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CN-119619230-B - Detection method for single-molecule fluorescent egg white photoisomerization

CN119619230BCN 119619230 BCN119619230 BCN 119619230BCN-119619230-B

Abstract

The invention provides a method for detecting white light isomerization of a single-molecule fluorescent egg, which comprises the steps of loading a prepared solid nano-pore membrane into a detection platform, dissolving a GFP (green fluorescent protein) test sample into a buffer solution, respectively irradiating the solution under a visible light source to obtain a sample, then irradiating the visible light source to obtain the sample, adding the sample irradiated by an ultraviolet light source into a detection pond, respectively collecting electrical signals obtained by the GFP sample under the irradiation of the visible light source and the alternate irradiation of the visible light source/ultraviolet light source, and analyzing the difference of the two electrical signals. According to the invention, photoisomerized green fluorescent protein entering the nanopore under bias is analyzed by an electrical method, and the information of the molecular size, the polarity of the surface charge, the shape and the like of the protein can be obtained by analyzing the difference of ion blocking current amplitude, translocation time and blocking rate and the change of molecular perforation orientation, which are generated by the heterogeneous surface charge distribution and conformational change of the protein.

Inventors

  • LIANG LIYUAN
  • XIANG LIMIN
  • CHEN XUAN
  • TIAN RONG
  • FANG SHAOXI
  • WENG TING
  • WANG DEQIANG

Assignees

  • 中国科学院重庆绿色智能技术研究院

Dates

Publication Date
20260508
Application Date
20241118

Claims (9)

  1. 1. A method for detecting white light isomerization of single-molecule fluorescent egg white light is characterized in that a prepared solid nano-pore membrane is filled into a detection platform, GFP test samples are dissolved in buffer solution, samples obtained by irradiating the solution under a visible light source and samples obtained by irradiating the solution with an ultraviolet light source are added into a detection pool, electrical signals obtained by irradiating the GFP samples under the visible light source and alternately irradiating the visible light source/the ultraviolet light source are respectively collected, and differences of the two electrical signals are analyzed.
  2. 2. The method for detecting white light isomerism of a single-molecule fluorescent egg according to claim 1, comprising the steps of: 1) Preparing a Sample 1, namely dissolving GFP in a buffer solution to obtain a GFP solution of 10-50 nM, and then irradiating the GFP solution under the wavelength of 450nm of a visible light source; 2) Preparing a Sample 2, namely irradiating the Sample 1 obtained in the step 1) with an ultraviolet light source with the wavelength of 365nm in a light-proof environment; 3) The assembly of the solid nano-pore detection platform, namely transferring the prepared solid nano-pore membrane into a detection pool, and connecting two chambers to a patch clamp through an electrode; 4) The method comprises the steps of collecting electrical signals, adding a Sample 1 Sample to the Cis side of a detection cell, adding the buffer solution in the step 1) to the Trans side, applying a bias voltage on a patch clamp to obtain electrical signals under visible light, adding a Sample2 Sample to the Cis side of the detection cell, adding the buffer solution in the step 1) to the Trans side, applying a bias voltage on the patch clamp to obtain electrical signals under visible/ultraviolet alternating light, and analyzing the difference of the two electrical signals.
  3. 3. The method for detecting white light isomerization of single-molecule fluorescent eggs of claim 2, wherein the buffer solution is 1M KCl,0.5mM CaCl 2 , 10mM Tris and pH 7.4 mixed solution.
  4. 4. The method for detecting white light isomerization of single molecule fluorescent egg according to claim 1 or 2, wherein the irradiation time is the same as the irradiation time of the visible light source and the ultraviolet light source.
  5. 5. The method for detecting white light isomerization of single-molecule fluorescent eggs according to claim 4, wherein the visible light source and the ultraviolet light source are respectively irradiated for 30-60 min.
  6. 6. The method for detecting white light isomerization of single-molecule fluorescent egg white according to claim 1 or 2, wherein the solid nano-pore membrane is a SiN x membrane with a membrane thickness of 15-20nm and is suspended on a silicon substrate with a window of 4-10 μm 2 .
  7. 7. The method for detecting white light isomerization of single-molecule fluorescent egg white light as set forth in claim 1 or 2, wherein the method for preparing the solid nano-pore film is characterized in that a SiN x film is fixed in a detection pool, a mixed solution with 1M KCl,10mM Tris,1mM EDTA,pH of 8 is injected, and dielectric breakdown is carried out at the defect of the SiN x film structure under current pulse to form nano-pores.
  8. 8. The method for detecting white light isomerization of single-molecule fluorescent eggs of claim 7, wherein the SiN x film is obtained by immersing SiN x chips in ethanol, water and acetone solution, removing pollutants on the surface of the film, cleaning the film with plasma water, and performing oxygen plasma oxygen injection treatment on the surface of the SiN x film.
  9. 9. The method for detecting white light isomerization of single-molecule fluorescent eggs of claim 7, wherein the diameter of a nano pore canal in the obtained SiN x nano pore membrane is 5-6 nm.

