Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow

We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.     

Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

Design and analysis of single-cell capture microfluidic device

The aim of our study was to develop microfluidic devices using microchannel technology with the capability of capturing single cells. We analyzed and compared the cell-capturing efficiencies of series-loop microchannel and parallel-loop microchannel devices that were produced using polydimethylsiloxane (PDMS). Each set of microchannels was composed of a main flow channel and several branch channels with capturing zones. The microfluidic devices were designed to use the differences in flow rates between the main flow channel and the branch channels as a means of capturing single cells based on size and sequestering them within the microstructure of multiple capture zones. The data indicated that the flow medium encountered significant resistance in the series-loop microchannel device, which resulted in an inability to hold the captured cells within any of the capture zones. Flow resistance was, however, greatly reduced in the parallel-loop microchannel device compared to the series-loop device, and single cells were captured in all the capturing zones of the device. Our data suggest that the parallel-loop microchannel technology has significant potential for development toward high-throughput platforms capable of capturing single cells for physiological analyses at the single-cell level.     

Passive microfluidic control of two merging streams by capillarity and relative flow resistance

In the progress of microfluidic devices, a simple and precise control of multiple streams has been essential for complex microfluidic networks. Consequently, microfluidic devices, which have a simple structure, typically use external energy sources to control the multiple streams. Here, we propose a pure passive scheme that uses capillarity without using external force or external regulation to control the merging of two streams and even to regulate their volumetric flow rate (VFR). We accomplish this process by controlling the geometry of two inlets and a junction, and by regulating the hydrophilicity of a substrate. Additionally, we use the relative flow resistance to control the VFR ratio of the merged two streams. Our results will significantly simplify the control of multiple streams without sacrificing precision.     

Pyrosequencing in a microfluidic flow-through device

To explore genome variation meaningfully, there is a critical need for a high-throughput and inexpensive platform for DNA analysis. Pyrosequencing is a nonelectrophoretic bioluminometric DNA sequencing method that uses a four-enzyme mixture reaction to monitor nucleotide incorporation in real time. Currently, the commercialized pyrosequencing technique is limited to a 96-microtiter plate format. However, high throughput and inexpensive pyrosequencing is required to meet the need of screening large numbers of samples. We present here DNA pyrosequencing on a nanoliter-volume microfluidic platform. The microfluidic approach involves the trapping of the DNA on microbeads in an on-chip filter chamber and flow-through of the pyrosequencing reagents to monitor the reaction in real time. Two single-nucleotide polymorphisms were successfully scored to evaluate the microfluidic platform. In addition to significantly reducing reagent costs, microfluidic systems promise to improve the read length by eliminating intermediate product accumulation by constant removal of unincorporated nucleotides and elimination of dilution effects at each reaction cycle in the current plate format. Although only one filter chamber was used in this study, the platform should be readily adaptable to parallel analyses of nanoliter samples using filter chamber arrays to obtain high-throughput DNA analysis.     

Discrimination and analysis of phytoplankton using a microfluidic cytometer

Identification and analysis of phytoplankton is important for understanding the environmental parameters that are influenced by the oceans, including pollution and climate change. Phytoplanktons are studied at the single cell level using conventional light-field and fluorescence microscopy, but the technique is labour intensive. Flow cytometry enables rapid and quantitative measurements of single cells and is now used as an analytical tool in phytoplankton analysis. However, it has a number of drawbacks, including high cost and portability. We describe the fabrication of a microfluidic (lab-on-a-chip) device for high-speed analysis of single phytoplankton. The device measures fluorescence (at three wavelength ranges) and the electrical impedance of single particles. The system was tested using a mixture of three algae (Isochrysis Galbana, Rhodosorus m., Synechococcus sp.) and the results compared with predictions from theory and measurements using a commercial flow cytometer (BD FACSAria). It is shown that the microfluidic flow cytometer is able to distinguish and characterise these different taxa and that impedance spectroscopy enables measurement of phytoplankton biophysical properties.     

Electrokinetically based approach for single-nucleotide polymorphism discrimination using a microfluidic device

In this work, we describe and implement an electrokinetic approach for single-nucleotide polymorphism (SNP) discrimination using a PDMS/glass-based microfluidic chip. The technique takes advantage of precise control of the coupled thermal (Joule heating), shear (electroosmosis), and electrical (electrophoresis) energies present at an array of probes afforded by the application of external electrical potentials. Temperature controllers and embedded thermal devices are not required. The chips can be easily and inexpensively fabricated using standard microarray printing methods combined with soft-lithography patterned PDMS fluidics, making these systems easily adaptable to applications using higher density arrays. Extensive numerical simulations of the coupled flow and thermal properties and microscale thermometry experiments are described and used to characterize the in-channel conditions. It was found that optimal conditions for SNP detection occur at a lower temperature on-chip than for typical microarray experiments, thereby revealing the importance of the electrical and shear forces to the overall process. To demonstrate the clinical utility of the technique, the detection of single-base pair mutations in the survival motor neuron gene, associated with the childhood disease spinal muscular atrophy, is conducted.     

Single-molecule DNA amplification and analysis in an integrated microfluidic device

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.     

