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Blood plasma separation in microfluidic channels using flow rate control.

Several studies have clearly shown that cardiac surgery induces systemic inflammatory responses, particularly when cardiopulmonary bypass (CPB) is used. CPB induces complex inflammatory responses. Considerable evidence suggests that systemic inflammation causes many postoperative complications. Currently, there is no effective method to prevent this systemic inflammatory response syndrome in patients undergoing CPB. The ability to clinically intervene in inflammation, or even study the inflammatory response to CPB, is limited by the lack of timely measurements of inflammatory responses. In this study, a microfluidic device for continuous, real-time blood plasma separation, which may be integrated with downstream plasma analysis device, is introduced. This device is designed to have a whole blood inlet, a purified plasma outlet, and a concentrated blood cell outlet. The device is designed to separate plasma with up to 45% hematocrit of the inlet blood and is analyzed using computational fluid dynamics simulation. The simulation results show that 27% and 25% of plasma can be collected from the total inlet blood volume for 45% and 39% hematocrit, respectively. The device's functionality was demonstrated using defibrinated sheep blood (hematocrit=39%). During the experiment, all the blood cells traveled through the device toward the concentrated blood outlet while only the plasma flowed towards the plasma outlet without any clogging or lysis of cells. Because of its simple structure and control mechanism, this microdevice is expected to be used for highly efficient, realtime, continuous cell-free plasma separation.

Animals↗

PubMed2006

Periodic microfluidic bubbling oscillator: insight into the stability of two-phase microflows.

This Letter describes a periodically oscillating foam flow in a microfluidic device. For constant input parameters, both the produced bubble volume and the flow rate vary over a factor of two. We make explicit the link between the foam topology alternance and flow rate changes, and construct a retroaction model where bubbles still present downstream determine the volume of new bubbles, in agreement with experiment. This gives insight into the various parameters important to maintain monodispersity and at the same time shows a method to achieve controlled polydispersity.

Microfluidic Analytical Techniques↗

PubMed2006

Application of a microfluidic device for counting of bacteria.

AIMS: To develop a miniaturized analytical system for counting of bacteria. METHODS AND RESULTS: Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 x 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer.

Colony Count, Microbial↗

PubMed2006

Microfluidic digital PCR enables multigene analysis of individual environmental bacteria.

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

Animals↗

PubMed2005

Microfluidic tool box as technology platform for hand-held diagnostics.

BACKGROUND: Use of microfluidics in point-of-care testing (POCT) will require on-board fluidics, self-contained reagents, and multistep reactions, all at a low cost. Disposable microchips were studied as a potential POCT platform. METHODS: Micron-sized structures and capillaries were embedded in disposable plastics with mechanisms for fluidic control, metering, specimen application, separation, and mixing of nanoliter to microliter volumes. Designs allowed dry reagents to be on separate substrates and liquid reagents to be added. Control of surface energy to +/-5 dyne/cm2 and mechanical tolerances to < or = 1 microm were used to control flow propulsion into adsorptive, chromatographic, and capillary zones. Fluidic mechanisms were combined into working examples for urinalysis, blood glucose, and hemoglobin A(1c) testing using indicators (substances that react with analyte, such as dyes, enzyme substrates, and diazonium salts), catalytic reactions, and antibodies as recognition components. Optical signal generation characterized fluid flow and allowed detection. RESULTS: We produced chips that included capillary geometries from 10 to 200 microm with geometries for stopping and starting the flow of blood, urine, or buffer; vented chambers for metering and splitting 100 nL to 30 microL; specimen inlets for bubble-free specimen entry and containment; capillary manifolds for mixing; microstructure interfaces for homogeneous transfer into separation membranes; miniaturized containers for liquid storage and release; and moisture vapor barrier seals for easy use. Serum was separated from whole blood in <10 s. Miniaturization benefits were obtained at 10-200 microm. CONCLUSION: Disposable microchip technology is compatible with conventional dry-reagent technology and allows a highly compact system for complex assay sequences with minimum manual manipulations and simple operation.

Blood Glucose↗

PubMed2005

Using microfluidic channel networks to generate gradients for studying cell migration.

In this chapter, we will discuss a method for the generation of gradients that can be quantitatively used for studying directional cell migration. Microfluidic networks, which serially split and remix small volumes of solutions under laminar flow conditions to generate a series of microchannels of increasing protein concentration. At a juncture of these microchannels, where a single broad channel is formed, a protein concentration gradient can be easily achieved. This method is highly useful because of the ability with which we can control, manipulate and analyze chemical gradients and cells' chemotactic behavior in a quantitative manner.

Animals↗

PubMed2006

Recent advances of microfluidics in Mainland China.

The history and current status of research on microfluidics in China is summarized in this review. The recent representative contributions in this field by Chinese scientists are cited. A perspective on some trends in future development of this field in China is presented.

