Synthesis Of Polydopamine Nanocomposites For Ratiometric Fluorescence Detection Of Nucleic Acid

Nucleic acides play important roles in various biological processes and its abnormal expression is closely related to the occurrence of many deseases.The detection of nucleic acides shows great potentials in the prediction,diagnosis and prognosis of diseases.Among the nucleic acid detection methods,ratiometric fluorescence method is a valuable tool for the accurate detection of analytes owing to its self-calibration feature,which can decrease the errors from light excitation and scattering,complex microenvironment,and so on.Regarding this,this thesis developed three kinds of polydopamine（PDA）based ratiometric fluorescence nanoprobes for nucleic acid detection,which have achieved high selectivity and sensitivity,and good accuracy in serum and cell samples.The detail works are as follows:Taking advantage of PDA’s high quenching efficiency,a core-shell structure with[Ru（bpy）₂（dcbpy）]²⁺encapsulated Si O₂ nanoparticle as the core（Ru-Si O₂）and PDA as the shell was synthesized（Ru-Si O₂@PDA）and used as a universal ratiometric fluorescence sensing nanoplatform for ratiometric detection of DNA and protein.The Ru-Si O₂@PDA nanocomposites have narrow size distribution,and can emit a stable luminescence at 650 nm under irradiation and simultaneously quench the fluorescence emitted from the various fluorophores getting close to them.The nanocomposites showed strong adhesion to fluorophore labelled DNA and could resist the actack from nuclease enzyme and proteins.The bio-recognition behaviors change the probe’s configuration and bring the fluorophore far away from the PDA shell.Correspondingly,the fluorescence recovers and its ratio to the constant fluorescence reference is linear to the targets’concentration.Using DNA and protein as model analytes,the Ru-Si O₂@PDA based nanoplatform showed high sensitivity and good accuracy in the ratiometric analysis of serum samples.Taking advantage of PDA’s high photothermal property,a highly sensitive ratiometric fluorescence nanoprobe for intracellular mi RNA imaging was fabricated by integrating a Ru-Si O₂@PDA nanoplatform with a near infrared light（NIR）assisted DNA strand displacement signal amplification strategy.The detection process was simple,which needed one-step incubation,without the additional transfection of fuel DNA.When the target was absent,the green fluorescence of signal DNA was quenched and Ru-Si O₂@PDA emited stable red fluorescence and weak green fluorescence.Once the target mi RNA was present,the target displaced the signal DNA from the capture DNA,released the signal DNA far away from the Ru-Si O₂@PDA and recovered the green fluorescence.Under NIR light irradiation,the Ru-Si O₂@PDA increased the local temperature around the probe and triggered the release of fuel DNA,which thus recycled the target mi RNA and effectively amplified the ratiometric signal.The value of I_green/I_red was linear to mi RNA let-7a ranging from 0.8-100 nM,and realized the fluorescence imaging of let-7a in A549 cells,as well as its in vivo up-and down-regulation expressions.The properties of PDA can be adjusted by controlling the polymerization degree.Using polyethyleneimine（PEI）to control the polymerization degree of PDA,a PDA and polyethyleneimine（PEI）copolymerized nanoparticles with fluorescence emission property were synthesized.The PDA-PEI nanoparticles have a narrow size distribution,emit stable green fluorescence,and can be stably dispersed in aqueous solution for a long time of at least 6 months.The PDA-PEI nanoparticles are full of functional groups of-OH,-NH₂,and aromatic ring,which can facilitate the assembly of DNA through hydrogen bond,electrostatic,andπ-πstacking.They can also well quench the fluorescence of the fluorophores getting close to them.By well control the chain length and doping amunt of PEI,the quenching efficiency of PDA-PEI to AMCA,TAMRA,and Cy5 all reached above 99%.Based on the stable green fluorescence and high quenching properties of PDA-PEI,a ratiometric nanoplatform for nucleic acid detection was constructed with the assistance of DSN enzyme,and achieves the highly sensitive and accurate detection of nucleic acid in serum.