植物细胞钙离子检测与成像技术研究进展

钙离子是一种重要的第二信使,参与调节植物多种生理和发育过程.胞内钙离子呈现复杂的时空动态变化,只有实时捕获钙离子在植株整体水平以及亚细胞水平的变化,才能深入理解钙信号的重要功能.过去几十年中,人们在钙离子检测与成像技术方面进行了大量探索,本文总结了近年来植物科学研究中常用的钙离子检测与成像技术,着重介绍了化学荧光探针和基于荧光蛋白钙离子探针的原理、特点、浓度计算方法、在细胞质以及细胞器钙离子检测与成像中的应用等,并总结了化学荧光探针的装载方法.     

Ag纳米粒子修饰多模光纤的拉曼增强特性实验

为了分析研究光纤表面增强拉曼散射(SERS)探针 的拉曼增强特性,采用简单廉价的化学方法制备Ag纳米颗粒修饰 光纤SERS探针,采用罗丹明6G(R6G,Rhodamine 6G)作为探针分子进行了不同浓度R6G溶 液(10－7~10－9M)的拉曼测试实 验和浓度为10－6M的R6G溶液的Time-course SERS Mapping实验,研究了该光纤 SER S探针的拉曼增强特性,制备的光纤SERS 探针样品检测R6G的极限浓度低至10－9M;在Time-course SE RS Mapping实验中,分 析验证了探针分子溶液蒸发过程对光纤 SERS探针的拉曼增强特性的影响。研究表明,由于制备的光纤SERS探针对荧光噪声没有抑 制作用,导致所测得探针分子 的拉曼光谱受探针分子溶液状态、测试方式的极大影响。当溶液充足时(浸泡状态),所测 光谱信号荧光噪声与有效拉曼光 谱信号都比较大;在溶液干燥过程中,所测光谱信号荧光噪声与有效拉曼光谱信号都减少, 但荧光噪声减小幅度远大于拉曼光谱信号。     

FCS/FCCS技术及其在植物细胞生物学中的应用

荧光相关光谱(fluorescence correlation spectroscopy,FCS)是一种单分子荧光检测技术,能够灵敏地检测荧光分子的浓度、运动参数及构象变化等数据。在此技术基础上发展起来的荧光互相关光谱(fluorescence crosscorrelation spectroscopy,FCCS)突破了FCS技术只能探测单种荧光分子的限制,可以通过分析两种及以上荧光信号来研究分子间的相互作用。由于FCS和FCCS检测的快速精确性,这两种技术被越来越多的应用于生命科学的研究。本文详细阐述了荧光相关光谱和荧光互相关光谱的原理,并对其在生物学尤其是植物细胞生物学中的应用进行了介绍和总结。随着检测技术的改进和数据分析技术的完善,荧光相关光谱和荧光互相关光谱技术将在植物生物学的研究中得到越来越多的应用。     

一种基于激光加工微型通道的光纤生物传感器

提出一种新型结构的光纤生物传感器。利用的Nd:YAG激光(波长1064nm)在PMMA基片上加工制作合成的微米级的微型通道作为光纤生物传感器的样品检测微流路;利用化学腐蚀的方法在多模光纤上制作具有圆锥表面结构的分子识别探针和荧光接收探针。将分子识别探针、荧光接收探针和样品检测微流路组装成光纤生物传感器。在理论上,分析了荧光接收探针的圆锥形接收面的锥度角与接收到的荧光光线最小入射角之间的函数关系;在实验上,分析了分子识别探针附近的荧光强度与荧光接收探针接收到的荧光强度不同的原因。结果表明,该传感器在理论上和实验上是可行的;荧光接收探针的锥度角和分子识别探针与荧光接收探针的中心能否对准是影响接收到荧光多少的主要因素;同时,在检测溶液的浓度降低时,单个荧光分子的发光变得显著。     

eATP在植物生长发育及逆境胁迫中的作用

5'-三磷酸腺苷(ATP)是生物体各种生命活动所需能量的直接来源.研究表明,ATP在植物细胞线粒体、叶绿体等细胞器中合成后,可以被转运到胞外.胞外ATP(extracellular ATP,eATP)作为一种信号分子,在植物生长发育、逆境胁迫等多种生理过程中发挥重要功能.2014年,植物细胞中发现了第一个ATP受体,为阐明植物细胞eATP信号传导过程奠定了基础.本文中,作者将对植物生长发育及胁迫响应过程中产生的eATP及其功能进行总结,对eATP的发现、释放、识别、调控与信号传导、检测方法等进行论述,并对eATP在植物中的研究提出展望.     

