基于功能核酸的细胞荧光成像

细胞是生物体基本的结构和功能单元,对活细胞中特定生物组分进行动态分析,将为相关生命活动过程的研究提供重要信息。荧光成像为细胞分析提供了一种操作简单、灵敏度高、可实时监测细胞微观动态分子过程的光学生物成像技术。发展高性能的荧光探针用于活细胞成像已成为研究热点。功能核酸是一类具有特殊化学和生物学功能的寡核苷酸分子,除了天然存在的核酶(Ribozyme)和核糖开关(Riboswitch)之外,还包括通过指数富集的配体系统进化技术(SELEX)筛选获得的核酸适体和脱氧核酶(DNAzyme)。功能核酸由于具有合成简单、免疫原性低、相对分子质量小、化学稳定性高、易于修饰等优点,在生物成像领域受到广泛关注。本文主要综述了基于功能核酸的荧光探针在细胞成像领域中的应用研究,总结了该领域面临的挑战,并对其未来发展方向进行了展望。     

基于Cell-SELEX的核酸适配体在生化分析与生物成像中的应用

基于Cell-SELEX的核酸适配体是指以活细胞为靶标物,通过指数富集的配基系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的DNA/RNA文库中筛选得到的单链寡核苷酸.它能够与靶标细胞高亲和性、高特异性结合,具有分子量低、合成简单、化学稳定性好、免疫原性低、易于功能化修饰等优点,已广泛应用于生命科学研究领域.本文综述了基于Cell-SELEX技术筛选的核酸适配体在肿瘤细胞检测、分析和成像方面的研究进展,并对核酸适配体研究的发展前景和趋势进行了展望.     

核酸适配体荧光探针在生化分析和生物成像中的研究进展

核酸适配体是指通过体外筛选技术从核酸文库中筛选出来,能够高特异性、高亲和力识别靶标物的寡核苷酸序列,具有靶标类型广泛、合成简单、相对分子质量小、化学稳定性高、易于进行生物化学修饰等优点。 核酸适配体能够通过折叠成特定的二维或三维构型与靶标物特异性结合,加上合适的信号转导机制,为重要靶标物的研究提供理想的分子识别与分子检测探针。 荧光检测技术具有高灵敏、高分辨率、易于实现多元分析等优点。 将核酸适配体的分子识别特性与荧光优异的光学检测性能相结合,在生命科学研究领域有着广泛的应用空间。 本文主要综述了核酸适配体荧光探针常见的分子设计和信号响应方式,及其在细胞成像、亚细胞成像中的应用研究,并对核酸适配体探针目前面临的一些挑战进行了讨论,最后对其未来的发展方向进行了展望。     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

光子晶体薄膜的制备以及在编码载体中的应用

通过将由聚苯乙烯纳米粒子构成的光子晶体膜镶嵌在聚二甲基硅氧烷(PDMS)薄膜中制备得到了具有PDMS/光子晶体/PDMS夹心结构的可用于多重生物分析的光子晶体编码载体. 用编码载体进行了大肠杆菌3种基因的杂交检测: 以3种光子晶体膜作为编码载体固定核酸探针, 然后在含有荧光标记的目标分子的缓冲液中进行杂交反应. 杂交反应后以光子晶体膜的特征反射谱为核酸编码, 以荧光信号的有无来确定目标分子的存在与否. 实验结果表明PDMS/光子晶体/PDMS夹心结构是一种有效的构建悬浮载体的方法.     

核酸适配体功能化的稀土上转换纳米材料在生物检测中的应用

稀土上转换纳米材料可以吸收近红外光并发射出可见光或紫外光,在生物传感领域得到了广泛研究。核酸适配体能高特异性和高亲和性地与靶标物结合,被广泛应用于生物传感、疾病诊断等领域。将稀土上转换纳米材料与核酸适配体结合构建的检测体系,可实现对目标物灵敏、高选择性的检测。本文介绍了近几年核酸适配体功能化的稀土上转换纳米材料在生物小分子、蛋白质、核酸、病原微生物、细胞等方面的应用,并展望了其在分析检测领域的发展前景。     

