Target Binding Inductive Effect-based Fluorescence Biosensors For The Detection Of Protein

Protein is an important component of cell,and its sensitive and specific detection is of great significance for molecular diagnosis and medical research.Transcription factors are DNA binding proteins that regulate gene expression by binding to specific regions.Abnormal expression of transcription factors can cause inflammation,cancer and other related diseases,which have become a biomarker in diagnostic research.Terminal deoxynucleotidy transferase is a nontemplate-dependent polymerase protein that can add deoxyribonucleoside triphosphates to the end of the DNA strand and it is a biomarker of acute leukemia.Therefore,the sensitive and specific detection of transcription factor and terminal deoxynucleotidy transferase are of great significance for clinical diagnosis and pathological studies.Target binding inductive effect is a strategy that the binding of the target and DNA can protect or trigger next reaction due to space steric hindrance or the change of DNA structure.In this strategy,detection of protein can be transferred specifically to nucleic acid detection and it is easy to combine with amplification method,which can improve the utilization rate of the target and provide a platform for specific and sensitive detection of proteins.DNA nanomachine is a DNA nano-assembly technology that can achieve sustained movement through energy conversion based on DNA structure advantage of the programmatically,multivariat and controllability.This energy conversion is often caused by chain substitution,enzymatic reactions and environment change.DNA nanomachine research is of great significance in the field of targeted drug delivery,intracellular material transport and nano-scale assembly and can be used as biosensors for target analysis.Based on target binding inductive effect,we developed fluorescence biosensor for specific and sensitive detection of transcription factor and terminal deoxynucleotidy transferase.The main contents of this dissertation are presented as follows:Chapter one is the introduction to summarize the significance and traditional detection method of transcription factor and terminal deoxynucleotidy transferase as well as the basic principle and classification about target binding inductive effect.The concept of DNA nanomachine and its development are introduced and DNA nanomachine can be divided into environmental driving,enzymes driving and chain replacement driving based on three different driving mechanisms.In chapter two,a fluorescent biosensor is developed for sensitive detection of transcription factors based on target binding protection mediated rolling circle amplification.The binding of transcription factors with specific DNA sequences can cause steric hindrance to inhibit the cutting of Nt.BbvCI,which can guarantee the specificity of the detection.In addition,the multifunctional hairpin probe designed can be used for binding target,identifying Nt.BbvCI,outputting signal and serving as templates,which can avoid complex design of probe and nonspecific amplification.This method realizes the sensitive detection of TATA-binding protein with the detection limit of 88 pM and the linear range of 100 pM to 40 nM,exhibiting high specificity.In chapter three,a DNA walker nanomachine is developed based on target binding induced extension to initiate for terminal deoxynucleotidy transferase detection.By the binding of target and 3' hydroxyl end of DNA chain,DNA chain is induced to extend sequence and produced the active site,activating DNA nanomachine.At the same time,the fluorescence groups are continuously released by the cutting of lamada exonuclease and the detection signal was amplified to realize the sensitive detection of target.In the absence of target,the active site cannot be generated,avoiding effectively the background signal caused by non-specific activation.This method exhibits high specificity and sensitivity with the detection limit of 7 U/mL.