New advances in microfluidic flow cytometry

In recent years, researchers are paying the increasing attention to the development of portable microfluidic diagnostic devices including microfluidic flow cytometry for the point‐of‐care testing. Microfluidic flow cytometry, where microfluidics and flow cytometry work together to realize novel functionalities on the microchip, provides a powerful tool for measuring the multiple characteristics of biological samples. The development of a portable, low‐cost, and compact flow cytometer can benefit the health care in underserved areas such as Africa or Asia. In this article, we review recent advancements of microfluidics including sample pumping, focusing and sorting, novel detection approaches, and data analysis in the field of flow cytometry. The challenge of microfluidic flow cytometry is also examined briefly.     

Design and 3D modeling investigation of a microfluidic electrode array for electrical impedance measurement of single yeast cells

High-resolution microscopic imaging may cause intensive image processing and potential impact of light irradiation on yeast replicative lifespan (RLS). Electrical impedance spectroscopy (EIS) could be alternatively used to perform high-throughput and label-free yeast RLS assays. Prior to fabricating EIS-integrated microfluidic devices for yeast RLS determination, systematic modeling and theoretical investigation are crucial for device design and optimization. Here, we report three-dimensional (3D) finite-element modeling and simulations of EIS measurement in a microfluidic single yeast in situ impedance array (SYIIA), which is designed by patterning an electrode matrix underneath a cell-trapping array. SYIIA was instantiated and modeled as a 5 × 5 sensing array comprising 25 units for cell immobilization, culturing, and time-lapse EIS recording. Simulations of yeast growing and budding in a sensing unit demonstrated that EIS signals enable the characterization of cell growth and daughter-cell dissections. In the 5 × 5 sensing array, simulation results indicated that when monitoring a target cell, daughter dissections in its surrounding traps may induce variations of the recorded EIS signals, which could cause mistakes in identifying target daughter-cell dissections. To eliminate the mis-identifications, electrode array pitch was optimized. Therefore, the results could conduct the design and optimization of microfluidic electrode-array-integrated devices for high-throughput and accurate yeast RLS assays.     

Size-resolved counting of circulating tumor cells on pinched flow-based microfluidic cytometry

Precise cell detecting and counting is meaningful in circulating tumor cells (CTCs) analysis. In this work, a simple cyclic olefin copolymer (COC) microflow cytometer device was developed for size-resolved CTCs counting. The proposed device is constructed by a counting channel and a pinched injection unit having three channels. Through injection flow rate control, microspheres/cells can be focused into the centerline of the counting channel. Polystyrene microspheres of 3, 9, 15, and 20 µm were used for the microspheres focusing characterization. After coupling to laser-induced fluorescence detection technique, the proposed device was used for polystyrene microspheres counting and sizing. A count accuracy up to 97.6% was obtained for microspheres. Moreover, the proposed microflow cytometer was applied to CTCs detecting and counting. To mimic blood sample containing CTCs and CTCs mixture with different subtypes, an MDA-MB-231 (human breast cell line) spiked red blood cells sample and a mixture of MDA-MB-231 and MCF-7 (human breast cell line) sample were prepared, respectively, and then analyzed by the developed pinched flow-based microfluidic cytometry. The simple fabricated and easy operating COC microflow cytometer exhibits the potential in the point-of-care clinical application.     

微流控阻抗流式细胞仪在单细胞检测中的应用

单细胞水平的检测能够在细胞群中分辨出稀有的异常细胞,在生物医学领域如疾病的早期诊断和治疗评估等方面有着至关重要的作用.通过整合微流控技术、电阻抗技术与流式细胞术,微流控阻抗细胞仪能够在微流体精确操控条件下,实现流动态单细胞的连续、无损阻抗检测.与传统的单细胞检测方法相比,微流控阻抗细胞仪具有非标记、多参数、低污染和检测...     

Resistive pulse sensing as particle counting and sizing method in microfluidic systems: Designs and applications review

Resistive pulse sensing is a well‐known and established method for counting and sizing particles in ionic solutions. Throughout its development the technique has been expanded from detection of biological cells to counting nanoparticles and viruses, and even registering individual molecules, e.g., nucleotides in nucleic acids. This technique combined with microfluidic or nanofluidic systems shows great potential for various bioanalytical applications, which were hardly possible before microfabrication gained the present broad adoption. In this review, we provide a comprehensive overview of microfluidic designs along with electrode arrangements with emphasis on applications focusing on bioanalysis and analysis of single cells that were reported within the past five years.     

