On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology

Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.     

用于细胞破裂的微流控生物芯片的研制

基于微电子机械系统(MEMS)技术,研制成一种夹流式血细胞破裂微流控生物芯片。细胞样品在破胞试剂夹流作用下导入芯片并在微沟道中流动,两种液体在流动过程中充分混合,导致细胞破裂。采用抗凝全血为细胞样品,比较胍盐和曲拉通的破胞效果;并分析在胍盐破裂细胞条件下,细胞浓度和流速对破胞效果的影响。控制破胞试剂流速远大于样品流速,可在几秒钟内完成细胞的破裂;保持破胞试剂与样品流速的比例,同时提高流速可在芯片上实现细胞的快速破裂。夹流式细胞破裂芯片具有与细胞分离芯片和脱氧核糖核酸(DNA)提取芯片相集成的潜力,可实现对复杂生物样品预处理操作,为实现微全分析系统打下良好基础。     

Microfluidic cell disruption system employing a magnetically actuated diaphragm

A microfluidic cell lysis chip equipped with a micromixer and SPE unit was developed and used for quantitative analysis of intracellular proteins. This miniaturized sample preparation system can be employed for any purpose where cell disruption is needed to obtain intracellular constituents for the subsequent analysis. This system comprises a magnetically actuated micromixer to disrupt cells, a hydrophobic valve to manipulate the cell lysate, and a packed porous polymerized monolith chamber for SPE and filtering debris from the cell lysate. Using recombinant Escherichia coli expressing intracellular enhanced green fluorescent protein (EGFP) and lipase as model bacteria, we optimized the cell disruption condition with respect to the lysis buffer composition, mixing time, and the frequency of the diaphragm in the micromixer, which was magnetically actuated by an external magnetic stirrer in the micromixer chamber. The lysed sample prepared under the optimal condition was purified by the packed SPE in the microfluidic chip. At a frequency of 1.96 Hz, the final cell lysis efficiency and relative fluorescence intensity of EGFP after the cell disruption process were greater than 90 and 94%, respectively. Thus, this microfluidic cell disruption chip can be used for the efficient lysis of cells for further analysis of intracellular contents in many applications.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.     

An ultra‐high temperature flow‐through capillary device for bacterial spore lysis

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195°C) an ethylene glycol lysis buffer to perform rapid flow‐through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow‐through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.     

Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation

A highly effective, reagentless, mechanical cell lysis device integrated in microfluidic channels is reported. Sample preparation, specifically cell lysis, is a critical element in 'lab-on-chip' applications. However, traditional methods of cell lysis require purification steps or complicated fabrication steps that a simple mechanical method of lysis may avoid. A simple and effective mechanical cell lysis system is designed, microfabricated, and characterized to quantify the efficiency of cell lysis and biomolecule accessibility. The device functionality is based on a microfluidic filter region with nanostructured barbs created using a modified deep reactive ion etching process. Mechanical lysis is characterized by using a membrane impermeable dye. Three main mechanisms of micro-mechanical lysis are described. Quantitative measurements of accessible protein as compared to a chemically lysed sample are acquired with optical absorption measurements at 280 and 414 nm. At a flow rate of 300 microL min(-1) within the filter region total protein and hemoglobin accessibilities of 4.8% and 7.5% are observed respectively as compared to 1.9% and 3.2% for a filter without nanostructured barbs.     

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.     

Continuous dielectrophoretic cell separation microfluidic device

We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.     

Isolation of plasma from whole blood using planar microfilters for lab-on-a-chip applications

Researchers are actively developing devices for the microanalysis of complex fluids, such as blood. These devices have the potential to revolutionize biological analysis in a manner parallel to the computer chip by providing very high throughput screening of complex samples and massively parallel bioanalytical capabilities. A necessary step performed in clinical chemistry is the isolation of plasma from whole blood, and effective sample preparation techniques are needed for the development of miniaturized clinical diagnostic devices. This study demonstrates the use of passive, operating entirely on capillary action, transverse-flow microfilter devices for the microfluidic isolation of plasma from whole blood. Using these planar microfilters, blood can be controllably fractionated with minimal cell lysis. A characterization of the device performance reveals that plasma filter flux is dependent upon the wall shear rate of blood in the filtration channel, and this result is consistent with macroscale blood filtration using microporous membranes. Also, an innovative microfluidic layout is demonstrated that extends device operation time via capillary action from seconds to minutes. Efficiency of these microfilters is approximately three times higher than the separation efficiencies predicted for microporous membranes under similar conditions. As such, the application of the microscale blood filtration designs used in this study may have broad implications in the design of lab-on-a-chip devices, as well as the field of separation science.     

