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PubMed2005

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants.

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.

Adsorption↗

PubMed2003

Millisecond kinetics on a microfluidic chip using nanoliters of reagents.

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.

Biocompatible Materials↗

PubMed2001

An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

Electrochemistry↗

PubMed2004

Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities.

An introductory overview of the use of microfluidic devices for tissue engineering is presented. After a brief description of the background of tissue engineering, different application areas of microfluidic devices are examined. Among these are methods for patterning cells, topographical control over cells and tissues, and bioreactors. Examples where microfluidic devices have been employed are presented such as basal lamina, vascular tissue, liver, bone, cartilage and neurons. It is concluded that until today, microfluidic devices have not been used extensively in tissue engineering. Major contributions are expected in two areas. The first is growth of complex tissue, where microfluidic structures ensure a steady blood supply, thereby circumventing the well-known problem of providing larger tissue structures with a continuous flow of oxygen and nutrition, and withdrawal of waste products. The second, and probably more important function of microfluidics, combined with micro/nanotechnology, lies in the development of in vitro physiological systems for studying fundamental biological phenomena.

Animals↗

PubMed2005

Digital microfluidics using soft lithography.

Although microfluidic chips have demonstrated basic functionality for single applications, performing varied and complex experiments on a single device is still technically challenging. While many groups have implemented control software to drive the pumps, valves, and electrodes used to manipulate fluids in microfluidic devices, a new level of programmability is needed for end users to orchestrate their own unique experiments on a given device. This paper presents an approach for programmable and scalable control of discrete fluid samples in a polydimethylsiloxane (PDMS) microfluidic system using multiphase flows. An immiscible fluid phase is utilized to separate aqueous samples from one another, and a novel "microfluidic latch" is used to precisely align a sample after it has been transported a long distance through the flow channels. To demonstrate the scalability of the approach, this paper introduces a "general-purpose" microfluidic chip containing a rotary mixer and addressable storage cells. The system is general purpose in that all operations on the chip operate in terms of unit-sized aqueous samples; using the underlying mechanisms for sample transport and storage, additional sensors and actuators can be integrated in a scalable manner. A novel high-level software library allows users to specify experiments in terms of variables (i.e., fluids) and operations (i.e., mixes) without the need for detailed knowledge about the underlying device architecture. This research represents a first step to provide a programmable interface to the microfluidic realm, with the aim of enabling a new level of scalability and flexibility for lab-on-a-chip experiments.

Computers↗

PubMed2003

A new tool for routine testing of cellular protein expression: integration of cell staining and analysis of protein expression on a microfluidic chip-based system.

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work "on-chip," thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.

Antibodies↗

PubMed2005

[Laboratory on a microfluidic chip].

The recent achievements of microfluidic chip and its applications, based on the works mainly carried out in the authors' lab are reviewed. The chip fabrication capabilities have been extended into design and fabricate chips with higher degree of complexity in different materials, such as quartz, glass, polymethyl methacrylate (PMMA), and polydimethyl siloxane (PDMS). A set of methods for surface modification of micro-channels on such materials have been developed, which results in better reproducibility and higher efficiency in protein and peptide analysis. The use of novel materials for chip fabrication is also under investigation. A series of microfluidic workstations with integrated chip manipulation as well as laser induced fluorescence (LIF), ultraviolet (UV), electrochemical and chemiluminescence detection modules have been developed to attain the abilities of complex microfluidic control and data acquisition schemes. A single cell/single molecule imagining system was built up for dynamic analysis of molecular or cellular events too. Based on the work mentioned above, different functional units, such as membrane, monolithic, isotachophoresis (ITP) etc were set up and integrated. Glycoform separation of turkey ovalbumin in a lectin monolithic column and an electrophoresis channel was performed on an integrated microchip. And a novel technique has been developed that allows for the coupling of ITP and non-gel sieving electrophoresis for protein analysis in a single microchip and resulting in - 50 fold increase of the sensitivity in comparison with the use of gel electrophoresis only. A single molecule detection (SMD) based technique was developed for simultaneously measuring both bulk flow and near-wall flow velocity in the microchannels. And more recently, an SMD based technology was developed for observing molecular interactions at single molecule level. An ultra-rapid microchip electrophoresis method was established for simultaneous determination intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) related to apoptosis and oxidative stress. In an effort to develop a novel microfluidic based drug screening platform, systematic studies on the interaction between granulocyte colony-stimulating factor (G-CSF) and sulfated oligosaccharides were carried out at both molecular and cellular levels. Doxorubicin induced apoptosis of human hepatocellular carcinoma (HepG2) was studied using the integrated microfluidic device with concentration generator. In the application phase, severe acute respiratory syndrome (SARS) diagnosis based on reverse transcription-polymerase chain reaction (RT-PCR) and microfluidic chip electrophoresis (MCE) with 18 cases, methylation analysis of the P16 gene in 159 samples of patients and references for cancer diagnosis and polymorphism analysis of angiotenigen gene in 226 patients and references with essential hypertension are described. Forty-three up to date references are

