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PubMed2007

Design and testing of a disposable microfluidic chemiluminescent immunoassay for disease biomarkers in human serum samples.

This paper presents the development of a plastic microfluidic immunosensor for rapid, reliable and on-the-spot detection of disease biomarkers in human sera. The microfluidic chips were fabricated in cyclic polyolefin by hot-embossing with a silicon master mold. The master itself was made using photolithographic techniques and Deep Reactive Ion Etching (DRIE). As a platform model, serum concentrations of C-reactive protein (CRP), a cardiac and inflammation marker, was measured on-chip using chemiluminescence based immunoassay. The assay results were read via an on-board instant photographic film, and with an imager capable of detecting chemiluminescent signals. The on-board detection module obviates the need for any dedicated bench-top analyzer for reading the immunoassay results, and therefore makes the device self-sufficient for point-of-care diagnostics when simple positive/negative results are sought. The microfluidic chemiluminescence results were compared with standard microplate ELISA analysis to assess the accuracy of the developed microfluidic immunoassay. Screening of CRP in human serum samples showed good correlation with ELISA analysis and the mean difference between the two methods using the Bland and Altman method was -0.079 +/- 0.858 mg/L for hsCRP. With approximate assay times of 25 min, the developed microfluidic immunoassay approach shows great potential for rapid plus sensitive detection of disease markers at the point-of-care.

Biomarkers↗

PubMed2007

A microfluidic platform for sequential ligand labeling and cell binding analysis.

Developing biochemical and cell biological assay for screening biomolecules, evaluating their characteristics in biological processes, and determining their pharmacological effects represents a key technology in biomedical research. A PDMS-based integrated microfluidic platform was fabricated and tested for facilitating the labeling of ligand on the nanogram scale and sequential cell binding analysis in a manner that saves both time and reagents. Within this microfluidic platform, ligand labeling, cell immobolization, and optical analysis are performed in a miniaturized, continuous and semi-automated manner. This microfluidic device for ligand labeling and cell analysis is composed of two functional modules: (i) a circular reaction loop for fluorophore-labeling of the ligand and (ii) four parallel-oriented incubation chambers for immobilization of cells, binding of ligand to different cell populations, and optical evaluation of interactions between the labeled ligand and its cell targets. Epidermal growth factor (EGF) as the ligand and different cell lines with various levels of EGF receptor expression have been utilized to test the feasiblity of this microfluidic platform. When compared to studies with traditional Petri dish handling of cells and tissues, or even microwell analyses, experiments with the microfluidic platform described here are much less time consuming, conserve reagents, and are programmable, which makes these platforms a very promising new tool for biological studies.

Animals↗

PubMed2005

Microfluidic reactor for the radiosynthesis of PET radiotracers.

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term "microfluidic PET". The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate "proof of principle" we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[(18)F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1 min) and the rapid radiofluorination of 2-[(18)F]FDG (60% radiochemical yield within 4s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Bioreactors↗

PubMed2006

Microfluidic electroporation of robust 10-microm vesicles for manipulation of picoliter volumes.

We present a new way to transport and handle picoliter volumes of analytes in a microfluidic context through electrically monitored electroporation of 10-25 microm vesicles. In this method, giant vesicles are used to isolate analytes in a microfluidic environment. Once encapsulated inside a vesicle, contents will not diffuse and become diluted when exposed to pressure-driven flow. Two vesicle compositions have been developed that are robust enough to withstand electrical and mechanical manipulation in a microfluidic context. These vesicles can be guided and trapped, with controllable transfer of material into or out of their confined environment. Through electroporation, vesicles can serve as containers that can be opened when mixing and diffusion are desired, and closed during transport and analysis. Both vesicle compositions contain lecithin, an ethoxylated phospholipid, and a polyelectrolyte. Their performance is compared using a prototype microfluidic device and a simple circuit model. It was observed that the energy density threshold required to induce breakdown was statistically equivalent between compositions, 10.2+/-5.0 mJ/m2 for the first composition and 10.5+/-1.8 mJ/m2 for the second. This work demonstrates the feasibility of using giant, robust vesicles with microfluidic electroporation technology to manipulate picoliter volumes on-chip.

Electroporation↗

PubMed2006

Merging microfluidics with microarray-based bioassays.

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.

