基于纳米金和脱氧核酶荧光探针检测铅离子

本文以"8-17"脱氧核酶为识别靶标的配体,设计了一种基于纳米金(AuNPs)和脱氧核酶的荧光信号放大法检测Pb2+的探针。"8-17"脱氧核酶由一条底物链(17DS)和一条酶链(17E)组成。在底物链的一端修饰荧光基团6-羧基荧光素(FAM),而酶链通过巯基修饰到AuNPs表面。当底物链与酶链相混合时,酶链与底物链杂交,AuNPs与FAM靠近,导致FAM的荧光被AuNPs猝灭。当向该体系加入Pb2+时,"8-17"脱氧核酶被Pb2+激活,酶链将底物链剪切为两段,破坏了杂交的刚性结构,从而使得FAM的荧光恢复。基于此原理,构建了一种定量检测Pb2+的高灵敏传感器,该传感器对Pb2+的检测限达0.6nmol/L。     

核酸荧光探针检测铅离子的研究

将8-17 DNAzym e增加2个G-C碱基对进行增强热稳定性的结构修饰,并标记上1个荧光基团FAM和2个荧光猝灭基团Dabcyl,设计成双猝灭Pb2+荧光探针。研究了该探针对Cd2+、Zn2+、Mg2+、Cu2+、Mn2+、Pb2+6种二价金属离子的响应,结果表明探针对Pb2+具有很强的特异性,在探针浓度为2.5×10-7mol/L时,Pb2+浓度在8.5×10-8~7.5×10-6mol/L范围内和探针的荧光强度呈线性关系,检出限为8.5×10-8mol/L。该探针可用于Pb2+的定性和定量检测。     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

基于外源荧光探针的脱氧核酶切割RNA反应动力学参数测定方法的改进

对以EB作为外源荧光探针测定脱氧核酶切割RNA反应的动力学参数的方法进行了改进。实验结果表明：对于目前应用最为广泛的10-23脱氧核酶和8-17脱氧核酶,在生理条件下(150mmol／L KCl,2mmol／L MgCL2,pH7．5,37℃)采用改进方法测得的反应动力学参数(kobs 10-23=0．12min^-1,kobs 8-17=0．08min^-1)与原方法相比更接近于放射性标记方法的测定结果(k2 10-23=0．13min^-1,k2 8-17=0．08min^-1)。     

基于脱氧核酶的重金属离子荧光生物传感器的研究进展

近年来,重金属离子造成严重的水体和土壤污染,极大威胁着人类健康。因此,开发快速简便的重金属离子检测新技术对于食品工业、环境监测等具有重要的意义。基于脱氧核酶的重金属离子荧光生物传感器因其灵敏度高、检测快速且能实时现场进行检测等优点引起国内外研究者的广泛关注。本文就近年来文献中报道的基于脱氧核酶的各类重金属离子荧光生物传感器的构建及其在实际中的应用进行了综述,并对其未来的发展前景进行了展望。     

脱氧核酶传感器研究进展

脱氧核酶是利用体外分子进化技术获得的一类具有高效催化活性和结构识别能力的单链DNA片段。本文主要介绍了两类脱氧核酶,即RNA切割型脱氧核酶和G-四聚体型脱氧核酶在生物传感体系中的发展和应用。     

基于核酸外切酶Ⅲ及DNAzyme的铅离子荧光传感器的研究

利用DNAzyme及核酸外切酶Ⅲ(ExoⅢ)构建了荧光生物传感器用于检测铅离子(Pb²⁺)。DNAzyme与底物探针结合并在Pb²⁺辅助作用下切割底物,使发夹结构的底物探针在环上修饰有RNA碱基处断裂,切割断裂底物探针后,DNAzyme被释放,继续与下一个底物探针结合并切割,利用DNAzyme可循环反复催化裂解底物的特性实现循环反应。底物探针被切割断裂后,形成的Y字形探针可与信标探针结合并打开其发夹结构,产生荧光信号,同时在ExoⅢ的作用下降解,从3′端开始切割水解被打开的信标探针,释放出底物探针,继续与下一个信标探针结合切割,形成第二步的循环信号放大。经过两步的循环反应,荧光信号得到不断增强,从而达到高灵敏检测的目的。200μL反应体系在37℃反应60 min后,其荧光信号与Pb²⁺浓度在0.05～200 nmol/L范围内呈良好的线性关系,检出限为0.01 nmol/L。用于实际样品中Pb²⁺的检测,加标回收率为96.3%～108.3%。本方法具有简单、快速、高选择性、高灵敏的特点,在Pb     

