A method for detecting photoisomerization of a single molecule fluorescent protein

Abstract

The application provides a detection method of single-molecule fluorescent protein photoisomerization, wherein a prepared solid-state nanopore membrane is loaded into a detection platform, a GFP test sample is dissolved in a buffer solution, and a sample obtained by irradiating the solution under a visible light source and a sample obtained by irradiating the sample under the visible light source again under an ultraviolet light source are added into a detection cell, electrical signals of the GFP sample obtained under the visible light source and under the visible light source / ultraviolet light source alternating irradiation are collected respectively, and differences of the two kinds of electrical signals are analyzed. The application can obtain information of a molecular size, a surface charge polarity and a shape of a protein by analyzing differences of ion blocking current amplitude, translocation time and pore blocking rate and changes of a molecular perforation orientation of the green fluorescent protein entering the nanopore due to surface charge distribution and conformational changes of protein isomerization under the electrical method and bias voltage.

Description

Technical Field

[0001] This invention belongs to the field of single-molecule protein detection technology, specifically relating to a method for detecting photoisomerization of single-molecule fluorescent proteins. Background Technology

[0002] Enhanced green fluorescent protein (eGFP) is a broad protein composed of 11 retrograde β-sheet strands. high eGFP is a tightly packed, barrel-shaped protein composed of 238 amino acids with a molecular weight of approximately 26.9 kDa. It is a mutant based on green fluorescent protein (GFP) with higher fluorescence intensity and stability, making it an ideal probe for medical detection. eGFP has wide applications in molecular-level labeling and tracing. By fusing the eGFP gene with specific genes through genetic engineering and re-expression, real-time observation of dynamic intracellular processes can be achieved. By labeling analytes, eGFP can be used in cancer research to monitor the proliferation, migration, and death of tumor cells, and can also precisely track the transmission pathways of labeled viruses to study viral infection mechanisms.

[0003] Traditional methods for detecting green fluorescent protein (eGFP) include fluorescence microscopy, Western blotting, quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. While these techniques have proven successful in detecting eGFP, they each have drawbacks: they typically require long reaction times, involve complex reaction steps, and cannot achieve single-molecule-scale detection. Solid-state nanopore technology, with its advantages of label-free operation, low cost, high speed, and single-molecule detection, along with its high stability and integrability, has become a new research focus and hot topic. Summary of the Invention

[0004] To address the problems of long reaction times, complex reaction steps, and inability to achieve single-molecule-scale detection in existing green fluorescent protein detection methods, this invention provides a method for detecting single-molecule photoisomerism of fluorescent proteins. Utilizing solid-state nanopore single-molecule electrical detection technology, this method analyzes and identifies the photoresponse isomerism of proteins by collecting the electrical signal characteristics of fluorescent proteins passing through the nanochannel after switching between visible and ultraviolet light irradiation within the same pore. This novel method for detecting photoisomerism of functional proteins provides a simple characterization approach for fluorescent protein-based labeling and tracing applications.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] The purpose of this invention is to provide a method for detecting photoisomerism of single-molecule fluorescent proteins. The method involves loading a prepared solid nanoporous membrane into a detection platform, dissolving the GFP test sample in a buffer solution, and adding samples obtained by irradiating the solution under a visible light source and samples obtained by irradiating the solution under a visible light source followed by an ultraviolet light source into a detection cell. The electrical signals of the GFP samples obtained under visible light source irradiation and under alternating visible / ultraviolet light source irradiation are collected respectively, and the differences between the two electrical signals are analyzed.

[0007] Furthermore, a method for detecting single-molecule fluorescent protein photoisomerism includes the following steps:

[0008] 1) Prepare Sample 1: Dissolve GFP in a buffer solution to obtain a 10-50 nM GFP solution, and then irradiate it under a visible light source at a wavelength of 450 nm;

[0009] 2) Configure Sample 2: Irradiate the Sample 1 obtained in step 1) with an ultraviolet light source with a wavelength of 365nm in a light-protected environment;

[0010] 3) Assembly of the solid nanopore detection platform: The prepared solid nanopore membrane is transferred to the detection cell, and the two chambers are connected to the patch clamp through electrodes;

[0011] 4) Electrical signal acquisition: Sample 1 was added to the Cis side of the detection cell, and the buffer solution from step 1) was added to the Trans side. A bias voltage was applied to the patch clamp to obtain the electrical signal under visible light. Sample 2 was added to the Cis side of the detection cell, and the buffer solution from step 1) was added to the Trans side. A bias voltage was applied to the patch clamp to obtain the electrical signal under alternating visible / ultraviolet light. The difference between the two electrical signals was analyzed.