A single-view field filter device for rare tumor cell filtration and enumeration

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 μL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.     

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

Integrated microfluidic cell culture and lysis on a chip

We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated.     

Continuous blood cell separation by hydrophoretic filtration

We propose a new hydrophoretic method for continuous blood cell separation using a microfluidic device composed of slanted obstacles and filtration obstacles. The slanted obstacles have a larger height and gap than the particles in order to focus them to a sidewall by hydrophoresis. In the successive structure, the height and gap of the filtration obstacles with a filtration pore are set between the diameters of small and large particles, which defines the critical separation diameter. Accordingly, the particles smaller than the criterion freely pass through the gap and keep their focused position. In contrast, the particles larger than the criterion collide against the filtration obstacle and move into the filtration pore. The microfluidic device was characterized with polystyrene beads with a minimum diameter difference of 7.3%. We completely separated polystyrene microbeads of 9 and 12 microm diameter with a separation resolution of approximately 6.2. This resolution is increased by 6.4-fold compared with our previous separation method based on hydrophoresis (S. Choi and J.-K. Park, Lab Chip, 2007, 7, 890, ref. 1). In the isolation of white blood cells (WBCs) from red blood cells (RBCs), the microfluidic device isolated WBCs with 210-fold enrichment within a short filtration time of approximately 0.3 s. These results show that the device can be useful for the binary separation of a wide range of biological particles by size. The hydrophoretic filtration as a sample preparation unit offers potential for a power-free cell sorter to be integrated into disposable lab-on-a-chip devices.     

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.     

用于提取外周血DNA微流控样品预处理芯片的研制

基于固相萃取原理和微电子机械系统(Micro-Electro-Mechanical System, MEMS)技术研制了一种多孔氧化硅微流控样品预处理芯片, 并利用具有大比表面积的多孔氧化硅作为提取DNA的固相载体, 从而大大提高了DNA的提取产率. 分析了影响DNA提取产率的因素, 改进了芯片制备工艺和DNA提取实验方案, 成功地提取了小鼠外周血DNA, 提取产率为24 ng/(μL全血), 达到商用试剂盒水平. 同时以该DNA作为PCR扩增模板, 扩增效果良好.     

Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies

This review describes microfluidic systems in poly(dimethylsiloxane) (PDMS) for biological studies. Properties of PDMS that make it a suitable platform for miniaturized biological studies, techniques for fabricating PDMS microstructures, and methods for controlling fluid flow in microchannels are discussed. Biological procedures that have been miniaturized into PDMS-based microdevices include immunoassays, separation of proteins and DNA, sorting and manipulation of cells, studies of cells in microchannels exposed to laminar flows of fluids, and large-scale, combinatorial screening. The review emphasizes the advantages of miniaturization for biological analysis, such as efficiency of the device and special insights into cell biology.     

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.     

Double spiral microchannel for label-free tumor cell separation and enrichment

This work reports on a passive double spiral microfluidic device allowing rapid and label-free tumor cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. A numerical model is developed to simulate the Dean flow inside the curved geometry and to track the particle/cell trajectories, which is validated against the experimental observations and serves as a theoretical foundation for optimizing the operating conditions. Results from separating tumor cells (MCF-7 and Hela) spiked into whole blood indicate that 92.28% of blood cells and 96.77% of tumor cells are collected at the inner and the middle outlet, respectively, with 88.5% tumor recovery rate at a throughput of 3.33 × 10(7) cells min(-1). We expect that this label-free microfluidic platform, driven by purely hydrodynamic forces, would have an impact on fundamental and clinical studies of circulating tumor cells.