Developing biosensors for Ca²⁺ detection is an important yet challenging analytical task, since it is quite difficult to distinguish Ca²⁺ from Mg²⁺, Cd²⁺ and even lanthanides in some cases. In addition to small molecule and protein based Ca²⁺ probes, DNA has recently emerged as a powerful platform for metal sensing. DNAzymes are DNA-based catalysts, and DNAzyme-based sensors are highly attractive due to their high stability, low cost and excellent sensitivity. EtNa is a recently reported RNA-cleaving DNAzyme that has high selectivity for Ca²⁺ over Mg²⁺ and Na⁺. In this work, a systematic mutation study was performed on EtNa, aiming to further understand its biochemical features and improve sensor performance. Among the 45 tested mutants, EtNa-C5T showed higher activity and even better Ca²⁺ selectivity compared to the original EtNa. The selectivity for Ca²⁺ over Na⁺ improved by ∼20-fold. In the presence of 1 mM Ca²⁺, C5T has a cleavage rate of 0.07 min⁻¹. A catalytic beacon sensor was designed based on the C5T mutant with a detection limit of 11 μM Ca²⁺. This sensor was used to measure Ca²⁺ in five commercial milk samples and the results were compared to those from ICP analysis.

中文翻译：

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DNAzyme突变体的筛选，用于高灵敏度和选择性检测牛奶中的钙

开发用于Ca ²⁺检测的生物传感器是一项重要而又具有挑战性的分析任务，因为在某些情况下很难将Ca ²⁺与Mg 2 ⁺，Cd ²⁺甚至镧系元素区分开。除了基于小分子和蛋白质的Ca ²⁺探针外，DNA最近还成为金属感测的强大平台。DNAzyme是基于DNA的催化剂，基于DNAzyme的传感器具有高度的稳定性，低成本和出色的灵敏度，因此具有很高的吸引力。EtNa是最近报道的一种RNA裂解DNA酶，对Ca ^2+的选择性高于Mg ²⁺和Na ⁺。在这项工作中，对EtNa进行了系统的突变研究，旨在进一步了解其生化特征并改善传感器性能。在45个测试的突变体中，与原始EtNa相比，EtNa-C5T表现出更高的活性，甚至具有更好的Ca ²⁺选择性。Ca ²⁺对Na ⁺的选择性提高了约20倍。在1 mM Ca ²⁺存在下，C5T的裂解速率为0.07 min ^-1。基于C5T突变体设计了催化信标传感器，检测限为11μMCa ²⁺。该传感器用于测量五个商业牛奶样品中的Ca ²⁺，并将结果与​​ICP分析的结果进行比较。