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。     

单细胞毛细管电泳分析研究进展

本文从细胞悬浮液制备、单个细胞进样技术、细胞溶解技术、分离模式、检测方法和应用新进展6个方面对单细胞毛细管电泳分析进行了全面评述.重点介绍了单个细胞进样技术及检测方法的最新进展,并对单细胞毛细管电泳分析的未来发展方向进行了展望.     

微流控芯片单细胞进样和溶膜

单细胞分析对重大疾病的早期诊断、治疗和药物筛选以及细胞生理、病理过程的研究有重要意义.将毛细管电泳用于单细胞多组分的测定已取得一些成果,但受毛细管的一维结构限制,单细胞进样和溶膜操作较复杂.微流控分析芯片的网络结构和微米级的通道尺寸使简化单细胞分析成为可能.     

毛细管电泳在完整哺乳动物细胞分析中的应用

作为人体形态结构、生理功能和生长发育的基本单位,细胞的结构功能及行为分析具有重要的研究价值。毛细管电泳作为日渐成熟的微量分析技术在细胞分析及应用方面已取得显著进展。本文综述了毛细管电泳在完整哺乳动物细胞分析中的应用,包括群体细胞分析和完整单细胞分析。所述内容涉及血红细胞、公猪精子、人宫颈癌细胞、人神经母细胞瘤细胞、人结直肠腺癌细胞、人慢性髓系白血病细胞以及鼠小脑颗粒细胞。总结了完整哺乳动物细胞分析的毛细管电泳方法和条件,归纳分析了完整细胞分析中存在的细胞破碎、聚集、沉降、吸附及电泳异质性等关键问题及解决方法。对毛细管电泳在细胞分析方面未来的应用方向进行了展望。综述文献49篇。     

单细胞分析中的荧光标记化学*

细胞内组分复杂、含量低,因此测定单细胞内化学组分的分析方法必须具有灵敏度高、选择性好和分辨率高的特点。高灵敏度的荧光检测技术是单细胞分析中应用最多的检测方法之一。但是细胞内绝大部分物质其天然态是没有荧光的,且由于细胞膜的阻碍,衍生试剂不能自由地进入细胞内。为了使衍生试剂透过细胞膜标记细胞内待测物质而不引起显著的稀释效应,已进行了大量的研究工作。本文综述了在单细胞分析中常用的荧光标记方法,包括细胞作为微反应器的衍生法,借助于脂质体与聚乙二醇（PEG）等增加细胞膜通透性的衍生方法和在毛细管/芯片毛细管电泳分析单细胞时柱上衍生和柱后衍生法以及量子点的标记法等。对这些方法的原理、特点和在单细胞分析中的应用也做了较为详细的阐述。     

微流控芯片系统在单细胞研究中的应用

微流控芯片具有网络式通道结构,扩展了在细胞和亚细胞水平进行生命科学研究的能力,为单细胞研究提供了一个新的平台.在微流控芯片通道中,人们利用气压、液压和电压,或利用介电电泳、光学陷阱、行波介电电泳以及磁场等技术,可以操纵细胞通过或驻留在通道内的任意位置,从而使单细胞计数、筛选以及胞内组分分析等操作大大简化.本文对微流控芯片系统在血液流变学、单细胞操纵与计数以及单细胞胞内组分分析中的应用进行了综述,介绍了用于单细胞研究的多种微芯片系统,讨论了芯片上进行单细胞操纵的各种方法     

毛细管电泳技术和应用新进展

毛细管电泳（CE）是当前分析化学前沿领域和研究重点之一。本文对细管电泳技术－分离模式、进样技术和检测器为线索对最近的研究进展进行了综述,探讨了各种模式和技术的分析效率。并介绍了CE很有实用价值的几个方面的应用,包括在基因工程和单细胞分析中的突出表现,在手性分离和小分子离子分析方面的广泛应用。     

毛细管电泳的原理及应用(第五讲)毛细管电泳在医药领域中的应用

简单介绍了毛细管电泳在医药领域中的应用，包括药物分析、中药成分分析、手性对映体分离分析和临床化学、法医及单细胞分析等。药物分析中包括主药成分分析、相关杂质检测、药物计量离子配比测定和定量分析等。中药成分分析中包括各类药效成分、中药材中主要成分及中药复方制剂成分分析。手性对映体分离中包括机理研究、新型手性选择剂。临床化学中包括临床疾病诊断、临床蛋白分析、临床药物监测、药物代谢研究和分子生物学测定。法庭科学中包括毒物分析、枪击残余物分析、炸药分析、笔迹墨水分析。单细胞分析中包括神经细胞分析、红细胞分析和胚胎细胞分析。     

单细胞的毛细管电泳分析

对单细胞分析化学中毛细管电泳分离技术的进样方式、检测器、应用和前景予以综述,４６篇。     

单细胞视角下铅的体内代谢与检测

在全球工业需求和储能需求持续增加的背景下,铅污染仍然是当前主要的环境问题之一。持续的铅暴露导致了复杂的体内毒理效应,其临床治疗效果受限于铅检测技术的缺乏与铅动力学研究的不足。单细胞水平上铅的检测技术,可以揭示动力学相关的细胞机制,从微观角度考量细胞异质性对人体内铅运输和铅分布的影响。分析了铅污染现状并归纳了铅暴露源特征,总结了单细胞水平上铅代谢与检测方面的研究进展。细胞机制与细胞异质性对完善铅的动力学模型和理解有关机制的作用不容忽视。单细胞检测技术作为有效的研究工具,包括流式细胞技术、质谱流式细胞技术以及基于电感耦合等离子体质谱的单细胞电感耦合等离子体质谱技术、电感耦合等离子体飞行时间质谱技术和激光烧蚀-电感耦合等离子体质谱技术等,需要更加标准化与体系化的检测方案。基于生物数学模型与单细胞检测技术的发展,单细胞视角下铅的动力学研究有望进一步细化与完善。     

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.     