固相纳米孔对核酸组装的“免标记、均相”表征和应用探索

随着核酸自组装领域的飞速发展,除了作为遗传信息的载体外,核酸成为了一种具有高操作自由度和无限可能性的功能材料.基于核酸自组装原理的DNA纳米技术凭借其强大的可编辑性已经广泛应用于生物传感、纳米材料工程、医学诊疗以及分子计算机等领域.纳米孔作为一种新兴的单分子分析技术具有高分辨、高通量、免标记等特点,近年来在基因测序、分子物理化学性质分析等领域展示出了极大的应用潜力.作为一种新型高分辨表征技术,纳米孔已经在DNA纳米技术研究中崭露头角,被用于原位追踪和分析核酸分子的自组装行为.另一方面,DNA纳米技术也为纳米孔传感所面临的技术瓶颈提供了更多样化的解决思路,如借助功能核酸(Aptamer或DNAzyme)和无酶扩增核酸分子线路实现纳米孔对待测物的特异性增敏检测.本专论旨在通过对近期纳米孔技术与核酸自组装的跨领域研究成果进行系统性回顾,总结并展望纳米孔传感领域内核酸自组装的研究进展,以期为单分子生物分析、信息检索、基因分型和临床诊断等领域提供新思路和新方法.     

环介导扩增与石英晶体微天平快速检测核酸

建立环介导恒温扩增(LAMP)-石英晶体微天平(QCM)原位快速检测核酸的方法。将环介导恒温核酸扩增(LAMP)技术与石英晶体微天平(QCM)技术相结合,采用巯基化试剂分子组装方法,将LAMP反应体系中的4个引物之一固定于QCM电极上,在安装所述电极的QCM检测池中配置LAMP反应体系并进行环介导恒温核酸扩增,用QCM仪器在线原位检测频率变化,判断LAMP反应是否发生,进而判断体系中是否存在目标核酸特异基因。该方法检测核酸特异性强、灵敏度高,并且操作简便,有望发展成为快速筛查检测核酸的有效手段。     

核酸适体与互补核酸和目标蛋白之间竞争结合的热力学特性研究

以一种基于目标蛋白凝血酶的取代反应来研究核酸适体与互补核酸和核酸适体与目标蛋白之间竞争结合的热力学特性, 研究了核酸适体-互补核酸的解离反应和在目标蛋白存在下取代反应的平衡常数、标准焓变和标准熵变等热力学参数, 结果表明该取代反应是一个熵驱动的自发过程, 熵驱动从双链的核酸转变为核酸适体-目标蛋白的复合物. 该热力学研究会对核酸适体-互补核酸和核酸适体-凝血酶之间的结合过程的机理有一个更深的理解, 将有助于进一步揭示核酸与蛋白这两种生命中最关键物质之间的相互作用和关系, 为更好地理解基本的生物过程和预测设计适体生物传感器, 发展用于疾病诊断的方法有着重要的意义.     

稀土配合物荧光探针在活细胞成像研究中的新进展

稀土配合物荧光探针在生物学研究中,尤其在活细胞成像方面的应用渐已引起人们的关注.本文就稀土配合物的发光特点及其作为荧光探针在活细胞成像研究领域的进展进行了评述.引用文献70篇.     

In Situ Amplification‐Based Imaging of RNA in Living Cells

Owing to its important physiological functions, especially as molecular biomarkers of diseases, RNA is an important focus of biomedicine and biochemical sensing. Signal amplification detection has been put forward because of the need for accurate identification of RNA at low expression levels, which is significant for the early diagnosis and therapy of malignant diseases. However, conventional amplification methods for RNA analysis depend on the use of enzymes, fixation of cells, and thermal cycling, which confine their performance to cell lysates or dead cells, thus the imaging of RNA in living cells remained until recently little explored. In recent years, the advance of isothermal amplification of nucleic acids has opened paths for meeting this need in living cells. This minireview tracks the development of in situ amplification assays for RNAs in living cells, and highlights the potential challenges facing this field, aiming to improve the development of in vivo isothermal amplification as well as usher in new frontiers in this fertile research area.     

The challenge of long-term stability for nucleic acid-based electrochemical sensors

Nucleic acid-based electrochemical sensors are a versatile technology enabling affinity-based detection of a great variety of molecular targets, regardless of inherent electrochemical activity or enzymatic reactivity. Additionally, their modular interface and ease of fabrication enable rapid prototyping and sensor development. However, the technology has inhibiting limitations in terms of long-term stability that have precluded translation into clinically valuable platforms like continuous molecular monitors. In this opinion, we discuss published methods to address various aspects of sensor stability, including thiol-based monolayers and anti-biofouling capabilities. We hope the highlighted works will motivate the field to develop innovative strategies for extending the long-term operational life of nucleic acid-based electrochemical sensors.     

Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA‐Programmed Nanoparticle Disassembly

Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low‐abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal‐to‐background ratio of conventional molecular imaging probes. Herein, we report a class of DNA‐templated gold nanoparticle (GNP)–quantum dot (QD) assembly‐based probes for catalytic imaging of cancer‐related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA‐programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non‐catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells.     

Nucleic acids induced peptide-based AIE nanoparticles for fast cell imaging

Herein, we utilized nucleic acids induced peptide co-assembly strategy to develop novel nucleic acids induced peptide-based AIE (NIP-AIE) nanoparticles. Strong fluorescent of AIE could be observed when a little amount of nucleic acids was added into the peptide solution, and the intensity could be regulated by the concentration of nucleic acids. This AIE nanoparticle with good biocompatibility could achieve fast cell imaging. It is also proved that the fluorescence intensity of AIE decreased with time, which indicates that the reducible cross-linkers of Wpc peptide by GSH and nanoparticles gradually disintegrate in cell. Based on the different of AIE fluorescence signals which regulated by the formation and disintegration of nanoparticles, this AIE system is expected to be used for real-time monitoring of drug release from peptide-based nano carriers in vivo or in vitro, and may provide a new platform for the construction of other organic AIE nanoparticles.     

细胞内源性分子辅助荧光信号放大策略及细胞成像

细胞内原位信号放大策略是检测低丰度内源性目标物的有效手段, 但多数信号放大策略依赖于外源性辅助物, 不可避免地改变细胞内微环境, 进而对机体造成一定干扰. 针对此问题, 可利用细胞内源性物质(如金属离子、 核酸、 蛋白酶等)实现原位荧光信号放大, 对不同生物标志物进行荧光成像, 此方法对低丰度靶分子检测及成像具有重要意义. 本文对内源性物质辅助信号放大及细胞内荧光成像相关研究进行了归纳整理, 介绍了内源性核酸、 酶、 蛋白质、 三磷酸腺苷(ATP)和金属离子辅助信号放大策略, 并探讨了其信号放大机理; 总结了内源性物质辅助信号放大探针在低丰度物质检测及成像方面的研究进展; 最后展望了该策略在细胞成像方面的优势及应用前景.     

Bioanalytical applications of isothermal nucleic acid amplification techniques

The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.     

Probe Design for the Effective Fluorescence Imaging of Intracellular RNA

Over the past two decades, the spatiotemporal analysis of fluorescently labeled single RNA species has provided a broad insight into the synthesis, localization, degradation, and transport of RNA. To elucidate the dynamic behavior of functional RNAs in living cells, researchers throughout the world have proposed numerous fluorometric strategies for intracellular RNA imaging. Because, like most other biological molecules, RNA is intrinsically nonfluorescent, the development of methods for the labeling of RNAs of interest with fluorescent molecules is essential. Several artificial tag sequences have been attached onto the 3′ end of target RNAs and used as scaffolds for interacting with their fluorescent counterparts. In this Personal Account, we focus on the methods that have been developed to show how RNAs expressed in cells can be labeled and visualized by fluorescent proteins, small molecules, or nucleic acids. Each of these methods is designed to increase the sensitivity and specificity for imaging or to decrease the background fluorescence.     

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets’ copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.     

Oligonucleotide optical switches for intracellular sensing

Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or l-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.     

Recent advances in nucleic acid amplification-based optical biosensors for disease diagnosisEI北大核心CSCD

Optical biosensors have been widely used in the detection of biomarkers due to their advantages of simple operationquick responsehigh sensitivity and visualization. When constructing optical biosensors nucleic acid amplification technology can be used to improve the analytical performance of optical biosensor which can further realize the highly sensitive detection of biomarkers and provide more accurate information for disease diagnosis. In this reviewrecent advances in nucleic acid amplification-based optical biosensors for disease diagnosis were reviewed the possible problems may exist in practical applications and future development trends were proposed. © 2022, Youke Publishing Co.,Ltd. All rights reserved.