Integrated continuous microfluidic liquid-liquid extraction

We describe continuous flow liquid-liquid phase separation in microfluidic devices based on capillary forces and selective wetting surfaces. Effective liquid-liquid phase separation is achieved by using a thin porous fluoropolymer membrane that selectively wets non-aqueous solvents, has average pore sizes in the 0.1-1 microm range, and has a high pore density for high separation throughput. Pressure drops throughout the microfluidic network are modelled and operating regimes for the membrane phase separator are determined based on hydrodynamic pressure drops and capillary forces. A microfluidic extraction device integrating mixing and phase separation is realized by using silicon micromachining. Modeling of the phase separator establishes the operating limits. The device is capable of completely separating several organic-aqueous and fluorous-aqueous liquid-liquid systems, even with high fractions of partially miscible compounds. In each case, extraction is equivalent to one equilibrium extraction stage.     

A microfluidic chip based liquid-liquid extraction system with microporous membrane

A robust and simple approach for microfabricated chip based liquid-liquid extraction was developed for on-chip sample pretreatment. The chip based extraction system was composed of two microfabricated glass plates with a microporous membrane sandwiched in between. A simple bonding approach using epoxy was used to achieve bonding and sealing of the L-L extraction chip. Gravity was employed to drive the aqueous and organic flows through separate channels in the extraction system, separated by the membrane. During extraction, the analyte in an aqueous sample stream was transferred through the membrane into the organic stream. The fluorescence intensity of the analyte extracted into the organic stream was monitored in situ by a laser induced fluorescence detection system. The performance of the system was demonstrated using an aqueous solution of butyl rhodamine B (BRB) and isobutanol as sample and extractant, respectively. The system proved to be an efficient means for achieving chip based microporous membrane liquid-liquid extraction. The precision of fluorescence measurements was 1.5% R.S.D. (n = 4). A linear response range of 1 × 10⁻⁷ to 1 × 10⁻⁴ M BRB was obtained with a regression equation: I = 8.00 × 10⁶ C + 4.91. An enrichment factor of ca. 3 was obtained with an extraction efficiency of 69%.     

Improved microfluidic chip-based sequential-injection trapped-droplet array liquid-liquid extraction system for determination of aluminium

An improved microfluidic chip-based sequential-injection trapped-droplet array liquid-liquid extraction system with chemiluminescence (CL) detection was developed in this work. Two recess arrays were fabricated on both sides of the extraction channel to produce droplet arrays of organic extractant. A chip integrated monolithic probe was fabricated at the inlet of the extraction channel on the glass chip instead of the capillary probe connected to the microchannel, in order to improve the system stability and reliability. A slotted-vial array system coupled with the monolithic probe was used to sequentially introduce sample and different solvents and reagents into the extraction channel for extraction and CL detection. The performance of the system was demonstrated in the determination of Al³⁺ using Al³⁺-dihydroxyazobenzene (DHAB) and tributyl phosphate (TBP) extraction system. The operation conditions, including extraction time, concentration and flow rate of the CL reagents, were optimized. Within one analysis cycle of 12 min, an enrichment factor of 85 was obtained in the extraction stage with a sample consumption of 1.8 μL. The consumption of CL reagent, bis(2-carbopentyloxy-3,5,6-trichlorophenyl)oxalate (CPPO), was 120 nL/cycle. The detection limit of the system for Al³⁺ was 1.6 × 10⁻⁶ mol/L with a precision of 4.5% (R.S.D., n = 6).     

Surfactant-enhanced liquid-liquid extraction in microfluidic channels with inline electric-field enhanced coalescence

Continuous microfluidic liquid-liquid extraction is realized in a microfluidic device by generating emulsions with large interfacial areas for mass transfer, and subsequently breaking these emulsions using electric fields into easily separated segments of immiscible liquids (plugs). The microfluidic device employs insulated electrodes in a potassium hydroxide-etched channel to create large electric fields (100 kV m(-1)) that drive coalescence of the emulsion phase. The result is a transition from disperse to slug flow that can then readily be separated by gravity. Extractions of phenol and p-nitrophenol from an aqueous to hexane-surfactant solution serve as model systems. In addition to the increased surface area in the emulsion, extraction efficiency is enhanced by reverse micelles resulting from the presence of surfactants. The surfactant concentration is varied approximately 1-10 wt% and a general two-parameter model is developed to quantify the extraction behavior and demonstrate the effectiveness of reverse micelle enhanced extraction.     

An integrated microfluidic device for two-dimensional combinatorial dilution

High-throughput preparation of multi-component solutions is an integral process in biology, chemistry and materials science for screening, diagnostics and analysis. Compact microfluidic systems enable such processing with low reagent volumes and rapid testing. Here we present a microfluidic device that incorporates two gradient generators, a tree-like generator and a new microfluidic active injection system, interfaced by intermediate solution reservoirs to generate diluted combinations of input solutions within an 8 × 8 or 10 × 10 array of isolated test chambers. Three input solutions were fed into the device, two to the tree-like gradient generator and one to pre-fill the test chamber array. The relative concentrations of these three input solutions in the test chambers completely characterized device behaviour and were controlled by the number of injection cycles and the flow rate. Device behaviour was modelled by computational fluid dynamics simulations and an approximate analytic formula. The device may be used for two-dimensional (2D) combinatorial dilution by adding two solutions in different relative concentrations to each of its three inputs. By appropriate choice of the two-component input solutions, test chamber concentrations that span any triangle in 2D concentration space may be obtained. In particular, explicit inputs are given for a coarse screening of a large region in concentration space followed by a more refined screening of a smaller region, including alternate inputs that span the same concentration region but with different distributions. The ability to probe arbitrary subspaces of concentration space and to control the distribution of discrete test points within those subspaces makes the device of potential benefit for high-throughput cell biology studies and drug screening.     

