背景:将底物转化为比色读数的G-四链体(G4)/血红素DNA酶在传感器开发中被公认为方便的生物催化工具。然而,先前开发的比色G4/血红素DNA酶是基于扩散底物的DNA酶(DSBD)。目前的比色DSBD有几个缺点,包括高剂量(~mM)的扩散基质(DS),比色产品毒性,和单一比色读数,不容忍实验因素和背景的波动。此外,使用高剂量DS可以涂抹G4折叠,丢弃它们对环境更有害。因此,迫切需要探索具有克服DSBD的这些缺点的潜力的替代DNAzyme。
结果:我们在此开发了基于缔合底物的DNA酶(ASBD)。选择花青染料作为缔合底物(AS),因为它们与G4/血红素DNA酶的结合能力。关于DSBD,ASBD只需要低剂量(~10μM)的AS就能够引起快速和可见的底物转化。此外,因为花青染料是具有高消光系数的近红外染料并且它们的转化产物在较短波长处具有吸收带。因此,可以开发比色比反应以跟踪G4/血红素DNA酶的活性,具有耐受实验因素和背景的波动的能力。特别是,本文开发的ASBD可以承受一定程度的H2O2浓度波动。ASBD能够与其他酶协同工作(例如,葡萄糖氧化酶)实现级联传感。
结论:开发的ASBD可以在低剂量的底物下操作,具有比色比响应,并且可以克服DSBD中遇到的缺点。我们期望,通过在未来设计具有丰富色彩面板的AS,我们的工作将激发更有趣的开发环境良性和低碳G4/血红素DNAzymes和所需的丰富多彩的高性能传感器。
BACKGROUND: G-quadruplex (G4)/hemin DNAzymes with conversion of substrates into colorimetric readouts are well recognized as convenient biocatalysis tools in sensor development. However, the previously developed colorimetric G4/hemin DNAzymes are diffusive substrate-based DNAzymes (DSBDs). The current colorimetric DSBDs have several drawbacks including high dosage (∼mM) of diffusive substrates (DSs), colorimetric product toxicity, and single colorimetric readout without tolerance to fluctuation of experimental factors and background. In addition, the usage of high-dosage DSs can smear the G4 foldings and their discard is more harmful to environment. Therefore, exploring alternative DNAzymes with potential to overcome these drawbacks of DSBDs is urgently needed.
RESULTS: We herein developed associative substrate-based DNAzymes (ASBDs). Cyanine dyes were selected as associative substrates (ASs) due to their binding competency with G4/hemin DNAzymes. With respect to DSBDs, ASBDs needed only low dosage (∼10 μM) of ASs to be able to cause a rapid and visible substrate conversion. In addition, since cyanine dyes are NIR dyes with high extinction coefficients and their conversion products have absorption bands at shorter wavelength. Therefore, a colorimetric ratio response can be developed to follow activities of G4/hemin DNAzymes with competency to tolerate fluctuation of experimental factors and background. In particular, herein developed ASBDs can endure somewhat concentration fluctuation of H2O2. ASBDs are able to cowork with other enzymes (for example, glucose oxidase) to realize cascade sensing.
CONCLUSIONS: The developed ASBDs can operate at low dosage of substrates with a colorimetric ratio response and can overcome the drawbacks met in DSBDs. We expect that, by designing ASs with fruitful color panel in the future, our work will inspire more interesting in developing environment-benign and low-carbon G4/hemin DNAzymes and desired colorful high-performance sensors.