A simple, disposable microfluidic device for rapid protein concentration and purification via direct-printing

A facile and disposable microfluidic device for rapid protein concentration was fabricated by using a direct printing process. Two printed V-shaped microchannels in mirror image orientation were separated by a 100 mum wide toner gap. When a high electric field was applied across the two channels, nanofissures were formed by electric breakdown at the junction toner gap. This microfluidic device with nanofissures was used as a concentrator for protein. Negatively charged proteins were observed to concentrate at the anode side of the nanofissures upon application of an electric field across this junction. Using this device, about 10(3)-10(5)-fold protein concentration was achieved within 10 min. Systematic investigation showed that the concentration mechanism could be explained by the ion exclusion-enrichment effect of the nanofissures. In addition, the present microchip device integrated both functions of concentration and purification were confirmed. This simple on chip protein preconcentration and purification device could be a disposable sample preparation component in printed microfluidic systems used for practical biochemical assays.     

Patterned Colloidal Photonic Crystals

Colloidal photonic crystals (PCs) have been well developed because they are easy to prepare, cost‐effective, and versatile with regards to modification and functionalization. Patterned colloidal PCs contribute a novel approach to constructing high‐performance PC devices with unique structures and specific functions. In this review, an overview of the strategies for fabricating patterned colloidal PCs, including patterned substrate‐induced assembly, inkjet printing, and selective immobilization and modification, is presented. The advantages of patterned PC devices are also discussed in detail, for example, improved detection sensitivity and response speed of the sensors, control over the flow direction and wicking rate of microfluidic channels, recognition of cross‐reactive molecules through an array‐patterned microchip, fabrication of display devices with tunable patterns, well‐arranged RGB units, and wide viewing‐angles, and the ability to construct anti‐counterfeiting devices with different security strategies. Finally, the perspective of future developments and challenges is presented.     

A new on-chip ESI nozzle for coupling of MS with microfluidic devices

This paper presents a new on-chip electrospray ionisation (ESI) nozzle, which can be used as an interface for coupling microfluidic devices with mass spectrometric (MS) detection. The nozzle was micromilled in a polymer foil (polymethylmethacrylate (PMMA) 750 microm thick), normally used as a cover for microfluidic chips. The performance of this device was examined in the ESI-MS analysis of the tetrapeptide MRFA (methionine-argenine-phenylalanine-alanine). The spray quality is basically dependent on the inner diameter of the nozzle, beside the part of the organic modifier in the solution to be sprayed. Three different inner nozzle diameters (30, 50, 100 microm) and two different apex angles were investigated. Stable electrospray conditions can be generated with a relative standard deviation less than 10% of the total ion current, and down to a concentration of 0.01 micromol L(-1). The production of this ESI interface is relatively simple for the purpose of a low-cost batch fabrication of miniaturized analytical instruments.     

Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices

Cell-based microfluidic devices have attracted interest for a wide range of applications. While optical cell counting and flow cytometry-type devices have been reported extensively, sensitive and efficient non-optical methods to detect and quantify cells attached over large surface areas within microdevices are generally lacking. We describe an electrical method for counting cells based on the measurement of changes in conductivity of the surrounding medium due to ions released from surface-immobilized cells within a microfluidic channel. Immobilized cells are lysed using a low conductivity, hypotonic media and the resulting change in impedance is measured using surface patterned electrodes to detect and quantify the number of cells. We found that the bulk solution conductance increases linearly with the number of isolated cells contributing to solution ion concentration. The method of cell lysate impedance spectroscopy is sensitive enough to detect 20 cells microL(-1), and offers a simple and efficient method for detecting and enumerating cells within microfluidic devices for many applications including measurement of CD4 cell counts in HIV patients in resource-limited settings. To our knowledge, this is the most sensitive approach using non-optical setups to enumerate immobilized cells. The microfluidic device, capable of isolating specific cell types from a complex bio-fluidic and quantifying cell number, can serve as a single use cartridge for a hand-held instrument to provide simple, fast and affordable cell counting in point-of-care settings.     

