Development of a fully integrated microfluidic system for sensing infectious viral disease

An active micromixer system utilizing the magnetic force was developed and examined for its ability to facilitate the mixing of more than two fluid flows. The mixing performance of the active micromixer was evaluated in aqueous-aqueous systems including dyes for visual observation. A complete analytical microfluidic system was developed by integrating various functional modules into a single chip, thus allowing cell lysis, sample preparation, purification of intracellular molecules, and subsequent analysis. Upon loading the cell samples and lysis solution into the mixing chamber, the integrated microfluidic device allows efficient cell disruption by rotation of a micromagnetic disk and control of mixing time using the Teflon-coated hydrophobic film as a microvalve. This inflow is followed by separating the cell debris and contaminated proteins from the cell lysate sample using the acrylamide (AAm)-functionalized SPE. The inflow of partially purified cell lysate sample containing the gold binding polypeptide (GBP)-fusion protein was bound onto the gold micropatterns by means of its metal binding affinity. The GBP-fusion method allows immobilization of proteins in bioactive forms onto the gold surface without surface modification suitable for studying antigen-antibody interaction. It was used for the detection of severe acute respiratory syndrome (SARS), an infectious viral disease, as an example case.     

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.     

Microchip based sample treatment device interfaced with ICP-MS for the analysis of transition metals from environmental samples

A downscaled solid phase (SPE) device applicable for sample preparation prior to ICP-MS monitoring, have been constructed making use of the lab on a chip concept. Standard photolithography and wet chemical etching were used to fabricate glass microfluidic devices accommodating three microchannels, each of them incorporating a defined section that could be packed with SPE materials; selective chelating resin. The microfluidic device was interfaced with the ICP-MS instrument throughout a low flow rate concentric nebuliser using a Teflon connector, and coupled with a flow injection manifold delivering samples and reagents via a manually operated splitting valve. The feasibility of the miniaturized prototype to perform SPE of trace metals was proved by analyzing trace metals, Cd, Co, and Ni, in seawater reference materials; CASS-2 and SLEW-1. The obtained result was in good agreement with the certified values. The device could be used as a remote miniaturized sample treatment for field work.     

Cell lysis on a microfluidic CD (compact disc)

Cell lysis was demonstrated on a microfluidic CD (Compact Disc) platform. In this purely mechanical lysis method, spherical particles (beads) in a lysis chamber microfabricated in a CD, cause disruption of mammalian (CHO-K1), bacterial (Escherichia coli), and yeast (Saccharomyces cerevisiae) cells. Interactions between beads and cells are generated in the rimming flow established inside a partially filled annular chamber in the CD rotating around a horizontal axis. To maximize bead-cell interactions in the lysis chamber, the CD was spun forward and backwards around this axis, using high acceleration for 5 to 7 min. Investigation on inter-particle forces (friction and collision) identified the following parameters; bead density, angular velocity, acceleration rate, and solid volume fraction as having the most significant contribution to cell lysis. Cell disruption efficiency was verified either through direct microscopic viewing or measurement of the DNA concentration after cell lysing. Lysis efficiency relative to a conventional lysis protocol was approximately 65%. In the long term, this work is geared towards CD based sample-to-answer nucleic acid analysis which will include cell lysis, DNA purification, DNA amplification, and DNA hybridization detection.     

A microfluidic fluorous solid-phase extraction chip for purification of amino acids

An electrokinetically-driven microfluidic chip was developed to realize beads-based solid-phase extraction (SPE) of amino acids. This chip uses a two-level (deep/shallow) poly(dimethylsiloxane) (PDMS) microchannel network to confine the fluorous reversed-phase silica beads within the SPE chamber. The mixture of fluorous tagged and non-tagged amino acids was carried into the fluorous solid-phase extraction (F-SPE) chamber by electrokinetic pumping and was successfully separated and extracted. By adding a reference material to the sample, the extraction efficiency of the eluted fluorous-tagged amino acid was calculated using the detection results from mass spectrometry (MS). The F-SPE microchips showed good reproducibility and efficiency, yielding an average extraction efficiency of 55% with a RSD of 10.6% under the typical experimental conditions.     

Microfluidics technology for manipulation and analysis of biological cells

Analysis of the profiles and dynamics of molecular components and sub-cellular structures in living cells using microfluidic devices has become a major branch of bioanalytical chemistry during the past decades. Microfluidic systems have shown unique advantages in performing analytical functions such as controlled transportation, immobilization, and manipulation of biological molecules and cells, as well as separation, mixing, and dilution of chemical reagents, which enables the analysis of intracellular parameters and detection of cell metabolites, even on a single-cell level. This article provides an in-depth review on the applications of microfluidic devices for cell-based assays in recent years (2002–2005). Various cell manipulation methods for microfluidic applications, based on magnetic, optical, mechanical, and electrical principles, are described with selected examples of microfluidic devices for cell-based analysis. Microfluidic devices for cell treatment, including cell lysis, cell culture, and cell electroporation, are surveyed and their unique features are introduced. Special attention is devoted to a number of microfluidic devices for cell-based assays, including micro cytometer, microfluidic chemical cytometry, biochemical sensing chip, and whole cell sensing chip.     

