| Escherichia coli(E.coli)can cause gastrointestinal infection,arthritis,and sepsis and other diseases,which seriously endanger human health.Traditional detection methods for Escherichia coli are not only time-consuming and prone to contamination but also need expensive regents and professional operation.In recent years,DNAzymes with horseradish peroxidase activity have attracted much attention in the field of biosensors due to their advantages of simple synthesis,low cost,and stable activity.However,at present,the biosensors based on DNAzymes have the problems of low catalytical activity and high detection background value.Through the research discovery adjacent base pairs of DNAzymes can modulate ribozyme activity.Herein,two DNAzymes biosensors based on the activity regulation of adjacent bases were constructed for the detection of E.coli.The results showed that the above sensors have the advantages of simplicity,rapidity,low cost,high sensitivity,and high specificity,which provide a new idea for the detection of E.coli.The research contents of this paper are as follows:1.Construction and application of adjacent base pair inhibitory DNAzyme biosensors:4 types of DNAzymes were formed by changing the types of base pairs at the tail of G4(G-quadruplex).When the base pair is A:T(hybridization of adenine and thymine),the original catalytic activity of G4 DNAzyme can be significantly reduced,and the activity inhibition factor is as high as 17 times.This discovery is called the effect of adjacent base pairs inhibiting DNAzymes activity.Subsequently,the universality of this effect in various buffers,a wide pH range,and various reaction substrates was verified.Molecular dynamics simulations and adenine analogs were used to analyze the mechanism,and the possibility of this phenomenon was proposed:the carbonyl group on the hexatomic ring of the complementary base plays an important role.This effect retains the original structure of G4,and the sensor constructed based on this phenomenon has real-time analysis capability.Based on the above phenomenon,combined with Escherichia coli aptamer,a proximity base pair inhibitory DNAzyme sensor was constructed to detect Escherichia coli,and the working performance of the sensor was explored through specific detection,working curve,and standard addition experiments.The experimental results showed that the sensor exhibited a good linear relationship at 2×102-2×106 CFU/mL,the detection limit was 21 CFU/mL,and had good specificity.2.Construction and application of adjacent base pair enhanced DNAzyme biosensors:Combined with the above research ideas,16 types of DNAzymes were formed by changing the G4 tail adjacent to the first base and the second base pair.It was found that the DNAzyme activity of which the base pair at the end of G4 is AA:T(the first base is A,the second base pair is A:T)was increased by 3 times compared with the original DNAzyme activity.This discovery was called the effect of adjacent base pairs enhancing DNAzyme activity,which also existed in G4-AG:C.Subsequently,the universality of the enhancement of DNAzyme activity by adjacent base pairs was verified in different buffers,pH,and reaction-substrate systems.In addition adenine analogs can produce this phenomenon.Ultraviolet spectroscopy indicated that the phenomenon preserved the original structure of DNAzyme.Combining this phenomenon with Escherichia coli aptamer,an enhanced DNAzyme biosensor based on adjacent base pairs was constructed and applied to the detection of Escherichia coli.The specificity analysis and working curve showed that the sensor had good detection performance.The experimental results showed that the sensor exhibited a good linear relationship at 2×1022×107 CFU/mL,and the detection limit was 16 CFU/mL. |
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