单细胞分析的研究

单细胞分析是分析化学、生物学和医学之间渗透发展形成的跨学科前沿领域。近年来,毛细管电泳及微流控芯片用于单细胞分析已取得显著进展,特别表现在微流控芯片用于细胞的培养、分选、操纵、定位、分离及检测细胞的组分,实时监测细胞释放,及高通量阵列检测等方面。芯片的单元操作可根据需要灵活组合,显示出其独特的优点。本文重点介绍作者研究组的工作,并对近三年来国内外在毛细管电泳及芯片毛细管电泳用于单细胞分析的新进展进行评论。最后从毛细管电泳与微流控芯片、微流控芯片与细胞界面以及量子点用于探测活细胞等方面,展望了单细胞分析的发展前景。     

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.     

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.     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

Analytical detection techniques for droplet microfluidics—A review

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.     

The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities

Optical microscopy of single bacteria growing on solid agarose support is a powerful method for studying the natural heterogeneity in growth and gene expression. While the material properties of agarose make it an excellent substrate for such studies, the sheer number of exponentially growing cells eventually overwhelms the agarose pad, which fundamentally limits the duration and the throughput of measurements. Here we overcome the limitations of exponential growth by patterning agarose pads on the sub-micron-scale. Linear tracks constrain the growth of bacteria into a high density array of linear micro-colonies. Buffer flow through microfluidic lines washes away excess cells and delivers fresh nutrient buffer. Densely patterned tracks allow us to cultivate and image hundreds of thousands of cells on a single agarose pad over 30-40 generations, which drastically increases single-cell measurement throughput. In addition, we show that patterned agarose can facilitate single-cell measurements within bacterial communities. As a proof-of-principle, we study a community of E. coli auxotrophs that can complement the amino acid deficiencies of one another. We find that the growth rate of colonies of one strain decreases sharply with the distance to colonies of the complementary strain over distances of only a few cell lengths. Because patterned agarose pads maintain cells in a chemostatic environment in which every cell can be imaged, we term our device the single-cell chemostat. High-throughput measurements of single cells growing chemostatically should greatly facilitate the study of a variety of microbial behaviours.     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

A single-cell encapsulation method based on a microfluidic multi-step droplet splitting system

Single cell analysis is of great significance to understand the physiological activity of organisms.Microfluidic droplet is an ideal analytical platform for single-cell analysis. We developed a microfluidic droplet splitting system integrated with a flow-focusing structure and multi-step splitting structures to form 8-line droplets and encapsulate single cells in the droplets. Droplet generation frequency reached1021 Hz with the aqueous phase flow rate of 1 m L/min and the oil phase flow rate of 15 mL /min. Relative standard deviation of the droplet size was less than 5% in a single channel, while less than 6% in all the8 channels. The system was used for encapsulating human whole blood cells. A single-cell encapsulation efficiency of 31% was obtained with the blood cell concentration of 2.5× 10~4cells/mL, and the multicellular droplet percentage was only 1.3%. The multi-step droplet splitting system for single cell encapsulation featured simple structure and high throughput.     

A modular single-cell pipette microfluidic chip coupling to ETAAS and ICP-MS for single cell analysis

Accurate single-cell capture is a crucial step for single cell biological and chemical analysis. Conventional single-cell capturing often confront operational complexity, limited efficiency, cell damage, large scale but low accuracy, incompetence in the acquirement of nano-upgraded single-cell liquid. Flow cytometry has been widely used in large-scale single-cell detection, while precise single-cell isolation relies on both a precision operating platform and a microscope, which is not only extre...     

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100?000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.     

Microreactors as Tools for Synthetic Chemists—The Chemists' Round‐Bottomed Flask of the 21st Century?

Will microreactors replace the round‐bottomed flask to perform chemical reactions in the near future? Recent developments in the construction of microstructured reaction devices and their wide‐ranging applications in many different areas of chemistry suggest that they can have a significant impact on the way chemists conduct their experiments. Miniaturizing reactions offers many advantages for the synthetic organic chemist: high‐throughput scanning of reaction conditions, precise control of reaction variables, the use of small quantities of reagents, increased safety parameters, and ready scale‐up of synthetic procedures. A wide range of single‐ and multiphase reactions have now been performed in microfluidic‐based devices. Certainly, microreactors cannot be applied to all chemistries yet and microfluidic systems also have disadvantages. Limited reaction‐time range, high sensitivity to precipitating products, and new physical, chemical, and analytical challenges have to be overcome. This concept article presents an overview of microfluidic devices available for chemical synthesis and evaluates the potential of microreactor technology in organic synthesis.     

Analysis of intercellular calcium signaling using microfluidic adjustable laminar flow for localized chemical stimulation

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300 μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26 μm with a total flow rate of 20 μL min⁻¹, which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell–cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening.     

High‐throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps

The ability to accurately control fluid transport in microfluidic devices is key for developing high‐throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time‐consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low‐cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T‐Lymphocyte cells loaded with Oregon green and 6‐carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single‐cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady‐state population of immortalized cells.     

探索脑化学纳米电化学监测单细胞、单囊泡、突触间隙释放化学信号分子及形貌分析

针对已有的微米及纳米电化学监测单囊泡、单突触及突触间隙释放, 扫描电化学显微镜用于单细胞释放前后形貌变化的定量分析, 微流控与阵列电极集成芯片, 用于细胞灌注培养及监测释放化学信号分子的研究工作进行了评述. 同时, 对近几年此领域的前沿研究进行了简要评论, 并对其未来发展提出了一些新的观点.     

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.     

High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 μm diameter, 8 μm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 μm diameter and 8 μm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 μM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.     

An integrated microfluidic device for two-dimensional combinatorial dilution

High-throughput preparation of multi-component solutions is an integral process in biology, chemistry and materials science for screening, diagnostics and analysis. Compact microfluidic systems enable such processing with low reagent volumes and rapid testing. Here we present a microfluidic device that incorporates two gradient generators, a tree-like generator and a new microfluidic active injection system, interfaced by intermediate solution reservoirs to generate diluted combinations of input solutions within an 8 × 8 or 10 × 10 array of isolated test chambers. Three input solutions were fed into the device, two to the tree-like gradient generator and one to pre-fill the test chamber array. The relative concentrations of these three input solutions in the test chambers completely characterized device behaviour and were controlled by the number of injection cycles and the flow rate. Device behaviour was modelled by computational fluid dynamics simulations and an approximate analytic formula. The device may be used for two-dimensional (2D) combinatorial dilution by adding two solutions in different relative concentrations to each of its three inputs. By appropriate choice of the two-component input solutions, test chamber concentrations that span any triangle in 2D concentration space may be obtained. In particular, explicit inputs are given for a coarse screening of a large region in concentration space followed by a more refined screening of a smaller region, including alternate inputs that span the same concentration region but with different distributions. The ability to probe arbitrary subspaces of concentration space and to control the distribution of discrete test points within those subspaces makes the device of potential benefit for high-throughput cell biology studies and drug screening.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Recent advances in microchip electrophoresis for amino acid analysis

With the maturation of microfluidic technologies, microchip electrophoresis has been widely employed for amino acid analysis owing to its advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. In this article, we review the recent progress in amino acid analysis using microchip electrophoresis during the period from 2007 to 2012. Innovations in microchip materials, surface modification, sample introduction, microchip electrophoresis, and detection methods are documented, as well as nascent applications of amino acid analysis in single-cell analysis, microdialysis sampling, food analysis, and extraterrestrial exploration. Without doubt, more applications of microchip electrophoresis in amino acid analysis may be expected soon.