Trapping and releasing of single microparticles and cells in a microfluidic chip

A microfluidic device was designed and fabricated to capture single microparticles and cells by using hydrodynamic force and selectively release the microparticles and cells of interest via negative dielectrophoresis by activating selected individual microelectrodes. The trap microstructure was optimized based on numerical simulation of the electric field as well as the flow field. The capture and selective release functions of the device were verified by multi-types microparticles with different diameters and K562 cells. The capture efficiencies/release efficiencies were 95.55% ± 0.43%/96.41% ± 1.08% and 91.34% ± 0.01%/93.67% ± 0.36% for microparticles and cells, respectively. By including more traps and microelectrodes, the device can achieve high throughput and realize the visual separation of microparticles/cells of interest in a large number of particle/cell groups.     

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300 amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

High-throughput dielectrophoretic manipulation of bioparticles within fluids through biocompatible three-dimensional microelectrode array

Dielectrophoresis (DEP) has been deemed as a potential and ideal solution for bioparticle manipulation. A 3-D carbon micro-electro-mechanical system (MEMS) fabricated from the latest developed carbon-MEMS approach has advantages of offering low-cost, biocompatible and high-throughput DEP manipulation for bioparticles. In this paper, a typical process for fabrication of various 3-D microelectrode configurations was demonstrated; accurate numerical analysis was presented on electric field gradient distribution and DEP force based on various microelectrode array configurations. The effects of electrode edge angle, electrode edge-to-edge spacing and electrode height on the electric field distributions were investigated, and optimal design considerations and rules were concluded through analysis of results. The outcomes demonstrate that the sharp edge electrode is more effective in DEP manipulation and both electrode edge-to-edge spacing and electrode height are critical design parameters for seeking optimal DEP manipulation. The gradient magnitude increases exponentially as the electrode spacing is reduced and the electric field extends significantly as the electrode height increases, both of which contribute to a higher throughput for DEP manipulation. These findings are consistent with experimental observations in the literature and will provide critical guidelines for optimal design of DEP devices with 3-D carbon-MEMS.     

Interdigitated array microelectrode based impedance immunosensor for detection of avian influenza virus H5N1

Continuous outbreaks of avian influenza (AI) in recent years with increasing threat to animals and human health have warranted the urgent need for rapid detection of pathogenic AI viruses. In this study, an impedance immunosensor based on an interdigitated array (IDA) microelectrode was developed as a new application for sensitive, specific and rapid detection of avian influenza virus H5N1. Polyclonal antibodies against AI virus H5N1 surface antigen HA (Hemagglutinin) were oriented on the gold microelectrode surface through protein A. Target H5N1 viruses were then captured by the immobilized antibody, resulting in a change in the impedance of the IDA microelectrode surface. Red blood cells (RBCs) were used as biolabels for further amplification of the binding reaction of the antibody-antigen (virus). The binding of target AI H5N1 onto the antibody-modified IDA microelectrode surface was further confirmed by atomic force microscopy. The impedance immunosensor could detect the target AI H5N1 virus at a titer higher than 10³ EID₅₀/ml (EID₅₀: 50% Egg Infective Dose) within 2 h. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear range for the titer of H5N1 virus was found between 10³ and 10⁷ EID₅₀/ml. Equivalent circuit analysis indicated that the electron transfer resistance of the redox probe [Fe(CN)₆]^3−/4− and the double layer capacitance were responsible for the impedance change due to the protein A modification, antibody immobilization, BSA (bovine serum albumin) blocking, H5N1 viruses binding and RBCs amplification. No significant interference was observed from non-target RNA viruses such as Newcastle disease virus and Infectious Bronchitis disease virus. (The H5N1 used in the study was inactivated virus.)     

医用微流控芯片研究进展

近年来,随着社会经济的飞速发展,新型科学技术层出不穷,微流控芯片因具有试剂消耗量少、能耗低、反应速度快、高通量化、液体自驱等独特优势,已经发展成为集生化、医学、电子、材料及其交叉学科的研究热点.微流控技术(microfluidics)是在微电机加工系统(MEMS)技术基础上发展而来的,是在微米级微管中精确操纵微量流体的...     

Sensitive immunodetection through impedance measurements onto gold functionalized electrodes

This article deals with a direct electrochemical method of detecting antigens using new methods of functionalization of gold electrodes. Based on the reacting ability of gold with sulfhydryl groups, three protocols for the fixation of antibodies have been explored. They are based on either the self-assembling properties of functional thiols bearing long alkyl chains or the possibility of a direct coupling of antibody moieties. Coverage rates as high as 97% can be reached. The analysis of the electrochemical impedance behavior of such layers can lead to a sensitive method for the direct detection of the antibody/antigen interaction. The addition of a redox couple in the tested solution, acting as an amplifier, allowed detection limits for the antigens as low as a few picograms/milliliter to be reached.     

