| Peptide and aptamer are two kinds of important synthetic molecular receptors that possess high affinity and specificity to target molecules,such as ions,organic small molecules,peptides and protein.Peptide and aptamer have several advantages as compared to antibody and enzyme,including small size,easy synthesis and modification,low cost,good stability and wide applicability.Because of these unique advantages,peptide and aptamer appear as attractive alternatives to traditional molecular receptors or offset the shortcomings of traditional molecular receptors to be a new generation of molecular recognition element,which provides a new effective research tool for the development of highly sensitive and specific sensing technologies.Recently,peptide and aptamer have been used for the construction various biosensors,which shows the great application prospect in a lot of fileds,such as the diagnosis and treatment of diseases,food analysis,drug toxicology testing and environmental monitoring.Nevertheless,research of biosensor based on peptide and aptamer is not mature,especially the aspects of poor sensitivity,narrow dynamic range,low anti-interference ability and flux analysis.Taking advantages of nanomaterials,DNA signal amplification,several highly sensitive and selective biosensors based peptide and aptamer for the detection of proteins and small molecules were developed in this dissertation.The main contents include the following four parts:1.Using aptamer-based receptor molecules,carcinoembryonic antigen(CEA)as the fluorescence polarization enhancements,polymerase and Nb.BbvCI as biocatalysts,a new type of the autonomous protein-encoded aptamer nanomachine for amplified fluorescence polarization sensing of small molecules are developed based on target recognition,protein enhancement and isothermal exponential amplification.The DNA hairpin binds CEA to form a CEA functionalized DNA hairpin,and the FAM-labeled primer as signal probe.In the absence of target,the CEA-functionalized DNA hairpin is unable to bind with FAM-labeled primer,and cannot trigger isothermal exponential amplification.In this case,the FAM dye exhibits relative low FP value due to the small size of the FAM-labeled primer.Upon binding of the target with its aptamer sequences,the stem of DNA hairpin is opened.Subsequently,the opened stem anneals with FAM-labeled primer and proceeds with polymerization by the assistance of DNA polymerase and dNTPs,which displaces the target and leads to the generation of CEA-functionalized DNA duplex carrying the FAM dye,this results in an increase in the FP value.The displaced target again binds with the DNA hairpin to initiate target recycling amplification cycle,which leads to a further increase in the FP value.Moreover,the polymerization-generated DNA duplex can be cleaved by Nb.BbvCI,which leads to initiation of a circular replication-scission-displacement reaction,and the continuous generation and recycling of DNA trigger.This EXPA results in the generation of numerous DNA-protein complexes with the fluorophore dye,provides a readout signal for the amplified sensing of the target.With the use of aflatoxin B1(AFB1)as model analytes,we demonstrate selective detection of AFB1 with a detection limit of 0.24 pM.The method is flexible,and we also show ultrasensitive detection of cocaine by switching the corresponding aptamer sequence in the nanomachine,the detection limit for cocaine is 18 pM.These results demonstrate that the developed protein-encoded nanomachine promises a general protocol for ultrasenstive analysis of various types of target molecules.2.A novel fluorescence aptasensing method based on structure-switching aptamer triggering quadratic amplification and the universal quenching property of GO for simple,rapid,highly sensitive detection of carcinoembryonic antigen(CEA)is developed.This system mainly consists of an aptamer hairpin probe containing a recessed 3’ end and an overhanging 5’-FAM labeled.In the absence of target,the aptamer hairpin probe is unable to bind with the aptamer hairpin probe and cannot trigger the quadratic signal amplifications.In this case,the FAM-labeled DNA hairpin would be adsorbed by GO,resulting in an effective fluorescence quenching.However,upon recognition and binding with a specific target,the stem of DNA hairpin is opened,which change the conformation of DNA hairpin and reform a new hairpin DNA.Then the KF polymerase recognizes the recessed 3’ terminus of the new hairpin DNA and initiates the elongation of the duplex stem until the 5’ terminus becomes blunt.At the same time,the target is displaced by DNA polymerization and the replaced target can then bind with the aptamer hairpin probes to initiate the a new amplification reaction.Next,the resulting hairpin DNA is specifically hydrolyzed by T7 exonuclease in the direction from 5’ to 3’,resulting in the release of a FAM-labeled mononucleotide and the liberation of the single stranded DNA.Moreover,the released single stranded DNA partially hybridizes with another DNA hairpin to form a duplex DNA,in which,the strand with the blunt 5’terminus is selectively digested by T7 exonuclease to release single stranded DNA initiate the subsequent cycling cleavage process.Eventually,the cleavage of the aptamer hairpin probes release a large amount of FAM-labeled mononucleotides.The FAM-labeled mononucleotides will not be adsorbed by GO,leading to the recovery of fluorescence.The proposed sensing method achieves quadratic via sharing an aptamer hairpin probe,and reaches the low detection limit as 28 fg/mL of CEA.The results show that detection method with good specificity,and aptasensing method was successfully used for the determination of CEA level in human serum,which was consistent with that of results of the traditional enzyme-linked immunoassay.3.A novel fluorescence aptasensing method for protein detection was developed based on structure-switching aptamer triggering exponential amplification reaction(EXPAR)and DNAzyme.This system mainly consists of an aptamer hairpin probe,primer and molecular beacon(MB).In this assay,upon recognition and binding with a specific target,the stem of the aptamer hairpin probe is opened.After which the opened hairpin probe anneals with the primer,which initiates DNA polymerization.The polymerization will also displace the target during synthesis of the long duplex DNA with two nicking sites of Nb.BbvCI.The displaced target again binds with another the aptamer hairpin probe to initiate a target recycling amplification cycle and leads to the generation of duplex DNA.Moreover,the polymerization-generated duplex DNA can be cleaved by Nb.BbvCI,resulting in new sites for initiation of DNA replication.The reactivated DNA replication displaces the already synthesized DNAzyme sequence and DNA triggers,and egenerates DNA duplexes to initiate a circular replication-scission-displacement reaction."DNA triggers" can hybridize with hairpin probes to initiate a circular polymerization reaction,and subsequently initiate the circular replication-scission-displacement reaction.By following this mechanism,the presence of the target results in an EXPA format and generation of massive DNAzyme sequence.Moreover,the cleavage of two different fluorophore/quencher-modified substrates by the DNAzyme leads to the generating an amplified fluorescence signal.With the use of CEA as model analytes,we demonstrate selective detection of CEA with a detection limit of 5.2fg/mL.In addition,the method can be used in the analysis of complex biological samples.4.A novel graphene oxide(GO)sensing platform based on enzymatic phosphorylation for the multiplexed detection of protein kinases is developed.The GO is modified with streptavidin(SA).In the absence of protein kinases,these dye-labeled peptides are not assembled on GO,emitting strong fluorescence.However,in the presence of protein kinases,specific phosphorylation reactions occur,and the phosphorylated peptides carrying the biotin site and the dyes are generated.The phosphorylated peptide products can be assembled onto the GO through the SA-biotin binding.This assembly process brings the dyes and GO into close proximity,which leads to fluorescence quenching.This GO sensing platform can simultaneously and selectively detect protein kinase A,protein kinase Abl and protein kinase Src with low detection limits of 0.005 U/mL,0.02 U/mL and 0.05 U/mL,respectively.Moreover,it has been sucessfully used for the detection of multiple protein kinases in complex biological samples.In addition,this assay can be easily adapted to screening inhibitors for protein kinase because the phosphorylation of protein kinases is restrained in the presence of inhibitors. |