NH4OH-Based pretreatment for improving the nutritional quality of single-cell protein (SCP)

The potentiality of treatments with NH₄OH solutions for improving the quality of protein concentrates fromCandida utilis biomass was studied. The effects of NH₄OH concentration, reaction time, and temperature on both biomass recovery and composition of processed samples (including nucleic acid and protein contents) were studied. The results obtained were used to develop empirical models providing a quantitative interpretation of the interrelationships among the variables involved. Additional discussion of the reaction selectivity is provided. Under selected conditions, 96% of nucleic acid removal was achieved with 88% protein recovery. The treated cells were high in vitro digestibility and showed an amino acid profile similar to that of untreated biomass.     

Rhodococcus as Biofactories for Microbial Oil Production

Bacteria belonging to the Rhodococcus genus are frequent components of microbial communities in diverse natural environments. Some rhodococcal species exhibit the outstanding ability to produce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presence of an excess of the carbon source and limitation of the nitrogen source. For this reason, they can be considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO) producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summarized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production. This review provides a comprehensive and integrating vision on the potential of oleaginous rhodococci to be considered as microbial biofactories for microbial oil production.     

Enhancing signals of microfluidic impedance cytometry through optimization of microelectrode array

Microfluidic impedance cytometry shows a great value in biomedical diagnosis. However, the crosstalk between neighboring microelectrodes strongly weakens the impedance signal. Hereby, we demonstrate a novel microfluidic impedance cytometer consisted of sensing electrodes and ground electrodes (GNDs). The simulation reveals a signal enhancement by more than five times with GNDs compared to that without ones. We also found that the linear correlation between the impedance at a high frequency and that at a low frequency varies as microparticle size changes, which can be used for microparticle classification. The study can help with microelectrode optimization and signal processing for microfluidic impedance analysis.     

Electrochemiluminescence Single-cell Analysis on Nanostructured Interface

Due to the heterogeneity of single cells, the development of single-cell analysis methods is conducive to the research of cellular mechanisms, clinical diagnosis, and treatment. Electrochemiluminescence (ECL) has the advantage of being controllable in time and space. Compared with spectroscopy, ECL does not require a light source, thus avoiding the interference of scattered light and luminescent impurities. Therefore, ECL is playing an increasingly important role in the field of single-cell analysis. In ECL single-cell analysis, it is important to construct a suitable nanostructure interface to realize signal conversion. This review first briefly introduced the ECL system commonly used in single-cell analysis, then focused on the recent developments in ECL single-cell analysis on nanostructure interface, finally discussed the future challenges and outlooks of ECL single-cell analysis.     

单细胞分析研究进展

单细胞分析可以揭示生命基本单元——细胞物质组成、生理行为的多样性和差异性,是当今生命分析的主流前沿技术。同时,也对分析技术的定量检测极限、分辨率和精密操控能力提出了新挑战。文章着重从单细胞分离、检测、成像等几个主要方面,综述了单细胞技术研究的最新进展,并对分离和检测技术的发展作了展望。     

From Oncogenic Signaling Pathways to Single-Cell Sequencing of Immune Cells: Changing the Landscape of Cancer Immunotherapy

Over the past decade, there have been remarkable advances in understanding the signaling pathways involved in cancer development. It is well-established that cancer is caused by the dysregulation of cellular pathways involved in proliferation, cell cycle, apoptosis, cell metabolism, migration, cell polarity, and differentiation. Besides, growing evidence indicates that extracellular matrix signaling, cell surface proteoglycans, and angiogenesis can contribute to cancer development. Given the genetic instability and vast intra-tumoral heterogeneity revealed by the single-cell sequencing of tumoral cells, the current approaches cannot eliminate the mutating cancer cells. Besides, the polyclonal expansion of tumor-infiltrated lymphocytes in response to tumoral neoantigens cannot elicit anti-tumoral immune responses due to the immunosuppressive tumor microenvironment. Nevertheless, the data from the single-cell sequencing of immune cells can provide valuable insights regarding the expression of inhibitory immune checkpoints/related signaling factors in immune cells, which can be used to select immune checkpoint inhibitors and adjust their dosage. Indeed, the integration of the data obtained from the single-cell sequencing of immune cells with immune checkpoint inhibitors can increase the response rate of immune checkpoint inhibitors, decrease the immune-related adverse events, and facilitate tumoral cell elimination. This study aims to review key pathways involved in tumor development and shed light on single-cell sequencing. It also intends to address the shortcomings of immune checkpoint inhibitors, i.e., their varied response rates among cancer patients and increased risk of autoimmunity development, via applying the data from the single-cell sequencing of immune cells.     

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.