Quantification of bacterial cells based on autofluorescence on a microfluidic platform

Bacterial counts provide important information during the processes such as pathogen detection and hygiene inspection and these processes are critical for public health and food/pharmaceutical production. In this study, we demonstrate the quantification of the number of bacterial cells based on the autofluorescence from the cell lysate on a microfluidic chip. We tested three model pathogenic bacteria (Listeria monocytogenes F4244, Salmonella Enteritidis PT1 and Escherichia coli O157:H7 EDL 933). In the experiment, a plug of approximately 150 pL containing lysate from 240 to 4100 cells was injected into a microfluidic channel with downstream laser-induced fluorescence detection under electrophoresis conditions. We found that the autofluorescence intensity increased with the number of cells almost linearly for all three bacteria. The autofluorescence remained a single peak when the cell lysate contained a mixture of different bacterial species. We also demonstrate a simple microfluidic device that integrates entrapment and electrical lysis of bacterial cells with fluorescence detection. Such a device can carry out the quantification of bacterial cells based on lysate autofluorescence without off-chip procedures. This study offers a simple and fast solution to on-chip quantification of bacterial cells without labeling. We believe that the method can be extended to other bacterial species.     

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.     

Design of passive mixers utilizing microfluidic self-circulation in the mixing chamber

This paper proposes the design of a passive micromixer that utilizes the self-circulation of the fluid in the mixing chamber for applications in the Micro Total Analysis Systems (microTAS). The micromixer with a total volume of about 20 microL and consisting of an inlet port, a circular mixing chamber and an outlet port was designed. The device was actuated by a pneumatic pump to induce self-circulation of the fluid. The self-circulation phenomenon in the micromixer was predicted by the computational simulation of the microfluidic dynamics. Flow visualization with fluorescence tracer was used to verify the numerical simulations and indicated that the simulated and the experimental results were in good agreement. Besides, an index for quantifying the mixing performance was employed to compare different situations and to demonstrate the advantages of the self-circulation mixer. The mixing efficiencies in the mixer under different Reynolds numbers (Re) were evaluated numerically. The numerical results revealed that the mixing efficiency of the mixer with self-circulation was 1.7 to 2 times higher than that of the straight channel without a mixing chamber at Re= 150. When Re was as low as 50, the mixing efficiency of the mixer with self-circulation in the mixing chamber was improved approximately 30% higher than that in the straight channel. The results indicated that the self-circulation in the mixer could enhance the mixing even at low Re. The features of simple mixing method and fabrication process make this micromixer ideally suitable for microTAS applications.     

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

A microchamber array for single cell isolation and analysis of intracellular biomolecules

We present a microfluidic device that enables the determination of intracellular biomolecules in multiple single cells. The cells are individually trapped and isolated in a microchamber array. Since the microchambers can be opened and closed reversibly, the cells can be exposed to different solutions sequentially, e.g. for incubation, washing steps, labelling and finally, for lysis. The tightly sealed microchambers enable the retention and analysis of cell lysate derived from single cells. The performance of the device is demonstrated by monitoring the levels of the cofactors NADPH and NADH both in healthy mammalian cells and in cells exposed to oxidative stress. The platform was also used to determine the toxic impact of the alkaloid camptothecin on the intracellular enzyme glucose-6-phosphate dehydrogenase levels. In general, the device is applicable for the analysis of cell auto-stimulation and the detection of intracellular metabolite concentration or expression levels of proteins.     

A novel, compact disk-like centrifugal microfluidics system for cell lysis and sample homogenization

In this paper, we present the design and characterization of a novel platform for mechanical cell lysis of even the most difficult to lyse cell types on a micro or nanoscale (maximum 70 microL total volume). The system incorporates a machined plastic circular disk assembly, magnetic field actuated microfluidics, centrifugal cells and tissue homogenizer and centrifugation system. The mechanism of tissue disruption of this novel cell homogenization apparatus derives from the relative motion of ferromagnetic metal disks and grinding matrices in a liquid medium within individual chambers of the disk in the presence of an oscillating magnetic field. The oscillation of the ferromagnetic disks or blades produces mechanical impaction and shear forces capable of disrupting cells within the chamber both by direct action of the blade and by the motion of the surrounding lysis matrix, and by motion induced vortexing of buffer fluid. Glass beads or other grinding media are integrated into each lysis chamber within the disk to enhance the transfer of energy from the oscillating metal blade to the cells. The system also achieves the centrifugal elimination of solids from each liquid sample and allows the elution of clarified supernatants via siphoning into a collection chamber fabricated into the plastic disk assembly. This article describes system design, implementation and validation of proof of concept on two samples--Escherichia coli and Saccharomyces cerevisiae representing model systems for cells that are easy and difficult to lyse, respectively.     

Single-cell analysis by electrochemical detection with a microfluidic device

A novel electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electric field. Then, the docked cell was lysed by a direct current electric field strength of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

Sample pre-concentration by isotachophoresis in microfluidic devices

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

Continuous microfluidic DNA extraction using phase-transfer magnetophoresis

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 μl s(-1) and an eluate flow rate of 0.35 μl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.