A valve‐based microfluidic device for on‐chip single cell treatments

Assays toward single‐cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single‐cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two‐layer pneumatic valve‐based platform to implement cell immobilization and treatment on‐chip simultaneously, and cells after treatment could be collected non‐destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca²⁺. The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on‐chip capture and release of hydrogel beads was implemented. As a proof of concept for on‐chip single‐cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.     

Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter

The integration of complete analyses systems "on chip" is one of the great potentials of microfabricated devices. In this study we present a new pressure-driven microfabricated fluorescent-activated cell sorter chip with advanced functional integration. Using this sorter, fluorescent latex beads are sorted from chicken red blood cells, achieving substantial enrichments at a sample throughput of 12000 cells s(-1). As a part of the sorter chip, we have developed a monolithically integrated single step coaxial flow compound for hydrodynamic focusing of samples in flow cytometry and cell sorting. The structure is simple, and can easily be microfabricated and integrated with other microfluidic components. We have designed an integrated chamber on the chip for holding and culturing of the sorted cells. By integrating this chamber, the risk of losing cells during cell handling processes is eliminated. Furthermore, we have also developed integrated optics for cell detection. Our new design contributes to the ongoing efforts for building a fully integrated micro cell sorting and analysing system.     

A cell electro‐rotation micro‐device using polarized cells as electrodes

Cell rotation is widely required in various fields as an important technique for single cell manipulation. Usually, the electro‐rotational manipulation of single cells by dielectrophoresis technologies requires at least three electrodes to generate rotating electric fields which induce cells to rotate. Here, we present a novel microfluidic chip capable of rotating single cell using only two planar electrodes by taking polarized cells as the extra electrodes with phase‐shifted signal. To demonstrate this idea, we configured two parallel and planar electrodes as basic dielectrophoresis elements and placed trenches above these electrodes to attract cells, which were in turn polarized to be electrodes. Through simulation, we confirmed the functional structure of the device works well to generate proper rotating electric fields for cell rotation. Through experiment, we successfully demonstrated controlled electro‐rotation of HeLa and HepaRG cells. The novel electro‐rotation mechanism not only simplifies the micro‐device structure but also reduces the complexity of single cell rotation operation which will be a benefit to the potential users.     

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.     

Integrated cell manipulation system--CMOS/microfluidic hybrid

Manipulation of biological cells using a CMOS/microfluidic hybrid system is demonstrated. The hybrid system starts with a custom-designed CMOS (complementary metal-oxide semiconductor) chip fabricated in a semiconductor foundry. A microfluidic channel is post-fabricated on top of the CMOS chip to provide biocompatible environments. The motion of individual biological cells that are tagged with magnetic beads is directly controlled by the CMOS chip that generates microscopic magnetic field patterns using an on-chip array of micro-electromagnets. Furthermore, the CMOS chip allows high-speed and programmable reconfiguration of the magnetic fields, substantially increasing the manipulation capability of the hybrid system. Extending from previous work that verified the concept of the hybrid system, this paper reports a set of manipulation experiments with biological cells, which further confirms the advantage of the hybrid approach. To enhance the biocompatibility of the system, the microfluidic channel is redesigned and the temperature of the device is monitored by on-chip sensors. Combining microelectronics and microfluidics, the CMOS/microfluidic hybrid system presents a new model for a cell manipulation platform in biological and biomedical applications.     

微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

A single cell electroporation chip

Increasing the cell membrane's permeability can be accomplished via single cell electroporation. Polar substances that cannot otherwise permeate the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell. We developed a polymeric chip that can selectively immobilize and locally electroporate single cells. This easy-to-use chip focuses the electric field, eliminating the need to manipulate electrodes or glass pipettes. Moreover, this device allows parallel single cell electroporation. We demonstrate the effectiveness of our device design by electroporating HeLa cells using low applied voltages (< 1 V). We found the average transmembrane potential required for electroporation of HeLa cells to be 0.51 +/- 0.13 V. Membrane permeation is assessed electrically by measuring characteristic 'jumps' in current that correspond to drops in cell resistance, and microscopically by recording either the escape of cytoplasmic dye Calcein AM or the entrance of Trypan blue stain.     

Magnetic domain wall conduits for single cell applications

The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.     

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.     

A novel chip-based parallel transfection assay to evaluate paracrine cell interactions

The speed of gene function analyses in mammalian cells was significantly increased by the introduction of cell chip technology (reversely transfected cell microarray). However, the presently available technique is restricted to the analysis of autocrine effects of genes in the transfected cells. This limits the power of this method, as many genes are involved in heterotypic signaling both in physiologic and pathologic processes. At present, analyses of paracrine effects of transfected genes require trans-well or conditioned media approaches which are costly and time-consuming. Here, we present a novel method for the highly parallel analysis of paracrine gene functions on a chip. The basic idea was to adapt the cell chip technology to be performed with two different cell types which are differentially transfected: (1) an effector cell which is transfected with the genes of interest, and (2) an indicator cell in order to detect specific paracrine effects exerted from the transfected effector cells. Spot-to-spot diffusion of the paracrine mediators was prevented by matrix overlay, ultimately allowing 192 parallel tests for paracrine gene activations on one chip. In addition, we demonstrate the broad applicability and robustness of this technique using (1) various responder cell types, (2) various paracrine inducers, and (3) various indicator genes. The herein described approach allows for the first time a highly parallel analysis of paracrine gene functions and thus facilitates the characterization of genes involved in heterotypic cell communication in a broad range of research areas.     

High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel

In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.     

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。     

微流控芯片电泳/电化学检测人单个肝癌细胞中的谷胱甘肽

采用微流控装置结合电化学检测研究了测定人单个血红细胞中谷胱甘肽(GSH)的方法。在该方法中,细胞的进样、定位、溶膜以及细胞中谷胱甘肽的转移和检测都在配有通道端安培检测器的双T形芯片中完成。单个细胞用液压导入到双T的交界面,在电泳缓冲液中毛地黄皂苷的作用下,细胞膜被穿孔。再施加直流电压,细胞被溶膜。释放出来的GSH被此直流电压电迁移至通道端并在Au/Hg电极上被检测。用校正曲线法可以定量测定单个细胞中的GSH。     

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chips

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.     

Electronic sorting and recovery of single live cells from microlitre sized samples

Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.     

Real-time PCR of single bacterial cells on an array of adhering droplets

Real-time PCR at the single bacterial cell level is an indispensable tool to quantitatively reveal the heterogeneity of isogenetic cells. Conventional PCR platforms that utilize microtiter plates or PCR tubes have been widely used, but their large reaction volumes are not suited for sensitive single-cell analysis. Microfluidic devices provide high density, low volume PCR chambers, but they are usually expensive and require dedicated equipment to manipulate liquid and perform detection. To address these limitations, we developed an inexpensive chip-level device that is compatible with a commercial real-time PCR thermal cycler to perform quantitative PCR for single bacterial cells. The chip contains twelve surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. A one-step process that premixed reagents with cell medium before loading was applied, so no on-chip liquid manipulation and DNA purification were needed. To validate its application for genetic analysis, Synechocystis PCC 6803 cells were loaded on the chip from 1000 cells to one cell per droplet, and their 16S rRNA gene (two copies per cell) was analyzed on a commercially available ABI StepOne real-time PCR thermal cycler. The result showed that the device is capable of genetic analysis at single bacterial cell level with C(q) standard deviation less than 1.05 cycles. The successful rate of this chip-based operation is more than 85% at the single bacterial cell level.     

Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.