G-quadruplex DNAzyme Catalyzed Luminol Chemiluminescence And Its Application In The Analysis Of Potassium And Lead Ions

Due to the unique advantages of chemiluminescence (CL) analytical technology, such as low interference of scattered light background, high sensitivity, fast analysis speed, wide linear range and simple, cheap and easy miniaturized apparatus, it has been widely applied in trace and ultra-trace the analysis of inorganic, and organic compounds. Luminol, as a luminophor with high luminescence quantum yield and good water solubility, is a class of most widely studied chemiluminescent reagents currently. Traditional CL reaction system of luminol-H2O2 is catalyzed by horseradish peroxidase (HRP), gold-, carbon-and metal chalcogenide nanomaterials. However, natural protease is easy to lose its activity and its modification needs high-cost and complicated technology, In addition, these nanomaterials are also not easy to functionalize, so their applications in luminol CL analysis are limited.As a new type of DNA enzyme, G-quadruplex DNAzyme has received widespread attention in recent years. Such artificial mimicking-HRP is easy to prepared and functionalize and performs stably, so it has been wildly applied in the construction of nano-devices and biosensors. It is ideal alternative for protease to catalyze the oxidation reaction to achieve signal amplification. Currently, the reported G-quadruplex DNAzyme are mostly used for catalyzing hydrogen peroxide mediated oxidation of TMB, ABTS and luminol, but there still seems to be limitation of the application in luminol luminescence system compared to TMB and ABTS.The innovation of this paper is the combination of CL technique and the DNAzyme formed by the DNA aptamers of potassium (K+) and lead (Pb2+). We detected K+and Pb2+ with high sensitivity and successfully applied these strategies in the analysis of biological samples. The range of application of CL analysis has been expanded and new CL analytical method has been developed in biochemical analytical field. Specific content includes the following three aspects:(1) Highly sensitive detection of K+ We have developed CL analytical strategy to detect K+ sensitively base on K+-induced G-quadruplex DNAzyme. The investigation indicate, that only K+ can induce rich-G sequence to form parallel G-quadruplex compared to other metal ions. Parallel G-quadruplex can efficiently bind to hemin, which enables the formation of activated DNAzyme to enhance CL intensity. The results shows that the intensity of CL shows a linear dependence on the concentration of K+ within a range of 2-120μ.M with a limit of detection (3σ) of 1.66μM, giving a vital clue to quantify K+ content in urine samples. This strategy firstly opens up CL as an effective and facile approach to detect K+ with high selectivity.(2) Highly sensitive detection of Pb2+ The binding constant of Pb2+-G-quadruplex complex is greater than that of K+-G-quadruplex complex, so K+-stabilized parallel G-quadruplex will be induced to transform into Pb+-stabilized anti-parallel G-quadruplex with higher stability but poor DNAzyme activity upon addition of Pb2+, which result in sharply declining CL readout signal. The results indicate that the changes of CL intensity shows a linear dependence on the logarithm of Pb2+ with a range of 0.4-10 nM and a limit of detection (3σ) of 0.06 nM. Thus, this method with advantaes of simple, low-cost, effective, isothermal and facile for ultra-sensitive detection of Pb2+ holds a great potential for clinical plumbism diagnosis by testing hair.(3) A K+-Pb2+ switched DNA logic gate PW17 can be induced to form different conformations of G-quadruplex by K+ and Pb+ respectively, leading to changes of catalytic activity of DNAzyme. K+-induced G-quadruplex DNAzyme can dramatically enhance CL while Pb2+-induced G-quadruplex has no catalytic activity. We have constructed a dual input INHIBIT DNA logic gate, setting K+ and Pb2+ as the two input and CL signal as the output. The establishment of the logic gate can provide reference for the program of CL detection of K+ and Pb+ simultaneously.The above research have successfully developed highly sensitive method for detection of K+ and Pb2+ based on K+- or Pb2+-induced G-quadruplex DNAzyme, exploring the application of DNAzyme in CL analysis, enriching the targets of CL analysis, and providing more highly sensitive detection methods for different biomarkers.