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PubMed2003

Biochemical analysis with microfluidic systems.

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Chemistry Techniques, Analytical↗

PubMed2002

Microfluidic technology for assisted reproduction.

The physical tools used in assisted reproduction have changed little over several decades. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review our work to develop microfluidic devices to perform the functions required in assisted reproduction. These functions include loading/unloading, culture, chemical manipulation, and mechanical manipulation of embryos and oocytes. Basic microfluidic theory and microfluidic device design and operation are discussed. Results are presented for mechanical removal of cumulus cells and for embryo culture. Results suggest that microfluidic systems will lead to improved efficiencies in assisted reproduction.

Animals↗

PubMed2003

A smart microfluidic affinity chromatography matrix composed of poly(N-isopropylacrylamide)-coated beads.

The efficient upstream processing of complex biological or environmental samples for subsequent biochemical analysis remains a challenge in many analytical systems. New microfluidic platforms that provide multidiagnostic capabilities on single chips face a similar challenge in getting specific analytes purified or contaminants removed in different fluid streams. Here, stimuli-responsive polymers have been used to construct "smart" beads that can be reversibly immobilized on microfluidic channel walls to capture and release targets. The 100-nm latex beads were surface-modified with the temperature-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm). At room temperature, a suspension of these beads flows through a microfluidic channel constructed of poly(ethylene terephthalate). However, when the temperature in the channel is raised above the lower critical solution temperature (LCST) of PNIPAAm, the beads aggregate and adhere to the walls of the channel. The adhered beads are stable for long durations on the channel walls (demonstrated up to 70 min) in the presence of flow. The beads were further modified with the affinity moiety biotin, which tightly binds streptavidin. The dual-modified beads were adhered to the channel walls and functioned as a chromatographic affinity separation matrix, capable of binding streptavidin that was flowed through the microfluidic channel. Upon the reverse thermal stimulation to below the PNIPAAm LCST, the beads and captured streptavidin were observed to quickly dissolve and elute from the channel walls. This temperature-responsive affinity chromatography matrix can thus be flowed into a column and aggregated via temperature change, followed by the controlled release of affinity-captured targets back into the microfluidic flow stream.

Acrylamides↗

PubMed2005

Multilevel microfluidics via single-exposure photolithography.

This communication reports a new method to form multilevel features in a single layer of SU-8 photoresist to facilitate the generation of 3D microfluidic chips. The method utilizes the spatial dependence of diffracted light intensity to selectively overexpose masked regions of photoresist and requires only a UV light source and a single transparency mask. 3D structures are formed within microfluidic channels using this selective overexposure method, with feature sizes being determined by the exposure dose and mask feature sizes. The dimensions of the internal features and the microfluidic channels can be varied independently according to these parameters, and any number of different heights can be obtained in a single exposure step. The method provides a simple means of forming 3D microfluidic structures with integrated features, including mixing structures, flow stabilization ridges, and separation weirs to increase the capabilities of microfluidic chips in a variety of microchemical applications.

Journal Article↗

PubMed2005

Continuous microfluidic reactors for polymer particles.

This article provides an overview of our work in the area of the synthesis of polymer particles in continuous microfluidic reactors. The method includes (a) the generation of highly monodisperse monomer droplets in a microfluidic flow-focusing device and (b) in-situ solidification of these droplets by means of photopolymerization. We discuss the effect of monomer properties on the emulsification process, the effect of the polymerization rate on the production of high-quality particles, the role of the material of the microfluidic device in droplet formation, and the synthesis of particles with different shapes and compositions. We also demonstrate the production of highly ordered arrays of polymer particles achieved by photopolymerization of the dynamic lattices of monomer droplets in microfluidic channels. The article is concluded with a summary of future research directions in the production of polymer colloids in microfluidic reactors.

Journal Article↗

PubMed2005

Multipurpose microfluidic probe.

Microfluidic systems allow (bio)chemical processes to be miniaturized with the benefit of shorter time-to-result, parallelism, reduced sample consumption, laminar flow, and increased control and efficiency. However, such miniaturization inherently limits the size of the solid objects that can be processed and entails new challenges such as the interfacing of macroscopic samples with microscopic conduits. Here, we report a microfluidic probe (MFP) that overcomes these problems by combining the concepts of 'microfluidics' and of 'scanning probes'. Here, liquid boundaries formed by hydrodynamic forces underneath the MFP confine a flow of processing solution and replace the solid walls of closed microchannels. The MFP is therefore mobile and can be used to process large surfaces and objects by scanning across them. We illustrate the versatility of this concept with several examples including protein microarraying, complex gradient-formation, multiphase laminar-flow patterning, erasing, localized staining of cells and the contact-free detachment of a single cell. Many constraints imposed by the monolithic construction of microfluidic channels can now be circumvented using an MFP, opening up new avenues for microfluidic processing.