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.     

An integrated microfluidic device for influenza and other genetic analyses

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.     

An integrated optics microfluidic device for detecting single DNA molecules

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.     

The liquid-liquid diffusive extraction of hydrocarbons from a North Sea oil using a microfluidic format

Extraction of a GC-amenable hydrocarbon fraction from oil by liquid-liquid diffusion across a laminar interface can be performed in a microfluidic format. Analysis of figures of merit, determined using standard analytical techniques, show this method to be an effective new tool for rapidly processing small quantities of oil and petroleum for GC analysis. Methods based upon similar microsystems devices could find widespread use in a variety of fields, including those associated with organic geochemistry and oil exploration and production, where the manipulation of petroleum constituents (greater than C14) is necessary for analytical purposes.     

A microfluidic chip based sequential injection system with trapped droplet liquid-liquid extraction and chemiluminescence detection

A microfluidic chip-based sequential injection system with trapped droplet liquid-liquid extraction preconcentration and chemiluminescence detection was developed for achieving high sensitivity with low reagent and sample consumption. The microfabricated glass lab-chip had a 35 mm long extraction channel, with 134 shrunken opening rectangular recesses (L 100 microm x W 50 microm x D 25 microm) arrayed within a 1 mm length on both sides of the middle section of the channel. Ketonic peroxyoxalate ester solution was filled in the recesses forming organic droplets, and keeping the aqueous sample solution flowing continuously in the extraction channel; analytes were transferred from the aqueous phase into the droplets through molecular diffusion. After liquid-liquid extraction preconcentration, catalyst and hydrogen peroxide solutions were introduced into the channel, and mixed with analytes and peroxyoxalate ester to emit chemiluminescence light. The performance of the system was tested using butyl rhodamine B, yielding a precision of 4% RSD (n = 5) and a detection limit of 10(-9) M. Within a 17 min analytical cycle, the consumptions of sample and peroxyoxalate solutions were 2.7 microL and 160 nL, respectively.     

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health. Therefore, an on-site visualization method for antibiotic detection is required. However, the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-care-testing detection of suspected samples. Herein, we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk. The rapid qualitative and quantitative analysis of four types of antibiotics (sulfonamides, β-lactams, streptomycin, and tetracyclines) was performed using mobile phone photography and mobile phone application analysis. The detection time was maintained at 10 min. The limits of detection (LODs) for the four antibiotics were 0.15, 0.12, 0.25, and 0.29 ng/mL, respectively, and the selectivity for the different antibiotics was observed even in a highly complex matrix. This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.     

Droplet-based microfluidic device for multiple-droplet clustering

We present a multiple-droplet clustering device that can perform sequential droplet trapping and storing. Shape-dependent droplet manipulation in forward and backward flows has been incorporated to achieve high trapping and storing efficiency in a 10 × 12 array of clustering structures (e.g., storing well, storing chamber, trapping well, and guiding track). In the forward flow, flattened droplets are trapped in each trapping well. In the backward flow, the trapped droplets are released from the trapping well and follow the guiding tracks to their corresponding storing wells. The guided droplets float up out of the confining channel to the super stratum of the storing chamber due to interfacial energy and buoyancy effects. This forward/backward flow-based trapping/storing process can be repeated several times to cluster droplets with different contents and samples in the storing chambers. We expect that the proposed platform will be a valuable tool to study complex droplet-based reactions in clustered droplets.     

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.     

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells’ autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.     

An integrated microfluidic device in polyester for electrophoretic analysis of amino acids

A precolumn reaction chamber was integrated into a polyester microfluidic device with a miniaturized detection system. The reaction chamber was designed to be a zigzag channel, 70 microm in width, 8 mm in length, followed by a wider straight channel, 150 microm in width, 2 mm in length. The detection system is composed of an embedded light-emitting diode (LED), an integrated optical fiber, and a photomultiplier tube (PMT). A success in amino acid analysis using the integrated microchemical analysis device proved that the precolumn reaction chamber was compatible with the integrated detection system. Three kinds of amino acids, arginine, glycine, and phenylalanine, mixed and reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) in the precolumn reaction chamber to produce fluorescent products, were separated by micellar eletrokinetic chromatography (MEKC) and detected by LED-excited fluorescence. The detection limits for arginine, glycine, and phenylalanine were 1, 1, and 0.5 mM, respectively, which can be improved by further optimizations of the reaction system and detection system.     

A polymer-based microfluidic device for immunosensing biochips

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.     

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.     

A microfluidic electroporation device for cell lysis

We demonstrate a micro-electroporation device for cell lysis prior to subcellular analysis. Simple circuit models show that electrical lysis method is advantageous because it is selective towards plasma membrane while leaving organelle membrane undamaged. In addition, miniaturization of this concept leads to negligible heat generation and bubble formation. The designed microdevices were fabricated using a combination of photolithography, metal-film deposition, and electroplating. We demonstrate the electro-lysis of human carcinoma cells in these devices to release the subcellular materials.     

Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.