[0012] Furthermore, the buffer solution is a mixed solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.4.

[0013] Furthermore, the duration of irradiation using visible light sources is the same as that using ultraviolet light sources.

[0014] Furthermore, the light source was irradiated with visible light and ultraviolet light for 30–60 minutes respectively.

[0015] Furthermore, the light was irradiated with visible light and ultraviolet light for 30 minutes each.

[0016] Furthermore, the solid nanoporous membrane is SiN. x membrane.

[0017] Furthermore, the method for preparing the solid nanoporous membrane is as follows: SiN xThe thin film was immobilized in the detection cell, and a mixed solution of 1 mM KCl, 10 mM Tris, 1 mM EDTA, and pH 8 was injected. The SiN film was then subjected to a current pulse. x Nanopores are formed by dielectric breakdown at defects in the thin film structure.

[0018] Furthermore, the SiN x The thin film is SiN x The chip is immersed in ethanol, water, and acetone solutions to remove contaminants from the film surface, then cleaned with plasma water, and finally the SiN is treated. x The thin film was obtained by oxygen plasma injection treatment on its surface.

[0019] Furthermore, the SiN x The film thickness is 15–20 nm, and the film is suspended in a window with an area of ​​4–10 μm. 2 On a silicon substrate.

[0020] Furthermore, the soaking time is 0.5 to 1 hour.

[0021] Furthermore, the obtained SiN x The diameter of the nanopores in the nanoporous membrane is 5–6 nm.

[0022] Furthermore, the electrode used in step 3) is an Ag / AgCl electrode.

[0023] Furthermore, the bias voltage applied during detection in step 4) is 100-200mV.

[0024] Furthermore, the electrical signal includes translocation time, blocking amplitude, and orifice ratio.

[0025] Furthermore, the GFP is eGFP.

[0026] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0027] This invention is based on single-molecule solid-state nanopore detection technology, using silicon-based silicon nitride thin films as nanopore carrier materials to detect the electrical signals of fluorescent proteins after switching between visible and ultraviolet light irradiation. Through electrical methods, the amplitude of ion blocking current, translocation time, porosity differences, and changes in molecular perforation orientation caused by the surface charge distribution and conformational changes of photo-induced isomerized green fluorescent proteins entering the nanopore under bias voltage are analyzed. This yields information such as the protein's molecular size, surface charge polarity, and shape. This invention provides important reference for studying the conformational changes and functional mechanisms of functional proteins under photoinduced conditions, and proposes a novel analytical method for the accurate detection of photoinduced isomerism in enhanced green fluorescent proteins and other functional proteins, which is significantly superior to existing traditional detection techniques.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the nanopore detection of green fluorescent protein in the single-molecule fluorescent protein photoisomerization detection method of the present invention.

[0030] Figure 2 This is a schematic diagram illustrating the switching of visible / ultraviolet light sources to irradiate the sample in a single-molecule fluorescent protein photoisomerization detection method according to the present invention.

[0031] Figure 3 The images show the detection data of sample translocation time, blocking current amplitude, and porosity under visible light irradiation and under alternating visible / ultraviolet light irradiation in Example 1 of this invention. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0033] Furthermore, unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be obtained commercially or prepared using existing methods. In the embodiments of this invention, SiN... x The chip was purchased from Suzhou Nanotech Co., Ltd. The chip film thickness is 20 nm, and the film is suspended in a window area of ​​4 μm. 2 On a silicon substrate.

