The Application Of Dnazyme Based Biosensor Technology In The Detection Of Pb²⁺ And RNase H

Deoxyribozymes?DNAzymes?are single stranded DNA molecules which have high catalytic and structural recognition capabilities for this reason it is widely used in biological and chemical fields.Our work mainly focused on the replacement of partial base of DNAzyme that has been successfully altered its cofactor.On the basis of this alteration the fluorescent molecular beacon is combined to realize the high sensitivity detection of metal ions and enzymes in vitro.In addition,we also explored the effects of some drugs on catalytic efficiency of DNAzyme.Following are the main contents of our work:1.Ultra-sensitive detection of Pb²⁺ions based on DNAzyme and r GOAccording to the ability of DNAzyme to cut RNA,we constructed a detection system for Pb²⁺by designing a single-stranded fluorescent probe which containing RNA base and by using r GO which has excellent quenching capacity and different adsorption characteristics to different lengths of DNA chains.In order to maximize the activity of DNAzyme which increase the final detection limit.We carried out the optimization of the experimental conditions to select the best condition for DNAzyme activity.In addition,due to the high quenching efficiency of r GO,we optimized its dosage and compared its effect with ordinary GO.The experimental results showed that:?a?r GO used in the Pb²⁺system have a stronger quenching ability than GO,because less than 4?g/ml of rGO was required for quenching the fluorescent probe compared with 10?g/ml GO was required for quenching the fluorescent probe.The?F is larger for rGO than GO so the detection limit was increased.?b?The detection limit of Pb²⁺was 100p M.?c?The detection limit of Pb²⁺in serum was 500p M?0.1?g/L?,which was lower than normal blood value?<99?g/L?.2.Construct Mg²⁺-dependent DNAzyme,and achieve RNase H avtivity detection systemIn this system,we replaced the partial base of Pb²⁺-depend DNAzyme that has been previously reported and successfully altered its cofactor form Pb²⁺to Mg²⁺,the new DNAzyme has highly specific for Mg²⁺.We designed the dual standard hairpin probe which could be cut by the Mg²⁺dependent DNAzyme.We embedded the DNAzyme sequence into a hairpin DNA-RNA hybrid chain to inhibit its activity.In the presence of RNase H,the DNA-RNA hybrid chain was cleaved so that the DNAzyme was released and regained its activity.We established the RNase H activity detection system and also complete the DNAzyme-based detection system.The experimental results showed that:?a?The detection limit of RNase H was 0.01 U/m L which can also be detected in serumand cell extract;?b?We studied the effects of 11different drugs on RNase H activity and we found that the 11 drugs can increase the RNase H activity;?c?The effects of six antibiotics on the catalytic efficiency of DNAzyme were analyzed and we found that gentamicin and sulfur streptomycin have inhibitory effect on DNAzyme;?4?This method was also used to detect Mg²⁺and DNAzyme and the detection limit of Mg²⁺and DNAzyme was 5n M,5p M,respectively.