综述或非传感器论文 2008 非传感器论文

Catalytic DNAzymes: derivations and functions.

Expert opinion on biological therapy Pan W, Clawson GA
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Catalytic DNAzymes: derivations and f... 传感器构成示意图

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传感器类型

综述或非传感器论文

检测对象

铅离子(Pb2+,水样/溶液)、铀酰离子(UO2 2+,水样)、铜离子(Cu2+,水溶液)、腺苷酸(AMP,溶液)、溶菌酶(lysozyme,溶液)、葡萄糖(glucose,溶液)、凝血酶(thrombin,溶液)、DNA(溶液)、端粒酶活性(telomerase activity,HeLa细胞)

检测原理

本文综述的DNAzyme传感器通常以催化DNA酶(CDz)为核心识别/催化元件。金属离子(如Pb2+、UO2 2+)可直接结合并激活17E、39E等DNAzyme,使其切割含单RNA连接的底物;小分子或蛋白(如AMP、lysozyme)则先与DNA适配体(aptamer)结合,引起构象变化并释放/暴露DNAzyme底物。被激活的DNAzyme催化切割荧光-淬灭剂标记底物,使荧光基团与淬灭剂分离,荧光增强;或hemin-过氧化物酶DNAzyme催化化学发光底物产生发光信号。由于DNAzyme可多次周转切割,信号随被测物浓度增加而增强,部分体系还通过PCR诱导DNAzyme生成或适配体-DNAzyme自组装实现放大。

检测灵敏度

17E-Pb2+:线性范围: 10 nM–4 µM;39E-UO2 2+:LOD: 45 pM;动态范围: up to 400 nM;Cu2+:LOD: 35 nM;peroxidase CDz-DNA:LOD: 1 × 10^-9 M;telomerase:LOD: ~1000 cells;glucose:LOD: 5 × 10^-6 M;thrombin:LOD: 10^-8 M;M13 phage DNA:LOD: 10^-18 M;AMP:LOD: 10^-6 M;lysozyme:LOD: 10^-13 M

效应效果

综述报道的DNAzyme传感器选择性/灵敏度较高:17E对Pb2+选择性>80倍;39E检测UO2 2+ LOD 45 pM、动态范围至400 nM;Cu2+灵敏度35 nM。过氧化物酶DNAzyme可检测DNA(1×10^-9 M)、M13 DNA(10^-18 M)、AMP(10^-6 M)、lysozyme(10^-13 M)、glucose(5×10^-6 M)、thrombin(10^-8 M)及HeLa端粒酶(约1000细胞)。10-23可区分单碱基错配,kcat/Km 4×10^9 M^-1 min^-1,较锤头/发夹核酶高1-2个数量级。作者认为CDz临床前景有限,但稳定、可体外选择、适合芯片化与严苛环境,传感/纳米应用潜力大。

传感器的构成

  • 固定化基底:金表面(gold surfaces)、碳纳米管(CNTs)、金涂层纳米毛细管膜(gold-coated nanocapillary membranes)或溶胶-凝胶材料(sol-gel-derived materials),用于固定DNAzyme/催化信标。
  • 催化/识别元件:17E、39E、DET22-18、MgZ等催化DNA酶(CDz/DNAzyme),结合金属离子或经适配体变构后切割单RNA连接。
  • 识别辅助元件:DNA适配体(aptamer,如ATP结合适配体、anti-AMP/lysozyme aptamer),与目标结合后释放或激活DNAzyme底物。
  • 信号标记物:荧光基团-淬灭剂对(fluorophore–quencher pair)或hemin-过氧化物酶CDz(PS2.M/PS5.ST1),产生荧光增强或化学发光。
  • 换能读出:荧光或化学发光信号(fluorescence/chemiluminescence),随被测物浓度变化而增强。

中文摘要

催化RNA酶(CRz)在多种生物体中天然存在,而催化DNA酶(CDz/DNAzyme)则通过随机序列库的体外选择/进化获得。本文简要概述CDz的体外直接选择方法、理化性质与功能,并讨论其未来应用。作者重点介绍直接选择策略,即利用目标催化事件本身从随机DNA库中筛选具有催化活性的序列,并讨论序列多样性引入、突变、重组、DNA再扩增及进化群体分析。CDz可催化RNA或DNA切割、自磷酸化、自帽化、自脱嘌呤、N-糖苷键切割、胸腺嘧啶二聚体光修复、过氧化物酶反应以及DNA/RNA连接等反应。虽然CDz曾用于下调靶mRNA,但作为核酸药物的临床前景已被反义寡核苷酸和小干扰RNA(siRNA)取代;作者认为其临床价值有限,但在传感器和纳米技术领域具有显著潜力。

英文摘要

BACKGROUND: Although catalytic RNA enzymes (CRzs) are naturally occurring in many organisms, their DNA counterparts (CDzs) were developed by in vitro selection/evolution from random sequence libraries. OBJECTIVE: To provide a brief overview of how CDzs have been selected in vitro, and of their properties and functions, as well as their possible future utility. METHODS: We concentrated on examples of 'direct' selection of CDzs. Many CDzs have been used in biological settings, for example downregulation of target mRNAs, while many more recent applications use CDzs in biosensor and nanotechnology settings. CONCLUSIONS: Although much work has concentrated on using CDzs for regulating gene expression, their potential as nucleic acid medicines has diminished substantially, supplanted by simple antisense oligonucleotides and, more recently, by small interfering RNAs (siRNAs). It seems unlikely that CDzs will have clinical utility. In contrast, they are likely to have significant potential in the sensor/nanotechnology arena.

关键词

催化DNA酶DNAzyme体外选择SELEX生物传感器核酸催化