Animals↗

PubMed2004

A self-priming microfluidic diaphragm pump capable of recirculation fabricated by combining soft lithography and traditional machining.

Fluid transport is crucial in the development of microanalytical devices. While there are many micropump designs available, most are incapable of sustaining recirculation of fluid at microL/min to mL/min levels. We have designed and fabricated a positive displacement micropump by combining soft lithography with traditional bulk machining. The micropump is actuated through pneumatic pressure. The pump is self-priming and is suitable for recirculating fluid through a microfluidic device containing mammalian cell culture. By custom designing the volume of the pumping chamber, tight control of the output flow rate can be obtained by changing the actuation frequency. It can also be fabricated easily on plastic substrates without access to expensive microfabrication equipment.

Cell Culture Techniques↗

PubMed2004

Continuous cell partitioning using an aqueous two-phase flow system in microfluidic devices.

We present a novel microfluidic system in which an aqueous two-phase laminar flow is stably formed, and the continuous partitioning of relatively large cells can be performed, eliminating the influence of gravity. In this study, plant cell aggregates whose diameters were 37-96 microm were used as model particles. We first performed cell partitioning using a simple straight microchannel having two inlets and two outlets and examined the effects of the flow rate and the phase width on partitioning efficiency. Second, by using a microchannel with a pinched segment, the partitioning efficiency was successfully improved. This microscale aqueous two-phase flow system can further be incorporated into micro total analysis systems (microTAS) or lab-on-a-chip technology, owing to its simplicity, applicability, and biocompatibility.

Cell Aggregation↗

PubMed2006

In situ fabrication of cross-linked protein membranes by using microfluidics.

We report a novel technique for preparing cross-linked protein membranes within microchannels by using an interfacial cross-linking reaction. Glass microchannels with a Y input were assembled by using a simple adhesive bonding technique to achieve dual, parallel laminar flows. Membrane formation utilised an interfacial reaction at the liquid-liquid interface, which involved bovine serum albumin (aqueous solution with a flow rate of 300 microL min(-1)) and terephthaloyl chloride (xylene solution with a flow rate of 700 microL min(-1)), to form thin ( approximately 25 microm) cross-linked films along the length of the channel under the continuous pressure-driven laminar flow. Such microfabricated membranes could extend the separation potential of any microfluidic structure to provide a stable barrier layer. Furthermore, degradation of the membrane was possible by using an alkali sodium dodecyl sulfate solution, which led to the complete disappearance of the membrane. These membranes could facilitate additional modification to allow for different permeability properties by controlled degradation. The one-step in situ membrane-fabrication methodology reported here generated precisely localised membranes and avoided the complexities of subcomponent assembly, which require complicated alignment of small, preformed membranes. This methodology could become the basis for sophisticated microseparation systems, biosensors and several "lab-on-a-chip" devices.

Alkalies↗

PubMed2006

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system.

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.

Benzoin↗

PubMed2003

Coated microfluidic devices for improved chiral separations in microchip electrophoresis.

Chiral separations of fluorescein isothiocyanate-labeled amines have been performed in poly(vinyl alcohol) (PVA)-coated microfluidic glass chips. Baseline separation of enantiomers could be realized in coated devices while they could not be resolved in uncoated chips. The electroosmotic flow (EOF) in PVA-coated channels is suppressed over a wide pH range which leads to a considerable improved reproducibility of migration times in repetitive analysis. Due to the high resolution obtained in such devices, it was possible to reliable determine the enantiomeric purity with high accuracy. One percent of the minor enantiomer could be determined in the presence of large excess of the other enantiomer. As the EOF was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Applying PVA-coated channels considerable improved precision of migration times was found. The relative standard deviation of migration times was below 1% in PVA-coated devices. Accordingly, excessive rinsing or etching steps in order to stabilize the EOF could be omitted while this was necessary for a reliable operation of uncoated devices.

Electrophoresis, Capillary↗

PubMed2003

Monolithic valves for microfluidic chips based on thermoresponsive polymer gels.

The direct preparation of thermoresponsive monolithic copolymers by photopatterning of a liquid phase consisting of an aqueous solution of N-isopropylacrylamide, N-ethylacrylamide, N,N'-methylenebisacrylamide, and 4,4'-azobis(4-cyanovaleric acid) has been studied and the products used as valves within the channels of microfluidic devices. The volume change associated with the polymer phase transition at its lower critical solution temperature (LCST) leads to the rapid swelling and the deswelling of the 2.5% cross-linked monolithic gel thus enabling the polymer to close or open the channel and to function as a nonmechanically actuated valve. The LCST at which the valve switches was easily adjusted within a range of 35 degrees C-74 degrees C by varying the proportions of the monovinyl monomers in the polymerization mixture. The closed valve holds pressures of up to 18 MPa without noticeable dislocation, structural damage, or leakage. In contrast, following deswelling by raising the temperature above LCST the valve offers no appreciable flow resistance since its large, micrometer-size pores are open. Laser-triggered photobleaching of a fluorescent dye contained in the liquid phase enabled monitoring of flow through the device and determination of the times required to open and close the valve. The valves are characterized by very fast actuation times in a range of 1-4 s depending on the type of device. No changes in performance were observed even after repeated open-close cycling of the valves.