泵浦/探测增益光谱学

近些年来,采用两束不同频率的激光作为泵浦/探测技术来研究分子的激发态光谱,愈来愈为人们广泛应用。而对于光谱信号的检测,通常有两种不同的方法。一种是检测分子被激发后所发射的荧光。但是,检测荧光存在一个缺点是要想提高待测信号的光谱分辨率或时间分辨率或空间分辨率,都将使检测的灵敏度降低。另外,对于液态或溶液的有机分     

基于R6G探针表面增强拉曼散射的pH传感

用金纳米粒子(Au NPs)作为基底,基于罗丹明6G(R6G)探针分子拉曼特征峰随pH的变化,构建了一种具有表面增强拉曼散射(SERS)信号输出的pH传感器。由于(闭)开环形式下存在的(去)质子化R6G分子吸附于增强基底表面的倾斜度取向不同,R6G在不同pH下表现出了不同的SERS活性。根据寻找到的R6G在1363 cm-1和1314 cm-1位置处的SERS光谱峰面积比与pH的线性关系,设计出pH传感器。实验结果表明:R6G的SERS信号在室温下可以稳定2 h以上;当样品溶液pH在7和3之间转换时,传感器表现出了较好的恢复性。在pH检测过程中引入其他金属阳离子后,该探针表现出了对H+较好的选择性。通过检测实际样品的pH发现,该探针的分析性能良好,适于在酸性介质中检测pH。     

两种近红外荧光探针的合成及肿瘤靶向研究

合成了两种近红外有机荧光探针,即叶酸-PEG-ICG-Der-01和LDL-ICG-Der-02,它们分别以肿瘤表面高度表达的叶酸受体以及低密度脂蛋白(LDL)受体为靶点。探针复合物中的叶酸和低密度脂蛋白部分为探针分子提供靶向"导向",通过化学共价键分别与有机近红外染料ICG-Der-01和ICG-Der-02偶联,近红外染料则为探针分子的荧光信号输出端。利用紫外分光光度计、近红外荧光光谱仪及近红外荧光成像系统分析这两种荧光探针的光学性质,以及它们在叶酸受体及LDL受体过度表达的肿瘤鼠体内的成像过程。结果显示,所合成的叶酸-PEG-ICG-Der-01和LDL-ICG-Der-02探针的荧光强度及光稳定性都高于对应的染料单体。而体内成像结果则表明两种探针都保持了叶酸和LDL的生物活性,能有效地靶向到相关肿瘤部位,成像清晰,并且能最终代谢排出体外。比较这两种探针,叶酸-PEG-ICG-Der-01对肿瘤细胞的靶向性要优于LDL-ICG-Der-02,并能用于肿瘤早期诊断。     

多通道SPR影像传感器及其对DNA的特异性检测

SPR传感器的响应包括多种成份,除了待测分子与探针分子相互结合引起的响应(特异性响应)外,还有样液中其它成份及其浓度的变化、温度变化以及非待测分子与敏感膜的相互作用引起的响应(非特异性响应).后者的存在会严重影响SPR传感器的测量精度.本文提供了一种提高检测精度的方法,即采用多通道SPR影像传感器结构,通过不同通道之间的比较,提取出待测分子与探针分子之间相互作用所引起的响应,在真正意义上实现了对生物分子相互作用的实时、动态检测.通过对DNA的检测,证明这种方法对生物分子相互作用的特异性检测是行之有效的.     

一种基于激光诱导荧光法的心肌钙蛋白Ⅰ检测系统

基于激光诱导荧光的测量原理,研制了一种用于心肌钙蛋白Ⅰ(cTnⅠ)快速检测的便携式系统。首先在测试杯中cTnⅠ与荧光试剂发生免疫反应,经过分离除去多余的荧光试剂,测试杯中的荧光分子与cTnI的量成相关性,通过对荧光分子进行诱导激发,使其发出荧光信号,然后经过光电转换、微弱电信号放大、信号采集与处理,最终实现对cTnⅠ的快速定量检测。临床实验显示,在cTnT为1.5~12.2 ng/mL范围内,本系统与TosohAIA-360生化分析仪的检测结果具有很好的线性关系,样品数为6时两者的相关系数达到0.97。     

