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At least 361 records · Page 20Linked to original sources

PubMed2006

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay.

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-microm-wide and 800-microm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, beta-carotene was found to be the most efficient hydrogen peroxide scavenger (SAHP of 3.27x10(-3) micromol-1 L), followed by alpha-tocopherol (SAHP of 2.36x10(-3) micromol-1 L) and quercetin (SAHP of 0.31x10(-3) micromol-1 L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements.

Anthracenes↗

PubMed2007

Refolding of a membrane protein in a microfluidics reactor.

Membrane protein production for structural studies is often hindered by the formation of non-specific aggregates from which the protein has to be denatured and then refolded to a functional state. We developed a new approach, which uses microfluidics channels, to refold protein correctly in quantities sufficient for structural studies. Green fluorescent protein (GFP), a soluble protein, and bacteriorhodopsin (BR), a transmembrane protein, were used to demonstrate the efficiency of the process. Urea-denatured GFP refolded as the urea diffused away from the protein, forming in the channel a uniform fluorescent band when observed by confocal microscopy. Sodium dodecyl sulphate-denatured BR refolded within the channel on mixing with detergent-lipid mixed micelles. The refolding, monitored by absorbance spectroscopy, was found to be flow rate dependent. This potential of microfluidic reactors for screening protein-folding conditions and producing protein would be particularly amenable for high-throughput applications required in structural genomics.

Bacteriorhodopsins↗

PubMed2006

Microfluidic alignment of collagen fibers for in vitro cell culture.

Three dimensional gels of aligned collagen fibers were patterned in vitro using microfluidic channels. Collagen fiber orientation plays an important role in cell signaling for many tissues in vivo, but alignment has been difficult to realize in vitro. For microfluidic collagen fiber alignment, collagen solution was allowed to polymerize inside polydimethyl siloxane (PDMS) channels ranging from 10-400 microm in width. Collagen fiber orientation increased with smaller channel width, averaging 12+/-6 degrees from parallel for channels between 10 and 100 microm in width. In these channels 20-40% of the fibers were within 5 degrees of the channel axis. Bovine aortic endothelial cells expressing GFP-tubulin were cultured on aligned collagen substrate and found to stretch in the direction of the fibers. The use of artificially aligned collagen gels could be applied to the study of cell movement, signaling, growth, and differentiation.

Adsorption↗

PubMed2006

A parallel-gradient microfluidic chamber for quantitative analysis of breast cancer cell chemotaxis.

Growth factor-induced chemotaxis of cancer cells is believed to play a critical role in metastasis, directing the spread of cancer from the primary tumor to secondary sites in the body. Understanding the mechanistic and quantitative behavior of cancer cell migration in growth factor gradients would greatly help in future treatment of metastatic cancers. Using a novel microfluidic chemotaxis chamber capable of simultaneously generating multiple growth factor gradients, we examined the migration of the human metastatic breast cancer cell line MDA-MB-231 in various conditions. First, we quantified and compared the migration in two gradients of epidermal growth factor (EGF) spanning different concentrations: 0-50 ng/ml and 0.1-6 ng/ml. Cells showed a stronger response in the 0-50 ng/ml gradient. However, the fact that even a shallow gradient of EGF can induce chemotaxis, and that EGF can direct migration over a large dynamic range of gradients, confirms the potency of EGF as a chemoattractant. Second, we investigated the effect of antibody against the EGF receptor (EGFR) on MDA-MB-231 chemotaxis. Quantitative analysis indicated that anti-EGFR antibody impaired both motility and directional orientation (CI = 0.03, speed = 0.71 microm/min), indicating that cell motility was induced by the activation of EGFR. The ability to compare, in terms of quantitative parameters, the effects of different pharmaceutical inhibitors, as well as subtle differences in experimental conditions, will aid in our understanding of mechanisms that drive metastasis. The microfluidic chamber described in this work will provide a platform for cell-based assays that can be used to compare the effectiveness of different pharmaceutical compounds targeting cell migration and metastasis.

Breast Neoplasms↗

PubMed2006

Size-based microfluidic enrichment of neonatal rat cardiac cell populations.

