Western Blotting analysis method based on SERS (Surface Enhanced Raman Scattering) nano-label development

Abstract

The invention provides a Western Blotting analysis method based on SERS (Surface Enhanced Raman Scattering) nano-label development, and belongs to the technical field of molecular biology. The technical problems that traditional WB analysis is long in consumed time and high in cost and depends on expensive detection instruments are solved. According to the technical scheme, the method comprises the following steps: 1) electrophoretic separation of protein; 2) transferring the separated protein into a membrane; and 3) developing and detecting the protein based on the SERS nano-label. The method has the beneficial effects that the requirements of qualitatively detecting the protein through color development and quantitatively analyzing the protein through Raman spectrum can be met at the same time, and a new solution is provided for WB analysis commonly used in molecular biology.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a Western Blotting analysis method based on SERS nanoparticle label development. Background Art

[0002] Protein blotting (Western Blotting, WB) is an experimental technique widely used in the research of molecular biology, biochemistry and immunogenetics. The main function of this technique is to detect the presence, expression level and molecular weight of specific proteins in cell or tissue extracts. It is an important tool for studying gene expression and protein function at the protein level. The basic principle of WB is based on the specific binding of antigen and antibody. First, proteins in cells or tissues are separated by molecular weight using polyacrylamide gel electrophoresis (SDS-PAGE); then, the proteins on the gel are transferred to a solid-phase support, an NC membrane or a PVDF membrane, by blotting. The proteins on the solid-phase support serve as antigens, bind to specific primary antibodies, and then bind to secondary antibodies modified with labels; finally, through a substrate color reaction or fluorescence imaging, visible target protein bands are formed, thereby obtaining the presence and expression level of specific proteins. Currently, the display of protein bands in WB requires detection of specifically bound antibodies on the solid-phase support through a chemiluminescence or fluorescence imaging system. Chemiluminescence first requires incubating the membrane in a dark box containing a luminescent solution, and then using a chemiluminescence instrument or film for exposure detection. The operation is cumbersome and the detection signal is easily interfered by the environment; while fluorescence imaging requires expensive detection equipment, and the detection cost is relatively high; in addition, the sensitivity of traditional WB analysis methods is generally at the ng level, and it is impossible to quantitatively detect low-abundance target proteins. Therefore, developing a WB analysis method that does not rely on complex equipment, is easy to operate and has high detection sensitivity has important application prospects.

[0003] In recent years, with the further development of nanotechnology, surface-enhanced Raman scattering (SERS), as a highly sensitive analytical tool, has received extensive attention in the field of molecular detection, and its analytical sensitivity can reach the single-molecule level. The generation of SERS spectra requires, on the one hand, an enhancement substrate that can produce a high Raman enhancement effect; on the other hand, the target molecule needs to be adsorbed on the surface of the enhancement substrate, and the target molecule needs to have a large Raman scattering cross-section. For labeled detection, placing Raman signal molecules at the core-shell gap of single-metal or bimetal can ensure a high Raman signal intensity, so that the detection system can obtain a high detection sensitivity in molecular detection; in addition, based on the aggregation effect of the enhancement substrate, that is, the nanomaterial, a bright color can be presented at the molecular detection point, so the target molecule can be qualitatively or semi-quantitatively analyzed by a simple colorimetric method. This process is similar to the detection principle of early pregnancy test strips. Due to the aggregation of gold nanoparticles at the detection point, it is possible to judge whether the sample contains HCG and the concentration of HCG contained by the display and depth of the color.

[0004] Therefore, by combining SERS nanolabels based on the core-shell structure, a Western Blotting analysis method based on SERS nanolabel development is developed. On the one hand, the molecular weight of the target protein can be determined by the method of SERS nanolabel aggregation and color development, and the target protein molecule can be qualitatively or semi-quantitatively analyzed; on the other hand, by collecting the Raman spectra of the target protein bands, low-abundance target protein molecules can be quantitatively detected. This WB analysis scheme can effectively solve the difficulties and disadvantages of traditional WB analysis methods, thereby further expanding the application scope of WB. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a Western Blotting analysis method based on SERS nanolabel development, which can simultaneously meet the requirements of qualitative detection of proteins by color development and quantitative protein analysis by Raman spectroscopy; and solve the technical problems of long time consumption, high cost and dependence on expensive detection instruments in traditional WB analysis, and provide a new solution for the commonly used WB analysis in molecular biology.

