Screen-printed microfluidic device for electrochemical immunoassay

In this paper, a new microfluidic array device has been fabricated with screen printing technology. In contrast to traditional microfabrication processes, our method is simple, inexpensive and also suitable for mass production. The device is used for sandwich-type electrochemical immunoassay, in which probes are covalently attached to the electrode surface via electropolymerized polypyrrole propylic acid (PPA) film. This novel microfluidic system enables the whole array preparation and detection processes, including the probe immobilization, sample injection, enzyme incubation and electrochemical detection, to be conducted in the sealed microchannels. For a demonstration, mouse IgG is selected as the target analyte and its detection is realized by sandwich ELISA with goat anti-mouse IgG, rat anti-mouse IgG (conjugated to alkaline phosphatase) and p-aminophenyl phosphate (PAPP) as the primary antibody, second antibody, and enzyme substrate, respectively. A detection limit of 10 ng mL(-1) (67 pM) is achieved with a dynamic range of 100 ng mL(-1)-10 microg mL(-1). In addition, anti-goat IgG is also immobilized as an alternative probe to test mouse IgG in the solution, in order to demonstrate the multiplexing capability as well as the specificity of the device. As expected, the electrochemical responses are much lower than that using anti-mouse IgG as the probe, indicating good selectivity of the immunoassay device. These results indicate a great promise toward the development of miniaturized, low-cost protein biochips for clinical, forensics, environmental, and pharmaceutical applications.     

Magnetic bead based immunoassay for enumeration of CD4 T lymphocytes on a microfluidic device

Human immunodeficiency virus (HIV) diagnostics are urgently needed in resource-scarce settings. Monitoring of HIV-infected patients requires accurate counting of CD4⁺ T lymphocytes. However, the current methods for enumeration of CD4⁺ T lymphocytes are of high cost, technically complex and time-consuming. In this paper, we developed a simple, rapid and inexpensive one-step immunomagnetic method for separating and counting CD4⁺ T lymphocytes on microfluidic devices with enlarged reaction chambers. CD4⁺ T lymphocytes were successfully separated and captured from the cell suspension obtained from mouse thymus. CD4 counts were determined under an optical microscope in a rapid and simple format. In order to acquire the maximum efficiency of cell capture, relative parameters were investigated, including section area of the reaction chamber and injection flow rate of the cell suspension. The enlarged reaction chamber with two symmetrical cone-shaped ends was helpful for cell capture, and the maximum capability of captured CD4⁺ T lymphocytes was about 700 cells μL⁻¹. Our investigations avoided the complex sample pre-treatment, and the entire analysis time was significantly reduced to 15 min. This CD4 counting microdevice had the potential to reduce the cost for HIV diagnosis in resource-limited settings.     

Electrochemical immunoassay on a 3D microfluidic paper-based device

A high-throughput, simple, fast, low-cost and sensitive paper-based electrochemical immunodevice has been demonstrated based on a functionalized 3D paper-based device for point-of-care diagnosis.     

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.     

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318–1320, 1; Martinez et al., Anal Chem 10:3699–3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606–19611, 3; Lu et al., Electrophoresis 9:1497–1500, 4; Abe et al., Anal Chem 18:6928–6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min.     

A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device

We describe a fluorogenic two-site noncompetitive heterogeneous immunoassay with magnetic beads on a low-voltage digital microfluidic platform using closed electrowetting-on-dielectric (EWOD). All the steps of an enzyme-linked immunosorbent assay (ELISA) were performed on the device using 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) phosphate as the fluorogenic substrate for the enzyme alkaline phosphatase. The performance of the system was demonstrated with cardiac marker Troponin I (cTnI) as a model analyte in phosphate-buffered saline samples. cTnI was detected within the diagnostically relevant range with a limit of detection of 2.0 ng/mL (CV?=?6.47 %). Washing of magnetic beads was achieved by movement through a narrow region of buffer bridging one drop to another with minimal fluid transfer. More than 90 % of the unbound reagents were removed after five washes. Further experiments testing human blood serum on the same platform demonstrated a sample-to-answer time at ～18.5 min detecting 6.79 ng/mL cTnI.     

