生物电化学仪器的发展现状与展望

电荷传递是生命运动的基本过程之一,电化学方法在生命科学中的应用为相关生命现象的研究提供了一个有效而独特的物理化学视角,并带来超出常规生物学检测的丰富信息.随着生物电化学研究的不断扩展和深化,已从早期的生物分子电化学研究深入向活体、活细胞、单活细胞水平甚至活细胞中单分子水平发展.研究者对仪器设备性能如灵敏度、分辨率(时间分辨、空间分辨和能量分辨)和操作性等提出了越来越高的要求.本文综述了生物电化学仪器在应用领域和研究领域的现状,重点介绍单细胞电化学检测系统的构建,并初步探讨国内生物电化学研究仪器的发展趋势.     

NEWS

单细胞分析是实现生命科学精细化研究重要的技术方法之一。它可以使得我们了解复杂生命过程中细胞与细胞之间的相互作用,更加深入地揭示疾病细胞的分子机理。目前主要的单细胞分析方法包括光学分析和电化学分析。光学分析具有通量高的优点;但需要针对被分析分子设计特定探针实现高选择性的检测,无法构建统一化探针;而电化学分析则可利用氧化酶,转化各种被分析分子成具有电活性的活性氧分子,实现统一化的高时空分析;但该方法存在检测通量低的缺点,难以满足临床检测需求。在基金委重大科研仪器设备研制专项“单细胞时空分辨分子动态分析系统”的支持下,南京大学陈洪渊院士、江德臣副教授课题组系统的发展了单细胞电致化学发光检测平台,将单细胞电化学检测过程产生的电化学信息转化为光信号,从而有效的发挥了电化学检测和光学检测的优点,实现高通量的单细胞分析。该方法避免了传统光学方法需针对特定分子进行独特设计的不足,有望用于临床分析。     

单细胞分析技术研究进展

细胞是生命结构和生命活动的基本单位,基于单细胞的研究是生命研究的基础。由于细胞体积极小、细胞微环境复杂并且起到关键作用的组分含量往往较低,因此在很多方面单细胞分析仍然是一项具有挑战性的任务。该文对现有的单细胞分析技术进行归纳、总结,重点介绍了不同单细胞分析技术的特点及其应用的最新进展。     

基于质谱的单细胞蛋白质组学分析方法及应用

细胞是生命体的最小组成单位,遗传及外部环境等因素使单细胞异质性广泛存在于众多生物体中。传统的生物学实验获得的结果多是大量细胞的平均测量值,因此在单细胞层面开展研究对于精确理解细胞的生长发育以及疾病的诊断与治疗至关重要。而作为重要的细胞和生命活动的执行者,蛋白质由于其不具备扩增特性,且种类繁多、丰度低、动态分布范围宽,与核酸等其他生物大分子相比,其单细胞组学研究相对滞后。而在所有的检测手段中,荧光检测以及电化学分析方法具有极高的灵敏度,但是囿于其研究通量有限,以及电化学活性依赖,很难成为普适性的单细胞蛋白质组学研究方法。质谱分析作为传统蛋白质组学中最为核心的研究技术,由于其高灵敏、高通量、结构信息丰富等特点,在单细胞蛋白质组学研究中独树一帜。该文综述了近年来基于质谱的单细胞蛋白质组学研究中的代表性方法,根据质谱分析前蛋白质分离方式的差异,将其分为基于毛细管电泳分离、液相色谱分离和无分离手段的直接检测3类方法,在介绍研究现状的同时对这些方法在细胞通量、蛋白质鉴定数目、灵敏度以及方法应用方面进行了总结与比较。最后,基于目前研究中面临的挑战以及发展趋势对基于质谱的单细胞蛋白质组学的研究前景进行了展望。     

基于微流控芯片利用水凝胶微结构捕获单细胞的分析方法研究

细胞是生命活动的基本单位,为了掌握生命过程的规律,必须以研究细胞为基础,深入探索细胞的行为.单细胞分析在近些年作为研究细胞内部物质、细胞分泌物和代谢产物的重要手段,特别是在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.     

