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At least 667 records · Page 37Linked to original sources

PubMed2007

Modeling, design and validation of a novel microfluidic sensor for in-vitro isotonic measurement of microvessel contraction/dilation.

The paper presents an innovative fluidic microdevice for in-vitro isotonic measurements of microvessel contraction/dilation. The proposed microdevice is based on the well-known fluid dynamic principle which correlates a geometrical change of a solid body immersed in a constant flow, with the differential pressure change induced in the flow itself. Indeed, a biochemically-induced change of microvessel diameter lead to a change of its hydraulic resistance which can be in-vitro measured by a differential pressure sensor. The novel microfluidic sensor has been modeled, designed and fabricated in order to properly implement this working principle. The fluidic scheme of the sensor consists of two pressure-sensorized chambers which are connected by a calibrated channel where the microvessel is placed. Experimental tests have been performed by using three microvessel passive simulators with different inner diameter (300, 600 and 800 microm), in order to validate the expected sensor functioning characterized by two sensitivities: the pressure drop S (PL) and the overall S 2-3 ones. Their measured values (S (PL,m)=2.132 mV/V/Pa, S (2-3,m)=0.787 mV/V/(mm3/s)/mm) confirm the validity of the proposed working principle and permit to be confident in a future application of the sensor in a clinical context.

Blood Flow Velocity↗

PubMed2007

Microfluidic patterning for fabrication of contractile cardiac organoids.

The development of in vitro methods of engineering three-dimensional cardiac tissues can be useful for tissue replacement, diagnostics and drug discovery. Here, we introduce the use of patterned hyaluronic acid (HA) substrates generated using microfluidic patterning as a method of fabricating 3D cardiac organoids. HA micropatterns served as inductive templates for organoid assembly. Upon seeding, cardiomyocytes elongated and aligned along the pattern direction attaching preferentially to the glass substrate and the interface between HA patterns and glass substrate. After 3 days in culture, the linearly aligned myocytes detached from the surface and formed contractile cardiac organoids. The procedure can be utilized to simply, rapidly and inexpensively create in vitro cardiac tissue models.

Animals↗

PubMed2007

Simultaneous detection of five indices of hepatitis B based on an integrated automatic microfluidic device.

Immunophenotyping evaluation is of particular importance for the clinical diagnosis, therapy, and prognosis of viral hepatitis. In this study, an integrated micro flow device has been developed to detect the differentiated antigens/antibodies for immunophenotyping of viral hepatitis. The sensors were fabricated with plasma-polymerized ethylenediamine film (PPF) and nanometer-sized gold particles (nanogold) on which the different hepatitis B antigens/antibodies (markers) were subsequently immobilized. Monitoring the changes in the potential signals before and after the antigen-antibody interaction provides the basis for an immunoassay that is simple, rapid, and cost-effective. It permits the detection of hepatitis B in the dynamic concentration range of 2 orders of magnitude (10(-6) g x L(-1) - 10(-4) g x L(-1)). Up to 7 successive assay cycles with retentive sensitivity were achieved for the sensors regenerated by 8 M urea. Moreover, the microfluidic device was applied to evaluate a number of practical specimens with analytical results in acceptable agreement with those clinically classified. The newly proposed multiparameter analysis technique provides a feasible alternative tool for the diagnosis and monitoring of hepatitis B.

Equipment Design↗

PubMed2006

Precise and comparative pegylation analysis by microfluidics and mass spectrometry.

Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.

Binding Sites↗

PubMed2005

Study of osteoblastic cells in a microfluidic environment.

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.

3T3 Cells↗

PubMed2005

Rapid localized cell trapping on biodegradable polymers using cell surface derivatization and microfluidic networking.

