Development and characterization of a capillary-flow microfluidic device for nucleic acid detection

We have developed a capillary flow-driven microfluidic biosensor to meet the needs of diagnostics for resource-limited areas. The device combined elements of lateral flow assays and microfluidic technology resulting in a hybrid with benefits of both formats. The biosensor was achieved by bonding two pieces of polymethyl methacrylate with channels ablated by a CO₂ laser, and enclosing an absorbent pad. The channels were UV/ozone treated to increase hydrophilicity which enabled capillary flow. The absorbent pad allowed for continuous flow in the channels once filled. The application of biosensor was demonstrated by detection of DNA with a sandwich assay. The target DNA was hybridized with nucleic acid modified magnetic beads as well as Ru(bpy) ₃ ²⁺ doped silica nanoparticles. Fluorescent signals were quantified in a holder fabricated to fit in a fluorescent microtiter plate reader. The capillary flow microfluidic was capable to detect 1?pmol target. The assay format which features rapid analysis and does not require the use of pumps could allow for inexpensive point of care diagnostics in the future.     

A droplet microfluidic approach to single-stream nucleic acid isolation and mutation detection

In this work, a droplet microfluidic platform for genetic mutation detection from crude biosample is described. Single-stream integration of nucleic acid isolation and amplification is realized on a simple fluidic cartridge. Subsequent DNA melting curve is employed with signal normalizing algorithm to differentiate heterozygous K-ras codon 12 c.25G>A mutant from the wild type. This technique showcases an alternative to modular bench-top approaches for genetic mutation screening, which is of interest to decentralized diagnostic platforms.     

A microfluidic device for thermal particle detection

We demonstrate the use of heat to count microscopic particles. A thermal particle detector (TPD) was fabricated by combining a 500-nm-thick silicon nitride membrane containing a thin-film resistive temperature detector with a silicone elastomer microchannel. Particles with diameters of 90 and 200 μm created relative temperature changes of 0.11 and ?0.44 K, respectively, as they flowed by the sensor. A first-order lumped thermal model was developed to predict the temperature changes. Multiple particles were counted in series to demonstrate the utility of the TPD as a particle counter.     

A microfluidic microwell device for immunomagnetic single-cell trapping

Single-cell analysis has been widely applied in various biomedical applications, such as cancer diagnostics, immune status monitoring, and drug screening. To perform an accurate and rapid cellular analysis, various magnetic-activated cell sorting techniques are available in the markets. However, large sample requirement and uneven magnetic field distribution limit its application in single-cell trapping and following analysis. To address these problems, we developed a microfluidic microwell device for immunomagnetic single-cell trapping. By adding a microwell layer between the microchannel and magnet, the magnetic field along the device becomes more uniform. Besides, magnetic beads can be retained in the array of microwell after the high-speed washing step, whereas untrapped beads would be flushed away, resulting in high single-particle trapping efficiency (62%) and purity (99.6%). To achieve large-area single-cell trapping, we introduced a “sweeping” loading protocol to further expand the single-particle trapping range. In the microwell region near to the edge of the magnet, over 3000 single magnetic beads were trapped in a 10 mm² area. Finally, we demonstrated immunomagnetic-labeled THP-1 cells can successfully be trapped at single-cell level in the microwell. The cell trapping process can be done in 10 min. We believe the platform with an accurate and efficient single-cell trapping functionality could potentially be used for various cellular analyses at the single-cell level.     

A multilayer lateral-flow microfluidic device for particle separation

Particle separation technology plays an important role in a wide range of applications as a critical sample preprocessing step for analysis. In this work, we proposed and fabricated a multilayer lateral-flow particle filtration and separation device based on polydimethylsiloxane molding and transfer bonding techniques. Particle separation capability was demonstrated by 4.5-um polystyrene bead filtration and cancer cell (SK-BR-3) retrieving. This device exhibits higher throughput compared with most active particle separation methods and is less vulnerable to membrane clogging problem. This novel multilayer particle filtration and separation device is expected to find applications in biomedical, environmental and microanalysis fields.     

A suction-type, pneumatic microfluidic device for liquid transport and mixing

This study presents a new suction-type, pneumatically driven microfluidic device for liquid delivery and mixing. The three major components, including two symmetrical, normally closed micro-valves and a sample transport/mixing unit, are integrated in this device. Liquid samples can be transported by the suction-type sample transport/mixing unit, which comprised a circular air chamber and a fluidic reservoir. Experimental results show that volume flow rates ranging from 50 to 300 μl/min can be precisely controlled during the sample transportation processes. Moreover, the transport/mixing unit can also be used as a micro-mixer to generate efficient mixing between two reaction chambers by regulating the time-phased deformation of the polydimethylsiloxane (PDMS) membranes. A mixing efficiency as high as 98.4% can be achieved within 5 s utilizing this prototype pneumatic microfluidic device. Consequently, the development of this new suction-type, pneumatic microfluidic device can be a promising tool for further biological applications and for chemical analysis when integrated into a micro-total analysis system (μ-TAS) device.     