Native heart consists of myocytes and non-myocytes. We demonstrate here the feasibility of a size-based microfluidic separation of myocytes and non-myocytes from the neonatal rat myocardium. The device consists of a middle channel (50 microm wide, 200 microm tall, and 4 cm long) connected to adjacent side channels by microsieves (80 microm wide, 5 microm tall and 40 microm in length). The side channels increase in width in a flared shape along the length of the device to ensure constant pressure gradient across all sieves. In the first step, non-myocytes were removed from the myocytes by a conventional pre-plating method for 75 min. Subsequently, the non-myocytes were further enriched in a microfluidic device at 20 microl/min. We demonstrated that the cells in the middle and side channels maintained viability during sorting and the ability to attach and grow in culture. Upon culture for 48 h cardiomyocytes from the reservoir (control) and middle channel stained positive for cardiac Troponin I, exhibited a well developed contractile apparatus and contracted spontaneously and in response to electrical field stimulation. Most of the cells in the side channel expressed a non-myocyte marker vimetin. Fluorescent activated cell sorting indicated significant enrichment in the side channel (p < 0.001) for non-myocytes. Original cell suspension had a bimodal cell size distribution with the peaks in the range from 7-9 microm and 15-17 microm. Upon cell sorting the distribution was Gaussian in both side channel and middle channel with the peaks in the range 7-9 microm and 9-11 microm respectively, indicating that the separation by size occurred.

Animals↗

PubMed2007

Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture.

Microfluidic bioreactors fabricated from highly gas-permeable poly(dimethylsiloxane) (PDMS) materials have been observed, somewhat unexpectedly, to give rise to heterogeneous long term responses along the length of a perfused mammalian cell culture channel, reminiscent of physiologic tissue zonation that arises at least in part due to oxygen gradients. To develop a more quantitative understanding and enable better control of the physical-chemical mechanisms underlying cell biological events in such PDMS reactors, dissolved oxygen concentrations in the channel system were quantified in real time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye, ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP). The data indicate that despite oxygen diffusion through PDMS, uptake of oxygen by cells inside the perfused PDMS microchannels induces an axial oxygen concentration gradient, with lower levels recorded in downstream regions. The oxygen concentration gradient generated by a balance of cellular uptake, convective transport by media flow, and permeation through PDMS in our devices ranged from 0.0003 (mg/l)/mm to 0.7 (mg/l)/mm. The existence of such steep gradients induced by cellular uptake can have important biological consequences. Results are consistent with our mathematical model and give insight into the conditions under which flux of oxygen through PDMS into the microchannels will or will not contribute significantly to oxygen delivery to cells and also provide a design tool to manipulate and control oxygen for cell culture and device engineering. The combination of computerized microfluidics, in situ oxygen sensing, and mathematical models opens new windows for microphysiologic studies utilizing oxygen gradients and low oxygen tensions.

Animals↗

PubMed2007

Rapid circular microfluidic mixer utilizing unbalanced driving force.

This paper proposes a novel rapid circular microfluidic mixer for micro-total-analysis-systems (mu-TAS) applications in which an unbalanced driving force is used to mix fluids in a circular chamber at low Reynolds numbers (Re). The microfluidic mixer has a three-layered structure and is fabricated on low-cost glass slides using a simple and reliable fabrication process. Using hydrodynamic pumps, fluids are driven from two inlet ports into a circular mixing chamber. Each inlet port separates into two separate channels, which are then attached to opposite sides of the 3-dimensional (3-D) circular mixing chamber. The unequal lengths of these inlet channels generate an unbalanced driving force, which enhances the mixing effect in the mixing chamber. Numerical simulations are performed to predict the fluid phenomena in the mixing chamber and to estimate the mixing performance under various Reynolds number conditions. The numerical results are verified by performing flow visualization experiments. A good agreement is found between the two sets of results. The numerical and experimental results reveal that the mixing performance can reach 91% within a mixing chamber of 1 mm diameter at a Reynolds number of Re=3. Additionally, the results confirm that the unbalanced driving force produces a flow rotation in the circular mixer at low Reynolds numbers, which significantly enhances the mixing performance. The novel micromixing method presented in this study provides a simple solution for mixing problems in Lab-on-a-chip systems.

