The Study Of Novel Methods For Fluorescence Biosensing Based On Enzyme-free And Label-free Signal Amplification

Although the expression level of molecules that can be used as disease-related biomarkers is abnormally high,the total amount is still low in the organism.To achieve effective detection of low-dose disease-related biomarkers,the signal amplification process of biomolecules is essential.The enzyme-free signal amplification methods of biomolecules have more relaxed conditions and easier operations,which can also perform signal amplification from hundreds to thousands of times for target biomolecules when compared with the traditional enzymatic signal amplification methods.The fluorescence detection method is relatively mature in the field of life sciences,and it can be divided into labeling fluorescence detection and label-free fluorescence detection.Among them,label-free fluorescence detection avoids almost all complex covalent modifications and chemical labels,which has the advantages of simple operations,high specificity and economic effectiveness.Therefore,the development of a simple and economical fluorescent biosensor to realize the detection of specific biomarkers and enzymes is of great significance for the early intervention and prognosis of relevant tumors and nervous system diseases.And fluorescence detection method based on the catalytic hairpin assembly（CHA）has been studied in this thesis.Besides,a simple and economical fluorescence detection method has been developed for the detection of related enzymes in Alzheimer’s disease.The specific research content of this thesis is as follows:1.A method based on catalytic hairpin assembly amplification process for detecting the activity of Endo IV was constructed.In the initial stage of the detection method,Endo IV specifically acts on the AP site of the substrate hairpin structure to release the initiating fragments that sealed in the substrate hairpin and then initiates the signal amplification process of the catalyzed hairpin assembly,which enables the detection of Endo IV activity.The experimental results show that the method is highly sensitive,easy to operate and with a detection limit of 3.7×10^-7 U/m L,which is expected to be used for the detection of other base excision repair enzymes and the screening of related inhibitors.2.A target-triggered catalytic hairpin assembly fluorescent signal amplification detection method was fabricated.The introduction of DNA templated silver nanoclusters（DNA-Ag NCs）on the basis of enzyme-free signal amplification greatly reduces the background interference caused by fluorescent dyes and can provide more stable fluorescence output under the action of Guanine（G）-rich sequences.It has been found that the fabricated method has a detection limit of～45 p M.Of course,this method can also be extended to detect other disease biomarkers（such as micro RNA and other small biomolecules）through reasonable adjustment of the hairpin probe sequence or structure.This enzyme-free fluorescence signal amplification method based on target sequence triggering shows great potential in the early diagnosis and early intervention of related diseases.3.A fluorescence detection method for butyrylcholinesterase based on polydopamine（PDA）was developed.For this label-free detection method,PDA fluorescent nanoparticles are obtained by the conversion of dopamine under the action of potassium permanganate and then spontaneous agglomeration.Experimental results show that the detection limit of this simple and economical fluorescence detection method for butyrylcholinesterase can reach 0.47 ng/m L.And this butyrylcholinesterase fluorescent detection method based on PDA nanoparticles is expected to further explore the role of butyrylcholinesterase in Alzheimer’s disease and provide a certain clinical basis for the early diagnosis of Alzheimer’s disease.