[0034] Example 1:

[0035] See appendix Figure 1-2 A method for detecting photoisomerization of a single-molecule fluorescent protein, comprising the following steps:

[0036] 1) Pretreatment of silicon nitride chips and preparation of solid nanoporous membranes

[0037] SiN x The chip is immersed in ethanol, water, and acetone solutions to remove organic and inorganic contaminants from the film surface, then cleaned with plasma water, and finally the SiN... x Pretreated SiN is obtained by oxygen implantation treatment on the surface of the thin film. x Thin film. Pretreated SiN... xThe thin film was immobilized in the detection cell, and a mixed solution of 1M KCl, 10mM Tris, 1mM EDTA, and pH 8 was injected. SiN... x The thin film is exposed to an electric field with an intensity comparable to its dielectric strength. The charge accumulation and thermal effects of this strong electric field induce leakage currents at structural defects in the thin film, leading to SiN... x Nanopores are formed by dielectric breakdown at defects in the thin film structure.

[0038] 2) Preparation of test samples

[0039] Prepare Sample 1: Dissolve eGFP in a buffer solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.4 to obtain a 50nM GFP solution, and then irradiate it under a visible light source at a wavelength of 450nm for 30min.

[0040] Prepare Sample 2: Dissolve eGFP in a buffer solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.4 to obtain a 50nM eGFP solution. Then irradiate the solution with a visible light source at a wavelength of 450nm for 30min, and then irradiate it with a UV light source at a wavelength of 365nm for 30min in a dark environment.

[0041] 3) Assembly of the solid-state nanopore detection platform

[0042] The solid nanoporous membrane prepared in step 1) is transferred to the detection cell, and the two chambers of the detection cell are connected to the e-One-HS micro patch clamp via Ag / AgCl electrodes.

[0043] 4) Electrical signal acquisition

[0044] Sample 1 was added to the Cis side of the detection cell, and a buffer solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.4 was added to the Trans side. The e-One-HS micro-patch clamp was opened, and a bias voltage of 100-200mV was applied to obtain the electrical signal under visible light. Sample 2 was added to the Cis side of the detection cell, and a buffer solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.4 was added to the Trans side. The e-One-HS micro-patch clamp was opened, and a bias voltage of 100-200mV was applied to obtain the electrical signal under alternating visible / ultraviolet light irradiation. The differences between the two electrical signals were analyzed.

[0045] For Example 1, the translocation time, blocking current amplitude, and pore blockage rate were measured, and all measurements were performed in a dark Faraday cage.

[0046] See appendix Figure 3 It can be seen that the nanopore translocation signal of eGFP under different bias voltages has obvious characteristics, which conforms to the basic law that the translocation time decreases with the increase of voltage and the blocking current amplitude increases with the increase of voltage. When enhanced green fluorescent protein (eGFP) under visible light is irradiated with ultraviolet light, its pore-passing time decreases from 0.2222±0.0135 ms to 0.1753±0.0093 ms, showing a significant reduction in pore-passing time. This is because the conformational change after irradiation reduces the average spatial size and weakens the interaction with the nanopores. Furthermore, the blocking current amplitude and ΔI / Io distribution of eGFP change significantly before and after irradiation. The ΔI / Io of eGFP in longitudinal pore-passing decreases from 0.4182±0.0005 to 0.3971±0.0019, while the ΔI / Io of eGFP in transverse pore-passing increases from 0.6765±0.0125 to 0.9025±0.0305. This is because ultraviolet light provides energy to the eGFP molecules, causing some molecules to change their pore-passing mode, resulting in a decrease in the blocking current amplitude of longitudinal pore-passing and a significant increase in the blocking current amplitude of transverse pore-passing. Therefore, the conformation of eGFP changes after being irradiated with visible and ultraviolet light. By comparing the electrical signals before and after irradiation, the single-molecule information of enhanced green fluorescent protein can be accurately and clearly presented.

[0047] Example 2

[0048] Unlike Example 1, the irradiation time of the visible light source at a wavelength of 450nm and the irradiation time of the ultraviolet light source at a wavelength of 365nm in step 2) were adjusted to 40min. Electrical signal detection was performed under visible light irradiation and visible / ultraviolet alternating light irradiation, including displacement time, blocking current amplitude, and porosity.

[0049] The test results showed that even after the illumination time was adjusted, the displacement time, the amplitude of the blocking current, and the pore blockage rate still showed significant changes, indicating that the detection method of the present invention is simple and highly operable.