Animals↗

PubMed2004

Transport, location, and quantal release monitoring of single cells on a microfluidic device.

A novel microfluidic device has been developed for on-chip transport, location, and quantal release monitoring of single cells. The microfluidic device consists of a plate of PDMS containing channels for introducing cells and stimulants and a glass substrate into which a cell micro-chamber was etched. The two tightly reversibly sealed plates can be separated for respective cleaning, which significantly extends the lifetime of the microchip that is frequently clogged in cell analysis experiments. Using hydraulic pressure, single cells were transported and located on the microfluidic chip. After location of a single PC12 cell on the microfluidic chip, the cell was stimulated by nicotine that was also introduced through the micro-channels, and the quantum release of dopamine from the cell was amperometricly detected with our designed carbon fiber microelectrode. The results have demonstrated the convenience and efficiency of using the microfluidic chip for monitoring of quantal release from single cells and have offered a facile method for the analysis of single cells on microfluidic devices.

Journal Article↗

PubMed2004

Microfluidic laser embedded in glass by three-dimensional femtosecond laser microprocessing.

Microfluidic dye lasers three-dimensionally embedded in glass have been fabricated for what is believed to be the first time by integrating micro-optical and microfluidic components by use of a femtosecond laser. By pumping the microfluidic laser, in which the microfluidic chamber was filled with the laser dye Rhodamine 6G dissolved in ethanol, with a frequency-doubled Nd:yttrium aluminum garnet laser, lasing action was confirmed by analysis of the emission spectra at different pump powers. In addition, by arranging two microfluidic chambers serially in the glass, we built a microfluidic twin laser that produces an array of two simultaneous laser emissions with one pump laser.

Journal Article↗

PubMed1997

Effects of Microfluidizer technology on Bacillus licheniformis spores in ice cream mix.

We studied the effect of Microfluidizer technology (sometimes referred to as "microfluidization"), a new ultra-high pressure homogenization process, on spores of Bacillus licheniformis in ice cream mix. Four batches of pasteurized ice cream mix were preheated to 33, 36, 44, or 50 degrees C, and spores of B. licheniformis were added to yield an inoculum of 2.0 x 10(4) spores/ ml of mix. Samples were treated at 50,000, 100,000, 150,000, and 200,000 kPa. Respective percentages of spore destruction ranged from 6 to 68%. As process pressure in the Microfluidizer system increased, the temperature of the product also increased. At the Microfluidizer system outlet, temperatures ranged from 46 to 88 degrees C. Therefore, a combination of forces, including high pressure and temperature, likely had a multiplier effect on spore destruction during Microfluidizer processing of ice cream mix. Data suggest that it might be possible to design a pasteurizer-Microfluidizer system that would inactivate most bacterial spores in dairy foods without the extreme heat treatment currently required in commercial processing operations.

Bacillus↗

PubMed2007

Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC-labeled antibody.

Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 microm in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the microchannel. Due to strong non-specific adsorption of microbeads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C. parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.

Animals↗

PubMed2006

Microfluidic technologies for MALDI-MS in proteomics.

The field of microfluidics continues to offer great promise as an enabling technology for advanced analytical tools. For biomolecular analysis, there is often a critical need to couple on-chip microfluidic sample manipulation with back-end MS. Though interfacing microfluidics to MS has been most often reported through the use of direct ESI-MS, there are compelling reasons for coupling microfluidics to MALDI-MS as an alternative to ESI-MS for both online and offline analysis. The intent of this review is to provide a summary of recent developments in the integration of microfluidic systems with MALDI-MS, with an emphasis on applications in proteomics. Key points are summarized, followed by a review of relevant technologies and a discussion of outlook for the field.

Microfluidics↗

PubMed2006

Improvements on the electrokinetic injection technique for microfluidic chips.

