Methods for counting particles in microfluidic applications

Microfluidic particle counters are important tools in biomedical diagnostic applications such as flow cytometry analysis. Major methods of counting particles in microfluidic devices are reviewed in this paper. The microfluidic resistive pulse sensor advances in sensitivity over the traditional Coulter counter by improving signal amplification and noise reduction techniques. Nanopore-based methods are used for single DNA molecule analysis and the capacitance counter is useful in liquids of low electrical conductivity and in sensing the changes of cell contents. Light-scattering and light-blocking counters are better for detecting larger particles or concentrated particles. Methods of using fluorescence detection have the capability for differentiating particles of similar sizes but different types that are labeled with different fluorescent dyes. The micro particle image velocimetry method has also been used for detecting and analyzing particles in a flow field. The general limitation of microfluidic particle counters is the low throughput which needs to be improved in the future. The integration of two or more existing microfluidic particle counting techniques is required for many practical on-chip applications.     

无透镜微流控成像流动细胞检测与计数系统

在未来面向个人化的生物医疗诊断中,实时的细胞检测与计数具有重要需求.现有的细胞检测和计数系统例如流式细胞仪和血细胞计数器不适用于小型化流动细胞实时检测和计数.通过将CMOS图像传感器芯片和微流控芯片结合,提出了一种用于流动细胞检测和计数的无透镜微流控成像系统,与用于计数静态细胞的其它无透镜微流控成像系统不同,该系统可以通过基于时域差分的运动检测算法检测和计数微流体通道中连续流动的细胞样本.测试结果表明:该系统可以对微流控通道中流动的人体骨髓基质细胞实现自动检测和计数,并具有-6.53％的低统计错误率.该系统提供了面向未来即时应用的细胞检测和计数解决方案.     

Blister pouches for effective reagent storage on microfluidic chips for blood cell counting

We present the analysis of blister pouches for reagent storage and release into microfluidic devices towards point-of-care blood cell counting applications. Blister pouches provide an effective reagent storage mechanism and can be mounted onto microfluidic cartridges directly. Reagents can be released from blister pouches through automated or manual compression and consequent rupturing of the pouches. A microfluidic device for metering of blister pouch contents was developed and investigated as part of this work, as precise volumes of reagents are often required when performing reactions, and particularly for blood cell counting applications which are the focus of this study. The metering device shows high accuracy and repeatability with an error of 1.93% and standard deviation of 3.1% across 30 test results. This work also investigates important blister pouch characteristics for three different types of blister pouch foil materials, including forces required to burst the blister, as well as shelf life and reagent compatibility of the blisters. Typical forces required are in the range of 25–35 N depending on the blister foil material used. Blister shelf life can be greatly affected by the reagent being stored, and thus, the blister foil material choice is crucial. This work provides a clear understanding of the implementation required to ensure that the blister pouches can be effectively used on microfluidic chips, with an example application area being point-of-care diagnostics.     

Impedance spectra of patch clamp scenarios for single cells immobilized on a lab-on-a-chip

A simple method based on impedance spectroscopy (IS) was developed to distinguish between different patch clamp modes for single cells trapped on microapertures in a patch clamp microchannel array designed for patch clamping on cultured cells. The method allows detecting via impedance analysis whether the cell membrane is ruptured (and culturing prevented) or the cell is still in the attached mode. A modular microfluidic lab-on-a-chip device based on planar patch clamp technology was used to capture multiple individual cells on an array of microapertures. The comparison of the measured and simulated impedance spectra proved that the presented method could distinguish between a cell-attached mode and a whole-cell mode even with low-quality seals. In physiological conditions, the capacitance of HeLa cells was measured to ~38 pF. The first gigaseal was recorded and maintained for 40 min. Once whole-cell configurations were established, trapped cells were superfused with a 140 mM KCl aqueous solution: the change in the measured cell impedance revealed a capacitance decrease to ~27.5 pF that could be due either to a change in the cell size or to the reduced charge separation across the cell membrane. After incubating the chip for 24 h, HeLa cells adhered and grew on the chip surface but did not survive when trapped on the microapertures. The microfluidic system proved to work as a micro electrophysiological analysis system, and the IS-based method can be used for further studies on the post-trapping strength of the seal between the microapertures and the trapped cells to be cultured.     

