Biosensor Researchment Based On Aggregation-indeced Luminescence

At present,the aggregation-inducing luminescent compounds have developed rapidly,especially tetraphenylethylene compounds.Due to their advantages such as simple synthesis and convenient modification process,the researchers have attracted extensive attention.In this dissertation,tetraphenylethylene derivatives were used as fluorescent probes.Based on their aggregation-induced luminescence properties,three kinds of fluorescence sensors were designed to analyze and detect myoglobin,methylase and protein kinase.The specific ideas are as follows:?1?Based on the self-positive electroporation of tetraphenylethylene quaternary ammonium salt,an experimental protocol was designed by using the specific binding of myoglobin and its aptamer.When myoglobin is absent,the aptamer and tetraphenylethylene quaternary ammonium salt are combined by positive and negative electricity,aggregation induces luminescence effect,and the fluorescence signal is high;when myoglobin is present,aptamer and myoglobin were binding,and tetraphenylethylene quaternary ammonium salt can not be stacked on the aptamer,which lead to the lower fluorescence signal.In order to detect myoglobin,a simple,fast and sensitive fluorescence biosensing method was designed.When the myoglobin concentration was in the range between 0.5 to 10?g/mL,the fluorescence intensity was in a good linear relationship with the myoglobin concentration.?2?The length of the DNA sequence has a certain influence on the fluorescence intensity generated by the accumulation of the tetraphenylethylene quaternary ammonium salt.So,we designed the TdT enzyme to extend the DNA sequence without a template to enhance the fluorescence effect.The stem-loop DNA sequence containing a specific methylation site and a 3'-Dabcyl end modification was designed.After a series actions by the methylase,Dpn I,and TdT enzymes,the stem loop was cut and the 3'-Dabcyl end was released,and the rest of the DNA fragment was extended.Fluorescent probes that are free in solution accumulate on the extended DNA strands,causing aggregation-induced luminescence effects.Detection of methylation enzymes is achieved by collecting fluorescence signals.There is a good linear relationship between the fluorescence intensity and the concentration of Dam MTase in the range between 0.5 to 100 U/mL.?3?The tetraphenylvinyl group has strong hydrophobicity.Click reaction is used to bind the hydrophilic polypeptide and TPE-N₃,which leads to the good hydrophilicity of the peptide-TPE probe and the weak self fluorescence intensity in dilute solution.Based on the characteristics of carboxypeptidase cleavage and protein kinase phosphorylation of specific polypeptide sites,an experimental protocol for detecting protein kinase CKII activity was designed.Without CKII treatment,carboxylic acid residues can not be phosphorylated by the protein kinase and the peptide is completely digested by CPY.As a rerult,the fluorescent substance TPE is released into Dam MTase the solution.Because of its strong hydrophobicity,small molecules of TPE will aggregate and the fluorescence signal will increase.After the CKII treatment,the phosphorylated residues can prevent the completely digestion of polypeptide by carboxypeptidase.The hydrophilicity of the remained polypeptide is still strong,so that no aggregation-induced luminescence effect is generated and the fluorescence signal can not be enhanced.There is a good linear relationship between the fluorescence intensity and the concentration of target CKII in the range of 0.1-80m U/?L.