This paper presents a T-form electrokinetic injection system for the discrete time-based loading and dispensing of samples of variable-volume in a microfluidic chip. A novel push-pull effect is produced during the loading and dispensing processes by the application of an appropriate control voltage distribution. The experimental and numerical results show that this push-pull loading technique produces compact sample plugs and hence improves the detection resolution of the microfluidic device. The injection system is integrated with a microflow switch, and a suitable voltage control scheme is proposed to guide the sample to the desired outlet port such that the microfluidic device can function as a microdispenser. The time-based variable-volume T-form injection method presented in this study is performed using a compact geometry and a simple control scheme and can be readily integrated with other microfluidic devices to form a microfluidic system capable of continuous monitoring and analysis of bioreactions in the life science and biochemistry fields.

Electrophoresis, Microchip↗

PubMed2006

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications.

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

DNA↗

PubMed2005

Microfluidic genetic analysis with an integrated a-Si:H detector.

We have developed an integrated hydrogenated amorphous silicon (a-Si:H) fluorescence detector for microfluidic genetic analysis. It consists of a half-ball lens, a ZnS/YF3 multilayer optical interference filter with a pinhole, and an annular a-Si:H PIN photodiode allowing the laser excitation to pass up through the central aperture in the photodiode and the filter. Microfluidic separations of multiplex PCR products generated from methicillin-resistant/sensitive Staphylococcus aureus (MRSA/MSSA) DNA on microfluidic capillary electrophoresis (CE) devices are successfully detected with the integrated detector. Similarly, multiplex PCR amplicons from the kanamycin resistant and K12 serotype-specific genes of E. coli cells are detected. The direct detection of multiplex PCR amplicons indicates that the fluorescence detector can be successfully coupled with current microfluidic PCR-CE platforms. This work establishes that the integrated a-Si:H detector provides relevant limits of detection for point-of-care genetic and pathogen analysis with microfluidic devices.

DNA, Bacterial↗

PubMed2005

Continuous-flow fractionation of animal cells in microfluidic device using aqueous two-phase extraction.

Monitoring of live cells is important in the field of medical science, diagnostics, biology, and the pharmaceutical industry. In this study, live and dead CHO-K1 (Chinese Hamster Ovary) cells were fractionated by continuous-flow extraction in a microfluidic device using immiscible aqueous two-phase extraction technique. The polymer solutions offered stable two-phase flows in microchannel without diffusive mixing. The fundamentals of aqueous two-phase extraction can support stable and reproducible recovery and separation of biomolecules in microfluidic devices. Polyethylene glycol 8000 (PEG 8000, 4%) and dextran T 500 (5%) were selected as model polymer solutions. The appropriate flow rates of polymer and cell solutions were suggested. The fractionation efficiency of live and dead CHO K-1 cells from the culture broth was compared in normal macroscale system and microfluidic device. The optimum pH for the fractionation was 6.6 in both the normal and micro-scale systems. The loss of target live cells by sedimentation was circumvented in microfluidic device because of the negligible effect of gravity on the sedimentation. Most live cells were distributed to PEG-rich phase, while dead cells were found at the interface of two polymer solutions in microchannel. In this case, the recovery and fractionation efficiency of live cells in the PDMS-based microfluidic device was 100% and 97.0%, respectively.

Animals↗

PubMed2007

External force-assisted cell positioning inside microfluidic devices.

This paper describes straightforward approaches to positioning cells within microfluidic devices that can be implemented without special equipment or fabrication steps. External forces can effectively transport and position cells in preferred locations inside microfluidic channels. Except for centrifugal force-based positioning that can be used with any microfluidic channels, hydrodynamic and gravitational force-based positioning yield reproducible and biocompatible results when implemented with a microfluidic "module" that contains a barrier with embedded microgrooves. Primary rat cortical neurons, metastatic human breast cancer cells MDA-MB-231, NIH 3T3 mouse fibroblasts, and human umbilical vein endothelial cells (HUVECs) were compatible with the positioning processes. After positioning, cells attached, proliferated and migrated like control cells that were cultured on tissue culture dishes or glass coverslips. No apparent morphological differences were observed in positioned cells compared with control cells. Finally, to demonstrate a practical application of the methods, cells were placed in a single row along a wall inside a microfluidic chemotaxis chamber (MCC), and were exposed to stable concentration gradient of chemoattractant. Cell positioning allows that all cells get exposed to the same level of chemoattractant at the start of the experiment helping standardize cellular response.

Animals↗

PubMed2007

A microfluidic platform for 3-dimensional cell culture and cell-based assays.

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

Animals↗