Development of microfluidic device for electrical/physical characterization of single cell

A novel device with microchannels for flowing cells and twin microcantilever arrays for measuring the electrical impedance of a single cell is proposed. The fabrication process is demonstrated and the twin microcantilever arrays have been successfully fabricated. In our research, we measured the electrical impedance for normal and abnormal red blood cell over the frequency range from 1 Hz to 10 MHz. From the electrical impedance experiment of normal and abnormal red blood cell, it was examined that the electrical impedance between normal and abnormal red blood cells was significantly different in magnitude and phase shift. In this paper, we show that the normal cell can be taken apart from the abnormal cell by electrical impedance measurement. Therefore, it is expected that the applicability of this technology can be used in cellular studies such as cell sorting, counting or membrane biophysical characterization.     

Investigation of wax and paper materials for the fabrication of paper-based microfluidic devices

Paper-based microfluidic devices hold great potential in today’s microfluidic applications. They offer low costs, simple and quick fabrication processes, ease of uses, etc. In this work, several wax and paper materials are investigated for the fabrication of paper-based microfluidic devices. A novel method of using wax as a suitable backing to a paper-based analytical device has been demonstrated. Governing equations for the mechanics of the fluid flow in paper-based channels with constant widths have been experimentally validated. Experimental results showing deviations from the governing equations have been verified using fluidic channels with varying widths. There lies the possibility of manipulation of the fluid flow in paper-based microfluidic devices solely using geometric factors. This opens up many potential applications that may require sequential delivery of reagents or samples. Lastly, properties of paper such as the average pore diameter and permeability can be deduced from experimental results.     

Advances and applications on microfluidic velocimetry techniques

The development and analysis of the performance of microfluidic components for lab-on-a-chip devices are becoming increasingly important because microfluidic applications are continuing to expand in the fields of biology, nanotechnology, and manufacturing. Therefore, the characterization of fluid behavior at the scales of micro- and nanometer levels is essential. A variety of microfluidic velocimetry techniques like micron-resolution Particle Image Velocimetry (μPIV), particle-tracking velocimetry (PTV), and others have been developed to characterize such microfluidic systems with spatial resolutions on the order of micrometers or less. This article discusses the fundamentals of established velocimetry techniques as well as the technical applications found in literature.     

Soft lithography based on photolithography and two-photon polymerization

Over the past decades, soft lithography has greatly facilitated the development of microfluidics due to its simplicity and cost-effectiveness. Besides, numerous fabrication techniques such as multi-layer photolithography, stereolithography and other methods have been developed to fabricate moulds with complex 3D structures nowadays. But these methods are usually not beneficial for microfluidic applications either because of low resolution or sophisticated fabrication procedures. Besides, high-resolution methods such as two-photon lithography, electron-beam lithography, and focused ion beam are often restricted by fabrication speed and total fabricated volume. Nonetheless, the region of interest in typical microfluidic devices is usually very small while the rest of the structure does not require complex 3D fabrication methods. Herein, conventional photolithography and two-photon polymerization are combined for the first time to form a simple hybrid approach in fabricating master moulds for soft lithography. It not only benefits from convenience of photolithography, but also gives rise to complex 3D structures with high resolution based on two-photon polymerization. In this paper, various tests have been conducted to further study its performance, and a passive micromixer has been created as a demonstration for microfluidic applications.     

Method for flow measurement in microfluidic channels based on electrical impedance spectroscopy

We have developed and characterized two novel micro flow sensors based on measuring the electrical impedance of the interface between the flowing liquid and metallic electrodes embedded on the channel walls. These flow sensors are very simple to fabricate and use, are extremely compact and can easily be integrated into most microfluidic systems. One of these devices is a micropore with two tantalum/platinum electrodes on its edges; the other is a micro channel with two tantalum/platinum electrodes placed perpendicular to the channel on its walls. In both sensors the flow rate is measured via the electrical impedance between the two metallic electrodes, which is the impedance of two metal–liquid junctions in series. The dependency of the metal–liquid junction impedance on the flow rate of the liquid has been studied. The effects of different parameters on the sensor’s outputs and its noise behavior are investigated. Design guidelines are extracted and applied to achieve highly sensitive micro flow sensors with low noise.     

