Avidin Functionalized Thermo-responsive Materials And Its Application In The Fluorescence Analysis Of Methylase And Thrombin

It is very important to establish a highly efficient and sensitive method for detecting biological enzymes,which play a very important role in life activities.The fluorescence analysis method is simple in operation,high in sensitivity,low in detection limit,and often used for analysis and detection of enzymes.Thermosensitive polymers are a class of polymers with specific phase transition temperatures.Poly-N-isopropylacrylamide?PNIPAAm?is a typical type of temperature-sensitive polymer with a phase transition temperature of 32 ?,which is very close to the physiological temperature of the human body.When the temperature is lower than 32 ?,the polymer is uniformly dispersed in the aqueous solution,and the solvent forms a clear transparent solution.Once the temperature is higher than the phase transition temperature,the hydrophobic interaction in the polymer molecule plays a major role,and the phase transition occurs.The solution becomes milky white gel.Many bioassays can be performed using the reversible phase transition properties of PNIPAAm materials.There is a specific strong interaction between streptavidin?SA?and biotin.And the study of SA-functionalized PNIPAAm-type temperature-sensitive polymers has also been widely reported.In order to further expand the new method for detecting biomolecules with fluorescent signals based on the temperature-sensitive polymers,our work focused on the detection of methyltransferase and thrombin.The details are as follows:?1?Synthesis of PNIPAAm-co-SA material.The single-stranded DNA?S₁?was designed with methyltransferase M.Sss I and restriction endonuclease sites,which was modified by biotin.Due to the strong interaction force between SA-biotin,the ss DNA containing the enzyme site was captured on the temperature sensitive material,and then the single-stranded DNA?S₂?complementary to S₁ was designed,and S₂ was modified with fluorescent dye FAM.The methyltransferase M.Sss I can transfer the methyl group on S-adenosylmethionine?SAM?in solution to the specific cytosine of double-stranded DNA,thus affecting the restriction endonuclease Hpa II.The PNPIPAAm was used for separation and enrichment,and the fluorescence intensity of the solution changed with the change of M.Sss I.In order to further improve the sensitivity of detection,we have introduced hybridization chain reaction?HCR?based on the previous work to improve the fluorescence intensity of the system and reduce the detection limit.We designed two special hairpin structures for DNA.After methylation of the transferase M.Sss I and restriction endonuclease Hpa II,the hairpin DNA?H1/H2?was added to initiate the HCR reaction,and the signal was amplified.,thereby improving the sensitivity of the system to detect M.Sss I.The detection limit is only 0.003 U/m L.?2?Thrombin is a very important substance in the human coagulation system.Abnormal thrombin in the human body causes many diseases,so it is very important to establish a highly sensitive and sensitive method for detecting thrombin.A special DNA sequence was designed,which was a complementary pair of thrombin aptamers.By using the specific binding ability of thrombin and its nucleic acid aptamer to release the short-stranded DNA,the specific hairpin DNA can be complementary to the short-stranded DNA.Then the nicking enzyme was added to introduce the nickel enzyme signal amplification?NESA?,due to the strong binding effect of SA-biotin,the biotin-modified hairpin DNA was separated and enriched with the temperature sensitive material PNIPAAm-co-SA.The dye-FAM-modified DNA was subjected to DNA hybridization,and the temperature-sensitive property of the temperature-sensitive material is used for separation and enrichment to detect thrombin.The detection limit of thrombin has been calculated was as low as 0.044 p M.