Study And Application Of Fluorescence Polarization Analytical Methods Based On Aptamer Probes

Fluorescence polarization(FP)is a simple,rapid,high-throughout and automatical method which is widely accepted and applied in the fields of biosensors and immunoassays.Recently,increasing attention was paid to FP analytical method based on aptamer probes.Various FP methods based on aptamers were constructed to detect biomolecules.These studies mainly concentrated on the design of amplified methods on FP signal.In this paper,two novel amplified methods were founded and proved to enhance FP signal.And the detail was as follows:1.Here we report a novel type of aptasensor based on silver enhanced fluorescence polarization for detection of lactoferrin(Lac)in milk powder with high sensitivity and specificity.The novel two split aptamers were gotten from the aptamer reported in our previous SELEX(systematic evolution of ligands by exponential enrichment)selection and their minimal structural units were optimized based on their affinity and specificity.Two split aptamers were modified to be linked with signal molecule fluorescein isothiocyanate(FITC)and enhancer sliver decahedral nanoparticles(Ag10NPs)respectively.The split aptamers could bind to different sites of Lac and assembled into a split-aptamers-target complex,narrowing the distance between Ag10NPs and FITC dye.As a result,Ag10NPs could produce mass-augment and metal-enhanced fluorescence(MEF)effect.It was convinced that two split aptamerwere highly sensitive to Lac and bound to different sites of Lac.In general,ternary amplification based on Ag10oNPs,split aptamers and MEF effect all contributed to the significant increase of FP values.It was proved that the sensitivity of this assay was about three orders of magnitude over traditional aptamer-based homogeneous assays with detection limit of 1.25 pM.Furthermore,this design was examined by actual milk powder with rapid and high throughout detection2.In this work,a novel amplified FP aptasensor based on magnetic nanoparticles@polydopamine(MNP@PDA)was innovatively developed for the sensitive detection of recombinant human erythropoietin-alpha(rHuEPO-?).The amplified FP signal was due to the magnetic separation and large mass of protein and MNP@PDA.Briefly,rHuEPO-a and MNP@PDA were added successively to react with the labeled aptamer(FAM-P1),which both contributed to the increase of FP signal due to the formation of FAM-P1-rHuEPO-? and particularly FAM-P1-MNP@PDA complex.The strong interaction between MNP@PDA and FAM-P1 ensured the high efficiency of mass amplification and magnetic separation.As a result,the detection limit for rHuEPO-? was 0.12 pM,4 orders of magnitude lower than original assay.Besides,three kinds of rHuEPO-? injection were used to evaluate the selectivity of this assay in complex matrix with reasonable standard deviation.In a word,this work provides a simple,rapid and non-modified sensing platform for the detection of other biomolecules.