一种基于微流控芯片技术的流式细胞计数系统

采用聚二甲基硅氧烷（PDMS）材料制作微流控流式细胞计数芯片,利用负压驱动与鞘液夹流技术实现样品的水力聚焦,达到了10μm的样品聚焦宽度。基于激光诱导荧光技术,制作了小型化的检测装置。以488nm固体激光器为光源,激光束以45°方向穿过一个水平狭缝,以线光源形式汇聚到微流控芯片的检测区域,并与微通道垂直交叉,细胞样品的荧光信号通过光电倍增管收集。整个分析系统结构简单,操作方便,灵敏度高,可初步实现细胞的计数。     

Miniaturized octupole cytometry for cell type independent trapping and analysis

Microflow cytometry, including robust alignment, separation, and trapping of living cells, is on the verge of commercialization. Yet, the necessary equipment is frequently not applicable to certain biological questions as the products have been specifically developed for particular cell types. We present a versatile cell handling technology based on single miniaturized octupoles that enables the physical manipulation of a broad variety of different cell types via controlled negative dielectrophoresis force fields. The octupole technology allows contactless and time-resolved cell analysis in physicochemical controlled microenvironments. Contactless cell manipulation and trapping with the octupole technology were shown to be independent of cell size and morphology. This was demonstrated with nine different cell types of technical and medical relevance, ranging from motile bacteria over yeast and small platelets (thrombocytes) up to large cancer cells. We also demonstrate applications of octupole cytometry for controlled analyses of mechano-elastic properties of single cells, contactless cultivation and perfusion for perturbation studies, as well as studying the interaction of different cell types in physical proximity. These examples prove the miniaturized octupole format as a versatile, noninvasive, and robust tool for microfluidic single cell cytometry that complements existing hydrodynamic, optical, and acoustic technologies.     

Micro-injection moulding of polymer microfluidic devices

Microfluidic devices have several applications in different fields, such as chemistry, medicine and biotechnology. Many research activities are currently investigating the manufacturing of integrated microfluidic devices on a mass-production scale with relatively low costs. This is especially important for applications where disposable devices are used for medical analysis. Micromoulding of thermoplastic polymers is a developing process with great potential for producing low-cost microfluidic devices. Among different micromoulding techniques, micro-injection moulding is one of the most promising processes suitable for manufacturing polymeric disposable microfluidic devices. This review paper aims at presenting the main significant developments that have been achieved in different aspects of micro-injection moulding of microfluidic devices. Aspects covered include device design, machine capabilities, mould manufacturing, material selection and process parameters. Problems, challenges and potential areas for research are highlighted.     

Size-controlled synthesis of gold nanoparticles using a micro-mixing system

A new microfluidic reaction chip capable of mixing, transporting and controlling reactions has been developed for the size-tunable synthesis of gold nanoparticles. This chip allows for an accelerated and efficient approach for the synthesis of gold nanoparticles. The microfluidic reaction chip is made by computer-numerically controlled machining and PDMS casting processes, which integrate a micro-mixer, a normally closed valve and a micro-pump onto a single chip. The micro-mixer is capable of generating a vortex-type flow field, which achieves a mixing efficiency as high as 95% within 1 s. Successful synthesis of dispersed gold nanoparticles has been demonstrated within an 83% shorter period of time (13 min), as compared to traditional methods (around 2 h). By using different volumes of reagents, the dispersed gold nanoparticles are found to have average diameters of 19, 28, 37 and 58 nm. The optical absorption spectra indicate that these synthesized nanoparticles have different surface plasmon resonance peaks, which are 521, 525, 530 and 537 nm, respectively. The development of this microfluidic reaction system holds promise for the synthesis of functional nanoparticles for further biomedical applications.     

Development of a biological detection platform utilizing a modular microfluidic stack

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.     