Recent advances in electric analysis of cells in microfluidic systems

Microfluidics offers an ideal platform to integrate cell-based assays with electric measurements. The technological advances in microfluidics, microelectronics, electrochemistry, and electrophysiology have greatly inspired the development of microfluidic/electric devices that work with a low number of cells or single cells. The applications of these microfluidic systems range from the detecting of cell culture density to the probing of cellular functions at the single-cell level. In this review, we introduce the recent advances in the electric analysis of cells on a microfluidic platform, specifically related to the quantification and monitoring of cells in static solution, on-chip patch-clamp measurement, and examination of flowing cells. We also point out future directions and challenges in this field. Figure Different microfluidic devices applied to electrical analysis of cells     

Rapid, sensitive, and quantitative detection of pathogenic DNA at the point of care through microfluidic electrochemical quantitative loop-mediated isothermal amplification

Single-step DNA detection: a microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single-step process in less than an hour.     

Recent progress of microfluidic sample preparation techniques

Sample preparation turns out to be one of the important procedures in complex sample analysis by affecting the accuracy, selectivity, and sensitivity of analytical results. However, the majority of the conventional sample preparation techniques still suffer from time-consuming and labor-intensive operations. These shortcomings can be addressed by reforming the sample preparation process in a microfluidic manner. Inheriting the advantages of rapid, high efficiency, low consumption, and easy integration, microfluidic sample preparation techniques receive increasing attention, including microfluidic phases separation, microfluidic field-assisted extraction, microfluidic membrane separation, and microfluidic chemical conversion. This review overviews the progress of microfluidic sample preparation techniques in the last 3 years based on more than 100 references, we highlight the implementation of typical sample preparation methods in the formats of microfluidics. Furthermore, the challenges and outlooks of the application of microfluidic sample preparation techniques are discussed.     

High-throughput sorting of nanoliter droplets enabled by a sequentially addressable dielectrophoretic array

Droplet microfluidics has emerged as a powerful tool for a diverse range of biomedical and industrial applications such as single-cell analysis, directed evolution, and metabolic engineering. In these applications, droplet sorting has been effective for isolating small droplets encapsulating molecules, cells, or crystals of interest. Recently, there is an increased interest in extending the applicability of droplet sorting to larger droplets to utilize their size advantage. However, sorting throughputs of large droplets have been limited, hampering their wide adoption. Here, we report our demonstration of high-throughput fluorescence-activated droplet sorting of 1 nL droplets using an upgraded version of the sequentially addressable dielectrophoretic array (SADA), which we reported previously. The SADA is an array of electrodes that are individually and sequentially activated/deactivated according to the speed and position of a droplet passing nearby the array. We upgraded the SADA by increasing the number of driving electrodes constituting the SADA and incorporating a slanted microchannel. By using a ten-electrode SADA with the slanted microchannel, we achieved fluorescence-activated droplet sorting of 1 nL droplets at a record high throughput of 1752 droplets/s, twice as high as the previously reported maximum sorting throughput of 1 nL droplets.     

On hybrid electroosmotic kinetics for field‐effect‐reconfigurable nanoparticle trapping in a four‐terminal spiral microelectrode array

Induced‐charge electroosmosis (ICEO) has attracted tremendous popularity for driving fluid motion from the microfluidic community since the last decade, while less attention has been paid to ICEO‐based nanoparticle manipulation. We propose herein a unique concept of hybrid electroosmotic kinetics (HEK) in terms of bi‐phase ICEO (BICEO) actuated in a four‐terminal spiral electrode array, for effective electrokinetic enrichment of fluorescent polystyrene nanoparticles on ideally polarizable metal strips. First, by alternating the applied AC voltage waves between consecutive discrete terminals, the flow stagnation lines where the sample nanoparticles aggregate can be switched in time between two different distribution modes. Second, we innovatively introduce the idea of AC field‐effect flow control on BICEO; by altering the combination of gating voltage sequence, not only the number of circulative particle trapping lines is doubled, but the collecting locations can be flexibly reconfigured as well. Third, hydrodynamic streaming of DC‐biased BICEO is tested in our device design, wherein the global linear electroosmosis dominates BICEO contributed from both AC and DC components, resulting in a reduction of particle enrichment area, while with a sharp increase in sample transport speed inside the bulk phase. The flow field associated with HEK is predicted using a linear asymptotic analysis under Debye–Huckel limit, with the simulation results in qualitative agreement with in‐lab observations of nanoparticle trapping by exploiting a series of improved ICEO techniques. This work provides an affordable and field‐deployable platform for real‐time nanoparticle trapping in the context of dilute electrolyte.