Detection method for photoisomerization of single-molecule fluorescent protein

Abstract

The invention provides a monomolecular fluorescent protein photoisomerization detection method, which comprises the following steps: loading a prepared solid nanopore membrane into a detection platform, dissolving a GFP test sample into a buffer solution, and respectively adding a sample obtained by irradiating the solution under a visible light source and a sample obtained by irradiating the solution with an ultraviolet light source after the irradiation of the visible light source into a detection pool; the method comprises the following steps: respectively acquiring electrical signals of a GFP sample under the irradiation of a visible light source and the alternate irradiation of the visible light source / ultraviolet light source, and analyzing the difference between the two electrical signals; according to the invention, through an electrical method, difference of ion blocking current amplitude, translocation time and hole blocking rate and molecular perforation orientation change caused by surface charge distribution and conformational change of protein isomerism when photoisomerized green fluorescent protein enters nanopores under bias voltage are analyzed; and information such as molecular size, surface charge polarity, shape and the like of the protein can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of single molecule protein detection, and in particular relates to a method for detecting photoisomerization of single molecule fluorescent protein. Background Art

[0002] Enhanced green fluorescent protein (eGFP) is a broad fluorescent protein consisting of 11 inversely extending β-pleated strands. high A compact barrel-shaped protein composed of 238 amino acids with a molecular weight of approximately 26.9kDa. eGFP is a mutant based on green fluorescent protein (GFP) with higher fluorescence intensity and stability, making it an ideal probe for medical testing. eGFP has a wide range of applications in labeling and tracing at the molecular level. By fusing the eGFP gene with a specific gene and then expressing it through genetic engineering technology, real-time observation of dynamic processes within cells can be achieved. By labeling the analyte, eGFP can be used in cancer research to monitor the proliferation, migration, and death of tumor cells, and can also accurately track the transmission path of the labeled virus to study the infection mechanism of the virus.

[0003] Traditional methods for detecting green fluorescent protein include fluorescence microscopy, Western blotting, fluorescence quantitative PCR, enzyme-linked immunosorbent assay, flow cytometry, etc. These technologies have been proven to successfully detect eGFP, but they have different disadvantages. They usually require a long reaction time, more complicated reaction steps, and cannot achieve single-molecule scale detection. Solid-state nanopore technology has the advantages of label-free, low cost, fast speed and single-molecule detection, and has become a new research focus and hotspot due to its high stability and integration characteristics. Summary of the invention

[0004] In order to solve the problems that the detection method of green fluorescent protein in the prior art requires a long reaction time, relatively complicated reaction steps, and cannot realize single-molecule scale detection, the present invention provides a detection method for single-molecule fluorescent protein photoisomerization, which utilizes solid-state nanopore single-molecule electrical detection technology to collect the electrical signal characteristics of fluorescent protein passing through the nanochannel after switching between visible and ultraviolet light irradiation in the same channel to analyze and identify the photoresponse isomerism of the protein. It is a new functional protein photoisomerization detection method, which provides a simple characterization for fluorescent protein labeling and tracing applications.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The purpose of the present invention is to provide a detection method for single-molecule fluorescent protein photoisomerization. The prepared solid-state nanoporous 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 solution with a visible light source and then an ultraviolet light source are added to a detection pool, and electrical signals of the GFP sample obtained under the irradiation of the visible light source and the irradiation of the visible light source / ultraviolet light source alternately are collected, and the difference between the two electrical signals is analyzed.

[0007] Furthermore, a method for detecting single-molecule fluorescent protein photoisomerization comprises 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 with a visible light source at a wavelength of 450 nm;

[0009] 2) Prepare Sample 2: Place the Sample 1 obtained in step 1) in a light-proof environment and irradiate it with an ultraviolet light source with a wavelength of 365 nm;

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

[0011] 4) Electrical signal acquisition: add Sample 1 to the Cis side of the detection cell, add the buffer solution in step 1) to the Trans side, apply a bias voltage to the patch clamp, and obtain an electrical signal under visible light; add Sample 2 to the Cis side of the detection cell, add the buffer solution in step 1) to the Trans side, apply a bias voltage to the patch clamp, and obtain an electrical signal under visible / ultraviolet alternating light; analyze the difference between the two electrical signals.

