Concentration and Activation Biresponsive Strategy in One Analysis System with Simultaneous Use of G4 Structure-specific Signal Probe and Enzyme-catalyzed Reaction

Background 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 H2O2 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. Result 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. Significance 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.