A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm². This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

Decreasing microfluidic evaporation loss using the HMDL method: open systems for nucleic acid amplification and analysis

Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss (V _loss/V _ini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures (T _HMDL and T _TR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the V _loss/V _ini within 60 min is low to 5% (T _HMDL = 105°C, T _TR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp fragment from Listeria monocytogenes genomic DNA.     

A microfluidic device for chemical and mechanical stimulation of mesenchymal stem cells

Recently, there has been considerable interest in stem cells which have the potential to differentiate into multiple lineages for cell therapy. Special attention has been paid, in particular, to the use of alternative sources of stem cells with fewer ethical issues. For example, mesenchymal stem cells (MSCs) from bone marrow have been proven to be multipotent for transplantation and tissue engineering. In this study, an integrated microfluidic chip capable of chemically and mechanically stimulating human mesenchymal stem cells (hMSCs) for adipogenic differentiation was presented. It was composed of a dilution module for controlling the insulin concentrations, and pneumatically-modulated membrane structures for precisely applying shear stresses on cells to perform chemical and mechanical stimulation, respectively. With this approach, a long-term culture and differentiation of MSCs were performed in an automatic manner. The accumulation of lipids that represented the results of insulin simulation was evaluated by Oil Red O staining. The measurements including lipid droplet numbers, optical density (OD) values, and expression of the PPARγ2 gene were used to assess the level of differentiation of the MSCs. The experimental result showed that the maximum oil droplets were induced under an optimal insulin concentration of 10 μg/ml. It was also revealed that, under mechanical stimulation, adipogenesis was inhibited under stronger levels of applied shear stresses and at higher pulsation frequencies. This proposed microfluidic chip has great potential as a powerful tool for MSC studies.     

A novel microfluidic high-throughput resistive pulse sensing device for cells analysis

Microfluidic impedance-based devices offer a simple method for counting and sizing particles and cells in fields of biomedical research and clinical diagnosis. In this work, we present design, fabrication and operational characteristics of a novel high throughput original MEMS-based Coulter counter. This microfluidic device possesses two sub channels including two pairs of coplanar Au/Cr electrodes in each channels which allows double detection of the particles simultaneously and increases the throughput. The present design provides minimizing the cross talk and obviating the need for hydrodynamic focusing of the sample particles by adjusting Y shape insulation obstacle in direction of flow. Moreover, reducing coincidence events and removing electrode polarization effect were purposed by applying optimum sizes for electrodes considering the ease of fabrication and low costs. The reliability of the novel device was evaluated for polystyrene particles and cancer cells in conductive solutions. Results, which were recorded as relative resistance pulses across four sensing zones, illustrate the capability of the double-channel proposed device in detecting, counting and sizing 10 and 20 µm polystyrene particles. The superiority of present design was proved by relative counting error of below 3 and 11% for the 10 µm and 20 µm particles, respectively and a throughput of hundreds particles per second. Aiming at demonstrating the functionality of the proposed device in the biomedical area, counting of SP2/0 cells was performed. The measured counting outputs for cells in the size range of 5.63–17.6 µm were validated with results of hemocytometer cell counter, with relative error less than 7%.

     

A portable,hand-powered microfluidic device for sorting of biological particles

Manually hand-powered portable microfluidic devices are cheap alternatives for point-of-care diagnostics. Currently, on-field tests are limited by the use of bulky syringe pumps, pressure controller and equipment. In this work, we present a manually operated microfluidic device incorporated with a groove-based channel. We show that the device is capable to effectively sort particles/cells by manual hand powering. First, the grooved-based channel with differently sized polystyrene particles was characterized using syringe pumps to study their distributions under various flow rate conditions. Afterward, the particle mixtures were sorted manually using hand power to verify the capability of this device. Finally, the manually operated device was used to sort platelets from peripheral blood mononuclear cells (PBMCs). The platelets were collected with a purity of ~ 100%. The purity of PBMCs was enhanced from 0.8 to 10.4% after multiple processes which results in an enrichment ratio of 13.8. During the process of manual hand pumping, the flow fluctuation caused by unstable injection will not influence the sorting performance. Due to its simplicity, this manually operated microfluidic chip is suitable for outfield settings.     

Polymer-based microfluidic device for measuring membrane protein activities

Functional assays of membrane proteins are becoming increasingly important, both in research and drug discovery applications. The majority of current assays use the patch-clamp technology to measure the activity of ion channels which are over-expressed in cells. In future, in vitro assay systems will be available, which use reconstituted membrane proteins in free-standing lipid bilayers suspended in nano- or micrometer-sized pores. Such functional assays require (1) expression, purification and reconstitution of the membrane protein of interest, (2) a reliable method for lipid bilayer formation and membrane protein integration, and (3) a sensitive detection system. For practical applications, especially for automation, the reliable and controllable transport of fluids is essential. In order to achieve a stable free-standing lipid bilayer, a pore diameter in the micro- to nanometer range is essential. Novel microfluidic devices were developed by bonding a thick (300 μm) polyether ether ketone foil, bearing a channel structure, to a thin (12 μm) foil with a micropore of about 10 μm diameter and then utilized for the formation of stable, free-standing lipid bilayers within the pore. A bacterial voltage-gated potassium channel is integrated therein by fusion and the ion channel activity detected by voltage clamp.     