[0006] In order to achieve the above object of the invention, the technical solutions adopted by the present invention are specifically as follows:

[0007] A Western Blotting analysis method based on SERS nanolabel development, comprising the following steps:

[0008] S1. Electrophoretic separation of proteins;

[0009] S2. Transfer of the separated proteins;

[0010] S3. Protein development and detection based on SERS nanolabels.

[0011] Further, step S1 includes the following steps:

[0012] S11. Obtain a sample from cells or tissues, grind the tissue in liquid nitrogen or process it using a homogenizer;

[0013] S12. Lyse the sample with a lysis solution to release the proteins in the sample;

[0014] S13. Determine the protein concentration in the sample solution by the BCA method to determine the sample concentration for loading;

[0015] S14. After mixing the sample solution with the loading buffer, place it at 100 °C and boil for 5 - 10 min to fully denature the proteins;

[0016] S15. Prepare an SDS-PAGE gel according to the molecular weight of the target protein;

[0017] S16. Add the denatured protein solution to the loading wells;

[0018] S17. Perform electrophoresis in a constant voltage or constant current mode. First, apply a voltage of 80 V in the stacking gel to concentrate the sample to be tested into a line; then, when the bromophenol blue reaches the bottom of the separating gel, adjust the voltage to 120 V and continue electrophoresis until the experiment ends.

[0019] Further, step S2 includes the following steps:

[0020] S21. Place a sponge, filter paper, gel, PVDF membrane or NC membrane, filter paper, and sponge in sequence and ensure there are no air bubbles between layers;

[0021] S22. Transfer the membrane at a constant current with a current intensity of 200 - 300 mA for 1 - 2 h.

[0022] Further, step S3 includes the following steps:

[0023] S31. Place the PVDF membrane or NC membrane in a blocking solution of 5 - 10% skim milk powder or BSA solution and block it on a shaker at room temperature for 1 - 4 h to reduce background signals and non-specific binding;

[0024] S32. Dilute the SERS nanolabel solution with the blocking solution to 2 - 100 nM, place the PVDF membrane or NC membrane in the SERS nanolabel solution, and incubate it on a shaker overnight at 4 °C or for 2 - 3 h at room temperature;

[0025] S33. Wash the transfer membrane three times with TBST buffer at room temperature for 10 - 15 min each time;

[0026] S34. Obtain the molecular weight of the protein from the color display of the protein band, qualitatively judge the concentration of the target protein by the depth of the color, or quantitatively determine the amount of the target protein by collecting Raman spectra.

[0027] Furthermore, the preparation steps of the SERS nanolabels in the step S32 are as follows:

[0028] S321. Prepare gold nanoparticles or silver nanoparticles by reducing chloroauric acid or silver nitrate, and use them as the core of the SERS nanolabels.

[0029] S322. Modify Raman active molecules on the surface of the gold core or silver core by electrostatic or covalent connection.

[0030] S323. Add chloroauric acid or silver nitrate solution and a reducing agent to the solution in S322 to prepare a gold shell or a silver shell.

[0031] S324. Connect an antibody matching the target protein by electrostatic adsorption or covalent connection to prepare a complete SERS nanolabel.

[0032] Furthermore, the spectrometer used for collecting the Raman spectra in the step S34 is a micro-Raman spectrometer, a portable Raman spectrometer or a handheld Raman spectrometer.

[0033] Furthermore, the shape of the core in the step S321 is circular, and the size is 5 - 20 nm.

[0034] Furthermore, the Raman active molecules in the step S322 are 4-mercaptobenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid), Nile blue A, methylene blue, crystal violet, malachite green or R6G.