Journal Article↗

PubMed2003

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.

Journal Article↗

PubMed2004

In situ fabrication of macroporous polymer networks within microfluidic devices by living radical photopolymerization and leaching.

Novel fabrication techniques and polymer systems are being explored to enable mass production of low cost microfluidic devices. In this contribution we discuss a new fabrication scheme for making microfluidic devices containing porous polymer components in situ. Contact lithography, a living radical photopolymer (LRPP) system and salt leaching were used to fabricate multilayer microfluidic devices rapidly with various channel geometries and covalently attached porous polymer plugs made of various photopolymerizable substrates. LRPP systems offer the advantages of covalent attachment of microfluidic device layers and facile surface modification via grafting. Several applications of the porous plugs are also explored, including a static mixer, a high surface area-to-volume reactor and a rapidly responding hydrogel valve. Quantitative and qualitative data show an increase in mixing of a fluorescein and a water stream for channels containing porous plugs relative to channels with no porous plugs. Confocal laser scanning microscopy images demonstrate the ability to graft a functional material onto porous plug surfaces. A reaction was carried out on the grafted pore surfaces, which resulted in fluorescent labelling of the grafted material throughout the pores of the plug. Homogenous fluorescence throughout the depth of the porous plug and along pore surfaces indicated that the porous plugs were surface modified by grafting and that reactions can be carried out on the pore surfaces. Finally, porous hydrogel valves were fabricated which swelled in response to contact with various pH solutions. Results indicate that a porous hydrogel valve will swell and close more rapidly than other valve geometries made with the same polymer formulation. The LRPP-salt leaching method provides a means for rapidly incorporating porous polymer components into microfluidic devices, which can be utilized for a variety of pertinent applications upon appropriate selection of porous plug materials and surface treatments.

Journal Article↗

PubMed2002

A prototype microfluidic platform for miniaturization and automation of serial dilution and dose-response assays.

A novel microfluidic device was designed and developed to miniaturize, multiplex, and automate serial dilution and three-reagent dose-response assays using submicroliter quantities of reagents. This prototype microfluidic device can be used to measure enzyme kinetics and to test a chemical lead's response to a target by fluorescent readout using common plate readers and detection systems. The prototype microfluidic system yielded serial dilution and dose-response assay data comparable to results obtained from manual titrations and reagent additions performed using a microwell plate. Enzyme kinetics were highly reproducible using these devices, although Michaelis-Menten kinetics results differed from those obtained in the microwell plate. In all cases reported here, assays performed on the microfluidic format required lower volumes of reagents compared with the microwell plate. In addition to savings in reagent consumption, the microfluidic devices and bench-top instruments offer additional advantages over conventional solutions, including a small footprint and compatibility with commercially available fluorescence detectors. Future directions for the prototype technology are discussed.

Automation↗

PubMed2003

Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems.

BACKGROUND: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface. RESULTS: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated. CONCLUSION: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale.

Animals↗

PubMed2004

Microfluidics in biotechnology.

Microfluidics enables biotechnological processes to proceed on a scale (microns) at which physical processes such as osmotic movement, electrophoretic-motility and surface interactions become enhanced. At the microscale sample volumes and assay times are reduced, and procedural costs are lowered. The versatility of microfluidic devices allows interfacing with current methods and technologies. Microfluidics has been applied to DNA analysis methods and shown to accelerate DNA microarray assay hybridisation times. The linking of microfluidics to protein analysis techologies, e.g. mass spectrometry, enables picomole amounts of peptide to be analysed within a controlled micro-environment. The flexibility of microfluidics will facilitate its exploitation in assay development across multiple biotechnological disciplines.

Journal Article↗

PubMed2003

Properties of frozen dairy desserts processed by microfluidization of their mixes.

Sensory properties and rate of meltdown of nonfat (0% fat) and low-fat (2% fat) vanilla ice creams processed either by conventional valve homogenization or microfluidization of their mixes were compared with each other and to ice cream (10% fat) processed by conventional valve homogenization. Mixes for frozen dairy desserts containing 0, 2, and 10% fat were manufactured. Some of the nonfat and low-fat ice cream mixes were processed by microfluidization at 50, 100, 150, and 200 MPa, and the remaining nonfat and low-fat ice cream mixes and all of the ice cream mix were processed by conventional valve homogenization at 13.8 MPa, first stage, and 3.4 MPa, second stage. The finished frozen and hardened products were evaluated at d 1 and 45 for meltdown rate and for flavor and body and texture by preference testing. Nonfat and low-fat ice creams that usually had a slower meltdown were produced when processing their mixes by microfluidization instead of by conventional valve homogenization. Sensory scores for the ice cream were significantly higher than sensory scores for the nonfat and low-fat ice creams, but the sensory scores for the conventional valve homogenized controls for the nonfat ice cream and low-fat ice cream were not significantly different from the sensory scores for the nonfat ice cream and low-fat ice cream processed by microfluidization of the mixes, respectively. Microfluidization produced nonfat and low-fat ice creams that usually had a slower meltdown without affecting sensory properties.