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple "mixer module" with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.     

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.     

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.     

T cell chemotaxis in a simple microfluidic device

This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.     

PDMS-based microfluidic device with multi-height structures fabricated by single-step photolithography using printed circuit board as masters

We have developed a method for fabricating microfluidic devices with multi-height structures using single step photolithography. The whole fabrication process is executed by conventional printed circuit board (PCB) technology without the need of having access to clean room facilities. Specifically designed "windows" and "rims" architectures were printed on films that were used as photomasks. Different levels of protruding features on the PCB master were produced by exposing a photomask followed by chemical wet etching. Poly(dimethylsiloxane) (PDMS) was then moulded against the positive relief master to generate microfluidic structures. In this report, we described the fabrication of a microfluidic device featured with a multi-height "sandbag" structure for particle entrapment and peripheral microchannels. Controlled immobilization of biological cells and immunocytochemcial staining assays were performed to demonstrate the applicability of the microfluidic device for cellular analysis. The integrity of the microdevice remained stable under applied pressure, indicating the robustness of the elastic PDMS structures for analytical operation. The simple microfabrication process requires only low-cost materials and minimal specialized equipment and can reproducibly produce mask lines of about 20 microm in width, which is sufficient for most microfluidic applications.     

Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance

The design and characterization of two kinds of poly(dimethylsiloxane)(PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride)(PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 microL min(-1) for the EDC/NHS-made device and 0.8 microL min(-1) for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.     

Hydrophobic Poly(tert‐butyl acrylate) Photonic Crystals towards Robust Energy‐Saving Performance

Photonic crystals (PCs) have been widely applied in optical, energy, and biological fields owing to their periodic crystal structure. However, the major challenges are easy cracking and poor structural color, seriously hindering their practical applications. Now, hydrophobic poly(tert‐butyl acrylate) (P(t‐BA)) PCs have been developed with relatively lower glass transition temperature (T_g), large crack‐free area, excellent hydrophobic properties, and brilliant structure color. This method based on hydrophobic groups (tertiary butyl groups) provides a reference for designing new kinds of PCs via the monomers with relatively lower T_g. Moreover, the P(t‐BA) PCs film were applied as the photoluminescence (PL) enhanced film to enhance the PL intensity of CdSe@ZnS QDs by 10‐fold in a liquid‐crystal display (LCD) device. The new‐type hydrophobic force assembled PCs may open an innovative avenue toward new‐generation energy‐saving devices.     

Ionic current rectification at a nanofluidic/microfluidic interface with an asymmetric microfluidic system

A nanofluidic-microfluidic interface is reported that rectifies ionic current using uncoated symmetric nanocapillaries. Previously, ionic current rectification has been achieved by other groups with nanochannels with differential coatings and in nanopores that are conical in shape. This simple device uses nanocapillary membranes (NCMs) with uncoated symmetric channels to connect a microfluidic channel and a larger solution reservoir. The conductivity of the solution in the microchannel appears to be critical in the formation of the low "off" state current and the high "on" state current. It is hypothesized that the "off" state current is low due to the formation of an ion depletion zone in the microchannel while the higher "on" state currents are produced by a zone of enhanced ionic concentration in the microchannel.     

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman?, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.     

Mastering a double emulsion in a simple co-flow microfluidic to generate complex polymersomes

We show that the production and the geometrical shape of complex polymersomes can be predicted by varying the flow rates of a simple microdevice using an empirical law which predicts the droplet size. This device is constituted of fused silica capillaries associated with adjusted tubing sleeves and T-junctions. Studying the effect of several experimental parameters, double emulsions containing a controlled number of droplets were fabricated. First, this study examines the stability of a jet in a simple confined microfluidic system, probing the conditions required for droplets production. Then, multicompartmental polymersomes were formed, controlling flow velocities. In this work, poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) and poly(butadiene)-block-poly(ethyleneoxide) (PBut-b-PEO) amphiphilic copolymers were used and dissolved in chloroform/cyclohexane mixture. The ratio of these two solvents was adjusted in order to stabilize the double emulsion formation. The aqueous suspension contained poly(vinyl alcohol) (PVA), limiting the coalescence of the droplets. This work constitutes major progress in the control of double emulsion formation in microfluidic devices and shows that complex structures can be obtained using such a process.     

Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes

To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed a microfluidic device that allows a concentrated sample to be diluted by up to five orders of magnitude within milliseconds, at the physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and three-color Förster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from low nanomolar up to 10 μm , thus covering a wide range of biomolecular interactions. The design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results.     

Three-dimensional interconnected microporous poly(dimethylsiloxane) microfluidic devices

This technical note presents a fabrication method and applications of three-dimensional (3D) interconnected microporous poly(dimethylsiloxane) (PDMS) microfluidic devices. Based on soft lithography, the microporous PDMS microfluidic devices were fabricated by molding a mixture of PDMS pre-polymer and sugar particles in a microstructured mold. After curing and demolding, the sugar particles were dissolved and washed away from the microstructured PDMS replica revealing 3D interconnected microporous structures. Other than introducing microporous structures into the PDMS replica, different sizes of sugar particles can be used to alter the surface wettability of the microporous PDMS replica. Oxygen plasma assisted bonding was used to enclose the microstructured microporous PDMS replica using a non-porous PDMS with inlet and outlet holes. A gas absorption reaction using carbon dioxide (CO(2)) gas acidified water was used to demonstrate the advantages and potential applications of the microporous PDMS microfluidic devices. We demonstrated that the acidification rate in the microporous PDMS microfluidic device was approximately 10 times faster than the non-porous PDMS microfluidic device under similar experimental conditions. The microporous PDMS microfluidic devices can also be used in cell culture applications where gas perfusion can improve cell survival and functions.     

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).     

High-speed fabrication of patterned colloidal photonic structures in centrifugal microfluidic chips

In this paper, we report a rapid and facile method for fabricating colloidal photonic crystals inside microchannels of radially symmetric microfluidic chips which were made using soft-lithography. As the suspension of monodisperse silica or polystyrene latex spheres was driven to flow through the channels under the action of centrifugal force, the colloidal spheres were quickly assembled into face centered cubic arrangement which had a few photonic stop bands. The soft-microfluidic channels and cells confined the colloidal crystals into designed patterns. The optical reflectance was modulated by the refractive-index mismatch between the colloidal particles and the solvent in the interstices between the particles. Therefore, the present microfluidic chips with built-in colloidal photonic crystals can be used as in-situ optofluidic microsensors for high throughput screening or light filters in integrated adaptive optical devices.     

Green microfluidic devices made of corn proteins

An alternative green microfluidic device made of zein, a prolamin of corn, can be utilized as a disposable environmentally friendly microchip especially in agriculture applications. Using standard soft lithography and stereo lithography techniques, we fabricated thin zein films with microfluidic chambers and channels. These were bonded to both a glass slide and another zein film. The zein film with microfluidic features bonds irreversibly with other surfaces by vapor-deposition of ethanol to create an adhesive layer resulting in very little or no trapped air and small shape distortion. Zein-zein and zein-glass microfluidic devices demonstrated sufficient strength to facilitate fluid flow in a complex microfluidic design that showed no leakage under high hydraulic pressure. Zein-glass microfluidic devices with serpentine mixing design showed successful fluid manipulation as a concentration gradient of Rhodamine B solution was generated. The ease of fabrication and bonding and the flexibility and moldability of zein offer attractive possibilities for microfluidic device design and manufacturing. These devices can include several unit operations with mixing being one of the most commonly used. The zein-based microfluidic devices, made entirely from a biopolymer from agricultural origin, offer alternative environmentally friendly material choices that are less dependent on limited petroleum based polymer resources.     

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.