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At least 271 records · Page 15Linked to original sources

PubMed2004

Thermophoresis of DNA determined by microfluidic fluorescence.

We describe a microfluidic all-optical technique to measure the thermophoresis of molecules. Within micrometer-thick chambers, we heat aqueous solutions with a micrometer-sized focus of infrared light. The temperature increase of about 1 K is monitored with temperature-sensitive fluorescent dyes. We test the approach in measuring the thermophoresis of DNA. We image the concentration of DNA in a second fluorescence-color channel. DNA is depleted away from the heated spot. The profile of depletion is fitted by the thermophoretic theory to reveal the Soret coefficient. We evaluate the method with numerical 3D calculations of temperature profiles, drift, convection and thermophoretic depletion using finite element methods. The approach opens new ways to monitor thermophoresis at the single molecule level, near boundaries and in complex mixtures. The flexible microfluidic setting is a good step towards microfluidic applications of thermophoresis in biotechnology.

Bacteriophage lambda↗

PubMed2005

Microfluidic tools for studying the specific binding, adsorption, and displacement of proteins at interfaces.

A combination of temperature and concentration gradient microfluidic devices were employed to study the mechanistic details of biomacromolecule interactions at oxide interfaces. These lab-on-a-chip techniques allowed high-throughput, multiplexed data collection using only nanoliters of analyte. The three examples presented demonstrate rapid data acquisition relative to standard methods. First, we show ligand-receptor binding data for multivalent binding between membrane-bound ligands and incoming aqueous proteins with several binding pockets. A model is described for obtaining both the first and second dissociation constant for the reaction. The second example employs temperature gradient microfluidics to study the thermoresponsive properties of polymers and proteins. Both the folding mechanism and subsequent formation of an aqueous two-phase system were followed. Finally, these microfluidic techniques were combined with fluorescence microscopy and nonlinear optical spectroscopy to elucidate the mechanism of fibrinogen displacement from silica surfaces. This combination of methods enabled both direct and indirect observation of protein conformational changes.

Adsorption↗

PubMed2005

Magnetically actuated micromixing on an array-pattern microfluidic chip for immunoassay of human thyrotropin.

A novel array-pattern ring microfluidic chip has been designed and fabricated with ITO glass, and a low-cost, easy-to-use active magnetic mixer is developed. The mixer consists of externally driven magnetic stir plates and ferromagnetic microneedles as active mobile stirrers in the microchannel. Mixing performance in the microfluidic chip has been experimentally characterized by chemiluminescence immunoassay of human thyrotropin (TSH) and compared with tube-based chemiluminescence immunoassay. The mixing method proposed in this study can be readily applied to integrated microfluidic systems, such as micro-total-analysis systems, lab-on-a-chip, and so on.

Equipment Design↗

PubMed2006

Microfluidic ethanol biobatteries on a microchip.

This chapter outlines the methods and procedures for making a microfluidic and microfabricated biofuel cell. Commercially available screen-printing carbon inks are employed as electrodes by micromolding them onto glass microchips. The carbon ink electrodes are modified with methylene green and alcohol dehydrogenase immobilized within a modified Nafion membrane to act as bioanodes in the microfluidic system. The complete biofuel cell produces power using an external platinum cathode and an integrated microfluidic bioanode. Miniaturization of power sources, such as biofuel cells, is important in applications for implementation in small technologies (i.e., sensors, bioreactors, and lab-on-a-chip technology).

Electrochemistry↗

PubMed2006

Single cell analysis on microfluidic devices.

There is significant variability among cells of the same type at the single cell level. This variability may be because of external stimuli that vary temporally or spatially among a population of cells. It may also be owing to the nonsynchronized responses of cells to various stimuli. In addition, differences in otherwise similar cells may be generated by genetic mutations acquired by one or more of the cells. Often times multiple biochemical pathways and molecules are involved in such differences. In order to better understand these differences and to detect those rare cells in a large population that may be indicative of early disease states, methods that are capable of rapidly quantifying multiple molecular species in single cells are desired. Microfluidic devices may provide the optimal platform upon which to develop such methods. Microfluidics has the capability of combining the high-throughput manipulation and transport of cells with rapid, high-efficiency separations and high-sensitivity detection. This chapter describes how to fabricate microfluidic devices for the high-throughput manipulation and rapid electrical lysis of single, nonadherent (suspension) cells followed by the injection and separation of the fluorescently labeled cell contents.