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

[0013] Furthermore, the irradiation time of the visible light source is the same as that of the ultraviolet light source.

[0014] Furthermore, the visible light source and the ultraviolet light source are irradiated for 30 to 60 minutes respectively.

[0015] Furthermore, the visible light source and the ultraviolet light source were irradiated for 30 minutes respectively.

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

[0017] Furthermore, the preparation method of the solid nanoporous membrane is: xThe film was fixed in the detection pool and a mixed solution of 1M KCl, 10mM Tris, 1mM EDTA, pH 8 was injected. x Dielectric breakdown at film structural defects forms nanopores.

[0018] Furthermore, the SiN x The film is SiN x The chip was immersed in ethanol, water, and acetone solution to remove contaminants on the membrane surface, and then cleaned with plasma water. x The film surface is treated with oxygen plasma oxygen implantation.

[0019] Furthermore, the SiN x The film thickness is 15-20nm, and the film is suspended in the window area of ​​4-10μm 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 to 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 100-200 mV.

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

[0025] Furthermore, the GFP is eGFP.

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

[0027] The present invention is based on single-molecule solid-state nanopore detection technology, using silicon-based silicon nitride film as a nanopore carrier material to perform nanopore electrical signal detection on fluorescent proteins after switching between visible and ultraviolet light irradiation. By electrical methods, the amplitude of the ion blockage current, the difference in translocation time and porosity, and the change in molecular perforation orientation caused by the surface charge distribution and conformational changes of the photo-isomerized green fluorescent protein entering the nanopore under bias are analyzed, and information such as the molecular size, surface charge polarity, and shape of the protein can be obtained. The present invention has an important reference for studying the conformational changes of functional proteins under light induction and their functional mechanisms, and proposes a new analytical method for the precise detection of photo-isomerization of enhanced green fluorescent protein and even other functional proteins, which is significantly better than the traditional detection technology reported previously.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 The present invention is a schematic diagram of switching between visible / ultraviolet light sources to irradiate a sample in a method for detecting single-molecule fluorescent protein photoisomerization.

[0031] Figure 3 These are detection graphs of the translocation time, blocking current amplitude, and porosity of the sample under visible light irradiation and the sample under visible / ultraviolet alternating light irradiation in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations 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 included in the scope of protection that the present invention is intended to protect.

[0033] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. x The chip was purchased from Suzhou Nano Company. The chip film thickness is 20nm, and the film is suspended in the window area of ​​4μm 2 on a silicon substrate.

[0034] Embodiment 1:

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

[0036] 1) Silicon nitride chip pretreatment and preparation of solid-state nanoporous membrane

[0037] SiN x The chip was immersed in ethanol, water, and acetone solution to remove organic and inorganic pollutants on the membrane surface, and then cleaned with plasma water. x The surface of the film is oxygen-injected to obtain the pre-treated SiN x The pre-treated SiN xThe film was fixed in the detection pool and injected with a mixed solution of 1M KCl, 10mM Tris, 1mM EDTA, pH 8. x The film is exposed to an electric field with a strength comparable to the dielectric strength of the film. The charge accumulation and thermal effects of the strong electric field will generate leakage current at the structural defects of the film, causing SiN x Dielectric breakdown at film structural defects forms nanopores.

[0038] 2) Preparation of test samples

[0039] Prepare Sample 1: Dissolve eGFP in 1M KCl, 0.5mM CaCl2, 10mM Tris, pH 7.4 buffer solution to obtain a 50nM GFP solution, and then irradiate with 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, pH 7.4 to obtain a 50nM eGFP solution, then irradiate with a visible light source at a wavelength of 450nm for 30min, and then irradiate 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 nanopore 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] Add Sample 1 to the Cis side of the detection cell, add a buffer solution of 1M KCl, 0.5mM CaCl2, 10mMTris, pH 7.4 to the Trans side, turn on the e-One-HS micro patch clamp, apply a bias of 100-200mV, and obtain electrical signals under visible light; add Sample 2 to the Cis side of the detection cell, add a buffer solution of 1M KCl, 0.5mM CaCl2, 10mMTris, pH 7.4 to the Trans side, turn on the e-One-HS micro patch clamp, apply a bias of 100-200mV, and obtain electrical signals under visible / ultraviolet alternating light; analyze the difference between the two electrical signals.

