Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry , Sichuan University , Chengdu 610064 , China.
Analytical & Testing Center , Sichuan University , Chengdu 610064 , China.
Anal Chem. 2019 Jul 2;91(13):8691-8696. doi: 10.1021/acs.analchem.9b02467. Epub 2019 Jun 18.
Nature endows us with a unique toolbox of highly specific enzymes, while their detection is of great importance in biological processes. The label-free assay based on DNA-templated CuNPs is widely accepted for enzyme assay owning to its simple procedure, fast kinetic, high quantum yield, and large Stokes shift. A challenge in the application of them is the low fluorescent signal stability of DNA-templated CuNPs, whose signal sharply decreases in a few minutes after formation. In this work, a long-term stable nuclease assay is proposed by utilizing the elemental mass spectrometry detection of CuNPs. The high sensitivity was also realized, thanks to a great number of copper isotopes (Cu and Cu) intrinsically incorporated in CuNPs. The experimental conditions, including the length of polyT ssDNA template, the concentration of polyT template, the concentration of Cu, the sodium ascorbate concentration, the copper reduction reaction time, and the Exonuclease I (Exo I) digestion reaction time, were investigated in detail. The dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/μL was obtained using inductive coupled plasma mass spectrometry (ICPMS) Cu signal, with a detection limit (3σ) of 0.029 U/mL. The ICPMS Cu signal remained unchanged for at least 18 days. The spiked-recovery assay in RPMI 1640 cell medium also demonstrated the reliability of the proposed nuclease assay.
大自然赋予了我们一套独特的高度特异性酶工具包，而它们的检测在生物过程中非常重要。基于 DNA 模板化 CuNPs 的无标记测定法因其简单的程序、快速的动力学、高量子产率和大斯托克斯位移而被广泛用于酶测定。它们应用的一个挑战是 DNA 模板化 CuNPs 的荧光信号稳定性低，其信号在形成后几分钟内急剧下降。在这项工作中，通过利用 CuNPs 的元素质谱检测，提出了一种长期稳定的核酸酶测定法。由于 CuNPs 中固有地掺入了大量的铜同位素（Cu 和 Cu），因此也实现了高灵敏度。详细研究了实验条件，包括聚 T 短 ssDNA 模板的长度、聚 T 模板的浓度、Cu 的浓度、抗坏血酸钠的浓度、铜还原反应时间和 Exonuclease I（Exo I）消化反应时间。使用电感耦合等离子体质谱法（ICPMS）Cu 信号获得了 Exo I 浓度从 0.1 U/mL 到 20 U/μL 的动态范围，检测限（3σ）为 0.029 U/mL。至少 18 天内 ICPMS Cu 信号保持不变。在 RPMI 1640 细胞培养基中的加标回收测定也证明了所提出的核酸酶测定法的可靠性。