[0035] Furthermore, the reducing agent in the step S323 is sodium citrate, sodium borohydride or ascorbic acid.

[0036] Furthermore, the thickness of the gold shell or silver shell in the step S323 is 3 - 20 nm.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] 1. The present invention effectively combines the traditional WB with SERS nanolabels, can simultaneously meet the requirements of qualitatively detecting proteins by color development and quantitatively analyzing proteins by Raman spectra, solves the technical problems of long time consumption, high cost and dependence on expensive detection instruments in traditional WB analysis, and provides a new solution for the commonly used WB analysis in molecular biology.

[0039] 2. The SERS nanolabels in the present invention, as developers for protein bands, have high Raman signal responsiveness, thus enabling the detection of low-abundance target proteins in samples.

[0040] 3. The SERS nanolabels in the present invention are lower in price compared to the primary antibodies and labeled secondary antibodies used in traditional WB, thus effectively reducing the detection cost.

[0041] 4. The operation process of the present invention is similar to that of traditional WB and is less difficult in actual operation. Therefore, it is conducive to its application and popularization, and has broad application prospects in the field of molecular biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings are used to provide further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, but do not constitute a limitation to the present invention.

[0043] Figure 1 Schematic diagram of sample preparation and electrophoresis process in the present invention.

[0044] Figure 2 Schematic diagram of membrane transfer and incubation with SERS nanolabels in the present invention.

[0045] Figure 3 Schematic diagram of the synthesis of SERS nanolabels in the present invention.

[0046] Figure 4 TEM image of the gold-core silver-shell SERS nanolabels in the present invention.

[0047] Figure 5 Raman spectrum of the gold-core silver-shell SERS nanolabels in the present invention.

[0048] Figure 6 Optical photograph of the NC membrane after incubation with SERS nanolabels in the present invention.

[0049] Figure 7 Raman spectrum of the protein band at the NC membrane in the present invention.

[0050] Among them, the reference numerals are: 1. cell; 2. tissue; 3. grind in liquid nitrogen or process with a homogenizer; 4. lysis buffer; 5. protein; 6. loading buffer; 7. SDS-PAGE gel; 8. denatured protein solution; 9. electrophoresis; 10. sponge; 11. filter paper; 12. PVDF membrane or NC membrane; 13. membrane transfer; 14. skim milk powder or BSA solution; 15. SERS nanolabel; 16. qualitatively judge the concentration of the target protein by the depth of color; 17. Raman spectrum; 18. gold nanoparticles or silver nanoparticles; 19. Raman active molecule; 20. gold shell or silver shell; 21. antibody. Detailed implementation manners

[0051] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Of course, the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0052] Embodiment 1

[0053] As Figure 1 shown, in the Western Blotting analysis method based on SERS nanolabel development of this embodiment, the electrophoretic separation of proteins includes the following operating steps:

[0054] ① Obtain a sample from cell 1, and lyse the sample with lysis buffer 4 to release the protein 5 in the sample;

[0055] ② Determine the protein concentration in the sample solution by the BCA method to determine the sample concentration for loading;

[0056] ③ After mixing the sample solution with loading buffer 6, place it at 100 °C and boil for 5 min to fully denature the protein;

[0057] ④ Configure an SDS-PAGE gel 7 according to the molecular weight of the target protein GAPDH;

[0058] ⑤ Add the denatured protein solution 8 to the sample well;

[0059] ⑥ Perform electrophoresis 9 in a constant voltage or constant current manner. First, apply a voltage of 80 V in the stacking gel to concentrate the sample to be measured into a line; then, when the bromophenol blue reaches the bottom of the separating gel, adjust the voltage to 120 V and continue electrophoresis until the experiment ends.

[0060] As Figure 2 shown, the membrane transfer of the separated protein includes the following operating steps:

[0061] ① Place a sponge 10, a filter paper 11, a gel 7, an NC membrane 12, a filter paper 11 and a sponge 10 in sequence, and ensure that there are no air bubbles between each layer;

[0062] ② Under constant current conditions, perform membrane transfer 13 at a current intensity of 200 mA, and set the membrane transfer time to 1 h.