基于脱氧核酶的无标记端粒酶检测新方法

设计了一种发卡型核酸探针,结合脱氧核酶(DNAzyme)与支点介导链置换技术建立了一种检测端粒酶的新方法.该发卡型探针通过阻碍G-四链体的形成来抑制DNAzyme的过氧化物酶活性.当体系中的端粒酶引物TS被催化延伸后,可以通过链置换反应破坏该发卡结构,从而释放出自由的DNAzyme以催化过氧化氢氧化ABTS2-,产生可被检测的吸收信号变化.实验结果表明,应用该方法可以检测低至500个Hela细胞等当量的端粒酶,且该方法操作简单、不需要荧光标记和复杂的表面修饰,有望在肿瘤细胞端粒酶活性分析中获得广泛应用.     

多功能脱氧核酶用于生物医学分析的研究进展

脱氧核酶(DNAzymes)是一类人工合成的通过指数富集式配体系统进化技术(SELEX)筛选得到的具有催化功能的单链DNA分子. 由于DNAzyme具有易于合成和修饰、 化学结构稳定及催化活性高等优点, 近年来在生物传感和医学诊断领域备受关注. 对DNAzyme的活性进行调控是挖掘其多方面应用潜能的关键, 灵活的活性调控方式将促进DNAzyme在不同领域的应用. 本文综合评述了一些调控脱氧核酶活性的主要方法, 并对其在生物医学分析领域方面的应用进行了简要介绍.     

RNA切割型脱氧核酶在致病菌检测中的研究进展

脱氧核酶是通过体外筛选技术得到的具有催化功能的单链寡核苷酸分子. 随着体外筛选技术的发展, 越来越多的脱氧核酶被筛选出来. 其中, 靶向细菌的具有RNA切割作用的脱氧核酶(RNA-cleaving DNAzyme, RCD)同时拥有分子识别和催化RNA裂解的功能, 且具有设计灵活、 易于制备和修饰、 成本低廉等优势, 在致病菌检测领域获得了广泛的关注和应用, 为致病菌的早期发现和预防提供了全新的分子工具. 本文系统总结了RCD在致病菌检测中的研究和应用, 并对其面临的问题和未来发展前景进行了展望.     

基于核酸酶的新型阻抗传感器检测纺织品中的铅

建立一种基于核酸酶的电化学阻抗传感器检测纺织品中铅含量的方法。采用金纳米粒子修饰的金电极作为基底电极,以一种新型的核酸酶17EDNAzyme体系作为铅离子的生物识别元件,构建了一种新型的电化学阻抗传感器用于高灵敏选择性检测铅离子。在最佳实验条件下,传感器对溶液中铅离子的线性检测范围为1.0μg/L~10mg/L,检出限为0.6μg/L。测定结果的相对标准偏差小于5.4%(n=6),加标回收率在93.5%~106.0%之间。采用该法对实际纺织品样品进行测试,并与国标方法测定值进行对比,实验结果表明两种方法的测定值无显著性差异。该方法操作简便,对铅离子的检测具有良好的选择性,可用于现场样品的在线、快速测定。     

荧光法检测重金属铅离子的研究进展北大核心CSCD

铅作为一种重金属,广泛应用于工业生产,对环境和人体健康具有显著影响。因此,开发铅离子检测技术是一项具有重要意义的研究内容。荧光法与传统重金属离子检测方法相比,具有灵敏度高、选择性好等优点,故荧光法常用于水体等实际样品中重金属离子的定性或定量分析。本文围绕近几年报道的基于荧光法检测铅离子的研究现状进行介绍,包括荧光染料、荧光纳米材料、荧光生物材料包括荧光蛋白等3种检测材料,并在此基础上提出荧光法检测铅离子领域面临的主要挑战,对未来的研究趋势进行了展望。     

A signal-on electrogenerated chemiluminescent biosensor for lead ion based on DNAzyme

A highly reproducible and sensitive signal-on electrogenerated chemiluminescence (ECL) biosensor based on the DNAzyme for the determination of lead ion was developed. The ECL biosensor was fabricated by covalently coupling 5′-amino-DNAzyme-tagged with ruthenium bis (2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-ethylenediamine (Ru1-17E′) onto the surface of graphite electrode modified with 4-aminobenzoic acid, and then a DNA substrate with a ribonucleotide adenosine hybridized with Ru1-17E′ on the electrode. Upon binding of Pb²⁺ to the Ru1-17E′ to form a complex which catalyzed the cleavage of the DNA substrate, the double-stranded DNA was dissociated and thus led to a high ECL signal. The signal linearly increases with the concentration of Pb²⁺ in the range from 5.0 to 80 pM with a detection limit of 1.4 pM and a relative standard derivation of 2.3%. This work demonstrates that using DNAzyme tagged with ruthenium complex as an ECL probe and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity, good stability and significant regeneration ability is promising approach.     