Chemistry Techniques, Analytical↗

PubMed2007

Microtubule transport, concentration and alignment in enclosed microfluidic channels.

The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargo-functionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 microm wide and 1 microm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels (approximately 100 microm wide) serve as reservoirs and connect to 250 microm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 microm diameter circular ring functionalized with motors concentrates and aligns bundles of approximately 3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells.

Equipment Design↗

PubMed2006

A low temperature bonding of quartz microfluidic chip for serum lipoproteins analysis.

A low-temperature bonding method for microfabrication of quartz microfluidic chips has been developed. The bonding process involved two steps: pre-bonding and post-annealing at low temperature. The bonding quality was evaluated by measuring the shear force at bonding interface and the electrical properties of the chips. Shear force of 5.66 MPa (566 N/cm(2)) was obtained in a bonded chip after a post-annealing at 200 degrees C for 6 h. We owe the strong bonding strength to the formation of Si-O-Si bonds at the bonding interface during the post-annealing stage. The bonding procedures were not sensitive to surrounding and could be performed in a routine laboratory without clean room conditions. The performance of the fabricated microfluidic chips was tested by capillary zone electrophoresis (CZE) of serum lipoproteins with laser-induced fluorescence (LIF). The low-density (LDL) and high-density (HDL) lipoproteins in the serum was separated completely by using tricine buffer with methylglucamine.

Cold Temperature↗

PubMed2004

Direct observation of long-strand DNA conformational changing in microchannel flow and microfluidic hybridization assay.

Conformational control of macromolecules is useful for efficient chemical and biochemical reactions. This article reports a direct observation method for macromolecules, such as long-strand DNA, in microchannel flow as well as a simple method for stretching DNA strands by microfluidics. Stretching and orientation of DNA molecules by control of flow within a microchannel was observed by optical microscopy. This DNA stretching is explained by coil-stretch transition of polymer molecules. This technique is useful for creating chemical reactions with macromolecules. It offers high selectivity and efficiency that are impossible to achieve in bulk solution. We also demonstrate that our microfluidic stretching method can accomplish efficient hybridization of long-strand DNA. This method will be useful for direct hybridization assay of long-strand DNA.

DNA↗

PubMed2004

Whole-cell-reporter-gene-based biosensing systems on a compact disk microfluidics platform.

Biosensing systems such as reporter-gene-based whole-cell assays are increasingly finding applications in biological and environmental screening. A whole-cell approach to such analyses can provide valuable information about the bioavailable level of a compound of interest. These biosensing systems rely on the molecular recognition of a specific analyte by a regulatory protein and, therefore, can detect low levels of the target analyte. In this study, Escherichia coli cells containing plasmid pSD10 were engineered to sense the model target analytes arsenite and antimonite, the target analytes in this study. The biosensing system takes advantage of the recognition of the regulatory protein, ArsR, for arsenite and antimonite to produce the reporter protein, which in this case is GFPuv. The fluorescence emitted by the GFPuv in the cells can be directly related to the concentration of the analyte in the cell, making this biosensing system useful in the detection of arsenite and/or antimonite in a variety of samples. Miniaturization of biosensing systems can further enhance their utility by decreasing reagent consumption and analysis time and by allowing for the high-throughput screening of samples. To that end, we employed a microcentrifugal microfluidics platform that has low power, space, and reagent requirements, increased speed of detection, and the potential for portability. Herein, we demonstrate for the first time the adaptation of a whole-cell sensing system to a microcentrifugal microfluidics platform. Moreover, we were able to detect our target analytes in a rapid and sensitive manner compared to conventional sensing methods.

Antimony↗

PubMed2005

Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations.

We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90 degrees to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be approximately 41pmolcm(-2), near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.

Adsorption↗

PubMed2004

Microfluidic approaches to malaria detection.

Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.

Electrophoresis↗

PubMed2004

Poly(ethylene glycol) interfaces: an approach for enhanced performance of microfluidic systems.