Dairy Products↗

PubMed2005

Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices: cell culture and flow studies with glial cells.

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.

Animals↗

PubMed2006

Integrated polymerase chain reaction chips utilizing digital microfluidics.

This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated, transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips, a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required an operation voltage of 12V(RMS) at a frequency of 3 KHz for digital microfluidic actuation and 9V(DC) for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip (LOC) by utilizing EWOD-based digital microfluidics.

Dengue Virus↗

PubMed2005

An integrated microfluidic processor for single nucleotide polymorphism-based DNA computing.

An integrated microfluidic processor is developed that performs molecular computations using single nucleotide polymorphisms (SNPs) as binary bits. A complete population of fluorescein-labeled DNA "answers" is synthesized containing three distinct polymorphic bases; the identity of each base (A or T) is used to encode the value of a binary bit (TRUE or FALSE). Computation and readout occur by hybridization to complementary capture DNA oligonucleotides bound to magnetic beads in the microfluidic device. Beads are loaded into sixteen capture chambers in the processor and suspended in place by an external magnetic field. Integrated microfluidic valves and pumps circulate the input DNA population through the bead suspensions. In this example, a program consisting of a series of capture/rinse/release steps is executed and the DNA molecules remaining at the end of the computation provide the solution to a three-variable, four-clause Boolean satisfiability problem. The improved capture kinetics, transfer efficiency, and single-base specificity enabled by microfluidics make our processor well-suited for performing larger-scale DNA computations.

Biotinylation↗

PubMed2005

Mammalian electrophysiology on a microfluidic platform.

The recent development of automated patch clamp technology has increased the throughput of electrophysiology but at the expense of visual access to the cells being studied. To improve visualization and the control of cell position, we have developed a simple alternative patch clamp technique based on microfluidic junctions between a main chamber and lateral recording capillaries, all fabricated by micromolding of polydimethylsiloxane (PDMS). PDMS substrates eliminate the need for vibration isolation and allow direct cell visualization and manipulation using standard microscopy. Microfluidic integration allows recording capillaries to be arrayed 20 microm apart, for a total chamber volume of <0.5 nl. The geometry of the recording capillaries permits high-quality, stable, whole-cell seals despite the hydrophobicity of the PDMS surface. Using this device, we are able to demonstrate reliable whole-cell recording of mammalian cells on an inexpensive microfluidic platform. Recordings of activation of the voltage-sensitive potassium channel Kv2.1 in mammalian cells compare well with traditional pipette recordings. The results make possible the integration of whole-cell electrophysiology with easily manufactured microfluidic lab-on-a-chip devices.

Animals↗

PubMed2006

Microfluidic vias enable nested bioarrays and autoregulatory devices in Newtonian fluids.

We report on a fundamental technological advance for multilayer polydimethylsiloxane (PDMS) microfluidics. Vertical passages (vias), connecting channels located in different layers, are fabricated monolithically, in parallel, by simple and easy means. The resulting 3D connectivity greatly expands the potential complexity of microfluidic architecture. We apply the vias to printing nested bioarrays and building autoregulatory devices. A current source is demonstrated, while a diode and a rectifier are derived; all are building blocks for analog circuitry in Newtonian fluids. We also describe microfluidic septa and their applications. Vias lay the foundation for a new generation of microfluidic devices.

Dimethylpolysiloxanes↗

PubMed2003

Multienzyme catalysis in microfluidic biochips.

The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride-alt-alpha-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent-noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 microm wide (at the center), 15 microm deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 microg/channel. SBP-catalyzed oxidation of p-cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H(2)O(2), with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis-Menten kinetics were obtained with K(m) and V(max) values of 0.98 mM and 0.21 micromol H(2)O(2) converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous-DMF solutions in 20-microL volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B-SBP bienzymic system was used to convert tolyl acetate into poly(p-cresol), and an invertase-glucose oxidase SBP trienzymic system was used to take sucrose and generate H(2)O(2) for SBP-catalyzed synthesis of poly(p-cresol).

Catalysis↗