Spatial control over cell attachment is essential for controlling cell behavior and engineering cell-based sensor arrays. Here we report on a patterning procedure that can be utilized on a wide range of adherent and non-adherent cell types without the need to identify the exact peptide sequence or extracellular matrix (ECM) necessary for optimal cell attachment. This is achieved by converting native sialic residues present on the surface of most cells into non-native aldehydes using a mild sodium periodate treatment. The aldehyde groups are then reacted with biotin hydrazide to produce biotinylated cells. Avidin is patterned onto the surface of a biotinylated biodegradable block copolymer, polylactide-poly(ethylene glycol)-biotin (PLA-PEG-biotin) by microfluidic networking using a PDMS stamp. The biotinylated cells then bind specifically to the patterned avidin regions. The PEG that is presented from the PLA-PEG-biotin copolymer in the regions without avidin immobilization minimizes cell binding in the non-patterned regions.

Avidin↗

PubMed2004

Application of the synthetic jet concept to low Reynolds number biosensor microfluidic flows for enhanced mixing: a numerical study using the lattice Boltzmann method.

The concept of macro scale synthetic jets has been applied to the low Reynolds number (Re=10), two-dimensional channel flows which may be found in biosensor microfluidic systems. The current numerical investigation utilizes a hybrid approach of the lattice Boltzmann (LB) method for flow field computations and a finite-difference, convection-diffusion equation for passive scalar transport. The study presents the modified main channel flow results for various wall jet geometries (derived from synthetic jets), jet inlet conditions, scaling issues and Reynolds numbers. The results indicate limited effects due to jet cavity-slot geometry, and that the forced jet imparts momentum to the channel flow thus enhancing fluid mixing.

Biosensing Techniques↗

PubMed2004

Silicon chip-based patch-clamp electrodes integrated with PDMS microfluidics.

We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks.

Animals↗

PubMed2004

Thin-film IrOx pH microelectrode for microfluidic-based microsystems.

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.

Animals↗

PubMed2005

Microfluidic-based diagnostics for cervical cancer cells.

The use of biomarkers has facilitated the detection of specific tumor cells. However, the technology to apply these markers in a clinical setting has not kept pace with their increasing availability. In this project, we use an antibody-based microfluidics platform to recognize and capture cervical cancer cells. Because HPV-16 infection of cervical cells and up-regulation of alpha6-integrin cell surface receptors are correlated, we utilized alpha6-integrin as a capture antibody bound to the channel surface. Normal human glandular epithelial cells (HGEC), human cervical stromal cells (HCSC) and cervical cancer cells (HCCC) were suspended in PBS and flowed through the system. Greater than 30% of the cancer cells were captured while the capture of the normal cell types was less than 5%. The technique is sensitive and accurate. It is potentially useful in the detection of cervical cancer at all stages, as well as other of cancers with similar characteristics of cell surface antigen expression.

Biomarkers, Tumor↗

PubMed2005

Miniaturized immunoassay microfluidic system with electrokinetic control.

A portable heterogeneous immunoassay system is presented in this paper. It consists of a poly(dimethylsiloxane) (PDMS) based microfluidic chip as the immunoreactor, a miniaturized programmable high voltage sequencer as the power supply and the flow controller, and a laser-optical fiber fluorescence detection module as the signal reader. The operation of this immunoassay system is automatic. The sequential reagent dispensing and washing processes are controlled by the programmable sequencer. The reagent consumption was only 12 microL and the assay time was only 26 min. The detection limit for Escherichia coli O157:H7 bacterial antigen with this miniaturized system was 0.3 ng/muL, lower than that obtained using fluorescence microscope in previous studies.

Antigens, Bacterial↗

PubMed2006

Electrochemical microfluidic biosensor for nucleic acid detection with integrated minipotentiostat.