毛细管电泳单细胞分析*

细胞是生命活动的基本单位,单细胞分析能够阐明细胞的结构与功能,揭示细胞之间的差异性,在细胞分化、生理病理研究及疾病早期诊断中具有重要作用。毛细管电泳作为一种高效的分离手段,所需样品体积小,能与高灵敏度检测器联用,特别适于单细胞分析。本文从单细胞进样技术、单细胞裂解技术、检测方法及应用等方面,对2007年以来毛细管电泳用于单细胞分析的最新进展做了相应叙述,并对未来的发展方向进行了展望。表明毛细管电泳不仅适于单细胞分析,而且仍有巨大的发展潜力和广阔的应用空间。     

21世纪单细胞分析发展

细胞是生物体的形态结构和生命活动的基本单位。了解生物体生命活动的规律 ,必须以研究细胞为基础 ,探索细胞的生命活动 ,掌握和控制生、老、病、死的规律 ,造福于人。　　目前细胞研究已经从细胞整体 (单细胞 )深入到亚细胞 (局部细胞质、细胞膜、囊泡 )和分子水平 (DNA等生物大分子及单分子 )。因此 ,单细胞分析向分析化学提出了严峻的挑战 ,也带来众多机遇。由于细胞极小 (一般直径 7～ 10 0 μm) ,样品量很少 (体积fL～pL) ,胞内组分十分复杂 (最简单的红血球细胞含蛋白质上千种 ) ,胞内生化反应速度快 (ms～s级 ) ,因此 ,单细胞分析…     

基于纳米材料的细胞释放生物信号分子电化学传感器的研究进展

电化学传感器已被证实是一种检测细胞释放信号分子的有效方法.纳米材料如碳纳米材料、金属纳米颗粒,以及纳米复合材料等因其独特性质在电化学分析技术方面应用广泛.本文综述了近年来基于纳米材料的电化学传感器用于检测细胞中释放的信号分子的研究进展,并展望了其发展方向.     

单细胞分析技术在人类细胞图谱计划中的应用

2016年10月人类细胞图谱计划正式启动,该计划旨在区分不同类型的细胞及其在人体内的位置,研究激活、分化等细胞行为的分子机制,定义病理表征和标记分子.人类细胞图谱的建立将推动对人类健康和疾病诊断、监测、治疗的发展,从而促进对生命最基本单元的理解.建立系统的单细胞分析方法是完成人类细胞图谱的重要前提和保证.微流控细胞分离、单细胞DNA/RNA测序、细胞原位分析、质谱流式细胞分析等技术已经逐渐建立并用于单细胞分析.然而人类细胞图谱仍然面临诸多挑战,需要具有更高灵敏度和准确度的分析方法以及高效的数据处理技术.本文简要介绍了人类细胞图谱计划的发展现状及现有单细胞分析技术,并对挑战所需的新技术提出了展望.     

蛋白质磷酸化作用的电化学检测方法

蛋白质的磷酸化作用是一种可逆的翻译后修饰方式,几乎参与生物体内所有关键生命活动,在核内基因的特定表达、细胞的分裂分化以及各种生命活动的转导中发挥重要意义。基于磷酸化前后电化学信号的改变,利用电化学传感器的方法能快速简便地检测蛋白质的磷酸化作用,且具有较高的选择性和灵敏度。本文回顾了几种常用的电化学检测磷酸化作用的方法,基于电极材料的不同进行了分类与总结,汇总了每种方法中用到的修饰电极的纳米材料及生物分子,最后总结了电化学方法检测蛋白质磷酸化的优缺点,并对有效检测磷酸化作用进行了前景展望。     

Microfluidic techniques for dynamic single-cell analysis

Dynamic single-cell analysis is a very important and frontier research field of single-cell analysis. Microfluidic techniques have become new and effective tools for precise, high-throughput, automatic analysis of single-cell dynamic process. This review aims to give an overview of dynamic single-cell analysis methods based on microfluidic platforms, with emphasis on the recent developments of microfluidic devices and its application to real-time dynamic monitoring of the signal molecules release from single living cell with temporal and spatial resolution, dynamic gene expression in single cells, the cell death dynamic events at the level of a single cell, and direct cell—cell communication between individual cell pairs.     