Carboxylesterase↗

PubMed2006

Triggering and visualizing the aggregation and fusion of lipid membranes in microfluidic chambers.

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an approximately 6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.

Electrochemistry↗

PubMed2003

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Computer Simulation↗

PubMed2006

Microfluidic enzyme-linked immunosorbent assay technology.

In this chapter, we have presented an overview of microfluidic enzyme-linked immunosorbent assay (ELISA) by first introducing the principle of immunoassay, ELISA, and microfabricated devices, followed by a discussion of microfabrication technology and the characterization of microfluidic components. Significant advances in laboratory technology are contributing to the further understanding of microfluidic function, surface modification and immobilization, which lead to the development of improved biomolecule detection methods and prospective applications. For the future, the exploitation of more robust-manufacturing processes and integrated assay systems in an automatic fashion with much reduced assay time and reagent consumption will allow for the effective detection and quantification of biological agents that are of interest in medical diagnostics, food safety surveillance, and environmental monitoring.

Enzyme-Linked Immunosorbent Assay↗

PubMed2000

Fabrication of microfluidic systems in poly(dimethylsiloxane).

Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

Animals↗

PubMed2002

Velocity-independent microfluidic flow cytometry.

Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.

Bacteriophage lambda↗

PubMed2002

Components for integrated poly(dimethylsiloxane) microfluidic systems.

This review describes the design and fabrication of microfluidic systems in poly(dimethylsiloxane) (PDMS). PDMS is a soft polymer with attractive physical and chemical properties: elasticity, optical transparency, flexible surface chemistry, low permeability to water, and low electrical conductivity. Soft lithography makes fabrication of microfluidic systems in PDMS particularly easy. Integration of components, and interfacing of devices with the user, is also convenient and simpler in PDMS than in systems made in hard materials. Fabrication of both single and multilayer microfluidic systems is straightforward in PDMS. Several components are described in detail: a passive chaotic mixer, pneumatically actuated switches and valves, a magnetic filter, functional membranes, and optical components.

Dimethylpolysiloxanes↗

PubMed2003

Microfluidic peroxidase biochip for polyphenol synthesis.

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.

Bioreactors↗

PubMed2003

Cytometric analysis of protein expression and apoptosis in human primary cells with a novel microfluidic chip-based system.

BACKGROUND: Work with primary cells is inherently limited by source availability and life span in culture. Flow cytometry offers extensive analytical opportunities but generally requires high cell numbers for an experiment. METHODS: We have developed assays on a microfluidic system, which allow flow cytometric analysis of apoptosis and protein expression with a minimum number of fluorescently stained primary cells. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by two-channel fluorescence detection. For some assays the staining reactions can be performed on-chip and the analysis is done without further washing steps. RESULTS: We have successfully applied the assays to evaluate (a) activation of E-selectin (CD62E) expression by interleukin-1beta in human umbilical vein endothelial cells (HUVECs), (b) induction of CD3 by phorbol-12-myristate-13-acetate in freshly prepared human peripheral blood lymphocytes, and (c) staurosporine-induced apoptosis in HUVEC and normal human dermal fibroblasts. CONCLUSIONS: Results obtained with the microfluidic system are in good correlation with data obtained using a standard flow cytometer, but demonstrate new dimensions in low reagent and cell consumption.

Apoptosis↗

PubMed2005

A polymeric master replication technology for mass fabrication of poly(dimethylsiloxane) microfluidic devices.