[0063] In addition, the protein development and detection based on SERS nanolabels include the following operating steps:

[0064] ① Place the NC membrane 12 in a blocking solution of 5% skim milk powder 14, and block it on a shaker at room temperature for 1 h to reduce background signals and non-specific binding;

[0065] ②Dilute the SERS nanoprobe solution to 15 to 2 nM with the blocking solution, place the NC membrane 12 in the SERS nanoprobe solution 15, and incubate overnight on a shaker at 4 °C;

[0066] ③Wash the transfer membrane three times with TBST buffer at room temperature for 10 min each time;

[0067] ④Obtain the molecular weight of the protein from the color display of the protein band, and qualitatively judge the concentration of the target protein 16 by the depth of the color (as Figure 6 shown).

[0068] Among them, the preparation steps of the SERS nanoprobe are as Figure 3 shown, and specifically include the following steps:

[0069] ①Obtain gold nanoparticles 18 by reducing chloroauric acid, which are circular in shape and have a particle size of 16 nm, and use this as the core of the SERS nanoprobe 15;

[0070] ②Modify the surface of the gold core with the Raman active molecule 4-mercaptobenzoic acid 19 by covalent bonding;

[0071] ③Add silver nitrate solution and ascorbic acid to the solution in ② to prepare a silver shell 20 with a thickness of 4 nm;

[0072] ④Connect the antibody 21 matching GAPDH by electrostatic adsorption to prepare the SERS nanoprobe 15 as Figure 4 shown, whose Raman spectrum is as Figure 5 shown, with obvious Raman characteristic peaks and high intensity.

[0073] Example 2

[0074] As Figure 1 shown, for the Western Blotting analysis method based on SERS nanoprobe development in this example, the electrophoretic separation of proteins includes the following operating steps:

[0075] ①Obtain a sample from tissue 2 and grind tissue 2 in liquid nitrogen 3;

[0076] ②Lyse the sample with lysis buffer 4 to release the protein 5 in the sample;

[0077] ③Determine the protein concentration in the sample solution by the BCA method to determine the sample concentration for loading;

[0078] ④Mix the sample solution with the loading buffer 6 and boil it at 100 °C for 10 min to fully denature the protein;

[0079] ⑤According to the molecular weight of the target protein α-tubulin, prepare an SDS-PAGE gel 7;

[0080] ⑥Add the denatured protein solution 8 to the sample loading wells;

[0081] ⑦Perform electrophoresis 9 in a constant voltage or constant current mode. First, apply a voltage of 80 V in the stacking gel to concentrate the sample to be tested into a line; then, when the bromophenol blue reaches the bottom of the separating gel, adjust the voltage to 120 V and continue electrophoresis until the experiment ends.

[0082] As Figure 2 shown, the protein transfer after separation includes the following operating steps:

[0083] ①Place the sponge 10, filter paper 11, gel 7, PVDF membrane 12, filter paper 11, and sponge 10 in sequence and ensure there are no air bubbles between the layers;

[0084] ②Under constant current conditions, perform membrane transfer 13 at a current intensity of 300 mA, and set the membrane transfer time to 2 h.

[0085] In addition, the protein development and detection based on SERS nanolabels include the following operating steps:

[0086] ①Place the PVDF membrane 12 in a blocking solution of 10% BSA solution 14 and block it on a shaker at room temperature for 4 h to reduce background signals and non-specific binding;

[0087] ②Dilute the SERS nanolabel solution 15 with the blocking solution to 100 nM, place the PVDF membrane 12 in the SERS nanolabel solution 15, and incubate it at room temperature for 2 h;

[0088] ③Wash the transfer membrane three times with TBST buffer at room temperature for 15 min each time;

[0089] ④Obtain the molecular weight of the protein through the color display of the protein band, and quantitatively determine the amount of the target protein by collecting the Raman spectrum 17 with a micro-Raman spectrometer.

