A highly sensitive and selective catalytic DNA biosensor for lead ions
A highly sensitive and selective catalytic DNA biosensor for lead ions
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DOI：
10.1021/ja0021316
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发表时间：
2000-10-25
影响因子：
15
通讯作者：
Lu, Y
中科院分区：
文献类型：
--
作者：
Li, J;Lu, Y
最近催化活性dna（脱氧核酶）1的发现引起了广泛的兴趣，它们作为简单、稳定、经济的蛋白质和核酶的替代品在生化和制药领域的应用。我们在此报道了催化dna作为金属离子（特别是Pb2+）的一类独特生物传感器的新应用，其可量化的检测范围从10 nM到4 μM，对Pb2+的选择性是其他金属离子的80倍。铅是一种常见的环境污染物。低水平铅接触可导致许多不利的健康影响。4血液中的铅含量在480nm时被认为是有毒的。目前的铅检测方法，如原子吸收光谱法，6电感耦合等离子体质谱法，7和阳极溶出伏安法，8往往需要复杂的设备或样品处理。简单而廉价的实时Pb2+采样方法在环境监测、临床毒理学、废水处理和工业过程监测等领域具有重要意义。基于荧光标记的有机螯合剂、9、10蛋白、11-13或肽14、15的荧光传感器已成为实现上述目标的有力工具。虽然在开发用于Ca2+ 9、12和Zn2+等金属离子的氟传感器方面取得了显著进展，但设计和合成敏感和选择性金属离子氟传感器仍然是一个重大挑战。也许在氟传感器研究中最大的挑战是设计和合成一种能够特异和强金属结合的传感器。由于我们对金属结合位点构建的了解有限，因此以组合方式寻找传感器具有重要价值。在这方面，从1014-1015个随机DNA/RNA序列文库中体外选择DNA/RNA提供了相当大的机会。1,3与化学传感器和肽基传感器的组合搜索相比，体外选择DNA/RNA能够采样更大的序列池，通过聚合酶链反应（PCR）扩增所需序列，并通过诱变PCR引入突变以提高性能。例如，体外选择方法已被用于获得对小有机分子有反应的DNA/RNA适配体17、18和适配酶19。同样，也获得了对Pb2+， 1,20, Cu2+， 21和Zn2+ 22, 23具有高度特异性的核酶/脱氧核酶。利用DNA/RNA适体转导小有机分子的分子识别以改变荧光强度最近已被证明。18还设计了一种灵敏的脱氧核酶/荧光团系统，用于检测和定量临床标本中的核酸。这些结果为利用具有水解裂解活性的脱氧核酶检测金属离子奠定了基础。本研究选择脱氧核酶，因为它能够通过金属离子的分子识别和金属离子依赖的水解裂解活性来转导信号，从而具有潜在的宽检测范围和高灵敏度。本研究选择了一种体外选择的脱氧核酶（称为17E），它能够在DNA底物（称为17DS） 22中切割单个RNA连锁（图1a）。通过3种不同的体外筛选过程获得相同的17E序列基序，分别为10 mM Mg2+、2 0.5 mM Mg2+/50 mM组氨酸、25或100 μM Zn2+， 22种活性为Zn2+。Ca2+> Mg2+在相似条件下。22,26进一步的分析表明该酶具有高度依赖Pb2+的活性。
The recent discovery of catalytically active DNAs (deoxyribozymes) 1 has led to a wide-spread interest in their use as simple, stable, and cost-effective alternatives to proteins and ribozymes in biochemical and pharmaceutical applications. 2, 3 We report here a new application for catalytic DNAs as a unique class of biosensors for metal ions, specifically Pb2+, with a quantifiable detection range from 10 nM to 4 μM and a selectivity of> 80-fold for Pb2+ over other metal ions. Lead is a common environmental contaminant. Low-level lead exposure can lead to a number of adverse health effects. 4 The lead level in the blood is considered toxic when it is g480 nM. 5 Current methods for lead detection, such as atomic absorption spectrometry, 6 inductively coupled plasma mass spectrometry, 7 and anodic stripping voltammetry, 8 often require sophisticated equipment or sample treatment. Simple and inexpensive methods that permit real-time sampling of Pb2+ are important in the fields of environmental monitoring, clinical toxicology, wastewater treatment, and industrial process monitoring. Fluorosensors based on fluorescently labeled organic chelators, 9, 10 proteins, 11-13 or peptides14, 15 have emerged as powerful tools toward achieving the above goals. 16 While remarkable progress has been made in developing fluorosensors for metal ions such as Ca2+ 9, 12 and Zn2+, 13, 14 designing and synthesizing sensitive and selective metal ion fluorosensors remains a significant challenge. Perhaps the biggest challenge in fluorosensor research is the design and synthesis of a sensor capable of specific and strong metal-binding. Since our knowledge about the construction of metal-binding sites is limited, searching for sensors in a combinatorial way is of significant value. In this regard, in Vitro selection of DNA/RNA from a library of 1014-1015 randomDNA/RNA sequences offers considerable opportunity. 1, 3 Compared with combinatorial searches of chemosensors and peptidyl sensors, in Vitro selection of DNA/RNA is capable of sampling a larger pool of sequences, amplifying the desired sequences by the polymerase chain reaction (PCR), and introducing mutations to improve performance by mutagenic PCR. For example, the in Vitro selection method has been used to obtain DNA/RNA aptamers17, 18 and aptazymes19 that are responsive to small organic molecules. Similarly, ribozymes/deoxyribozymes that are highly specific for Pb2+, 1, 20 Cu2+, 21 and Zn2+ 22, 23 have been obtained. The use of DNA/RNA aptamers to transduce the molecular recognition of small organic molecules to a change in fluorescence intensity has been demonstrated recently. 18 A sensitive deoxyribozyme/fluorophore system was also designed to detect and quantify nucleic acids in clinical specimens. 24 These results set the stage for the utilization of deoxyribozymes with hydrolytic cleavage activity for detection of metal ions. A deoxyribozyme is chosen for this study because it is capable of transducing signals through both molecular recognition of metal ions and metal-iondependent hydrolytic cleavage activity, resulting in a potentially wide detection range and high sensitivity. An in Vitro-selected deoxyribozyme (termed 17E) that is capable of cleaving a single RNA linkage within a DNA substrate (termed 17DS) 22 (Figure 1a) was chosen for this study. The same 17E sequence motif was obtained from three different in Vitro selection processes involving 10 mM Mg2+, 2 0.5 mM Mg2+/50 mM histidine, 25 or 100 μM Zn2+, 22 with activity in the order of Zn2+. Ca2+> Mg2+ under similar conditions. 22, 26 Further assays of this enzyme indicate a highly Pb2+-dependent activity …