Aptamer-based fluorometric determination of norovirus using a paper-based microfluidic device

Microchimica Acta - The authors describe a rapid and highly sensitive point-of-care device for rapid determination of noroviruses, a leading cause of acute gastroenteritis. The assay is based on...     

Development of a microfluidic paper-based analytical device for the determination of salivary aldehydes

A low cost, disposable and easy to use microfluidic paper-based analytical device (μPAD) was developed for simple and non-invasive determination of total aldehydes in saliva with a potential to be used in epidemiological studies to assess oral cancer risk. The μPAD is based on the colour reaction between aldehydes (e.g. acetaldehyde, formaldehyde), 3-methyl-2-benzothiazolinone hydrazone (MBTH) and iron(III) to form an intense blue coloured formazan dye. The newly developed μPAD has a 3D design with two overlapping paper layers. The first layer comprises 15 circular detection zones (8 mm in diameter), each impregnated with 8 μL of MBTH, while the second layer contains 15 reagent zones (4 mm in diameter). Two μL of iron(III) chloride are added to each one of the second layer zones after the addition of sample to the detection zones in the first layer. All hydrophilic zones of the μPAD are defined by wax printing using a commercial wax printer.     

Colorimetric determination of acidity constant using a paper-based microfluidic analytical device

Acid dissociation constant is an important chemical characteristic of organic and inorganic compounds and it affects both chemical properties and biological activities of the molecules. Herein, a very simple, fast and cost-effective method based on microfluidic technology has been reported for colorimetric determination of acidity constants. The designed device works based on pH-metric titration of colorful indicators followed by colorimetric measurements by a smart phone mobile device or a flatbed scanner. So, it does not need sophisticated instrumentation and is accomplished in a very short time (about 1 min). All titration steps are transferred on a star-like designed µPAD device: (1) spotting 0.3 µL buffers of different pHs at the end of the channels (reaction zones), (2) spotting a 30 µL portion of the indicator on the center of µPAD followed by movement of indicator solution toward the reaction zone by capillary action of the paper. The measured color change of the indicators at the reaction zone is fitted to the Henderson–Hasselbalch equation, through which acidity constants are calculated. The performance of the device was evaluated by measuring acidity constant of 4 indicators including bromothymol blue, bromocresol green, bromocresol purple and phenolphthalein. A very close agreement was achieved between those measured by the suggested device here and the previously reported values. The reproducibility of this method was lower than 5% for relative standard deviation of three replicate measurements.     

Rapid simultaneous determination of nitrate and nitrite on a centrifugal microfluidic device

A centrifugal microfluidic device was developed for the rapid sequential determination of two critical environmental species, nitrate and nitrite, in water samples. The nitrate is reduced to nitrite and the nitrite is derivatized. The analytes are determined spectrophotometrically through the disc with a 1.4 mm pathlength. The detection limits are 0.05 and 0.16 mg L⁻¹ for nitrite and nitrate respectively. The use of powdered reagents, the 100 μL sample required and the design of the device suggest that it would be suitable for field use.     

High-throughput rheology in a microfluidic device

High-throughput rheological measurements in a microfluidic device are demonstrated. A series of microrheology samples are generated as droplets in an immiscible spacer fluid using a microfluidic T-junction. The compositions of the sample droplets are continuously varied over a wide range. Rheology measurements are made in each droplet using multiple particle tracking microrheology. We review critical design and operating parameters, including the droplet size, flow rates and rapid fabrication methods. Validation experiments are performed by measuring the solution viscosity of glycerine and the biopolymer heparin as a function of concentration. Overall, the combination of microrheology with microfluidics maximizes the number of rheological measurements while simultaneously minimizing the sample preparation time and amount of material, and should be particularly suited to the characterization of scarce or expensive materials.     

