Designed Of Highly Sensitive Electrochemiluminescence Biosensor Based On DNAzyme And EXO ? Signal Amplification

Electrogenerated Chemiluminescence(also called electrochemiluminescence,abbreviated as ECL),is directly or indirectly initiated by the electrochemical and chemical luminescence.It has been widely applied in analytical science because of its advantages such as simple operation,good temporal and spatial control,and high sensitivity.Conventional luminescent materials are luminols,ruthenium bipyridyls,and aromatic hydrocarbons and so on.Quantum dots,semiconductor materials are newly developed branch of ECL system in recent years and rapidly established its important position in the ECL field.Molecular beacon was first proposed in 1996.It has the advantages of high selectivity and specificity in the conversion between its stem-loop structure and stretch structure.According to the different signal,molecular beacon technology can be divided into fluorescent molecular beacons and electrochemical molecular beacons.The fluorescent molecular beacon is a hairpin DNA functionalized at one end with a fluorophore and at the other end with a quenching agent to quench the fluorescent signals.Without the target DNA,the quencher is in close proximity to the fluorophore,no fluorescent signal is generated.By adding a complementary target DNA,the loop of hairpin DNA is unfolded,the fluorophore moves away from the quencher,resulting in fluorescent signal generation.Recently,novel fluorescent probe technology has already penetrated into many aspects of biological macromolecule detection and other fields based on its advantages of high sensitivity,specificity and simple operation.Based on the previous study of electroluminescence technology and molecular beacon technology in our laboratory,a novel electrochemically luminescent catalytic active switching molecular beacon(ECL-MB)has been designed.Combination with aptamers and dual amplification cycles using Exo ? and 8-17 DNAzyme,we created a highly sensitive electrochemiluminescence biosensor to sensitive and specific detection of thrombin and UDG.This scheme can also be used in a complex environment,and it is applicable to a large number of biological analyses for its simple operation,stable biological activity,and high sensitivity.So ECL-MB have important application prospect in the increasingly rigorous requirements of medical research and clinical diagnosis.Chapterl.IntroductionFirst of all,the basic principle and characteristics on electrochemiluminescence technology and molecular beacon technology are introduced.In addition to these,research background and the applications of ECL analysis combine with any other new techniques in analytical chemistry field are also reviewed.At the last,we describe a novel electrochemically luminescent catalytic active switching molecular beacon(ECL-MB)and its principle and characteristics.Chapter2.Design a highly sensitive electrochemiluminescence biosensor based on 8-17 DNAzyme signal amplification for thrombin detectionIn this chapter,we designed a highly sensitive electrochemiluminescence biosensor based on the 8-17 DNAzyme signal amplification technique for thrombin detection.The ECL-MB is a hairpin DNA functionalized at both ends with a catalytically active porphyrin(hemin).In the absence of the target sequence,the ECL-MB possesses a stem-loop conformation with the two identical hemin groups in close proximity to become dimeric forms,and the ECL-MB catalytic activity was switched off,unable to catalyze the electrochemical luminescence reaction of luminol,and the ECL signal was low.Upon the addition of the thrombin,it interacts with probe to release 8-17 DNAzyme chain,8-17 DNAzyme continues to react with ECL-MB,with the catalysis of Zn2+,ECL-MB cleavaged,hemin restores monomer morphology,catalyzes Luminol electrochemiluminescence reaction,ECL signal surge.The results showed that the ECL-MB assay could detect the thrombin in a simple,rapid and highly sensitive manner,and there was a good linear relationship between ECL signal and thrombin concentration between 10pM and InM.Chapter3.Design a highly sensitive electrochemiluminescence biosensor based on EXO ? signal amplification for UDG detectionIn this chapter,we designed a highly sensitive electrochemiluminescence biosensor based on the Exo ? signal amplification technique for UDG detection.In this scheme,we first designed a ds-DNA probe,named probe 1,which consisted of S1 and S2.The S2 chain contained U bases,with the target UDG cleavage,the double-stranded structure of probe1 was destroyed,release the S1 chain.The S1 chain is then reacted with probe2(consist of S3 and S4)to form two cycles under the action of Exonuclease ? to release the S3 chain.Then S3 chain and ECL-MB to form ECL-MB/S3 complex,ECL-MB from the hairpin structure into a double-stranded structure,hemin dimer form was destroyed,restored into hemin monomer morphology,electrochemical luminescence catalytic activity recovery,catalytic Luminol-H2O2 system,resulting in a strong ECL signal.The electrochemical luminescence biosensor is used for the detection of UDG enzymes,showing a highiy sensitive and selective,with a detection limit as low as 1.3×10-5U/mL.Moreover,even at a very low UDG concentration,the ECL-MB assay showed a good specificity in the presence of other potentially interfering components.At the same time,the ECL-MB assay was used to detect the UDG in Hela cells and MCF-7 cells,demonstrating that ECL-MB assay has potential in detect of biomarker variation in vivo.