A Novel Fluorescent Probe for Lead Ions

A new fluorescent probe for lead ions, p-nitrophenyl 3H-phenoxazin-3-one-7-yl phosphoric acid (NPPA), has been synthesized by linking resorufLn (serving as a fluorophore and electron acceptor) to p-nitrophenol (serving as a fluorescence quencher and electron donor) through phosphodiester bonds. When NPPA was irradiated with light, intramolecular fluorescence self-quenching took place due to the PET (photoinduced electron transfer) from the donor to the acceptor. However, upon addition of Pb^Ⅱ, the phosphate ester bonds in the probe were cleaved and the fluorophore was released, accompanying the retrievement of fluorescence.     

Photoactivatable Engineering of CRISPR/Cas9-Inducible DNAzyme Probe for In Situ Imaging of Nuclear Zinc Ions

DNAzyme-based fluorescent probes for imaging metal ions in living cells have received much attention recently. However, employing in situ metal ions imaging within subcellular organelles, such as nucleus, remains a significant challenge. We developed a three-stranded DNAzyme probe (TSDP) that contained a 20-base-pair (20-bp) recognition site of a CRISPR/Cas9, which blocks the DNAzyme activity. When Cas9, with its specialized nuclear localization function, forms an active complex with sgRNA within the cell nucleus, it cleaves the TSDP at the recognition site, resulting in the in situ formation of catalytic DNAzyme structure. With this design, the CRISPR/Cas9-inducible imaging of nuclear Zn²⁺ is demonstrated in living cells. Moreover, the superiority of CRISPR-DNAzyme for spatiotemporal control imaging was demonstrated by integrating it with photoactivation strategy and Boolean logic gate for dynamic monitoring nuclear Zn²⁺ in both HeLa cells and mice. Collectively, this conceptual design expands the DNAzyme toolbox for visualizing nuclear metal ions and thus provides new analytical methods for nuclear metal-associated biology.     

胞苷保护的铜纳米簇新型荧光探针检测重铬酸根离子

基于重铬酸根离子(Cr_2O_7~(2-))对胞苷保护的荧光铜纳米簇(Cu NCs)的猝灭作用,构建了一种可用于检测Cr_2O_7~(2-)的荧光传感器.实验结果表明,该传感体系检测Cr_2O_7~(2-)的线性范围为0.05~7.0μmol/L,检出限为24 nmol/L(S/N=3).该传感器对Cr_2O_7~(2-)的检测具有良好的选择性,可用于湖水样中Cr_2O_7~(2-)的检测.     

Paper-based scanometric assay for lead ion detection using DNAzyme

A facile and simple paper-based scanometric assay was developed to detect Pb²⁺ using GR5-DNAzyme. Magnetic beads (MBs) and gold nanoparticles (AuNPs) were used as a signal collector and a signal indicator, respectively. They were linked together by GR5-DNAzyme, comprising an enzyme and a substrate strand pairing up with each other. In the presence of Pb²⁺, the substrate strand is cut into two pieces, resulting in the disassembly of AuNPs from the MBs. These AuNPs were spotted on predefined areas on a chromatography paper, where signal is amplified through silver reduction. This sensing platform exhibits high sensitivity and selectivity toward Pb²⁺, giving a detection limit of 0.3 nM and a linear fitting range from 0.1 to 1000 nM. Testing of this biosensor in river water and synthetic urine samples also showed satisfying results. Besides offering simultaneous and multi-sample analysis, this paper-based sensing platform presented here could be potentially applied and served as a general platform for on-site, naked eyes, and low-cost monitoring of other heavy metal ions in environmental and body fluid samples.     

基于铜纳米簇的聚集诱导发光检测铅离子

基于谷胱甘肽保护的非贵金属铜纳米簇(Cu NCs@GSH)的聚集诱导发光现象,建立了快速检测铅离子(Pb2+)的“Turn on”型荧光分析方法.Cu NCs@GSH溶液荧光强度很弱,当存在pb2+时,荧光强度可显著增强,溶液显示明亮的橙黄色.基于此原理建立了检测pb2+的荧光方法,线性范围为200～700 μmol/L,检出限为106 μmol/L,常见金属离子不干扰pb2+的测定.本方法简单快速、选择性高,可实现对pb2+的可视化定性检测.