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PubMed2001

Concentration and separation of proteins in microfluidic channels on the basis of transverse IEF.

The use of microfluidic channels formed by two electrodes made of gold or palladium to perform transverse isoelectric focusing (IEF) is presented as a means for continuous concentration and fractionation of proteins. The microchannels were 40 mm long with an electrode gap of 1.27 mm and a depth of 0.354 mm. The properties of pH gradients formed as a result of the electrolysis of water were influenced by variation of parameters such as the initial pH, ionic strength, and flow rate. Transverse IEF in pressure-driven flow is demonstrated using bovine serum albumin in a single ampholyte buffer as well as in multiple-component buffers. Experimental results of protein focusing compare well to predictions of a mathematical model. Optimal conditions for efficient continuous fractionation of a protein mixture are summarized and discussed.

Animals↗

PubMed2001

Single-molecule DNA amplification and analysis in an integrated microfluidic device.

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.

Base Sequence↗

PubMed2001

Electroosmotically induced hydraulic pumping with integrated electrodes on microfluidic devices.

Electroosmotic manipulation of fluids was demonstrated using thin metal electrodes integrated within microfluidic channels at the substrate and cover plate interface. Devices were fabricated by photolithographically patterning electrodes on glass cover plates that were then bonded to polymeric substrates into which the channels were cast. Polymeric substrates were used to provide a permeable membrane for the transport and removal of gaseous electrolysis products generated at the electrodes. Electroosmotic flow between interdigitated electrodes was demonstrated and provided electric field-free pumping of fluids in sections of the channel outside of the electrode pairs. The resultant pumping velocities were shown to be dependent on the applied voltage, not on the applied field strength, and independent of the length of the electroosmotically pumped region.

Electrochemistry↗

PubMed2001

Microfluidic device for airborne BTEX detection.

We fabricated a microfluidic device for the optical detection of airborne benzene, toluene, ethylbenzene and xylenes (BTEX). The device consists of concentration and detection cells formed of 3 cm x 1 cm Pyrex plates. The concentration cell is composed of an adsorbent to concentrate the BTEX gases and a thin-film heater todesorb the concentrated gases from the adsorbent thermally. The collected gases are introduced into the detection cell, which is connected to optical fibers, to measure their absorption spectra. We optimized the device's operating conditions by studying the thermal characteristics of the concentration cell and the time profile of the gas concentration flowing in the detection cell. We used the device under optimized operating conditions to detect toluene gas as a typical example BTEX. The gas concentration amplification rate was approximately 2 orders of magnitude, and we successfully measured parts-per-million levels of toluene gas with this device.

Air Pollutants↗

PubMed2001

Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection.

Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described.

Dimethylpolysiloxanes↗

PubMed2002

Effect of analyte adsorption on the electroosmotic flow in microfluidic channels.

The predictability and constancy over time of the electroosmotic flow in microchannels is an important consideration in microfluidic devices. A common cause for alteration of the flow is the adsorption of analytes to channel walls, for example, during capillary electrophoresis of proteins. It is shown that certain experimental data, published by Towns and Regnier (Towns, J. K; Regnier, F. E. Anal. Chem. 1992, 64, 2473-2478.), on the anomalous elution times for proteins in capillary electrophoresis can be explained using a simple model for analyte adsorption that uses a result first reported by Anderson and Idol (Anderson, J. L.; Idol, W. K Chem. Eng. Commun. 1985, 38, 93-106.) on the electroosmotic flux in capillaries with axial variations in zeta-potential. It is suggested that it might be possible to use such a model to dynamically correct for altered elution times in capillary electrophoretic devices.

Adsorption↗

PubMed2001

Fabrication of a configurable, single-use microfluidic device.

This paper describes microfluidic devices that contain connections that can be opened by the user after fabrication. The devices are fabricated in poly(dimethylsiloxane) (PDMS) and comprise disconnected fluidic channels that are separated by 20 microm of PDMS. Applying voltages above the breakdown voltage of PDMS (21 V/microm) opened pathways between disconnected channels. Fluids could then be pumped through the openings. The voltage used and the ionic strength of the buffer in the channels determined the size of the opening. Opening connections in a specific order provides the means to control complex reactions on the device. A device for ELISA was fabricated to demonstrate the ability to store and deliver fluids on demand.

Dimethylpolysiloxanes↗

PubMed2002

A high-throughput continuous sample introduction interface for microfluidic chip-based capillary electrophoresis systems.

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with <4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.

