Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers
Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers
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DOI：
10.1021/ac034924r
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发表时间：
2003-12-01
影响因子：
7.4
通讯作者：
Lu, Y
中科院分区：
文献类型：
--
作者：
Liu, JW;Lu, Y
以前报道的一种基于DNAzyme的Pb2+生物传感器在4℃下表现出高的灵敏度和选择性。在该体系中，DNAzyme的底物和酶链分别标记有荧光团和猝灭剂。在Pb2+存在下，底物链被酶链切割，切割片段的释放导致荧光显著增强。但是，当温度升高到室温时，由于背景荧光很强，传感器的性能会大大降低。对传感器系统的仔细分析，包括熔化曲线的测量和自由底物的荧光共振能量转移(FRET)研究表明，部分荧光团标记的底物链从酶链上解离，导致室温下背景荧光信号增强。为了克服这个问题，我们设计了一种新的传感器系统，通过引入分子间和分子内猝灭剂。该设计得到了FRET研究的帮助，该研究表明，解离的底物保持随机卷曲构象，端到端的距离接近39埃，比完全延伸的DNA短得多。通过这种新的设计，背景荧光被显著抑制，与先前设计的60%相比，荧光强度增加了660%。这种背景荧光信号的抑制是在不损失传感器的选择性的情况下实现的。新的设计使得传感器可以在很宽的温度范围内用于实际应用。本文提出的设计原则也适用于其他核酸生物传感器，以降低背景荧光。
A previously reported DNAzyme-based biosensor for Pb2+ has shown high sensitivity and selectivity at 4degreesC. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb2+, the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of similar to39 Angstrom, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.