[0050] The detection method of this invention can provide more detailed information on the shape, surface charge, size, and conformation of enhanced green fluorescent protein (eGFP). It also allows for the control of single-molecule permeation rates and is simple and inexpensive. By capturing protein conformational changes in real time, it has broad application prospects in the fields of biomedicine and personalized medicine. Solid-state nanopore technology can detect the size and shape of eGFP, which can be used to construct highly sensitive biosensors, contributing to the development of devices for specific molecule detection. Furthermore, combining solid-state nanopores with optical methods such as fluorescence microscopy can further facilitate protein analysis studies, such as protein folding and protein-aptamer interactions, providing early diagnosis or prediction of disease progression for conditions like Parkinson's and Alzheimer's. In addition, combining eGFP with biomolecules or organelles, or introducing the eGFP gene into specific gene fragments through genetic engineering, allows for real-time, precise, and visual tracking of the dynamic processes of target substances within cells, providing efficient research tools for cell biology and drug development. This method provides a highly sensitive and high-resolution detection approach for single-molecule fluorescent proteins, especially eGFP, opening up new avenues for the study of conformational changes of functional proteins and their disease-related pathological findings, and has promising application prospects.

[0051] The detection method of this invention is also applicable to the detection of conformational changes in other biological functional proteins caused by mechanical force, salt concentration, light exposure, etc. It should be noted that the specific embodiments described above can enable those skilled in the art to more fully understand this invention, but do not limit this invention in any way.

[0052] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0053] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for detecting photoisomerization of a single-molecule fluorescent protein, characterized in that: The prepared solid nanoporous membrane was loaded into the detection platform. The GFP test sample was dissolved in the buffer solution. The sample obtained by irradiating the solution under a visible light source and the sample obtained by irradiating the solution under a visible light source and then under an ultraviolet light source were added to the detection cell. The electrical signals of the GFP sample obtained under visible light source irradiation and under alternating visible / ultraviolet light source irradiation were collected respectively, and the differences between the two electrical signals were analyzed.

2. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 1, characterized in that, Includes the following steps: 1) Prepare Sample 1: Dissolve GFP in a buffer solution to obtain a 10-50 nM GFP solution, and then irradiate it under a visible light source at a wavelength of 450 nm; 2) Configure Sample 2: Irradiate the Sample 1 obtained in step 1) with an ultraviolet light source with a wavelength of 365nm in a light-protected environment; 3) Assembly of the solid nanopore detection platform: The prepared solid nanopore membrane is transferred to the detection cell, and the two chambers are connected to the patch clamp through electrodes; 4) Electrical signal acquisition: Sample 1 was added to the Cis side of the detection cell, and the buffer solution from step 1) was added to the Trans side. A bias voltage was applied to the patch clamp to obtain the electrical signal under visible light. Sample 2 was added to the Cis side of the detection cell, and the buffer solution from step 1) was added to the Trans side. A bias voltage was applied to the patch clamp to obtain the electrical signal under alternating visible / ultraviolet light. The difference between the two electrical signals was analyzed.

3. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 2, characterized in that: The buffer solution is a mixture of 1M KCl, 0.5mM CaCl2, 10mM Tris, and pH 7.

4.

4. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 1 or 2, characterized in that: The duration of illumination using visible light sources is the same as that using ultraviolet light sources.

5. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 4, characterized in that: Irradiate with visible light and ultraviolet light for 30–60 minutes respectively.

6. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 1 or 2, characterized in that: The solid nanoporous membrane is SiN. x The membrane has a thickness of 15-20 nm and is suspended in a window of 4-10 μm. 2 On a silicon substrate.

7. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 1 or 2, characterized in that: The solid nanoporous membrane is prepared by: using SiN x The thin film was immobilized in the detection cell, and a mixed solution of 1M KCl, 10mM Tris, 1mM EDTA, and pH 8 was injected. The SiN film was then subjected to a current pulse. x Nanopores are formed by dielectric breakdown at defects in the thin film structure.

8. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 7, characterized in that: The SiN x The thin film is SiN x The chip is immersed in ethanol, water, and acetone solutions to remove contaminants from the film surface, then cleaned with plasma water, and finally the SiN is treated. x The thin film was obtained by oxygen plasma injection treatment on its surface.

9. The method for detecting single-molecule fluorescent protein photoisomerism as described in claim 7, characterized in that: The obtained SiN x The diameter of the nanopores in the nanoporous membrane is 5–6 nm.

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