Study On The Recognition And Detection Of Biomacromolecules Based On Upconversion Nanomaterials

Biomacromolecules like nucleic acids and proteins are closely related to life activities.They play different biological functions through the special structure and particular movement,sending out predictive signals about the normal or abnormal body status.The recognition and detection of biomacromolecules have great significance in genomics,proteomics and biomedical diagnosis and treatment.The optical detective technology has been established as important molecular detection methods because it is easy to operate and does not need separation procedures.Rare earth doped upconversion luminescence nanoparticle with long excitation(near infrared),short emission(ultraviolet,visible or near infrared)can effectively avoid the interference of the biological sample autofluorescence on the optical detection and improve the sensitivity and accuracy of the detection.Based on the excellent luminescence properties,the construction of macromolecule fluorescence probes of the rare earth doped upconversion luminescence nanoparticle has attracted much attention in recent years.In this theses,we studied the application of the rare earth doped upconversion luminescence nanoparticles as a novel fluorescent nano material for the identification and detection of biomacromolecules,like methyltansferase,modified nucleic acid base and biological enzyme.We established a series of new sensitive,rapid and reliable detection methods and provided new design ideas for the construction of biomacromolecule detection sensors.In chapter 1,the recognition of biomacromolecules was reviewed,including DNA and nucleic acid modification,the structure and function of proteins,the features of various detection technologies for biomacromolecules,the applications,advantages and disadvantages of the current common fluorescence nano materials in the biomacromolecule detection,the fabrication and surface modification of rare earth doped upconversion luminescence nanoparticle and its applications in the identification and detection of biomacromolecues.In chapter 2,we designed and constructed a DNA methylation transferase nanosensor based on the luminescence resonance energy transfer(LRET)between upconversion luminescence nanoparticles and gold nanorods.First,the uniform red emission rare earth doped oleic acid coated upconversion luminescence nanoparticles NaYbF4:20%Y,2%Er(OA-UCNPs)with good luminescence performance have been synthesized by thermal decomposition;secondly,a ligand exchange method has been used for the surface modification of OA-UCNPs with branched polyethyleneimine,which increasing the dispersibility of UCNPs in aqueous solution and providing a modified bio-functional groups on the surface of UCNPs at the same time;thirdly,streptavidin was conjugated with the amino modified UCNPs(PEI-UCNPs)through amide bond;finally,the DNA methylation transferase nanosensor has been successfully assembled by using streptavidin coupled UCNPs as donors,AuNRs as acceptors and biotin and-SH bi-modificated duplex as the linker through the specific binding ability between biotin and streptavidin and the covalent bond between-SH and Au0.The construction of the nanosensor has been characterized by TEM,XRD,FT-IR,UV-Vis,upconversion luminescence and Zeta potential.Upconversion luminescence spectral has been used to identify and quantitatively detect DNA methyltransferase M.HhaI in the presence of restriction endonuclease HhaI.The results showed the high sensitivity of the probe,linear interval for 0.8?24 U·mL-1 with the DOL of up to 0.04 U·mL-1.Based on the detection ability of methyltransferase activity of the nanosensor,we tested the activity of methyltransferase inhibitors.The results showed that 5-azacytidine and procaine have different performance on the inhibition of methyltransferase activity because their mechanisms are different.In chapter 3,a LRET rare earth doped upconversion nanosensor has been constructed using upconversion luminescence nanoparticles(NaYbF4:20%Y,2%Er),gold nanorods and rational designed aptamer.With the help of restriction endonucleases Hpa? and MspI,the nanosensor can identify the 5'-CCGG-3' DNA sequence and respond to methylcytosine in CpG dinucleotide.This nanosensor has been also used to detect the methylcytosine in the special sequence 5'-CCGG-3' of DNA with the LOD of 7 pM.The sensitivity of nanosensor is 0.25 mg·mL-1.In chapter 4,a trypsin-sensitive upconversion luminescence nanoprobe has been built using the green light emissive rare earth doped upconversion luminescence nanoparticles(NaYF4:20%Yb,2%Er),gold nanoparticles and a rational designed peptide.The construction of nanosensor has been characterized by TEM,XRD,FT-IR,UV-Vis,upconversion luminescence and Zeta potential.The nanosensor has been used as the substrate of trypsin.Acording to the FRET mechanism,the upconversion luminescence intensity would be significantly enhanced when the peptide in the nanosensor was hydrolyzed by trypsin.The nanosensor of trypsin has a high selectivity and wide detection linear interval in 12-208 ng·mL-1,and a LOD of 4.15 ng·mL-1.The initial of trypsin hydrolysis rate has a liner correlation with the concentration of BBI in the range of 0-10 ?g·mL-1.The levels of trypsin in patients can be used as one of the markers for pancreatic diseases or cancers.The designed nanosensor based on the UCNPs could be used for the detection of trypsin and screening of its inhibitors.Through the above study,it shows that upconvesion nanomaterials can be used in the recognition and detection of biomacromolecule,such as DNA methyltransferase,nucleic acid methylation and protease,et al.By selecting the right coupling group,this type of upconversion luminescence nanoprobe can be used to detect other biomacromolecules and has great prospect.