| Acute myocardial infarction(AMI)has a rapid onset and a high mortality rate,which is the main reason for the high mortality of cardiovascular and cerebrovascular diseases.Therefore,it is of great significance to develop an early diagnosis method for AMI.Studies have shown that when AMI occurs,the level of specific microRNAs(miRNAs),e.g.miRNA-499,in patients’ circulating blood is significantly increased.Therefore,these miRNAs can be used as a kind of biomarkers for disease detection and diagnosis,which is an advanced topic of bioanalysis.However,these miRNAs generally exist in circulating blood at ultralow levels,and so ultrasensitive analytical techniques are usually required to detect them.This urgent demand can be met by a spherical nucleic acid enzyme(SNAzyme)catalyzed chemiluminescence(CL)system that integrates high efficiency of enzymatic reaction with high sensitivity of CL detection.SNAzymes are nano-form G-quadruplex(G4)DNAzymes developed from spherical nucleic acids,showing great promise for applications in nucleic acid detection.For the construction of SNAzymes,gold nanoparticles(AuNPs)are usually used as the core,and thiol labeled G4 forms a dense DNAzyme monolayer on the surface of AuNPs through Au-S bonds(AuNPs-SNAzyme).In order to reduce the cost,this study attempted to explore a non-covalent method to prepare SNAzymes.Although there are many methods to modify unlabeled DNAs onto AuNPs,the unlabeled G-rich sequences are prone to fold into G4 structures during the modification process,which affects the modification efficiency.In addition,the G base of unlabeled G4s can interact with AuNPs,causing a decrease in the catalytic activity of SNAzymes.To overcome this trouble,a new dual-functional non-covalent SNAzymes based on SiO2(SiO2SNAzyme@ABEI)was developed here,and then used for the sensitive detection of AMI related nucleic acid markers.The main research contents include two parts:1.Construction of SiO2-SNAzyme@ABEI.First,by virtue of the versatility of SiO2,the CL reagent N-(4-aminobutyl)-N-ethylisoluminol(ABEI)was covalently bonded to the surface of SiO2(SiO2@ABEI)by amide reaction.At the same time,five DNA functionalization methods were compared in terms of modifying unlabeled G4s onto AuNPs or SiO2 to prepare AuNPs-SNAzyme and SiO2-SNAzyme@ABEI.The results show that,compared with AuNPs,SiO2@ABEI can efficiently adsorbe all of the used DNAs,independent of their sequences.Especially,the G4 sequences able to form secondary structures are also easily loaded onto the surface,and the resulting SNAzymes exhibit good catalytic activity.In addition,such a process is not limited to certain a functionalization strategy,namely,almost all methods for functionalizing AuNPs with DNA are suitable for the preparation of SiI2-SNAzyme@ABEI.Accordingly,SiO2-SNAzyme@ABEI that integrates the CL reagent and catalyst was developed.Compared with non-functionalized SiO2,the CL intensity of SiO2SNAzyme@ABEI was increased by about 1200 times,showing excellent CL analysis performance.It lays a solid foundation for its further application in biomarker analysis.2.Ultrasensitive CL detection of AMI related nucleic acid markers.On the basis of above work,we further constructed a dual-functional CL nanoprobe capable of analyzing target nucleic acids for the highly sensitive detection of AMI related nucleic acid markers.Using target nucleic acid molecules to induce the formation of a DNA three-arm junction,an ultrasensitive CL detection platform for AMI markers was constructed,with a low detection limit of 0.8 fM(S/N=3)and a broad linear range(10 fM-100 pM).In addition,this platform shows a good anti-interference ability in 1%human serum.Compared with the common AuNPs-SNAzymes,our non-covalent bifunctional CL nanoprobes simplify the preparation process and reduce the experimental cost,but not lose the excellent analytical performance.This feature greatly expands the application potential of SiO2-SNAzyme@ABEI in the analysis of AMI related nucleic acid markers. |
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