Key Laboratory of Coastal Zone Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS), Yantai, Shandong 264003, PR China.
Biosens Bioelectron. 2013 Sep 15;47:559-65. doi: 10.1016/j.bios.2013.03.066. Epub 2013 Apr 6.
A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities.
本文报道了一种简单、通用且无需标记的电势法，可在均相溶液中使用聚阳离子敏感膜电极测量核酸酶活性和氧化 DNA 损伤。鱼精蛋白是一种线性聚离子物质，可作为报告物来报告核酸酶（如限制性内切酶和非特异性核酸酶）对 DNA 的切割，以及羟基自由基对 DNA 的损伤。可以采用滴定模式或直接检测模式进行测量。在电势滴定模式下，酶促切割会显著影响 DNA 和鱼精蛋白之间的静电相互作用，从而改变用于鱼精蛋白对 DNA 的电势滴定的响应曲线。在优化条件下，S1 核酸酶的检测限为 2.7×10(-4)U/µL，DNase I 的检测限为 3.9×10(-4)U/µL，可实现对酶活性的电势检测。在直接检测模式下，鱼精蛋白与 DNA 的复合物可用作底物。感兴趣的核酸酶将 DNA 从鱼精蛋白/DNA 复合物中切割成较小的片段，从而产生游离的鱼精蛋白，并可通过聚阳离子敏感膜电极进行电势检测。采用直接测量法，S1 核酸酶的检测限为 3.2×10(-4)U/µL，DNase I 的检测限为 4.5×10(-4)U/µL，可快速检测核酸酶活性。此外，所提出的电势测定法证明了在检测羟基自由基方面的潜在应用。预计这种新的电势策略将为核酸酶、活性氧物种以及具有潜在抑制能力的药物的高通量筛选提供一个有前途的平台。
