适配体生物传感器在病原微生物检测中的应用

适配体是通过指数富集系统进化技术(SELEX)体外筛选得到的一类能够特异性地结合小分子物质、蛋白,甚至整个细胞的寡核苷酸序列.由于具有制备简便、易于修饰、稳定性好等特点,适配体已广泛应用于构建生物传感器,实现对病原微生物的识别和检测.本文在阐述适配体基本原理的基础之上,结合近年来病原微生物适配体研究领域的最新研究成果,综述以病原微生物为目标的适配体筛选技术的最新进展;列举目前已经筛选获得的病原微生物(原生生物、病毒、细菌)适配体;综述适配体生物传感器在病原微生物检测中的应用.并展望了适配体生物传感器在病原微生物检测领域的发展趋势.     

病原微生物的核酸检测分析研究进展

2019年12月,全球爆发新型冠状病毒肺炎疫情(COVID-19),使生物安全面临着巨大危机.病原微生物的快速、准确、低成本检测是保障生物安全的重要组成元素之一,是疫情预防、控制和诊断的关键.本综述介绍了核酸测序技术、等温扩增技术和基因编辑技术(CRISPR/Cas)于便携式一体化设备中的应用,为研发"低成本,高效率,...     

滚环扩增介导的核酸信号放大策略在生物传感器中的应用研究进展

滚环扩增(RCA)是一种等温核酸扩增技术,因其具有扩增效率高、保真度高等特点,在生物传感器领域得到了广泛的应用。该文介绍了RCA技术的基本原理、RCA环状模板环化方式和RCA的扩增类型,并对基于RCA技术的荧光、比色以及电化学生物传感器进行了介绍,旨在为RCA技术在生物传感器领域的进一步发展和应用提供参考。     

支化滚环扩增均相光散射检测单核苷酸多态性

结合简便、高效的支化滚环扩增反应与光散射技术,建立了一种均相、无标记检测单核苷酸多态性的方法.通过设计与突变型DNA完全匹配的锁式探针,突变型DNA可作为模板,使锁式探针在连接酶作用下于45℃连接成环,由Phi29 DNA聚合酶在33℃引发支化滚环扩增反应,生成大量的长链DNA产物.反应产物在0.2 mol/L HCl...     

荧光纳米材料在病原微生物检测中的应用

病原菌污染给人类的健康带来极大的安全隐患,对病原菌快速、准确和灵敏的检测是减少污染的重要手段。传统检测病原菌的方法存在耗时长和操作繁琐等缺点。荧光纳米材料具有荧光强度高、稳定性好以及良好的生物相容性等优势,为应用其构造传感器用于病原菌检测提供了新的研究途径。本文对近年来常见荧光纳米材料,包括半导体量子点、金属纳米簇、碳纳米材料、上转换纳米粒子和荧光硅纳米颗粒,在病原菌检测方面的应用进行了概述,着重将不同类别荧光纳米材料的光学性质和检测机理进行了分析和比较。纳米材料的生物修饰是实现病原菌特异性识别的重要环节,本文对抗体、适配体、噬菌体和抗生素等病原菌识别方式的特点及其与纳米材料的连接方式进行了介绍。最后对不同荧光纳米材料在检测病原菌中具有的优势和局限性进行了总结,并对其在未来的应用与研究重点进行了展望。     

滚环扩增型压电石英晶体传感器检测乙型肝炎病毒

通过捕获探针与纳米金膜之间的共价连接, 保证了滚环扩增(RCA)产物始终结合于金膜表面, Phi29 DNA聚合酶的高效扩增和Escherichia coli DNA链接酶的高度精确性使检测达到单碱基识别, 检测灵敏度达到10⁴ copies/mL. 实验结果表明, 与单碱基错配序列相比, RCA可明显增强检测的灵敏度. 该RCA基因传感器操作简单, 灵敏度和特异性较高, 在乙型肝炎病毒的快速检测方面具有一定的开发潜力.     

化学发光免疫分析技术在微生物检测中的应用

化学发光免疫分析技术凭借其化学发光的高灵敏性和免疫反应的高特异性在微生物快速检测中广泛应用。该文着重叙述了化学发光免疫分析技术核心检测体系在微生物检测中的研究进展,并对其涉及到的样品前处理和检测新方法进行了综述。化学发光免疫分析技术检测微生物用时短、成本低,但特异性识别方法和检测的灵敏度有待提升,新型的发光体系、发光放大方法、可替代抗体的识别分子以及相关的前处理技术是未来研究的重点。     

食源性致病菌等温扩增检测技术的研究进展

快速可靠的食源性致病菌检测方法是保障食品安全的关键。然而,传统培养法的检测周期过于冗长,无法适应对时间敏感度较高的现场或在线检测。为此,研究人员开发了多种以快速和高灵敏度为主要特征的等温扩增技术及相应的检测产品,为食源性致病菌的现场或在线检测提供了强有力的技术支撑。该文对环介导等温扩增、滚环扩增、链置换扩增、切口酶信号扩增、核酸外切酶Ⅲ辅助扩增5种等温扩增方法的原理及其优缺点进行了梳理和比较。在此基础上,分析了等温扩增方法在细菌活性识别、非特异性扩增、前处理效率、检测通量4个方面所遇到的共性问题,并结合作者已有工作,提出了解决这些问题的方法或策略。     

Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Self‐assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology, the emerging of scaffolded DNA origami is marvelous. It utilizes DNA double helix inherent specificity of Watson‐Crick base pairing and structural features to create self‐assembling structures at the nanometer scale exhibiting the addressable character. However, the assembly of DNA origami is disorderly and unpredictable. Herein, we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers. Firstly, long single‐stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs. Subsequently, the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons. By mixing them up, we illustrate the one‐dimensional even two‐dimensional assembly of DNA origami with good orientation.     

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.     

Optimal DNA Templates for Rolling Circle Amplification Revealed by In Vitro Selection

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC‐rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.     

DNA的滚环扩增合成研究

滚环扩增(RCA)反应作为一种简单高效的等温酶促反应,现已发展为核酸扩增领域的新技术,其产物在组装体搭建和多功能材料的制备方面有着广泛的应用。本文采用琼脂糖凝胶、紫外和透射电镜(TEM)等手段,探究了时间、三磷酸脱氧核糖核苷(dNTPs)、酶以及引物的浓度等因素对脱氧核糖核酸(DNA)滚环扩增产物的影响。结果表明:在反应开始的前30 min,RCA产物的长度受时间的影响比较明显;随着dNTPs浓度的提高,RCA产物的链长增长,浓度也不断提高;酶和引物的浓度对滚环扩增产物的长度没有明显影响,但对RCA产物浓度的影响较大,过量的酶致使RCA产物的含量显著下降。