Fluorescent Gold Nanoprobe Sensitive to Intracellular Reactive Oxygen Species

Gold nanoprobes immobilized with fluorescein‐hyaluronic acid (HA) conjugates are fabricated and utilized for monitoring intracellular reactive oxygen species (ROS) generation in live cells via nanoparticle surface energy transfer. A bio‐inspired adhesive molecule, dopamine, is used to robustly end‐immobilize HA onto the surface of gold nanoparticles (AuNPs) for securing intracellular stability against glutathione. ROS induces cleavage and fragmentation of the HA chains immobilized on the surface of the AuNPs allows rapid and specific detection of intracellular ROS by emitting strong fluorescence‐recovery signals. In particular, fluorescence‐quenched gold nanoprobes exhibit selective and dose‐dependent fluorescence‐recovery signals upon exposure to certain oxygen species such as superoxide anion () and hydroxyl radical (^·OH). The fluorescent gold nanoprobe is usefully exploited for real‐time intracellular ROS detection and antioxidant screening assay, and has exciting potential for various biomedical applications as a new class of ROS imaging probes.     

荧光金纳米团簇探针的生物成像应用进展（特邀）

金纳米团簇（AuNCs）因兼具优异荧光特性、超小尺寸、精确化学组成及良好生物相容性等优势，使其成为近些年备受关注的新型荧光探针。为了推动荧光AuNCs在成像领域的应用，研究者们一直致力于发展高性能荧光AuNCs的设计与制备策略。基于对AuNCs结构与发光机制的理解，诸如提高荧光量子产率和细胞摄取率等策略陆续被提出以增强AuNCs的细胞成像效果，极大提升了其作为荧光成像探针的潜力，并将AuNCs的应用推广至荧光寿命成像、多光子成像等新兴荧光成像技术。近些年发展的具有近红外二区荧光的AuNCs进一步推动了其在活体成像的应用。文中概述了AuNCs的制备方法、提高AuNCs细胞荧光成像效果的各种策略，以及AuNCs荧光成像应用的最新进展，并对该领域的挑战和未来发展进行了展望。     

Biocatalytic and Antioxidant Nanostructures for ROS Scavenging and Biotherapeutics

In human systems, reactive oxygen species (ROS) significantly affect different physiological activities and play critical roles in diverse living processes. It is widely known that excessive ROS generation in inflammatory tissues can further deteriorate the localized tissue injury and cause chronic diseases. Though promising for reducing ROS levels, many antioxidant molecules and natural enzymes suffer from abundant intrinsic limitations. Recently, a series of biocatalytic or antioxidant nanostructures have been designed with distinctive ROS scavenging capabilities, which show promising activities to overcome these kernel challenges. In this timely review, the most recent advances in engineering biocatalytic and antioxidant nanostructures for ROS scavenging are summarized. First, the ROS scavenging principles and corresponding methods for testing various enzymatic activities are carefully concluded. Subsequently, the rationally designed nanostructures with high ROS scavenging efficiencies are comprehensively discussed, especially on the catalytic activities, mechanisms, and structure-function relationships. After that, the representative applications of these ROS scavenging nanostructures for diverse biotherapeutics are summarized in detail. At last, the primary challenges and future perspectives in this emerging research frontier have also been outlined. It is believed that this progress review will offer a cutting-edge understanding and guidance to engineering future high-performance ROS scavenging nanostructures for broad biotherapeutic applications.     

Robust Organoalkoxysilanes as Red Unconventional Fluorescent Platform

Unconventional fluorescent materials have attracted intense and continuous attention due to the facile processability, excellent biocompatibility, and high availability. However, for the lack of suitable unconventional fluorescent platform, unconventional luminophore‐based fluorescent probes have not been applied in the biological field, especially in the detection of bioactive molecules. In this work, unconventional red fluorescence is observed from a series of organoalkoxysilanes for the first time. Particularly, the unique fluorescence derived from smart Si–O bridged structures prompt the fluorescent probe design strategy. The strategy involves applying the Si–O bridge to provide desirable red unconventional fluorescence, and ratiometric detection of endogenous nitric oxide in lysosomes and in vivo. It is expected that this novel strategy will expand the applications of unconventional fluorescence to the bioimaging field, and further provide valid approach for the future evolution of unconventional fluorescent probes.     

ROS-Catalytic Transition-Metal-Based Enzymatic Nanoagents for Tumor and Bacterial Eradication

The extensive research into developing new nanomedicines during the past few years has witnessed significant progress in diverse biomedical fields, especially for combating drug resistance in antitumor and antibacterial therapies. Recently, transition-metal-based enzymatic nanoagents (TM-EnzNAs) with catalytic production of reactive oxygen species (ROS) have been designed and intensively explored, which have become powerful nanoplatforms and exciting research frontiers in constructing next-generation nanotherapeutics to combat drug-resistant tumors and bacteria. Here, the focus is on the recent design, fundamental principles, and material chemistries in developing and applications of TM-EnzNAs. At first, the different ROS-producing mechanisms and the key factors to enhance ROS level are carefully concluded, and the analytic methods are systematically summarized. Then, the rationally engineered TM-EnzNAs via different synthetic approaches with high ROS producing efficiencies are comprehensively discussed, especially the catalytic activities, mechanisms, and structure–function relationships. After that, the representative applications of these ROS-catalytic TM-EnzNAs for antitumor and bacterial eradication are summarized in detail. Finally, the primary challenges and future perspectives have also been outlined. It is anticipated new therapeutic insights into combating drug-resistant tumors and bacteria will be provided, and significant new inspiration for designing future enzymatic nanoagents is offered.     