An electrochemical microfluidic biosensor with an integrated minipotentiostat for the quantification of RNA was developed based on nucleic acid hybridization and liposome signal amplification. Specificity of the biosensor was ensured by short DNA probes that hybridize with the target RNA or DNA sequence. The reporter probe was coupled to liposomes entrapping the electrochemically active redox couple potassium ferri/ferrohexacyanide. The capture probes were coupled to superparamagnetic beads that were isolated on a magnet in the biosensor. Upon capture, the liposomes were lysed to release the electrochemical markers that were detected on an interdigitated ultramicroelectrode array in the biosensor just downstream of the magnet. The current was measured, stored and displayed by miniaturized instrumentation (miniEC). The accuracy of the miniEC was evaluated by comparing its performance to a standard bench-top electrochemical workstation in static and dynamic DC amperometric experiments. In both sets of experiments, the inexpensive miniEC performance was comparable in signal strength to that of the electrochemical workstation. In fact, the miniEC achieved a detection limit of 0.01 microM combined ferri/ferrohexacyanide concentration which was 10 x lower than that of the standard lab-bench system. The response time of the miniEC system was the same for low concentrations taking about 10 s to steady state. It was, however, slower at higher concentrations, taking 5 s versus only 1 s for the bench-top system. Finally, the functionality of the miniEC was successfully demonstrated with the detection of Dengue virus RNA.

Biosensing Techniques↗

PubMed2006

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip.

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.

Acoustics↗

PubMed2006

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations.

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Animals↗

PubMed2006

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip.

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.

Electrochemistry↗

PubMed2006

Determination of the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in a microfluidic system.

A microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) was fabricated. The device consists of a glass chip with a micro-electrochemical L-glutamate sensor and a polydimethylsiloxane (PDMS) sheet with a Y-shaped micro-flow channel. A sample solution and a substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel. When the flows were stopped, substrates in a solution mixed immediately with either of the enzymes by diffusion in a mixing channel. L-glutamate produced by the enzymatic reaction of GOT or GPT in the flow channel was detected by using the L-glutamate sensor. A distinct current increase was observed immediately after mixing, and the initial slope of the response curve varied in proportion to the activity of GOT or GPT. The relation between the slope of the response curve and the enzyme activity was linear between 7 and 228 U l-1 for GOT and 9 and 250 U l-1 for GPT. The quality of the response curve was improved with an increase in the channel height. The measurement based on the rate analysis in the micro-flow channel facilitated the reduction of the influence of interferents. The influence of the viscosity of the sample solution was also checked for the analysis of real samples. The determination of the enzyme activities was also conducted in a system with micropumps fabricated for a sample injection. Two solutions could be mixed in the mixing channel, and the activity of the enzymes could be measured as in the experiments using microsyringe pumps.

Alanine Transaminase↗

PubMed2006

A novel microfluidic impedance assay for monitoring endothelin-induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is an established and independent risk factor for the development of heart failure and sudden cardiac death. At the level of individual cardiac myocytes (heart muscle cells), the cell morphology alters (increase in cell size and myofibrillar re-organization) and protein synthesis is activated. In this paper, a novel cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis cell concentration is reported to monitor the dynamic process of endothelin-1-induced cardiomyocyte hypertrophy. A dielectrophoresis (DEP) microfluidic device is fabricated capable of concentrating cells from a dilute sample to form a confluent cell monolayer on the surface of microelectrodes. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor. To examine the feasibility of this impedance sensing system, cardiomyocytes are treated with endothelin-1 (ET-1), a known hypertrophic agent. ET-1 induces a continuous rise in cardiomyocyte impedance, which we interpret as strengthening of cellular attachments to the surface substrate. An equivalent circuit model is introduced to fit the impedance spectrum to fully understand the impedance sensing system.

Animals↗

PubMed2007

Target delivery in a microfluidic immunosensor.

A study is presented that examines the effect of microfluidic mixing elements on direct and sandwich assays performed in microchannels. Patterned grooves were embossed in the top of microchannels made in PDMS using soft lithography. The grooves redirected the fluid flowing in the channel, enhancing delivery of the target from the bulk fluid to the surface and preventing the formation of a depletion layer at the surface. Comparing assays in grooved and plain channels demonstrated that the mixers improved assay results by 26-46%. A computational flow analysis showed that the grooves caused virtual particles in the bulk flow to come close to the surface ( approximately 11 microm) which is consistent with the signal increase seen experimentally. Direct assays for several concentrations of CY5-labeled biotin were performed in the microchannels. The mixers also improved signal intensity in sandwich assays for botulinum toxin which required mixing of the reagents as well as the direction of the target to the surface.

Biosensing Techniques↗