A Novel Method For The Detection Of Small Molecules Based On Cationic Conjugated Polymers And S1 Endonuclease Detection

Cationic conjugated polymers(CCPs)have attached much attention because of their good light-harvesting ability,fluorescent signal amplification and good water-solubility.CCPs exhibit the broad applications in biological detection,cell imaging,drug transport and release,and disease diagnosis.The graphene oxide has huge conjugated structure,making it as excellent electron or energy acceptor during electron transfer or energy transfer processes,and thus GO shows strong quenching effect on fluorescent probes.Aggregated perylene diimide(PDI)derivatives can efficiently quench a variety of adjacent fluorophores that emit over a wide wavelength range from the visible to NIR region,leading to a favorable application in biological sensing system.In this thesis,novel and universal fluorometric aptasensor platform and lable-free fluorometric assay method have been constructed based on fluorescent cationic conjugated polymers PFP and GO,or PFP and PDI,respectively,and their applications in Bisphenol A,dopamine and S1 nuclease were studied.1.Novel and Universal Fluorometric Aptasensor Platform based on Cationic Conjugated Polymer and AptamerA universal and sensitive fluorometric aptasensor was designed based on the fluorescence resonance energy transfer(FRET)between cationic conjugated poly(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorine phenylene(PFP)and the FAM-labeled aptamer..In the aptasensor,PFP,FAM-labeled aptamer and GO were used as the energy donor,the energy acceptor and probe,and the fluorescence quencher,respectively.In the absence of target,the FAM-aptamer was absorbed onto the surface of GO,resulting in the weak FRET from PFP to FAM because of FAM fluorescence being quenched by GO via ?-? stacking interaction upon excitation at 380 nm.In the presence of target,the aptamer can switch its configuration after recognizing ligands with high affinity to prevent the adsorption on the surface of GO.Furthermore,cationic PFP can form complex with aptamer through electrostatic interactions.In this case,the strong FRET was obtained once excitation at 380 nm owing to the desirable spectral overlap between PFP emission and FAM absorption.In this platform,cationic PFP not only is used as donor making FAM fluorescence amplification,but also can form complex with aptamer-target to prompt aptamer leaving the surface of GO,which enhances the sensitivity of assay.Herein,bisphenol A(a common pollutant that is hazardous to human and animals health)and dopamine(important molecule in human's biochemical processes)were used as model targets for the versatility application of the aptasensor platform.Our results demonstrated that this aptasensor platform achieved a universal,sensitive and selective sensing for targets.The detection limit(LOD)for detection BPA was determined to be 5 pg/mL,which is 10 times lower than reported method only using aptamer and GO.The LOD for DA was determined to be 1.0 nM,which is lower than most of previous reported methods.In addition,this aptasensor is applicable in detecting BPA in real water-samples and DA in diluted human plasma and serum.Importantly,it could be applied in detecting other contaminants and biomolecules only by changing the aptamer sequence.2.Lable-free Fluorometric Assay based on Water-soluble Cationic Conjugated Polymers and Perylene DiimideIt is well-known that cationic conjugated polymers own the excellent photoelectric property,and aggregated perylene diimide(PDI)has strong quenching ability.Taking advantage of these properties,we developed a new label-free platform for detection of S1 nuclease and BPA.Cationic conjugated polymer,ssDNA and PDI were used as optical transducer,the probe and the fluorescence quencher in the aptasensor,respectively.The PDI itself could not quench the fluorescence of PFP because of the electrostatic repulsion between the two.When ssDNA was added into the mixture solution,PFP/ssDNA/PDI complex can form due to stronger electrostatic interactions.The PDI aggregates on the ssDNA and quenches the fluorescence of PFP due to the adjacence between PDI and PFP.Upon addition of S1 nuclease,ssDNA is hydrolyzed into small fragment.The PFP/ssDNA/PDI complex thus is broken,resulting in PFP fluorescence recovering.Because the quenching ability of PDI is dependent on the length of ssDNA,it is reasonable to construct a label-free aptasensor based on aptamer configuration changing when recognizing ligands.Herein,BPA aptamer was used as a model to study the application.Since the BPA aptamer can bind BPA specifically which weakens the aggregation of PDI,the fluorescence of PFP recovers once exciting at 380 nm.The recovered fluorescence intensity of PFP is dependent on the concentration of S1 nuclease and BPA.The lable-free method could sense S1 nuclease and BPA with a detection limit of 1.0×10-6 U/mL and 0.05 ng/mL,respectively.In conclusion,our method is sensitive,cost-effective,simple,and provides a new platform for the detection of other biomolecules.