A massively parallel microfluidic device for long-term visualization of isolated motile cells

Visualizing the natural behavior of motile cells over many hours is a challenge, as cells can leave the field of view of a microscope in a matter of minutes. Many interesting cell behaviors—such as cell division, motility phenotype, cell–cell interactions, and multicellular colony formation—require hours of observation to characterize. We present a microfluidic device that traps hundreds of single motile cells in isolated chambers, thereby allowing observation over several days. This polydimethylsiloxane device features 400 circular chambers, connected to a central serpentine channel. Motile cells are loaded into these chambers through the serpentine channel. The channel is then purged with air, fluidically isolating the chambers from each other and effectively trapping the cells. We applied the device to observe the behavior of the choanoflagellate Salpingoeca rosetta. Because of its ability to live in both solitary and colonial forms, S. rosetta is a useful model organism for the study of the evolutionary origins of multicellularity. In particular, S. rosetta can take on two distinct colonial forms: chain colonies and rosette colonies. With our device, we are able to observe the formation of these colonies from single cells more easily and with higher throughput than ever before. This device has the potential to be a powerful tool for studying the long-term behavior of motile cells.     

A simple photolithography method for microfluidic device fabrication using sunlight as UV source

A straightforward method for microfluidic devices fabrication using sunlight as the ultraviolet (UV) source is established in this work. This method is based on photolithography, but obviates the need for specialized UV exposure facility. Substrates coated with photoresist were placed directly under sun in a perpendicular direction to the sunlight for exposure. Exposure conditions were optimized for patterning features with different kinds of photoresist, photoresist of different thicknesses and dimensions. Exposure time can be adjusted to obtain designed features on a mask with good lateral structure according to the energy measured by UV meter (with a constant intensity of UV in sunlight). Masters produced under optimum exposure conditions were used for the fabrication of several microfluidic devices with different materials, structures, or functions. Resultant devices were shown eminently suitable for microfluidic applications such as electrophoretic separation, multiple gradient generator, and pneumatic valve-based cell culture. This photolithographic method is simple, low cost, easy to operate, and environmental friendly. Especially, the masters can be obtained in parallel simultaneously, which is suitable for chip fabrication for mass production. It is also more attractive for the laboratories, in which the support for photolithographic facility is not available.     

An aptamer-based microfluidic device for thermally controlled affinity extraction

We present a microfluidic device for specific extraction and thermally activated release of analytes using nucleic acid aptamers. The device primarily consists of a microchamber that is packed with aptamer-functionalized microbeads as a stationary phase, and integrated with a micro heater and temperature sensor. We demonstrate the device operation by performing the extraction of a metabolic analyte, adenosine monophosphate coupled with thiazole orange (TO-AMP), with high selectivity to an RNA aptamer. Controlled release of TO-AMP from the aptamer surface is then conducted at low temperatures using on-chip thermal activation. This allows isocratic analyte elution, which eliminates the use of potentially harsh reagents, and enables efficient regeneration of the aptamer surfaces when device reusability is desired.     

A knowledge-based experimental design system for nucleic acid engineering

Presented in this paper is a knowledge-based experimental design system that incorporates the domain expertise used in nucleic acid engineering, thus automating the processing of error-prone, laborious low-level work, and many decision-making steps, and guiding the biologist toward a workable plan. This allows the biologist to work at a higher abstraction level, concentrating on more fundamental, difficult and challenging problems directly related to protein structure - function relationships. Cassette-based site-directed mutagenesis and synthetic gene designs are used as examples to illustrate the utility of the knowledge-based system approach to experimental design.     

Modeling and high performance simulation of electrophoretic techniques in microfluidic chips

Electrophoretic separations comprise a group of analytical techniques such as capillary zone electrophoresis, isoelectric focusing, isotachophoresis, and free flow electrophoresis. These techniques have been miniaturized in the last years and now represent one of the most important applications of the lab-on-a-chip technology. A 3D and time-dependent numerical model of electrophoresis on microfluidic devices is presented. The model is based on the set of equations that governs electrical phenomena, fluid dynamics, mass transport, and chemical reactions. The relationship between the buffer characteristics (ionic strength and pH) and surface potential of channel walls is taken into consideration. Numerical calculations were performed by using PETSc-FEM, in a Python environment, employing high performance parallel computing. The method includes a set of last generation preconditioners and solvers, especially addressed to 3D microfluidic problems, which significantly improve the numerical efficiency in comparison with typical commercial software for multiphysics. In this work, after discussing two validation examples, the numerical prototyping of a microfluidic chip for two-dimensional electrophoresis is presented.