Regulating oxygen levels in a microfluidic device

Microfluidic devices made from poly(dimethylsiloxane) (PDMS) are gas permeable and have been used to provide accurate on-chip oxygen regulation. However, pervaporation in PDMS devices can rapidly lead to dramatic changes in solution osmotic pressure. In the present study, we demonstrate a new method for on-chip oxygen control using pre-equilibrated aqueous solutions in gas-control channels to regulate the oxygen content in stagnant microfluidic test chambers. An off-chip gas exchanger is used to equilibrate each control solution prior to entering the chip. Using this strategy, problems due to pervaporation are considerably reduced. An integrated PDMS-based oxygen sensor allows accurate real-time measurements of the oxygen within the microfluidic chamber. The measurements were found to be consistent with predictions from finite-element modeling.     

Study of local intracellular signals regulating axonal morphogenesis using a microfluidic device

The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca²⁺ signals in axons. Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca²⁺ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.     

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.     

Formation of natural pH gradients in a microfluidic device under flow conditions: model and experimental validation

A new isoelectric focusing technique has been developed that incorporates natural pH gradient formation in microfluidic channels under flowing conditions. In conjunction, a one-dimensional finite difference model has been developed that solves a system of algebraic-ordinary differential equations that describe the phenomena occurring in the system, including hydrolysis at the electrodes, buffering effects of weak acids and bases, and mass transport due to both diffusion and electrophoresis. A quantitative, noninvasive, optically based method of monitoring pH gradient formation is presented, and the experimental data generated by this method are found to be in good agreement with model predictions. In addition, the model provides a theoretical explanation for initially unexpected experimental results. Model predictions are also shown to match well with experimental results of microfluidic isoelectric focusing of a single protein species. Accounting for the nonuniform velocity profile, characteristic of pressure-driven flow in microfluidic channels, is found to improve predictions of dynamic pH changes close to the electrodes and overall time required to reach steady state, but to reduce the accuracy of dynamic pH change predictions in other regions of the channel.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

Eight hundred-base sequencing in a microfabricated electrophoretic device

The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.     

Analysis of polychlorinated biphenyls in transformer oil by using liquid-liquid partitioning in a microfluidic device

Polychlorinated biphenyls (PCBs) that are present in transformer oil are a common global problem because of their toxicity and environmental persistence. The development of a rapid, low-cost method for measurement of PCBs in oil has been a matter of priority because of the large number of PCB-contaminated transformers still in service. Although one of the rapid, low-cost methods involves an immunoassay, which uses multilayer column separation, hexane evaporation, dimethyl sulfoxide (DMSO) partitioning, antigen-antibody reaction, and a measurement system, there is a demand for more cost-effective and simpler procedures. In this paper, we report a DMSO partitioning method that utilizes a microfluidic device with microrecesses along the microchannel. In this method, PCBs are extracted and enriched into the DMSO confined in the microrecesses under the oil flow condition. The enrichment factor was estimated to be 2.69, which agreed well with the anticipated value. The half-maximal inhibitory concentration of PCBs in oil was found to be 0.38 mg/kg, which satisfies the much stricter criterion of 0.5 mg/kg in Japan. The developed method can realize the pretreatment of oil without the use of centrifugation for phase separation. Furthermore, the amount of expensive reagents required can be reduced considerably. Therefore, our method can serve as a powerful tool for achieving a simpler, low-cost procedure and an on-site analysis system.     

Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 min was achieved. Further development of the system may allow application to high-throughput applications such as drug screening.     

A facile and fast approach for the synthesis of doped nanoparticles using a microfluidic device

The microfluidic approach emerges as a new and promising technology for the synthesis of nanomaterials. A microreactor allows a variety of reaction conditions to be quickly scanned without consuming large amounts of raw material. In this study, we investigated the synthesis of water soluble 1-thioglycerol-capped Mn-doped ZnS nanocrystalline semiconductor nanoparticles (TG-capped ZnS:Mn) via a microfluidic approach. This is the first report for the successful doping of Mn in a ZnS semiconductor at room temperature as well as at 80?°C using a microreactor. Transmission electron microscopy and x-ray diffraction analysis show that the average particle size of Mn-doped ZnS nanoparticles is ～3.0?nm with a zinc-blende structure. Photoluminescence, x-ray photoelectron spectroscopy, atomic absorption spectroscopy and electron paramagnetic resonance studies were carried out to confirm that the Mn(2+) dopants are present in the ZnS nanoparticles.     

Automated formation of lipid-bilayer membranes in a microfluidic device

Although membrane channel proteins are important to drug discovery and hold great promise as engineered nanopore sensing elements, their widespread application to these areas has been limited by difficulties in fabricating planar lipid-bilayer membranes. We present a method for forming these sub-5-nm-thick free-standing structures based on a self-assembly process driven by solvent extraction in a microfluidic channel. This facile automatable process forms high-quality membranes able to host channel proteins measurable at single-molecule conductance resolution.     

Synthesis of thiol functionalized gold nanoparticles using a continuous flow microfluidic reactor

Microfluidic devices show much promise for the controlled synthesis of materials on the nanoscale. In this work the first report of a synthetic method for the preparation of nanoparticles of gold stabilized by an adsorbed monolayer of a thiol (monolayer protected clusters) using a microfluidic device is described. Improvements in monodispersity are observed relative to bulk synthetic methods.