Study Of Novel Fluorescence Polarization Aptasensors Based On Nanomaterials And Enzyme Signal Amplification

Aptamers are in vitro selected single-stranded oligonucleotides that possess high affinity and specificity to target molecules.These oligonucleotide aptamers have the conformational interconversion properties of molecular recognition and the operating characteristics of nucleic acid tool enzymes.In addition,aptamers possess many advantages,such as good stability,easy synthesis and modification,and wide applicability.Because of these unique advantages,aptamers appear as attractive alternatives to antibodies or offset the shortcomings of antibodies 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.At present,aptamers have been used for the construction various biosensors involving various signal-transduction approaches,which shows the great application prospect in a lot of fileds,such as biomedical research,clinical diagnosis,environmental monitoring,and food analysis.Nevertheless,research of biosensor based on the unique biochemical properties of aptamers is not mature,especially the aspects of improving the sensitivity and the anti-interference ability,simplifying the assay procedure,improving speed and accuracy to get analytical informations are still faced great challenges.Based on the signal magnification of nanomaterials and artificial enzymes,in thesis,several novel fluorescence polarization aptasensor have been developed for the detection of proteins and small molecules.The details are summarized as follows:1.Two fluorescence polarization biosensor based on nicking enzyme signal amplification and graphene oxide(GO)-enhanced fluorescence polarization for the detection of target molecules are developed by using adenosine and thrombin as model analytes.FAM-labeled single-stranded DNA with a nicking enzyme recognition site can be adsorbed onto the GO surface through π-πstacking,thus the FAM dye exhibits very high FP value(FP)due to the extraordinarily larger volume of GO.When the nucleic acid aptamer binds with the target(single-stranded nucleic acid aptamer binds with the target or two nucleic acid aptamer subunits bind with the target),it leads to the enhancement of base stacking,resulting in the formation of a double-stranded DNA probe with the complete recognition site of the nicking enzyme.This triggers the selective enzymatic cleavage of the FAM-labeled single-stranded DNA by nicking enzyme,resulting in the release of the released target/aptamer complex and the short DNA fragment carrying the FAM dye from the GO surface and the decrease of the FP value.The released target/aptamer complex can hybridize with another FAM-labeled single-stranded DNA probe linked to the GO,thus achieving signal amplification.The detection limits obtained from adenosine and thrombin are 2 pmol/L and 1 fmol/L,respectively,which are at least four orders of magnitude lower than that of traditional homogeneous aptasensors.2.By coupling T7 exonuclease signal amplification with polystyrene nanoparticle-enhanced FP,we have developed a new FP aptasensing method for highly sensitive detection of ochratoxin A,adenosine and platelet-derived growth factor BB,respectively.In this system,FAM-labeled nucleic acid probe(DNA or nucleic acid aptamer fragment)is modified with polystyrene nanoparticles,and the FAM dye has high FP value.When aptamer associates with the target,a duplex DNA region at the surface of polystyrene nanoparticles can be formed by the following three ways:(1)The aptamer hairpin binds with ochratoxin A,and undergoes a conformational change,thus leading to stem separation.Then,the FAM-labeled nucleic acid probe can hybridize with the open hairpin probe.(2)Two aptamer subunits bind simultaneously to adenosine.(3)The single-stranded aptamer associates with platelet-derived growth factor BB and leads to an enhancement of base stacking,which promotes the hybridization of the target-aptamer complex with the FAM-labeled nucleic acid probe.The FAM-labeled nucleic acid probe in the duplex DNA regions can be digested by T7 Exo,resulting in the release of the target-aptamer complex or the target.The released target-aptamer complex or the target can then recognize and bind with another FAM-labeled nucleic acid probe,leading to the cyclic cleavage of the FAM-labeled nucleic acid probe and the release of many short DNA fragments carrying FAM dye from polystyrene nanoparticles,generating a substantial decrease of the FP value.Due to the introduction of both T7 Exo signal amplification and polystyrene nanoparticle amplification,the developed FP method exhibits extremely high sensitivity with the detection limits of 250 amol/L ochratoxin A,10.2 fmol/L adenosine,and 75 amol/L platelet-derived growth factor BB,respectively.Moreover,this method also exhibits high specificity.3.A new fluorescence polarization aptasensor based on polystyrene nanoparticle-enhanced FP and cascade circular strand-displacement amplification have been developed for ultrasensitive detection of proteins.This system mainly consists of an aptamer hairpin probe with FAM labeled on its stem region and a polystyrene nanoparticle-functionalized DNA duplex probe(assistant DNA/trigger DNA,the single-stranded part of assistant DNA that is complementary to the stem of the hairpin probe,and the trigger DNA is designed to be partly complementary to the aptamer hairpin probe).In the absence of target,the polystyrene nanoparticle-functionalized DNA duplex probe is unable to bind with the aptamer hairpin probe and cannot trigger the cascade circular strand-displacement amplification reaction.In this case,the FAM dyes exhibits relatively low background of the FP value.However,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 single-stranded part of the DNA duplex probe.In the presence of dNTPs and polymerase,a new long complementary DNA duplex carrying the FAM dye at the polystyrene nanoparticle surface is synthesized.This results in an increase in the FP value.At the same time,the target and the trigger DNA is displaced by DNA polymerization.Both the displaced target and trigger DNA can then bind with the aptamer hairpin probes to initiate the cascade circular strand-displacement amplification reaction,which leads to a substantial increase in the FP value.This method can ultrasensitively detect thrombin in 60 minutes with a detection limit of 28 amol/L.Moreover,it has been sucessfully used for the quantification of thrombin in human plasma.In addition,the developed method is also used for detection of human vascular endothelial growth factor-165.These results demonstrate that our proposed FP aptasensing method can be served as a general method for ultrasensitive analysis of various targets.