Stop-flow lithography in a microfluidic device

Polymeric particles in custom designed geometries and with tunable chemical anisotropy are expected to enable a variety of new technologies in diverse areas such as photonics, diagnostics and functional materials. We present a simple, high throughput and high resolution microfluidic method to synthesize such polymeric particles. Building off earlier work that we have done on continuous flow lithography (CFL) (D. Dendukuri, D. C. Pregibon, J. Collins, T. A. Hatton, P. S. Doyle, Nat. Mater., 2006, 5, 365-369; ref. 1), we have devised and implemented a new setup that uses compressed air driven flows in preference to syringe pumps to synthesize particles using a technique that we call stop-flow lithography (SFL). A flowing stream of oligomer is stopped before polymerizing an array of particles into it, providing for much improved resolution over particles synthesized in flow. The formed particles are then flushed out at high flow rates before the cycle of stop-polymerize-flow is repeated. The high flow rates enable orders-of-magnitude improvements in particle throughput over CFL. However, the deformation of the PDMS elastomer due to the imposed pressure restricts how quickly the flow can be stopped before each polymerization event. We have developed a simple model that captures the dependence of the time required to stop the flow on geometric parameters such as the height, length and width of the microchannel, as well as on the externally imposed pressure. Further, we show that SFL proves to be superior to CFL even for the synthesis of chemically anisotropic particles with sharp interfaces between distinct sections.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.     

An automated fluid-transport device for a microfluidic system

An automated fluid-transport device for a chip-based capillary electrophoresis system has been developed. The device mainly consists of six peristaltic micropumps, two vacuum micropumps, microvalves, multi-way joints, titanium tubes, and a macro-to-micro connector. Various solutions used for the cleaning and activation of chip channels, and electrophoresis separation, are allowed to automatically transport to chip reservoirs by the electric control module. The performance of the whole system was characterized by the analysis of fluorescein sodium using chip electrophoresis with LED-induced fluorescence detection. The peak-height variation (RSD) was 3.8% in six cycles of analyses. Additionally, compared with conventional manual operation, the developed device can spare 60% time for chip pretreatment. This microdevice offers high-efficiency pretreatment for microchips, thereby resulting in a remarkable improvement of analytical capacity for batch samples.     

Multilayer soft lithography of perfluoropolyether based elastomer for microfluidic device fabrication

The compatibility of microfluidic devices with solvents and other chemicals is extremely important for many applications such as organic synthesis in microreactors and drug screening. We report the successful fabrication of microfluidic devices from a novel perfluoropolyether based polymer utilizing the Multilayer Soft Lithography? (MSL) technique with simple, straightforward processing. The perfluorinated polymer SIFEL X-71 8115 is a highly chemically resistant elastomeric material. We demonstrate fabrication of a microfluidic device using an off-ratio bonding technique to bond multiple SIFEL layers, each patterned lithographically. The mechanical properties of the SIFEL MSL valves (including actuation pressures) are similar to PDMS MSL valves of the same geometry. Chemical compatibility tests highlight SIFEL's remarkable resistance to organic solvents, acids and alkalis.     

Design of a recursively-structured valveless device for microfluidic manipulation

A recursively-structured apparatus based on a pneumatic pumping structure has been investigated numerically and experimentally in the present study. For the T-connected channels, this apparatus demonstrated the ability to manipulate the liquid drop from a first channel to a second channel, while simultaneously preventing flow into the third channel. The microTAS research aimed at biochemical analysis miniaturization and integration has recently made explosive progress. However, there is still a considerable technical challenge in integrating these procedures into a multiple-step system. An important issue for this integration is microfluid management techniques. The microTAS method must be designed considering special transport mechanisms to move samples and reagents through the microchannels. The structure of this apparatus was simple and easily fabricated. Moreover, because there is a continuous airflow at the "outlet" during fluid manipulation, it is possible to avoid contamination of the air source similar to the "laminar flow hook" in biological experiments. Utilizing the concept of a recursive structure, one can easily design a device wherein more than three channels are included in the flow network, either intersecting in a single junction or in multiple junctions.     

A microfluidic device based on droplet storage for screening solubility diagrams

This work describes a new microfluidic device developed for the rapid screening of solubility diagrams. In several parallel channels, hundreds of nanolitre volume droplets of a given solution are first stored with a gradual variation in the solute concentration. Then, the application of a temperature gradient along these channels enables us to read directly and quantitatively phase diagrams, concentration vs. temperature. We show, using a solution of adipic acid, that we can measure ten points of the solubility curve in less than 1 hr and with only 250 microL of solution.     

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.