[0090] Among them, the preparation steps of the SERS nanolabel are as Figure 3 shown, and specifically include the following steps:

[0091] ①Obtain silver nanoparticles 18 by reducing silver nitrate, with a circular shape and a particle size of 5 nm, and use this as the core of the SERS nanolabel 15;

[0092] ②Modify the surface of the silver core with the Raman active molecule Nile blue A19 by electrostatic adsorption;

[0093] ③ Add chloroauric acid solution and sodium citrate into the solution in ② to prepare a gold shell 20 with a thickness of 3 nm;

[0094] ④ Connect the antibody 21 matching with α - tubulin by electrostatic adsorption method to prepare a complete SERS nanolabel 15.

[0095] Example 3

[0096] As Figure 1 shown, for the Western Blotting analysis method based on SERS nanolabel development in this example, the electrophoretic separation of proteins includes the following operating steps:

[0097] ① Obtain a sample from cell 1 and lyse the sample with lysis buffer 4 to release the protein 5 in the sample;

[0098] ② Determine the protein concentration in the sample solution by the BCA method to determine the sample concentration for loading;

[0099] ③ After mixing the sample solution with loading buffer 6, place it at 100 °C for 8 min to fully denature the protein;

[0100] ④ Prepare an SDS - PAGE gel 7 according to the molecular weight of the target protein GAPDH;

[0101] ⑤ Add the denatured protein solution 8 to the loading well;

[0102] ⑥ Perform electrophoresis 9 in a constant - voltage or constant - current manner. First, apply a voltage of 80 V in the stacking gel to concentrate the sample to be measured into a line; then, when the bromophenol blue reaches the bottom of the separating gel, adjust the voltage to 120 V and continue electrophoresis until the experiment ends.

[0103] As Figure 2 shown, the transfer of the separated protein includes the following operating steps:

[0104] ① Place the sponge 10, filter paper 11, gel 7, NC membrane 12, filter paper 11, and sponge 10 in sequence and ensure there are no air bubbles between each layer;

[0105] ② Under constant - current conditions, perform membrane transfer 13 at a current intensity of 250 mA, and set the membrane - transfer time to 1.5 h.

[0106] In addition, the protein development and detection based on SERS nanolabels include the following operating steps:

[0107] ① Place the NC membrane 12 in a blocking solution of 7% non - fat milk powder 14 and block it on a shaker at room temperature for 2 h to reduce background signals and non - specific binding;

[0108] ②Dilute the SERS nanoprobe solution to 15 - 50 nM with the blocking solution, place the NC membrane 12 in the SERS nanoprobe solution 15, and incubate at room temperature for 3 h;

[0109] ③Wash the transfer membrane three times with TBST buffer at room temperature, each time for 12 min;

[0110] ④Obtain the molecular weight of the protein from the color display of the protein band, and collect the Raman spectrum 17 with a handheld Raman spectrometer to quantitatively determine the amount of the target protein (as Figure 7 shown).

[0111] Among them, the preparation steps of the SERS nanoprobe are as Figure 3 shown, specifically including the following steps:

[0112] ①Obtain gold nanoparticles 18 by reducing chloroauric acid. The shape of the gold nanoparticles is round, and the particle size is 20 nm, which is used as the core of the SERS nanoprobe 15;

[0113] ②Modify the surface of the gold core with the Raman - active molecule 4 - mercaptobenzoic acid 19 by covalent bonding;

[0114] ③Add the chloroauric acid solution and sodium borohydride to the solution in ② to prepare a gold shell 20 with a thickness of 20 nm;

[0115] ④Connect the antibody 21 matching GAPDH by electrostatic adsorption to prepare the complete SERS nanoprobe 15.

[0116] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Western Blotting analysis method based on the development of SERS nanolabels, characterized in that, It includes the following steps: S1. Electrophoretic separation of proteins; S2. Transfer of the separated proteins to a membrane; S3. Protein imaging and detection based on SERS nanolabels.

