| Protein force spectroscopy measurement treats single protein molecule as research object to measure the force and displacement of the unfolding and folding process in real time,calculates and analyses the dynamic characteristic,and then is used in stability research,pathological analysis and so on.Now atomic force microscopy,magnetic tweezers and optical tweezers are the most widely used force spectroscopy techniques,among which optical tweezers show good applicability in protein force spectrum measurement due to its high mechanical,spatial and temporal resolution.The force spectrum measurement technique based on optical tweezers stretches and contracts the protein molecule by controlling the microsphere at both ends of the protein chain to move away and close to each other,and measure the force spectrum data in the process.In order to ensure the reliability and accuracy of measurement data and record the distribution of protein molecules in different states,it is necessary to count hundreds of force spectrum data in the process of unfolding and folding.However,due to the low content of effective protein molecular chain in the sample,it is difficult to form effective capture and easy to be broken after capture,which leads to the question of low measurement efficiency.Therefore,it is necessary to explore the methods to improve the efficiency of protein force spectroscopy measurement based on optical tweezers,in order to solve the contradiction between the requirement and the current situation.This paper focuses on this topic and covers three aspects: the coupling efficiency of protein molecular chain,the coupling strength of protein molecular chain,and the capturing efficiency of effective microsphere,which correspondingly solves the problem that the content of effective protein molecular chain is low in the early stage of force spectrum measurement,protein molecular chain is easily broken in the process of force spectrum measurement,and the microsphere connecting with protein molecular chain is hardly captured in the late stage of force spectrum measurement.Through the study of the whole process,a relatively complete system of high-efficiency protein force spectroscopy measurement is established,which has been verified by experiments.The main contents are as follows:1.The theory of optical tweezers,protein and force spectroscopy measurement involved in the protein force spectroscopy measurement based on optical tweezers are fully elaborated and analyzed,so that we can rightly use the system of optical tweezers,get the accurate recognition of protein molecular chains and provide the theoretical support for the analysis of force spectroscopy data in subsequent chapters.2.With respect to the problem that there is only low content of effective protein molecular chain in the sample,a protein molecular chain coupling method based on a single DNA chain is proposed from the design of biomolecular system,which can effectively avoid the interference of heteromolecular chain and provide a basic guarantee for improving the efficiency of force spectroscopy measurement.3.With respect to the problem that protein molecular chain is easily broken in the process of protein force spectroscopy measurement,a method of enhancing the coupling strength of protein molecular chain based on label modification is proposed,which can maintain the specific connection and enhance the coupling strength,realize the acquisition of high-frequency force spectroscopy data under the success of single capture,and provide a new idea for the study of force spectrum measurement of high unfolding force.4.With respect to the problem that the number of microspheres connected with protein molecular chain decreases and the efficiency of effective microsphere capture reduces,a method of effective microsphere capture based on the manipulation of micro droplets has been explored,and the original strategy of probability acquisition is changed to realize the controllable capture of effective microsphere. |
PDF Full Text Request