Microfluidic systems are extensively used platform for analytical and therapeutic applications. One of the major problems encountered in these systems is the loss of material due to non-specific surface interactions. When biological solutions are flowed through microchannels, they tend to adsorb on the surface due to the negative charge of the surface. This results in a reduced efficiency of the system which can be critical in sensitive biological analysis. Poly(ethylene glycol) (PEG) is known to form non-fouling interfaces on silicon and glass which are common materials used in microfluidic systems. The most common approach for modifying silicon/glass with PEG involves a solution phase protocol. Since the micro/nanofluidic systems have channel sizes ranging from hundreds of microns to submicron with variety of complicated network, this surface modification approach is not sufficient in forming uniform, conformal, and ultrathin films on the surface. Due to the enclosed features in these systems, the properties of liquids such as viscosity and surface tension play an important role in the clogging and eventually biofouling of these microchannels. Hence, we have developed a solvent-free vapor deposition protocol for modifying silicon/glass surfaces with PEG. Various concentrations of protein solutions were flowed through unmodified and PEG-modified glass microcapillaries of different lengths at different flow rates. PEG surfaces formed on silicon have shown 80% reduction in protein adsorption in static conditions.

Boranes↗

PubMed2004

Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks.

We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.

Antibodies↗

PubMed2005

An integrated microfluidic platform for magnetic microbeads separation and confinement.

An innovative microfluidic platform for magnetic beads manipulation is introduced, consisting of novel microfabricated 3D magnetic devices positioned in a microfluidic chamber. Each magnetic device comprises of an embedded actuation micro-coil in various design versions, a ferromagnetic pillar, a magnetic backside plate and a sensing micro-coil. The various designs of the micro-coils enable efficient magnetic beads trapping and concentration in different patterns. The finite element analysis (FEA) results show a significant increase of the developed force on suspended magnetic beads when the magnetic pillar and backside plate were integrated into the device structure. These simulation results were confirmed experimentally by measuring the magnetic beads trapping ratios for the different designs and structures of the devices under continuous flow conditions. The trapping ratios and profiles were studied using beads counting, measuring the change of inductance with the sensing micro-coil and by image processing. The devices have efficiently demonstrated a controlled and localized magnetic beads trapping and concentration at small spatial locations for the first time. The new results shown in this study demonstrate the feasibility of efficiently using these original devices as key elements in complex bio-analysis systems.

Computer-Aided Design↗

PubMed2006

Modeling micropatterned antigen-antibody binding kinetics in a microfluidic chip.

The reaction kinetics of antigen-antibody binding in the electrokinetically controlled microfluidic heterogeneous immunoassays has been investigated by numerical simulations. A two-dimensional computational model was employed to include the mass transport (convection and diffusion) and binding reaction between the antigen in the bulk flow and the immobilized antibody at the channel surface. The influence of the bulk velocity, the concentrations of the antibody and antigen, and the geometry of the microchips was studied for a variation of conditions and the guidance for designing of microfluidic immunoassay was provided. The model also shows that electrokinetically driven immunoassays have better reaction kinetics than pressure-driven ones, resulting from the plug-like velocity profile. Finally, a multi-patch immunoassay chip was analyzed and the reaction kinetics was optimized by rearranging the reaction patches at the channel surfaces.

Antigen-Antibody Complex↗

PubMed2006

Latex immunoagglutination assay for a vasculitis marker in a microfluidic device using static light scattering detection.

We have developed a microfluidic immunoassay device using fiber optics to detect static light scattering (SLS) of latex microsphere agglutination. A 400-mum silica fiber was used to deliver blue light emitting diode (LED) or red laser light sources. A miniature, portable spectrometer was used to measure forward light scattering intensity collected by the same type of multi-mode fiber. To first show feasibility, anti-mouse IgG were used as target biomolecules and highly carboxylated polystyrene latex microspheres (510 nm) coated with mouse IgG were used as probes. Next, we tested for the vasculitis marker, anti-PR3, using the same type of microspheres coated with PR3 proteins. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL(-1) was demonstrated for the vasculitis marker, anti-PR3. (Plasma samples from patients with vasculitis exhibited anti-PR3 at a median level of 380 ng mL(-1).) The optical detection system works without any fluorescence or chemiluminescence markers. The entire system proposed here is cost effective, small in size, and re-usable with simple rinsing. This may eventually lead to a portable, low-cost, re-useable, microfluidic, point of care immunoassay device.

Antibodies↗