Synthesis of Optically Complex Core–Shell Colloidal Suspensions: Pathways to Multiplexed Biological Screening

The ability to generate enormous random libraries of DNA probes via split‐and‐mix synthesis on solid supports is an important biotechnological application of colloids that has not been fully utilized to date. To discriminate between colloid‐based DNA probes each colloidal particle must be ‘encoded’ so it is distinguishable from all other particles. To this end, we have used novel particle synthesis strategies to produce large numbers of optically encoded particles suitable for DNA library synthesis. Multi‐fluorescent particles with unique and reproducible optical signatures (i.e., fluorescence and light‐scattering attributes) suitable for high‐throughput flow cytometry have been produced. In the spectroscopic study presented here, we investigated the optical characteristics of multi‐fluorescent particles that were synthesized by coating silica ‘core’ particles with up to six different fluorescent dye shells alternated with non‐fluorescent silica ‘spacer’ shells. It was observed that the diameter of the particles increased by up to 20 % as a result of the addition of twelve concentric shells and that there was a significant reduction in fluorescence emission intensities from inner shells as an increasing number of shells were deposited.     

Theoretical study on fluorescent probes for cyanide based on the indolium functional group

A series of fluorescent probes were investigated to explore the mechanism of the detection for CN⁻, and all of the probes have an indolium group. The interaction between the functional group and the cyanide ion was simulated to examine the selectivity of these probes. The influences of some other anions were also tested in the cyanide detecting process. The different binding abilities between CN⁻ and F⁻ in aqueous solution were distinguished using the potential energy calculations in consideration of hydrogen bonding interactions. The large change of the emitting wavelengths of the fluorescent probes before and after the addition of CN⁻ was explained through the analysis of the molecular orbital properties. The results indicate the large structural change results in a significant emission shift when each probe captures a cyanide ion. According to this conclusion, new fluorescent probes were designed.     

Laser diagnostics of flames, combustion products, and sprays

Recently, numerous laser-based diagnostic methods have been developed to probe sprays and combustion processes. These diagnostic probes encompass spontaneous Raman spectroscopy, coherent Raman spectroscopy, laser fluorescence and absorption for temperature and species concentration measurements; laser Doppler velocimetry for fluid flow measurements; and particle sizing and vapor concentration in sprays by numerous optical methods. Specific examples will be presented of species concentrations and temperatures in flames. These results have been compared to theoretical calculations for these properties and in many cases show excellent agreement between experiment and theory. Moreover, the spatial resolution offered by these laser techniques allows one to determine how a flame is changed by the presence of an adjacent cooled, metal wall. Two new Raman spectroscopic methods are also described for the direct spectroscopic measurements in a flame of atomic oxygen-an important radical in flame chemistry. Finally, a two-wavelength absorption/scattering measurment is described whereby vapor concentrations inside sprays may be measured. This technique has been applied to measuring the vapor concentration in an axisymmetric n-heptane fuel spray.     

Cellular fluorescent indicators and voltage/ion probe reader (VIPR) tools for ion channel and receptor drug discovery

High throughput functional assays are increasingly relied upon to generate early and novel discovery leads for drug development. Ion transport proteins including channels, transporters, and pumps play central roles in cellular bioenergetics, excitability, and a multitude of other biological functions. Facile, robust methods for detecting ion transport activity in both native and heterologous systems is desirable for rapid functional analysis and drug discovery for these difficult but important targets. Here we discuss cell-compatible fluorescent probes, functional assays, and VIPR instrumentation that are used to monitor real-time target activity and screen large chemical libraries for potent and selective modulators. Advances and issues for both exogenously applied and fluorescent protein probes of cellular membrane potential, Ca2+, Cl-, and pH are addressed. High throughput screening (HTS) compatible, rapid kinetic and fluorescence resonance energy transfer (FRET) assays are emphasized, in particular the use of voltage-sensitive FRET probes to assay ion channel activity in single cells and 96/384-well formats.