Study And Application Of Fluorescence Analytical Methods Based On Polydopamine Nanopartical Probes

Fluorescence biosensing is one of the most widely-used technology for the assay of biomolecules,with the inherent advantages of high sensitivity and selectivity,operational convenience,and in situ imaging properties.Owing to their unique physical and chemical properties,the application of nanomaterials in fluorescence biosensing has become an advanced research hotspot worldwidely.Polydopamine,a new kind of material,has broad applications in the fields of chemical,biological,medical,and materials science,due to the advantages of versatile adhesion capability,convenient chemical modification,and excellent biocompatibility.The study on the fluorescence related properties of polydopamine nanomaterials,and the application in biosensing,have important significance for the development of analytical chemistry for life science.In this work,the polydopamine nanospheres were prepared,and the fluorescence quenching ability of the polydopamine nanospheres and the quenching mechanism were studied.Some fluorescence biosensors based on polydopamine nanomaterials and nucleic acid were constructed and applied for the assay of some biomolecules.The main content includes the following aspects:1.Study on the fluorescence quenching ability of the polydopamine nanomaterialsFour kinds of polydopamine nanospheres with different sizes were prepared,the fluorescence quenching ability of the polydopamine nanospheres and the quenching mechanism were studied.The results indicated that the polydopamine nanospheres could quench the different wavelength fluorescence of the different fluorophores with high quenching efficiency.And the quenching ability was compared favorably with the commonly used nanoquenchers.The quenching was a combination of dynamic and static quenching,and through energy transfer and/or electron transfer.Further more,the prepared MNP@PDA also possessed high fluorescence quenching efficiency.The excellent fluorescence quenching ability of the polydopamine nanomaterials will lead to the construct of fluorescence biosensors for the assay of the biomolecules,employing the polydopamine nanomaterials as the universal nanoquencher.And the application of polydopamine will be broadened.2.Fluorescence biosensing platforms based on the polydopamine nanomaterialsBased on the variable affinities towards various ssDNA conformations and the high fluorescence quenching efficiency of the polydopamine nanospheres(PDANS),a biosensing platform was constructed by the combination of the PDANS and the DNA probe labelled with the fluorophore.Without the targets,the probe would be adsorbed on the surface of the PDANS,and the fluorescence was quenched through FRET.While,in the present of the targets,for the specific reaction with the targets,the conformation of the probe changed.The probe would be released from the PDANS due to the weakened interaction,resulting in the recovery of the fluorescence.Employing this platform,the sensitive and selective assay of DNA and thrombin was achieved,with the limit of detection of 0.1 nM and 0.5 nM,respectively.And the logic network based on the platform worked well.Another biosensing platform based on MNP@PDA was also developed for the sensitive assay of DNA and proteins.This sensing method is fast and simple,without the involvement of other reagents and further operations.This work provides opportunities to develop simple,rapid,and economical biosensors for molecular diagnostics.3.A fluorescence biosensing platform based on the polydopamine nanospheres intergrating with Exonuclease Ⅲ-assisted target recycling amplificationA fluorescence biosensing platform based on the polydopamine nanospheres(PDANS)intergrating with Exonuclease Ⅲ(Exo Ⅲ)was developed.Due to the interaction between the ssDNA and the PDANS,the fluorescence of 6-carboxyfluorescein(FAM)labelled in the probe would been quenched by PDANS through FRET.While,in the present of the target DNA,the probe DNA would hybridize with the target DNA to form the double-strand DNA complex.Thus,Exo Ⅲ could catalyze the stepwise removal of mononucleotides from 3 ’-terminus in the probe DNA,releasing the target DNA.As the FAM was released from the probe DNA,the fluorescence would no longer been quenched,led to the signal on.As one target DNA molecule could undergo a number of cycles to trigger the degradation of abundant probe DNA,Exo Ⅲ-assisted target recycling would led to the amplification of the signal.The detection limit for DNA was 5 pM,which was 20 times lower than that without Exo Ⅲ.And the assay time was largely shortened due to the faster signal recovery kinetics.What’s more,this target recycling strategy was also applied to conduct an aptamer-based biosensing platform.The fluorescence intensity was also enhanced for the assay of adenosine triphosphate(ATP).For the Exo Ⅲ-assisted target recycling amplification,DNA and ATP were fast detected with high sensitivity and selectivity.This work provides opportunities to develop simple,rapid,economical,and sensitive biosensing platforms for biomedical diagnostics.4.Aptamer/polydopamine nanospheres nanocomplex for in situ molecular sensing in living cellsA nanocomplex was developed based on the aptamer labelled with the fluorophore and the polydopamine nanospheres(PDANS)for molecular sensing in living cells.Due to the interaction between ssDNA and the PDANS,the aptamer was adsorbed on the surface of the PDANS,and the fluorescence was quenched by the PDANS through FRET.In vitro assay,the introduction of adenosine triphosphate(ATP)led to the release of the aptamer from the PDANS and the recovery of the fluorescence.The retained fluorescence of the nanocomplex was found to be linear with the concentration of ATP in the range of 0.01-2 mM,and the nanocomplex was highly selective towards ATP.For the excellent biocompatibility and the strong protecting ability to nucleic acids against enzymatic cleavage of the PDANS,the nanocomplex was transported into cells,and the "signal-on" sensing of ATP in living cells was realized,and the nanocomplex could even be employed for in situ ATP semi-quantification.This design provides a methodology model scheme for development of biosensors based on nanomaterials for intracellular molecules analysis.And the polydopamine nanomaterials could be a robust candidate for many biological fields,such as gene and drug delivery,intracellular imaging and in vivo monitoring.5.Polydopamine nanospheres signal amplification for sensitive fluorescence polarization detection of small moleculesFluorescence polarization(FP)is a reliable,sensitive,and robust assay approach for determination of many biological targets.However,it is generally not applicable for the assay of small molecules because their molecular masses are relatively too small to produce observable FP value changes.To address this issue,a FP signal amplification strategy was developed by employing polydopamine nanospheres(PDANS)as the signal amplifier.Because of the extraordinarily larger volume of PDANS,the fluorophore exhibits very high polarization when bound to PDANS.Conversely,low polarization is observed when the fluorophore is dissociated from the PDANS.As proof-of-principle,the approach was applied to FP detection of adenosine triphosphate(ATP)with a fluorescent aptamer.The aptamer exhibited very high polarization when bound to PDANS,while the FP was greatly reduced when the aptamer complexed with ATP.ATP could be detected down to 280 nM with high selectivity in 40 minutes.Moreover,because FP is less affected by environmental interferences,FP measurements could be conveniently used to directly detect as low as 1.02 μM ATP in human serum with high-throughput.The universality of the approach could be achieved to detect an array of biological analytes when complemented with the use of functional DNA structures.