Biological Assay↗

PubMed2005

PCR microfluidic devices for DNA amplification.

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.

DNA↗

PubMed2006

Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing.

Rapid detection and identification of influenza virus is becoming increasingly important in the face of concerns over an influenza pandemic. A fully integrated and self-contained microfluidic device has been developed to rapidly identify influenza A hemagglutinin and neuraminidase subtypes and sequence portions of both genes. The device consists of a DNA microarray with 12 000 features and a microfluidic cartridge that automates the fluidic handling steps required to carry out a genotyping assay for pathogen identification and sequencing. The fully integrated microfluidic device consists of microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows the detection and identification of influenza virus in a rapid and automated fashion.

DNA, Viral↗

PubMed2006

Electroporation of mammalian cells in a microfluidic channel with geometric variation.

Electroporation has been widely used to load impermeant exogenous molecules into cells. Rapid electrical lysis based on electroporation has also been applied to analyze intracellular materials at single-cell level. There has been increasing demand to implement electroporation in a microfluidic format as a basic tool for applications ranging from screening of drugs and genes to studies of intracellular dynamics. In this report, we have developed a simple technique to electroporate mammalian cells with high throughput on a microfluidic platform. In our design, electroporation only happened in a defined section of a microfluidic channel due to the local field amplification by geometric variation. The time of exposure of the cells to this high field was determined by the velocity of the cells and the length of the section. The change in the cell morphology during electroporation was observed in real time. We determined that electroporation of Chinese hamster ovary cells occurred when the local field strength was increased to approximately 400 V/cm. The internalization of membrane-impermeant molecules (SYTOX green) with cell viability preserved was also carried out to demonstrate transient electropermeabilization. The influence of the operational parameters of the device on cell viability was determined. A large percentage of cells remained viable after electroporation when the parameters were tuned. We also studied rapid cell lysis when the field intensity was in the range of 600-1200 V/cm. The rupture of cell membrane happened within 30 ms when the field strength was 1200 V/cm. Given the simplicity, high throughput, and high compatibility with other devices, this microfluidic electroporation technique may increase the application of microfluidic systems in screening of drugs and biomolecules and chemical cytometry.

Animals↗

PubMed2007

Microfluidic flow-flash: method for investigating protein dynamics.

We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or O2 binding to myoglobin after the laser-induced photolysis of CO from myoglobin by UV/visible absorbance spectral imaging.

Carbon Monoxide↗

PubMed2006

Shape-controlled production of biodegradable calcium alginate gel microparticles using a novel microfluidic device.

In this paper we describe a novel method of manufacturing shape-controlled calcium alginate gel microparticles in a microfluidic device. Both manufacturing shape-controlled microparticles and synthesizing hydrogel microparticles could be performed simultaneously in the microfluidic device. The novel microfluidic device comprised of two individual flow-focusing channels and a synthesizing channel was successfully applied as a continuous microfluidic reactor to synthesize gel microparticles with size and shape control. By passive control based on the microchannel geometric confinement and liquid-phase flow rates, we succeeded in producing monodisperse sodium alginate microparticles with diverse shapes (such as plugs, disks, microspheres, rods, and threads) in the flow-focusing channels of the microfluidic device. The shape and size of the sodium alginate microparticles could be tuned by adjusting the flow rates of the various streams. Further stages of the chemical reaction could be initiated by mixing sodium alginate microparticles and calcium chloride (CaCl2) solution in the synthesizing channel. The shapes of the sodium alginate microparticles could be permanently preserved by the synthesis of calcium alginate gel microparticles. The preparation conditions of size- and shape-controlled calcium alginate microparticles and influence factors were studied.

Alginates↗

PubMedDate not supplied

Titania and alumina sol-gel-derived microfluidics enzymatic-reactors for peptide mapping: design, characterization, and performance.