Acrylamides↗

PubMed2003

Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies.

This review describes microfluidic systems in poly(dimethylsiloxane) (PDMS) for biological studies. Properties of PDMS that make it a suitable platform for miniaturized biological studies, techniques for fabricating PDMS microstructures, and methods for controlling fluid flow in microchannels are discussed. Biological procedures that have been miniaturized into PDMS-based microdevices include immunoassays, separation of proteins and DNA, sorting and manipulation of cells, studies of cells in microchannels exposed to laminar flows of fluids, and large-scale, combinatorial screening. The review emphasizes the advantages of miniaturization for biological analysis, such as efficiency of the device and special insights into cell biology.

Biology↗

PubMed2003

Poly(dimethylsiloxane)-based microfluidic device with electrospray ionization-mass spectrometry interface for protein identification.

An easy method to fabricate poly(dimethylsiloxane) (PDMS)-based microfluidic chips for protein identification by tandem mass spectrometry is presented. This microchip has typical electrophoretic microchannels, a flow-through sampling inlet, and a sheathless nanoelectrospray ionization (ESI) interface. The surface of the microchannel was modified with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and the generated electroosmotic flow under acidic buffer condition used for the separation was found to be more stable compared to that generated by the microchannel without modification. The feasibility of the device for flow-through sampling, separation, and ESI-MS/MS analysis was demonstrated by the analysis of a standard mixture composed of three tryptic peptides. Results show that four peaks corresponding to three peptide standards and acetylated products of the standard peptide were well resolved and the deduced sequences were consistent with those expected. Furthermore, the compatibility of this device with other miniaturized devices to integrate the whole process was also explored by connecting a miniaturized enzymatic digestion cartridge and a desalting cartridge in series to the sampling inlet of the microchip for the identification of a model protein, beta-casein.

Amino Acid Sequence↗

PubMed2003

Detection of DNA fragmentation in a single apoptotic cardiomyocyte by electrophoresis on a microfluidic device.

The detection of doxorubicin-induced apoptosis in individual cardiomyocytes was performed on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells their lysis in an alkaline environment and the separation of released apoptotic DNA fragments. The fragments with typical 180 base pairs ladder pattern were electrophoretically resolved in a 2% solution of linear polyacrylamide with 0.1 M NaOH on a migration distance of 6 mm. The laser-induced fluorescence of fragments labeled by ethidium bromide was monitored by a photomultiplier tube mounted on a confocal microscope. The causal relation between the enhanced doxorubicin concentration and the extent of DNA fragmentation in a single cell was confirmed. The results show that the extent of DNA fragmentation is proportional to the time of a cell treatment. Onset of necrosis was evident in cardiomyocytes treated by doxorubicin for more than 24 h. The adverse effect of doxorubicin, an important cytostatics used for the treatment of many solid tumors, leads to the destruction of cardiomyocytes and, consequently, may result in the heart failure of treated individuals. Therefore, the monitoring of the extent of apoptotic DNA damage of cardiac myocytes represents critical step toward understanding of the development of chronic doxorubicin-induced cardiomyopathy.

Animals↗

PubMed2004

Zeta potential of microfluidic substrates: 1. Theory, experimental techniques, and effects on separations.

This paper summarizes theory, experimental techniques, and the reported data pertaining to the zeta potential of silica and silicon with attention to use as microfluidic substrate materials, particularly for microchip chemical separations. Dependence on cation concentration, buffer and cation type, pH, cation valency, and temperature are discussed. The Debye-Hückel limit, which is often correctly treated as a good approximation for describing the ion concentration in the double layer, can lead to serious errors if it is extended to predict the dependence of zeta potential on the counterion concentration. For indifferent univalent electrolytes (e.g., sodium and potassium), two simple scalings for the dependence of zeta potential on counterion concentration can be derived in high- and low-zeta limits of the nonlinear Poisson-Boltzman equation solution in the double layer. It is shown that for most situations relevant to microchip separations, the high-zeta limit is most applicable, leading to the conclusion that the zeta potential on silica substrates is approximately proportional to the logarithm of the molar counterion concentration. The zeta vs. pH dependence measurements from several experiments are compared by normalizing the zeta based on concentration.

Electric Capacitance↗

PubMed2004

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip.

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

Animals↗