[0045] The translocation time, blocking current amplitude and porosity of Example 1 were tested respectively, and the tests were all carried out in a dark Faraday cage.

[0046] See attached 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 pore translocation time decreases with the increase of voltage and the blocking current amplitude increases with the increase of voltage. When enhanced green fluorescent protein under visible light is irradiated by ultraviolet light, its transit time is reduced from the original 0.2222±0.0135ms to 0.1753±0.0093ms, and the transit time is significantly shortened. This is because the conformational change after illumination reduces the average spatial size and weakens the interaction with the nanopore. In addition, the blocking current amplitude and ΔI / Io distribution of eGFP before and after illumination change significantly. The ΔI / Io of eGFP during longitudinal transit decreases from 0.4182±0.0005 to 0.3971±0.0019, and the ΔI / Io during transverse transit increases from 0.6765±0.0125 to 0.9025±0.0305. This is because ultraviolet light provides energy for eGFP molecules, and some molecules change the way they transit, which reduces the longitudinal transit blocking current amplitude and significantly increases the transverse transit blocking current amplitude. Therefore, the conformation of eGFP changes after switching between visible and ultraviolet light. By comparing the electrical signals before and after illumination, the single molecule information of enhanced green fluorescent protein can be accurately and clearly presented.

[0047] Example 2

[0048] The difference from Example 1 is that 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) are adjusted to 40min, and electrical signal detection is carried out under visible light source irradiation and visible / ultraviolet alternating light, including translocation time, blocking current amplitude, and aperture resistance.

[0049] Through detection, after the illumination time was adjusted, the translocation time, blocking current amplitude and porosity still showed obvious changes, which shows that the detection method of the present invention has simple steps and strong operability.

[0050] The detection method of the present invention can provide more detailed information such as the shape, surface charge, size, conformation, etc. of enhanced green fluorescent protein, and can also regulate the single molecule permeation rate, and the steps are simple and the cost is low. By detecting and capturing the information of protein conformation 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 enhanced green fluorescent protein, which can be used to construct highly sensitive biosensors and help develop equipment for specific molecular detection. On this basis, combining solid-state nanopores with optical methods such as fluorescence microscopy can further carry out protein analysis research such as protein folding, protein-aptamer interaction, etc., to provide early diagnosis or predict the development of diseases such as Parkinson's disease and Alzheimer's disease. In addition, by compounding eGFP with biomacromolecules or organelles or introducing the eGFP gene into a specific gene fragment through genetic engineering technology, the dynamic process of the target substance in the cell can be tracked in real time, accurately and visually, providing efficient research tools for fields such as cell biology and drug development. This method provides a high-sensitivity, high-resolution detection method for single-molecule fluorescent proteins, especially eGFP, opens up new ideas for the study of conformational changes of functional proteins and their disease-related pathologies, and has good application prospects.

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

[0052] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0053] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

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

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

3. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 2, characterized in that: The buffer solution is a mixed solution of 1M KCl, 0.5mM CaCl2, 10mM Tris, with a pH of 7.

4.

4. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 1 or 2, characterized in that: The exposure time using the visible light source is the same as that using the ultraviolet light source.

5. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 4, characterized in that: The visible light source and ultraviolet light source were irradiated for 30 to 60 minutes respectively.

6. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 1 or 2, characterized in that: The solid nanoporous membrane is SiN x Film, film thickness 15-20nm, film suspended in the window 4-10μm 2 on a silicon substrate.

7. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 1 or 2, characterized in that: The preparation method of the solid nanoporous membrane is as follows: SiN x The film was fixed in the detection pool and a mixed solution of 1M KCl, 10mM Tris, 1mM EDTA, pH 8 was injected. x Dielectric breakdown at film structural defects forms nanopores.

8. A method for detecting photoisomerization of a single-molecule fluorescent protein as claimed in claim 7, characterized in that: The SiN x The film is SiN x The chip was immersed in ethanol, water, and acetone solution to remove contaminants on the membrane surface, and then cleaned with plasma water. x The film surface is treated with oxygen plasma oxygen implantation.

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

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