Polymer-based fabrication techniques for enclosed microchannels in biomedical applications

Investigations and analyses of body fluids like serum or whole blood are essential tasks in biomedical research in order to understand and diagnose diseases, to conduct pharmacological tests or to culture cells. Therefore, microfluidic systems provide a favorable tool for processing fluid samples as they allow downscaling of sample volumes and handling of single fluid components such as cells or proteins. For this reason, we present simple fabrication techniques for microchannel systems using polymer materials only. The demonstrated fabrication procedures are based on combinations of acrylic glass and the photo resists SU-8 and PerMX3020. On the one hand, these materials are low-priced compared to conventional silicon or glass. On the other hand, they have not shown any interaction with blood or other cell suspensions within the frame of our study. Furthermore, their transparency guarantees an easy observability of all processes within the system. Depending on the channel dimensions, different adhesion bonding techniques for closing of the systems are applied, whereas the fluidic interfaces are included by mechanical drilling. Summing up, we provide complete fabrication processes for fluidic systems which are simpler and more cost-effective than conventional methods and yet cope with all essential requirements for microfluidic applications.     

No more bonding,no more clamping,magnetically assisted membrane integration in microfluidic devices

The integration of porous membranes with microfluidic devices allows a simple but high-throughput mass transport control for numerous microfluidic applications, such as single-cell separation, sample analysis, and purification. In this study, we demonstrate a novel integration process of porous membranes into microfluidic devices by applying a magnetic field and hydrodynamically stabilizing them. This new approach simplifies the integration process by removing physicochemical bonding between membranes and microfluidic devices, but overcomes many practical issues observed in current methods, such as device leakage, membrane replacement, and membrane material selection. More importantly, our approach allows us to install membranes with diverse physicochemical features and spatial configurations into a single microfluidic device. This additional ability can significantly improve its performance and capability in applications. Finally, we successfully demonstrate the utilization of our membrane device for simple particle separation.     

Detection principles and development of microfluidic sensors in the last decade

Microfluidic sensor converts a physical quantity to useful signal with the help of microfluidic platform. Microfluidic sensors have got a wide attention in the last decade because of the increased demands from the automation and control in microsystems. This review on microfluidic sensors focuses on various types of sensors which have been developed for the microfluidic systems or applications based on the research contributions in the last decade. We start with a detailed comparison on the research developments in the last decade on microfluidic sensors with the help of year and country wise statistical charts on published works in the area. The review continues with the basics of microfluidic sensors and the working principles of microfluidic sensors by classifying various microfluidic sensors based on the parameter to be sensed. This review concludes with the attempt to provide an idea on research gap in the area of microfluidic sensors.     

Integration of impedance spectroscopy sensors in a digital microfluidic platform

Digital microfluidics combines the advantages of a low consumption of reagents with a high flexibility of processing fluid samples. For applications in life sciences not only the processing but also the characterization of fluids is crucial. In this contribution, a microfluidic platform, combining the actuation principle of electrowetting on dielectrics for droplet manipulations and the sensor principle of impedance spectroscopy for the characterization of the fluid composition and condition, is presented. The fabrication process of the microfluidic platform comprises physical vapor deposition and structuring of the metal electrodes onto a substrate, the deposition of a dielectric isolator and a hydrophobic top coating. The key advantage of this microfluidic chip is the common electric nature of the sensor and the actuation principle. This allows for fabricating digital microfluidic devices with a minimal number of process steps. Multiple measurements on fluids of different composition (including rigid particles) and of different conditions (temperature, sedimentation) were performed and process parameters were monitored online. These sample applications demonstrate the versatile applications of this combined technology.     

Prospects of low temperature co-fired ceramic (LTCC) based microfluidic systems for point-of-care biosensing and environmental sensing

Low temperature co-fired ceramic (LTCC) based microfluidic devices are being developed for point-of-care biomedical and environmental sensing to enable personalized health care. This article reviews the prospects of LTCC technology for microfluidic device development and its advantages and limitations in processing capabilities compared to silicon, glass and polymer processing. The current state of the art in LTCC-based processing techniques for fabrication of microfluidic components such as microchannels, chambers, microelectrodes and valves is presented. LTCC-based biosensing applications are discussed under the classification of (a) microreactors, (b) whole cell-based and (c) protein biosensors. Biocompatibility of LTCC pertaining to the development of biosensors and whole cell sensors is also discussed. Other significant applications of LTCC microfluidic systems for detection of environmental contaminants and toxins are also presented. Technological constraints and advantages of LTCC-based microfluidic system are elucidated in the conclusion. The LTCC-based microfluidic devices provide a viable platform for the development of point-of-care diagnostic systems for biosensing and environmental sensing applications.     

Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.