Description

Detection method for single-molecule fluorescent egg white photoisomerization Technical Field The invention belongs to the technical field of single-molecule protein detection, and particularly relates to a single-molecule fluorescent egg white light isomerism detection method. Background The enhanced green fluorescent protein (eGFP) is a broad band surrounded by 11 inverted-extending beta-sheet chainsHigh heightConsists of 238 amino acids and has a molecular weight of about 26.9kDa. eGFP is a mutant based on Green Fluorescent Protein (GFP), has higher fluorescence intensity and stability, and is an ideal probe in medical detection. The eGFP has wide application in the aspects of molecular level marking and tracing, and the eGFP gene and a specific gene are fused and re-expressed by a genetic engineering technology, so that the real-time observation of the intracellular dynamic process can be realized. By labeling the test substance, eGFP can be used for monitoring proliferation, migration and death processes of tumor cells in cancer research, and can precisely trace the transmission path of the labeled virus so as to study the infection mechanism of the virus. The traditional detection method for the green fluorescent protein comprises a plurality of methods such as a fluorescent microscope method, a Western blotting method, a fluorescent quantitative PCR method, an enzyme-linked immunosorbent assay method, a flow cytometry method and the like. These techniques have proven successful in detecting eGFP, but suffer from various drawbacks, they generally require long reaction times, relatively complex reaction steps, and do not allow for single-molecule scale detection. The solid-state nanopore technology has the advantages of no labeling, low cost, high speed, single molecule detection and the like, and becomes a new research key point and hot spot due to the characteristics of high stability, integration and the like. Disclosure of Invention In order to solve the problems that a detection method of green fluorescent protein in the prior art needs longer reaction time and more complex reaction steps, cannot realize detection of single molecular scale and the like, the invention provides a detection method of single molecular fluorescent egg white light isomerism, which utilizes a solid nano-pore single molecular electric detection technology to analyze and distinguish the photoresponse isomerism of the protein by collecting the electric signal characteristics of a nano channel after the fluorescent protein is subjected to visible and ultraviolet light switching irradiation in the same pore canal, and is a novel detection method of functional egg white light isomerism, thereby providing simple characterization for labeling and tracing application based on the fluorescent protein. The invention solves the technical problems by adopting the following technical scheme: the invention aims to provide a detection method for white light isomerization of single-molecule fluorescent eggs, which comprises the steps of loading a prepared solid nano-pore membrane into a detection platform, dissolving GFP test samples in buffer solution, respectively adding samples obtained by irradiating the solution under a visible light source and the samples obtained by irradiating the solution with an ultraviolet light source into a detection pond after the irradiation of the visible light source, respectively collecting electrical signals obtained by alternately irradiating the GFP samples under the irradiation of the visible light source and the visible light source/ultraviolet light source, and analyzing the difference of the two electrical signals. Further, the method for detecting the white light isomerism of the single-molecule fluorescent egg comprises the following steps: 1) Preparing a Sample 1, namely dissolving GFP in a buffer solution to obtain a GFP solution of 10-50 nM, and then irradiating the GFP solution under the wavelength of 450nm of a visible light source; 2) Preparing a Sample 2, namely irradiating the Sample 1 obtained in the step 1) with an ultraviolet light source with the wavelength of 365nm in a light-proof environment; 3) The assembly of the solid nano-pore detection platform, namely transferring the prepared solid nano-pore membrane into a detection pool, and connecting two chambers to a patch clamp through an electrode; 4) The method comprises the steps of collecting electrical signals, adding a Sample 1 Sample to the Cis side of a detection cell, adding the buffer solution in the step 1) to the Trans side, applying a bias voltage on a patch clamp to obtain electrical signals under visible light, adding a Sample2 Sample to the Cis side of the detection cell, adding the buffer solution in the step 1) to the Trans side, applying a bias voltage on the patch clamp to obtain electrical signals under visible/ultraviolet alternating light, and analyzing the difference of the two electrical signals. Further, the