Characterization of the Impact of Classical Cell-culture Media on the Response of Electrochemical Sensors

Biocompatibility testing is usually performed through staining and imaging of cell lines. We propose here to monitor cytotoxicity through real-time measurement of metabolites specifically issued from cell stress behaviour using a multiparametric electrochemical (bio)sensing platform. However, the composition of culture media varies widely according to the requirements of the utilized cell lines. This matter may have significant effects on the sensor's sensitivity. With this mind, the sensitivity of four electrochemical (bio)sensors (pH, hydrogen peroxide, nitric oxide/nitrite (NO and its by-product) and lactate) is investigated in different cell culture media. The main culture media studied were Minimum Essential Medium Eagle (MEM), Dulbecco's Modified Eagle Medium (DMEM), Williams’ Medium E and RPMI 1640 medium that were the recommended culture media for the cell types to be monitored. This work shows the impact of the different cell culture media on the performances of the different sensors (limit of detection, sensitivity, selectivity, response time and dynamic range). More particularly, FBS strongly impacts the response of the amperometric (bio)sensors. Then, cellular viability testing was effected within optimized medium (FBS content) for electrochemical sensor read-outs in the case of short-term cultures (one day) devoted to cytotoxicity testing. Real-time electrochemical monitoring provides important additional information about cell behaviour during biocompatibility testing that might be further implemented in different settings including pharmaceutical efficacy and biomaterials applications.     

Optical-facilitated single-entity electrochemistry

Single-entity electrochemistry focusing on the study of transient electrochemical process at the confined interface, has become a promising field that addresses questions from multi-disciplines such as cellular biology, material chemistry, organic chemistry, etc. It offers the fruitful information hidden in bulk electrochemical measurements. As the optical techniques improve in spatial and temporal resolution, the combination of electrochemistry with optical microspectroscopy provides more comprehensive information of single-entity electrochemistry. Herein, we review recent progress made in optical–electrochemical measurements covering three aspects from the precise localization and temperature measurements of single compartments, to the in-situ tracking of dynamic behaviors of single nanoparticles in electrochemical process, and to the monitoring confinement-controlled electrochemistry at the single molecule/ion level. The review demonstrates how these optical methods are innovatively integrated with single-entity sensing. It also reveals how these optical–electrochemical combinations push single-entity electrochemistry forward.     

Electrochemical Sensing in Contact Lenses

Electrochemical sensors embedded in hydrogel-based contact lenses provide valuable health-related information, enabling non-invasive and real-time continuous monitoring. Recently, considerable progress has been made in tear based electrochemical sensors. The scope of reported analytes is continuously expanding. This review identifies key chemical biomarkers (including metabolites, ions, proteins) that can be electrochemically detected in tears. The working principles of i) amperometric enzymatic biosensors, ii) ion-selective sensors for pH and ions, iii) voltammetric sensors and iv) affinity sensors are summarized. This review provides guidelines for the future development of contact lens based electrochemical sensors.     

Nanoelectrochemical architectures for high-spatial-resolution single cell analysis

The analysis of single cells instead of cell populations is important for characterizing cellular heterogeneity and elucidating the cellular signalling pathways. Nanoelectrodes have emerged as an increasingly important tool for biomolecule analyses at the single-cell level with high spatial or temporal resolution. Various electrochemical methods, such as amperometry and scanning electrochemical microscopy(SECM), have been applied. Research to date has focused on the development of new nanoelectrochemical architectures, such as arrays, to achieve higher spatial resolution and faster analysis rates for single-cell analysis. In this review, the fabrication of these new nanoelectrochemical architectures and their applications in high spatial resolution single-cell analyses are discussed. The recent progress of Chinese researchers is highlighted.     

Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells

Recently, stretchable electrochemical sensors have stood out as a powerful tool for the detection of soft cells and tissues, since they could perfectly comply with the deformation of living organisms and synchronously monitor mechanically evoked biomolecule release. However, existing strategies for the fabrication of stretchable electrochemical sensors still face with huge challenges due to scarce electrode materials, demanding processing techniques and great complexity in further functionalization. Herein, we report a novel and facile strategy for one-step preparation of stretchable electrochemical biosensors by doping ionic liquid and catalyst into a conductive polymer (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), PEDOT:PSS). Bis(trifluoromethane) sulfonimide lithium salt as a small-molecule plasticizer can significantly improve the stretchability and conductivity of the PEDOT:PSS film, and cobalt phthalocyanine as an electrocatalyst endows the film with excellent electrochemical sensing performance. Moreover, the functionalized PEDOT:PSS retained good cell biocompatibility with two extra dopants. These satisfactory properties allowed the real-time monitoring of stretch-induced transient hydrogen peroxide release from cells. This work presents a versatile strategy to fabricate conductive polymer-based stretchable electrodes with easy processing and excellent performance, which benefits the in-depth exploration of sophisticated life activities by electrochemical sensing.

A facile strategy for constructing stretchable sensors with excellent mechanical, electrochemical and biocompatible performance is developed, and in situ inducing and monitoring of stretch-evoked H₂O₂ release from cells has been successfully achieved.     

多荧光光纤传感器及其在混合溶剂检测中的应用

微纳光纤与其他微纳结构的集成可以拓展荧光光纤传感器检测范围和集成度,是光纤传感领域的研究热点。目前,国际上关于荧光光纤传感器这一领域的研究还处于单一检测物荧光响应的阶段,对多检测物的多通道荧光响应仍存在很大挑战。本文结合微纳光纤的光波导性能以及有机荧光材料的光功能特性,制备了能够同时激发和收集多种荧光的微纳光纤,并将之应用于高性能荧光光纤传感器的制备。通过选用不同荧光波长的有机材料与凝胶掺杂,制备了多荧光发射的光纤涂层材料,可控构筑了多组分荧光检测剂掺杂凝胶涂层。利用荧光光谱结合色度图分析,确定检测物与色坐标的关系,实现了多检测物的多通道荧光响应,为实现多荧光光纤传感器的可控构筑提供了有益的借鉴和指导意义。     

Phenotype-related drug sensitivity analysis of single CTCs for medicine evaluation

Due to the heterogeneous and variable drug sensitivity of tumor cells, real-time monitoring of a patient''s drug response is desirable for implementing personalized and dynamic therapy. Although considerable efforts have been directed at drug screening in living cells, performing repeated drug sensitivity analysis using patient-derived primary tumor cells at the single-cell level remains challenging. Here, we present an efficient approach to assess phenotype-related drug sensitivity at the single-cell level using patient-derived circulating tumor cells (CTCs) based on a drug sensitivity microfluidic chip (DS-Chip). The DS-Chip consists of a drug gradient generator and parallel cell traps, achieving continuous single CTC capture, drug gradient distributions, drug stimulation, fluorescent probe labeling and three-color fluorescence imaging. Based on the established DS-Chip, we investigated the drug sensitivity of single cells by simultaneously monitoring epithelial–mesenchymal transition (EMT) biomarkers and apoptosis in living cells, and verified the correlation between EMT gradients and drug sensitivity. Using the new approach, we further tested the optimal drug response dose in individual CTCs isolated from 5 cancer patients through fluorescence analysis of EMT and apoptosis. The DS-Chip allows noninvasive and real-time measurements of the drug sensitivity of a patient''s tumor cells during therapy. This developed approach has practical significance and can effectively guide drug selection and therapeutic evaluation for personalized medicine.

Due to the heterogeneous and variable drug sensitivity of tumor cells, real-time monitoring of a patient''s drug response is desirable for implementing personalized and dynamic therapy.     

基于不同工作电极的亚硝酸盐电化学传感器

亚硝酸盐是环境和生物循环中不可或缺的一部分,但其浓度超标不仅会污染环境,还会致癌。因此对亚硝酸盐实现高效、快速精密检测具有重要的科学和实际意义。基于电化学传感技术的污染物分析与检测已成为研究热点,尤其工作电极的选用和设计对提升响应速度、灵敏度、检测限及降低成本、简化操作等至关重要而备受研究者广泛关注。本文将以玻碳电极、碳糊电极、金属薄膜、聚合物薄膜及碳布电极为分类,针对亚硝酸盐的检测,详细综述基于不同电极的电化学传感器的研究进展。以期通过构建方法和电催化性能的对比为构建新型传感器提供理论与实际指导。