A protocol of producing multiple polymeric masters from an original glass master mold has been developed, which enables the production of multiple poly(dimethylsiloxane) (PDMS)-based microfluidic devices in a low-cost and efficient manner. Standard wet-etching techniques were used to fabricate an original glass master with negative features, from which more than 50 polymethylmethacrylate (PMMA) positive replica masters were rapidly created using the thermal printing technique. The time to replicate each PMMA master was as short as 20 min. The PMMA replica masters have excellent structural features and could be used to cast PDMS devices for many times. An integration geometry designed for laser-induced fluorescence (LIF) detection, which contains normal deep microfluidic channels and a much deeper optical fiber channel, was successfully transferred into PDMS devices. The positive relief on seven PMMA replica masters is replicated with regard to the negative original glass master, with a depth average variation of 0.89% for 26-microm deep microfluidic channels and 1.16% for the 90 mum deep fiber channel. The imprinted positive relief in PMMA from master-to-master is reproducible with relative standard deviations (RSDs) of 1.06% for the maximum width and 0.46% for depth in terms of the separation channel. The PDMS devices fabricated from the PMMA replica masters were characterized and applied to the separation of a fluorescein isothiocyanate (FITC)-labeled epinephrine sample.

Dimethylpolysiloxanes↗

PubMed2001

Detection of E. coli using a microfluidics-based antibody biochip detection system.

This work demonstrates the detection of E. coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays. The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform. The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors. The independently operating photodiodes allow simultaneous monitoring of multiple samples. In this study the sampling platform is a cellulosic membrane that is exposed to E. coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe. The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection. Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E. coli organisms. Selective detection of E. coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.

Animals↗

PubMed2001

Multi-layer microfluidic glass chips for microanalytical applications.

A new, versatile architecture is presented for microfluidic devices made entirely from glass, for use with reagents which would prove highly corrosive for silicon. Chips consist of three layers of glass wafers bonded together by fusion bonding. On the inside wafer faces a network of microfluidic channels is created by photolithography and wet chemical etching. Low dead-volume fluidic connections between the layers are fabricated by spark-assisted etching (SAE), a computer numerical controlled (CNC)-like machining technique new to microfluidic system fabrication. This method is also used to form a vertical, long path-length, optical cuvette through the middle wafer for optical absorbance detection of low-concentration compounds. Advantages of this technique compared with other, more standard, methods are discussed. When the new glass-based device for flow-injection analysis of ammonia was compared with our first-generation chips based on silicon micromachining, concentration sensitivity was higher, because of the longer path-length of the optical cuvette. The dependence of dispersion on velocity profile and on channel cross-sectional geometry is discussed. The rapid implementation of the devices for an organic synthesis reaction, the Wittig reaction, is also briefly described.

Journal Article↗

PubMed2005

Modeling of a microfluidic channel in the presence of an electrostatic induced cross-flow.

Amongst the processes that have been implemented in microfluidic devices, electrophoretic transport of charged molecules, along microfluidic channels, is one of the most commonly found. However, less work has been done about continuous, pressure gradient driven flow systems where an electric field is applied orthogonally with respect to the microchannel walls. The perspective applications of this technique, include continuous flow separation and concentration of analyte molecules, and the kinetic control of surface reactions. In order to dimensioning and optimizing such a device, a mathematical model has been formulated and analyzed both with numeric and analytic methods. The given solutions let the designer of microfluidic devices able to estimate the concentration profiles along the microchannel length, as a function of the main system parameters. As a practical example of application which could be of great interest in biotechnology applications, the results relative to the simulation of the electrostatic induced cross flow of single strand DNA oligonucleotides of about 20 bases has been reported.

Computer Simulation↗

PubMed2006

Flow sandwich-type immunoassay in microfluidic devices based on negative dielectrophoresis.

Microparticles have been manipulated in a microfluidic channel by means of negative dielectrophoresis (n-DEP), and the approach applied to a heterogeneous immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 microm diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area when surrounded by intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 6-15 V(peak) and frequencies over 500 kHz were applied to the two facing microelectrodes. A heterogeneous sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing a secondary antibody with a signal source (FITC-labeled anti-mouse IgG antibody), into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 microg/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. Thus, the automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP.

Biosensing Techniques↗