2. The Western Blotting analysis method based on SERS nanolabel development according to claim 1, wherein The step S1 includes the following steps: S11. Obtain a sample from cells (1) or tissues (2), grind the tissue (2) in liquid nitrogen or process it using a homogenizer (3); S12. Lyse the sample with a lysis buffer (4) to release the proteins (5) in the sample; S13. Determine the protein concentration in the sample solution by the BCA method to determine the sample concentration for loading; S14. After mixing the sample solution with a loading buffer (6), place it at 100 °C and boil for 5 - 10 min to fully denature the proteins; S15. Prepare an SDS - PAGE gel (7) according to the molecular weight of the target protein; S16. Add the denatured protein solution (8) to the sample wells; S17. Perform electrophoresis (9) in a constant - voltage or constant - current manner. First, apply a voltage of 80 V in the stacking gel to concentrate the sample to be tested into a line; then, when the bromophenol blue reaches the bottom of the separating gel, adjust the voltage to 120 V and continue electrophoresis until the experiment ends.

3. The Western Blotting analysis method based on SERS nanolabel development according to claim 1, characterized in that, The step S2 includes the following steps: S21. Place a sponge (10), filter paper (11), gel (7), PVDF membrane or NC membrane (12), filter paper (11), and sponge (10) in sequence, and ensure there are no air bubbles between layers; S22. Under constant - current conditions, perform membrane transfer (13) at a current intensity of 200 - 300 mA, and set the membrane - transfer time to 1 - 2 h.

4. The Western Blotting analysis method based on SERS nanolabel development according to claim 1, characterized in that, The step S3 includes the following steps: S31. Place the PVDF membrane or NC membrane (12) in a blocking solution of 5 - 10% skim milk powder or BSA solution (14), and block it on a shaker at room temperature for 1 - 4 h to reduce background signals and non - specific binding; S32. Dilute the SERS nanolabel (15) solution with the blocking solution to 2 - 100 nM, place the PVDF membrane or NC membrane (12) in the SERS nanolabel (15) solution, and incubate it on a shaker overnight at 4 °C or for 2 - 3 h at room temperature; S33. Wash the transfer membrane three times with TBST buffer at room temperature, each time for 10 - 15 min; S34. Obtain the molecular weight of the protein from the color display of the protein band, and qualitatively judge the concentration of the target protein by the depth of the color (16), or quantitatively determine the amount of the target protein by collecting Raman spectra (17).

5. The Western Blotting analysis method based on SERS nanoparticle label development according to claim 4, characterized in that, The preparation steps of the SERS nanolabel in the step S32 are as follows: S321. Prepare gold nanoparticles or silver nanoparticles (18) by reducing chloroauric acid or silver nitrate, and use this as the core of the SERS nanolabel (15); S322. Modify Raman - active molecules (19) on the surface of the gold or silver core by electrostatic or covalent linkage; S323. Add chloroauric acid or silver nitrate solution and a reducing agent to the solution in S322 to prepare a gold shell or silver shell (20); S324. Connect an antibody (21) that matches the target protein by electrostatic adsorption or covalent bonding to prepare a complete SERS nanolabel (15).

6. The Western Blotting analysis method based on SERS nanoparticle label development according to claim 4, characterized in that, The spectrometer used for collecting the Raman spectrum in step S34 is a microscopic Raman spectrometer, a portable Raman spectrometer, or a handheld Raman spectrometer.

7. The Western Blotting analysis method based on SERS nanoparticle label development according to claim 5, characterized in that, The shape of the core in step S321 is circular, and its size is 5 - 20 nm.

8. The Western Blotting analysis method based on SERS nanoparticle label development according to claim 5, wherein The Raman active molecule (19) in step S322 is 4-mercaptobenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid), Nile blue A, methylene blue, crystal violet, malachite green, or R6G.

9. The Western Blotting analysis method based on SERS nanoprobe development according to claim 5, wherein The reducing agent in step S323 is sodium citrate, sodium borohydride, or ascorbic acid.

10. The Western Blotting analysis method based on SERS nanoparticle label development according to claim 5, wherein The thickness of the gold shell or silver shell (20) in step S323 is 3 - 20 nm.

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