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.     

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。     

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。     

单核苷酸多态性电化学生物传感检测研究进展

作为第3代遗传标记物,单核苷酸多态性分析在实现遗传疾病的早期医学诊断以及发病机理研究等方面具有非常重要的意义,已成为当今分析化学和生命科学研究的热点领域。电化学生物传感器因具有灵敏、检测装置轻便价廉且易于微型化、自动化等优点,近年在单核苷酸多态性检测分析中得到了迅速的发展。该文简述了单核苷酸多态性生物传感检测的识别原理、电化学生物传感检测方法及信号放大策略,综述了近年来的研究进展,并对未来的发展方向作了展望。     

基于滚环合成的聚分子信标用于 凝血酶的靶向检测

采用滚环扩增(RCA)合成得到的DNA长链打开带适配体的分子信标, 由于RCA长链上带有多个与分子信标(MB)互补的重复序列, 其打开分子信标的能力比单一互补短链提高了上百倍. 所形成的聚多价分子信标组装体, 在分子信标浓度相同的情况下, 打开后的荧光强度也大幅上升; 并且由于组装体上多价适配体的存在, 聚分子信标对凝血酶的靶向能力显著增强. 实验结果表明, 聚分子信标结合凝血酶后, 其荧光信号与凝血酶浓度呈线性关系, 检测灵敏度达到0.2 nmol/L, 该体系的构建有利于实现对凝血酶的高灵敏、 特异性检测.     

电致化学发光检测单核苷酸多态性研究

单核苷酸多态性(Single nucleotide polymorphism,SNP),主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性,它是人类可遗传的变异中最常见的一种,占所有已知多态性的90%以上。SNP在人类基因组中广泛存在,平均每500～1000个碱基对中就有1个,估计其总数可达300万个,甚至更多。SNP是遗传疾病,肿瘤等疾病的主要诊断标记物,因此,     

基于滚环扩增技术的DNA水凝胶 用于大肠杆菌O157:H7的可视化快速检测

以大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)为研究模式菌,构建了基于醛基化磁珠(Aldehyde magnetic beads,AMBs)、滚环扩增技术(Rolling circle amplification,RCA)和DNA水凝胶可视化快速检测的适配体生...     

基于磁性颗粒微阵列与双色荧光杂交的单核苷酸多态性分型方法研究

基于磁性颗粒微阵列与双色荧光杂交,建立了单核苷酸多态性(Single nucleoitide polymorphism,SNP)分型方法。将利用不对称扩增得到的含有待检测位点生物素标记的单链PCR产物固定在链亲和素修饰的金磁纳米颗粒(Gold magnetic nanoparticles,GMNPs)表面;将ssDNA-GMNPs混合物点样在底部固定有磁铁的载玻片上构建磁性颗粒微阵列,然后在基因框中与双色荧光探针杂交;杂交完全后,充分洗涤,通过扫描获得分型结果。通过优化不对称PCR的扩增条件,直接扩增出产量较高的单链DNA作为靶序列用于分型。利用本方法对24个样本MTHFR基因的C677T位点多态性进行了检测。实验证明,本方法步骤简单,易实现自动化操作、非常适用于分子诊断与法医鉴定。     

滚环扩增技术在病原微生物检测中的应用

滚环扩增（RCA）技术是一种简单的恒温DNA扩增技术,在DNA聚合酶的催化下通过扩增闭合环状模板产生成千上万的重复序列。相较于变温核酸扩增技术如聚合酶链式反应（PCR）,RCA无需昂贵的变温仪器,更适合现场检测。该文介绍了RCA技术的原理和分类,综述了其在细菌、病毒以及其它病原微生物检测方面的应用现状,并展望了RCA检测病原微生物的应用前景。RCA在检测病原微生物领域有着巨大潜力,同时可为新型冠状病毒（SARS－CoV－2）的快速检测提供思路和补充。     

Highly Sensitive Determination of microRNA Using Target‐Primed and Branched Rolling‐Circle Amplification

One‐nucleotide differences in microRNAs (miRNAs) can be discriminated in an assay based on a branched rolling‐circle amplification (BRCA) reaction and fluorescence quantification. With the proposed method miRNA can be detected at concentrations as low as 10 fM , and the miRNA in a total RNA sample of a few nanograms can be determined.

     

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.     

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.     

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.     

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.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

Target detection by the naked eye : The action of an RNA‐cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single‐stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex‐binding dye, the color of the dye changed from blue to purple.