Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, China.
Department of Clinical Laboratory, Xinqiao Hospital, Army Medical University, Chongqing, 400037, China.
Anal Chim Acta. 2024 Nov 15;1329:343246. doi: 10.1016/j.aca.2024.343246. Epub 2024 Sep 14.
Enzymes with critical effects on life systems are regulated by expression and activation to modulate life processes. However, further insights into enzyme functions and mechanisms in various physiological processes are limited to concentration or activation analysis only. Currently, enzyme analysis has received notable attention, particularly simultaneous analysis of their concentration and activation in one system. Herein, N-methyl mesoporphyrin IX (NMM), a specific dye with notable structural selectivity for parallel G-quadruplex nucleic acid enzyme (G4h DNAzyme), is employed for the analysis of its concentration. In addition, the peroxidase activity of G4h DNAzyme is characterized based on G4h DNAzyme-catalyzed decomposition of HO to continuously consume luminol. Accordingly, an increased fluorescence (FL) response of NMM and a decreased FL response of luminol could be simultaneously employed to analyze the concentration and activation of G4h DNAzyme.
Herein, a novel concentration and activation biresponsive strategy is proposed using a G4h DNAzyme-based model that simultaneously employs a G4h structure-specific signal probe for enzyme concentration analysis and G4h DNAzyme-catalyzed reactions for enzyme activation analysis. Under optimal conditions, the biresponsive strategy can be effectively used for the simultaneous analysis of G4h DNAzyme concentration and activation, with detection limits of 718.7 pM and 233.4 nM respectively, delivering acceptable performances both in cell and in vitro.
This strategy can not only be applied to concentration and activation analyses of G4h DNAzyme but can also be easily extended to other enzymes by simultaneously combining concentration analysis via target-induced direct reaction and activation analysis via target-induced catalytic reaction, offering deeper insights into various enzymes and enabling their effective implementation in bioanalysis and biochemistry.
对生命系统具有关键影响的酶通过表达和激活进行调节，以调节生命过程。然而，人们对各种生理过程中酶的功能和机制的进一步了解仅限于浓度或激活分析。目前，酶分析受到了广泛关注，特别是在一个系统中同时分析其浓度和激活。在此，N-甲基甲川卟啉 IX（NMM）是一种对平行 G-四链体核酸酶（G4h DNAzyme）具有显著结构选择性的特定染料，用于分析其浓度。此外，基于 G4h DNAzyme 催化分解 HO 以连续消耗鲁米诺的特性，对 G4h DNAzyme 的过氧化物酶活性进行了表征。因此，可以同时使用 NMM 的荧光（FL）响应增加和鲁米诺的 FL 响应减少来分析 G4h DNAzyme 的浓度和激活。
在此，提出了一种基于 G4h DNAzyme 的新型浓度和激活双响应策略，该策略同时使用 G4h 结构特异性信号探针进行酶浓度分析和 G4h DNAzyme 催化反应进行酶激活分析。在最佳条件下，该双响应策略可有效用于同时分析 G4h DNAzyme 的浓度和激活，检测限分别为 718.7 pM 和 233.4 nM，在细胞和体外均具有可接受的性能。
该策略不仅可用于 G4h DNAzyme 的浓度和激活分析，还可通过同时结合目标诱导的直接反应进行浓度分析和目标诱导的催化反应进行激活分析，轻松扩展到其他酶，从而深入了解各种酶，并使其在生物分析和生物化学中得到有效应用。
