075 microM ATP. The largest decrease in oxygen saturation was found to be between 0 s (66.3% O(2) saturation) and 15 s (22.3% O(2) saturation). RBCs deoxygenated to a 22.3% O(2) saturation released 0.374 +/- 0.011 microM ATP when pumped through the microflow system. This value is an increase from 0.281 +/- 0.007 microM ATP in the presence of flow alone. The ATP release after exposure to hypoxia at 22.3% O(2) saturation was 0.381 +/- 0.014 microM ATP, a value statistically equivalent to that of hypoxia and flow combined. These data suggest that, at an oxygen saturation point of around 25.0% or above, deformation contributes to ATP release from the RBC; however, beyond this saturation point, the ATP release is largely due to hypoxia.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

A discrete dielectrophoresis device for the separation of malaria-infected cells

Malaria is a serious disease caused by Plasmodium parasites that infect red blood cells (RBCs). This paper presents the continuous separation of malaria-infected RBCs (iRBCs) from normal blood cells. The proposed method employed the discrete dielectrophoresis (DEP) in a microfluidic device with interdigitated electrodes. Our aim is to treat a sample having high concentration of cells to realize high throughput and to prevent the clogging of the microchannel with the use of the discrete DEP. The discrete DEP force for deflecting cells in the device was controlled by adjusting the magnitude, frequency, and duty cycle of the applied voltage. The effectiveness of the proposed method was demonstrated by separating the malaria-infected cells in samples having a cell concentration of 10⁶ cells/µl. From experimental results, we determined the enrichment that is needed to enhance the detection in the case of low parasitemia. The enrichment of the infected cells at the device output was 3000 times as high as that of the input containing 1 infected cell to 10⁶ normal cells. Therefore, the proposed method is highly effective and can significantly facilitate the detection of the infected cells for the identification of Malaria patients.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

High-throughput biophysical measurement of human red blood cells

This paper reports a microfluidic system for biophysical characterization of red blood cells (RBCs) at a speed of 100-150 cells s(-1). Electrical impedance measurement is made when single RBCs flow through a constriction channel that is marginally smaller than RBCs' diameters. The multiple parameters quantified as mechanical and electrical signatures of each RBC include transit time, impedance amplitude ratio, and impedance phase increase. Histograms, compiled from 84,073 adult RBCs (from 5 adult blood samples) and 82,253 neonatal RBCs (from 5 newborn blood samples), reveal different biophysical properties across samples and between the adult and neonatal RBC populations. In comparison with previously reported microfluidic devices for single RBC biophysical measurement, this system has a higher throughput, higher signal to noise ratio, and the capability of performing multi-parameter measurements.     

Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells

A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).     

Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

A novel microfluidic capture and monitoring method for assessing physiological damage of C. elegans under microgravity

Spatial microgravity is a significant factor affecting and causing physiological changes of organisms in space environment. On‐site assessment of the damage associated to microgravity is very important for future long‐term space exploration of mankind. In this paper, a new microfluidic device for analyzing the damage of microgravity on Caenorhabditis elegans (C. elegans) has been developed. This device is mainly composed of a microfluidic chip, a dual imaging module, and an imaging acquisition and processing module, which are integrated into a compact system. The microfluidic chip is designed as a platform for monitoring C. elegans, which is captured in an imaging region through a suction structure in the microfluidic chip. A dual imaging module is designed to obtain the images of bright field and fluorescence of C. elegans. The behaviors of C. elegans are analyzed based on the dual‐mode imaging of bright field and fluorescence to assess the degree of damage due to microgravity. A comparative study using a commercial microscope is also conducted to demonstrate the unique advantage of the developed system under the simulated microgravity. The results show that the developed system can evaluate the damage of C. elegans under microgravity accurately and conveniently. Furthermore, this device has compact size and weight, easy operation, and low‐cost, which could be highly advantageous for on‐site evaluation of the damage to microorganisms under microgravity in a space station.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems

Here, we report a microfluidic same‐single‐cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single‐cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P‐glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single‐cell increased dramatically over its same‐cell control on both fluorescence detection systems due to the inhibition by CsA.     

Monitoring erythrocytes in a microchip channel that narrows uniformly: towards an improved microfluidic-based mimic of the microcirculation

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 +/- 0.11, 1.92 +/- 0.14 and 2.09 +/- 0.10 microM ATP as the median cross-sectional area decreased from 4314 to 3192 to 2052 microm(2), respectively. Numerous samples (n = 6) displayed the same trend. Incubating a sample of RBCs with diamide, a substance known to stiffen cell membranes without harming the cell cytosol, provided evidence that no cell lysis occurred in the microchip device. This novel use of lab-on-a-chip technology allows for channel designs that enable an in vitro study of physiological events that occur in the microcirculation.     

Cell culture chip using low-shear mass transport

We have developed a flow cell that allows culturing adherent cells as well as suspended cells in a stable, homogeneous, and low-shear force environment. The device features continuous medium supply and waste exchange. In this paper, a simple and fast protocol for device design, fabrication, and assembly (sealing) based on a poly(dimethylsiloxane) (PMDS)/glass slide hybrid structure is described. The cell culture system performance was monitored, and the effective shear force inside the culture well was also determined. By manipulating the device dimensions and volumetric flow rate, shear stress was controlled during experiments. Cell adhesion, growth, proliferation, and death over long-term culture periods were observed by microscopy. The growth of both endothelial and suspension cells in this device exhibited comparable characteristics to those of traditional approaches. The low-shear culture device significantly reduced shear stress encountered in microfluidic systems, allowing both adherent and suspended cells to be grown in a simple device.     

Electrochemical hematocrit determination in a direct current microfluidic device

Hematocrit (HCT) tests are widely performed to screen blood donors and to diagnose medical conditions. Current HCT test methods include conventional microhematocrit, Coulter counter, CuSO₄ specific gravity, and conductivity‐based point‐of‐care (POC) HCT devices, which can be either expensive, environmentally inadvisable, or complicated. In the present work, we introduce a new and simple microfluidic system for a POC HCT determination. HCT was determined by measuring current responses of blood under 100 V DC for 1 min in a microfluidic device containing a single microchannel with dimensions of 180 μm by 70 μm and 10 mm long. Current responses of red blood cell (RBC) suspensions in PBS or separately plasma at HCT concentrations of 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70 vol% were measured to show feasibility of the microfluidic system for HCT determination. Key parameters affecting current responses included electrolysis bubbles and irreversible RBC adsorption; parameters were optimized via addition of nonionic surfactant Triton X‐100 into sample solution and carbonizing electrode surfaces. The linear trend line of current responses over a range of RBC concentrations were obtained in both PBS and plasma. This work suggested that a simple microfluidic device could be a promising platform for a new POC HCT device.