The design and characterization of titania-based and alumina-based Poly(dimethylsiloxane) (PDMS) microfluidics enzymatic-reactors along with their analytical features in coupling with MALDI-TOF and ESI-MS were reported. Microfluidics with microchannel and stainless steel tubing (SST) were fabricated using PDMS casting and O(2)-plasma techniques, and were used for the preparation of an enzymatic-reactor. Plasma oxidation for the PDMS microfluidic system enabled the channel wall of the microfluidics to present a layer of silanol (SiOH) groups. These SiOH groups act as anchors onto the microchannel wall linked covalently with the hydroxyl groups of trypsin-encapsulated sol matrix. As a result, the trypsin-encapsulated gel matrix was anchored onto the wall of the microchannel, and the leakage of gel matrix from the microchannel was effectively prevented. A feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by attached SST electrode and replaceable tip. The success of trypsin encapsulation was investigated by AFM imaging, assay of enzymatic activity, CE detection, and MALDI-TOF and ESI-MS analysis. The lab-made devices provide an excellent extent of digestion even at a fast flow rate of 7.0 microL/min, which affords the very short residence time of ca. 2 s. With the present device, the digestion time was significantly shortened compared to conventional tryptic reaction schemes. In addition, the encapsulated trypsin exhibits increased stability even after continuous use. These features are required for high-throughput protein identification.

Aluminum Oxide↗

PubMed2004

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids.

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices. We present an alternative paradigm--a fully integrated and reconfigurable droplet-based "digital" microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. The microdroplets, which act as solution-phase reaction chambers, are manipulated using the electrowetting effect. Reliable and repeatable high-speed transport of microdroplets of human whole blood, serum, plasma, urine, saliva, sweat and tear, is demonstrated to establish the basic compatibility of these physiological fluids with the electrowetting platform. We further performed a colorimetric enzymatic glucose assay on serum, plasma, urine, and saliva, to show the feasibility of performing bioassays on real samples in our system. The concentrations obtained compare well with those obtained using a reference method, except for urine, where there is a significant difference due to interference by uric acid. A lab-on-a-chip architecture, integrating previously developed digital microfluidic components, is proposed for integrated and automated analysis of multiple analytes on a monolithic device. The lab-on-a-chip integrates sample injection, on-chip reservoirs, droplet formation structures, fluidic pathways, mixing areas and optical detection sites, on the same substrate. The pipelined operation of two glucose assays is shown on a prototype digital microfluidic lab-on-chip, as a proof-of-concept.

Body Fluids↗

PubMed2005

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

Astrocytes↗

PubMed2005

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel.

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.

Animals↗

PubMed2005

World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays.

We report a practical world-to-chip microfluidic interfacing method with built-in valves suitable for microscale multichamber chip-based assays. One of the primary challenges associated with the successful commercialization of fully integrated microfluidic systems has been the lack of reliable world-to-chip microfluidic interconnections. After sample loading and sealing, leakage tests were conducted at 100 degrees C for 30 min and no detectable leakage flows were found during the test for 100 microchambers. To demonstrate the utility of our world-to-chip microfluidic interface, we designed a microscale PCR chip with four chambers and performed PCR assays. The PCR results yielded a 100% success rate with no contamination or leakage failures. In conclusion, we have introduced a simple and inexpensive microfluidic interfacing system for both sample loading and sealing with no dead volume, no leakage flow and biochemical compatibility.

DNA, Viral↗

PubMed2005

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm.

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.

Animals↗

PubMed2003

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device.

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.

Alleles↗

PubMed2004

Metal-polymer nanocomposites for integrated microfluidic separations and surface enhanced Raman spectroscopic detection.

The widespread development of microfluidics (microfluidics) has allowed the extension of efficient separations, fluid handling, and hyphenation with many detection modes to a small, portable, highly controllable physico-chemical platform. Surface enhanced Raman spectroscopy (SERS) offers the powerful advantage of obtaining vibrational spectroscopic information about analytes in an aqueous matrix with negligible background. The mating of electrophoretic separations with vibrational spectroscopy on a microfluidic device will allow the chromatographic efficiency of capillary electrophoresis (CE) with the unequivocal analyte "fingerprinting" capability of detailed structural information. By utilizing SERS as a means of detection, this work promises to yield redress for the hindrances of electrophoretic separations, including uncertainty in analyte band identification due to changing migration times as well as compromised detection sensitivity for non-fluorescent analytes. Our work represents the first steps toward developing CE-SERS on a microfluidic platform with a region of novel metal-pliable polymer nanocomposite SERS substrate fabricated directly into the device. The device fabrication material has been extensively employed by the microfluidics community for over five years. SERS detection can be achieved in real time or after the separations, with on-column laser-induced fluorescence employed as a secondary detection mode used for confirmation of efficiencies and band locations.

Journal Article↗