Journal Article↗

PubMed2002

Surface characterization of laser-ablated polymers used for microfluidics.

Fabrication of microfluidic devices by excimer laser ablation under different atmospheres may provide variations in polymer microchannel surface characteristics. The surface chemistry and electroosmotic (EO) mobility of polymer microchannels laser ablated under different atmospheres were studied by X-ray photoelectron spectroscopy and current monitoring mobility measurements, respectively. The ablated surfaces of PMMA were very similar to the native material, regardless of ablation atmospheres due to the negligible absorption of 248-nm light by that polymer. The substrates studied that exhibit nonnegligible absorption at this energy, namely, poly(ethylene terephthalate glycol), poly(vinyl chloride), and poly(carbonate), showed significant changes in surface chemistry and EO mobility when the ablation atmospheres were varied. Ablation of these three polymer substrates under nitrogen or argon resulted in low EO mobilities with a loss of the well-defined chemical structures of the native surfaces, while ablation under oxygen yielded surfaces that retained native chemical structures and supported higher EO mobilities.

Journal Article↗

PubMed2002

Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay.

A critical requirement for achieving a micro total analytical system for the analysis of cells and their constituent proteins is to integrate the lysis and fractionation steps on-chip. Here, an experimental microfluidic system integrating the lysis of bacterial cells and the extraction of a large intracellular enzyme, beta-galactosidase, is demonstrated. The beta-galactosidase is detected and quantified using a fluorogenic enzyme assay and a numerical model. While the focus is on the lysis of typical gram-negative bacterial cells (E. coli), the techniques described here could, in principle, be applied to a variety of different cell types.

Bacterial Proteins↗

PubMed2002

Fabrication of microchambers defined by photopolymerized hydrogels and weirs within microfluidic systems: application to DNA hybridization.

This paper describes fabrication of serial microchamber arrays within the channels of a microfluidic device. The chambers are defined using a combination of weirs and UV-cross-linked hydrogel plugs (poly(ethylene glycol) diacrylates). This approach permits the microchambers to be addressed by pump-driven pressure in one dimension and by electrophoresis in the other. The function of the device is demonstrated by detecting DNA targets. Single-strand DNA (ssDNA) probes labeled with biotin were immobilized onto microbeads coated with streptavidin. The DNA-functionalized microbeads were packed into each of three microchambers by injection through inlet wells. Three oligonucleotides were designed as probes and four as targets. Hybridization reactions were performed by moving the targets across the array of probe-containing microchambers by electrophoresis. The hybridization of fluorescein-labeled ssDNA targets to complementary probes was observed by fluorescence microscopy. These studies resulted in four key observations: (1) there was no detectable binding of targets to noncomplementary probes; (2) hybridization was 90% complete within 1 min; (3) once captured, the targets could be independently released and recovered from the microbeads by treatment with 0.1 N NaOH; (4) multiple analyses could be performed using a single bead set, but there was degradation in performance after each capture/release cycle.

DNA, Single-Stranded↗

PubMed2002

Separate detection of BTX mixture gas by a microfluidic device using a function of nanosized pores of mesoporous silica adsorbent.

We achieved separate detection of the components of 10 ppm of a benzene, toluene, and o-xylene mixture gas by using mesoporous silica powder incorporated in our microfluidic device. The device consists of concentration and detection cells formed of 3 cm x 1 cm Pyrex plates. We first introduced the mixture gas into the concentration cell where it was adsorbed on an adsorbent in a channel formed in the cell. We then raised the temperature using a thin-film heater and introduced the desorbed gas into the detection cell. Here, we measured the changes in the absorption spectra of the mixture gas in the detection cell. We found that the mixture ratio of the compounds in the desorbed gas varies with time because the thermal desorption property of each compound is different from that of the adsorbent. We analyzed the thermal desorption mechanism by comparing two types of silica adsorbents with different pore structures. We found that an adsorbent that has pores with a periodic and uniform nanosized column shape provides better component separation. We concluded that the uniform pore structure might cause the adsorbate molecules to exhibit a homogeneous adsorption state thus revealing the desorption properties of the gas more clearly.

Adsorption↗

PubMed2002

Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices.

Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties of the monoliths lead to very low back pressures enabling the use of simple mechanical pumping to carry out both the immobilization of the enzyme from its solution and the subsequent analyses of substrate solutions. The Michealis-Menten kinetic characteristics of the reactors were probed using a low molecular weight substrate: N-alpha-benzoyl-L-arginine ethyl ester. The effects of immobilization variables such as the concentration of trypsin in solution and percentage of azlactone functionalities in the monolith, as well as the effect of reaction time on the enzymatic activity, and of process variables such as substrate flow velocity and residence time in the reactor, were studied in detail. The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate. The excellent performance of the monolithic microreactor was also demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min, which affords a residence time of only 11.7 s. The digest was then characterized using MALDI-TOF MS, and 102 out of 153 possible peptide fragments were identified giving a sequence coverage of 67%.

Animals↗

PubMed2003

Passively driven integrated microfluidic system for separation of motile sperm.

This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.

Cell Separation↗

PubMed2003

Faceted design of channels for low-dispersion electrokinetic flows in microfluidic systems.

A novel methodology for designing microfluidic channels for low-dispersion, electrokinetic flows is presented. The technique relies on trigonometric relations that apply for ideal electrokinetic flows, allowing faceted channels to be designed using common drafting software and a hand calculator. Flows are rotated and stretched along the abrupt interface between adjacent regions having differing specific permeability--a quantity with dimensions of length that we introduce to derive the governing equations. Two-interface systems are used to eliminate hydrodynamic rotation of bands injected into channels. Regions bounded by interfaces form faceted flow "prisms" with uniform velocity fields that can be combined with other prisms to obtain a wide range of turning angles and expansion ratios. Lengths of faceted prisms can be varied arbitrarily, simplifying chip layout and allowing the ability to reduce unwanted effects such as transverse diffusion and Joule heating for a given faceted prism. Designs are demonstrated using two-dimensional numerical solutions of the Laplace equation.

Journal Article↗

PubMed2002

Coupled electrorotation of polymer microspheres for microfluidic sensing and mixing.

We show that coupled electrorotation (CER) of microscopic particles using microfabricated electrodes can be used for localized sensing and mixing. The effective use of microelectromechanical systems and micro total analysis systems requires many types of control. These include the abilityto (1) manipulate objects within microchannels by noncontact means, (2) mix fluids, and (3) sense local chemical parameters. Coupled electrorotation, in which the interactions between induced electric dipoles of adjacent particles lead to particle rotation, addresses aspects of all three challenges simultaneously. CER is a simple means of controlling the rotation of dielectric objects using homogeneous external radio frequency electric fields. CER is sensitive to several chemical and physical parameters such as the solution conductivity, pH, and viscosity. As a step toward integrating CER devices into microfluidic systems, a simple chip was designed to induce local mixing and to detect local changes in salt concentration, pH, and viscosity.

Journal Article↗

PubMed2003

Flow injection analysis in a microfluidic format.

A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation of a standard six-port, two-way valve used in conventional liquid chromatography and flow injection experiments. Analyte and carrier solutions continuously flow through this injection system allowing for measurements and sample changes to be performed rapidly and simultaneously. Injection volumes of 1.25 nL generated peak area reproducibility of better than 3% relative standard deviation. The flow injection device was evaluated with fluorescent dyes and demonstrated a detection limit of 400 zmol for fluorescein. A rudimentary sample selection system allowed calibration curves to be rapidly produced, often in less than 10 min. The hydrolysis of fluorescein diphosphate by alkaline phosphatase demonstrates that chemical assays can be carried out with this device in a manner characterized by short analysis times and low sample consumption.

Alkaline Phosphatase↗

PubMed2003

Flow control valves for analytical microfluidic chips without mechanical parts based on thermally responsive monolithic polymers.

Monolithic plugs of poly(N-isopropylacrylamide) cross-linked with 5% methylenebisacrylamide have been prepared by photoinitiated polymerization within the channel of a microfluidic device. The volume change associated with the polymer phase transition at its lower critical solution temperature of 32 degrees C allows both the rapid swelling and the deswelling of the monoliths enabling the polymer to close or open the channel as it functions as a nonmechanical valve. Thermoelectric elements capable of changing the temperature of the system between 17 and 57 degrees C were used to actuate the valve. Flow through the device was monitored by fluorescence measurements via the laser-triggered photobleaching of a dye contained in the liquid phase. Photobleaching occurs quickly once the flow is stopped, and the time required to open and close the valve was 3.5 and 5.0 s, respectively. No changes in function were observed even after 120 open-close cycles. Although the 2-mm-long valve was prepared from a polymerization mixture consisting of only a 5% aqueous solution of monomers, it resists pressures of up to 1.38 MPa (200 psi) without observable structural damage.

Acrylic Resins↗