Multicolor immunolabeling method based on Raman spectrum technology

Abstract

The invention discloses a multicolor immunolabeling method based on a Raman spectrum technology, and relates to the technical field of spectrum detection. Comprising the following steps: step 1, preparing nanogold, step 2, preparing polysalamann microspheres, namely modifying the surface of the nanogold, step 3, combining a nanogold marker with an antibody, step 4, combining the antibody with a Raman marker with an antigen of a to-be-detected object, and step 5, carrying out Raman spectrum detection on a final sample. The immunolabeling with more colors is realized. Nanogold is modified by self-assembly of sulfydryl and nanogold, and meanwhile, the nanogold and an antibody are connected by taking a substance containing sulfydryl and hydroxyl as a bridge. The specific recognition of the antibody and the specific spectrum of the modified gold nanospheres are used for recognizing substances, so that the distribution recognition of the antigen of the tissue slice can be achieved. Therefore, simultaneous expression and presentation of more types of tissue components are realized.

Description

Technical Field

[0001] The present application relates to the field of spectral detection technology, and in particular to a multicolor immunolabeling method based on Raman spectroscopy technology. Background Art

[0002] Multicolor immunofluorescence is currently a method for studying tissue morphology and in situ protein expression. With the help of this technology, multiple molecular markers can be achieved on a tissue sample, and the spatial distribution information of different components in the tissue can be presented on the same screen. Combined with quantitative analysis tools, it can comprehensively analyze the target molecules or cells and their related biomarkers in the tissue microenvironment.

[0003] However, due to the wide spectral peak of fluorescence and poor spectral signal specificity, it is generally only possible to present three-color fluorescence signals simultaneously, and it is impossible to achieve the simultaneous expression and presentation of more tissue components. Summary of the invention

[0004] In order to solve the problem that only three colors of fluorescence signals can be simultaneously presented due to the wide spectral peak of fluorescence and the poor specificity of spectral signals, and to achieve the simultaneous expression and presentation of more types of tissue components, the present application provides a multicolor immunolabeling method based on Raman spectroscopy technology.

[0005] The present application provides a multicolor immunolabeling method based on Raman spectroscopy technology, which adopts the following technical solution.

[0006] A multicolor immunolabeling method based on Raman spectroscopy technology, comprising:

[0007] Step 1: Preparation of multicolor Raman microspheres - surface modification of gold nanoparticles

[0008] Assemble the bridge substance and the labeling substance onto the surface of the gold nanoparticles through gold-sulfur bonds:

[0009] Dissolving the bridge material in an organic solvent to obtain a bridge material solution with a concentration of 1 mol;

[0010] Dissolving the marker substance in an organic solvent to obtain a marker substance solution with a concentration of 1 mol;

[0011] Pipette appropriate amount of bridge substance solution and marker substance solution with a concentration ratio of 0.5:1 into the test tube, and vortex to obtain substance A;

[0012] Nanogold and substance A were added into the test tube in a volume ratio of 2:1, vortexed and tightly mixed, sealed at a constant temperature of 28°C, and provided with a shaker for self-assembly for 2 hours to obtain substance B;

[0013] Place substance B in a centrifuge and centrifuge it three times to obtain a pink-brown turbid liquid purified substance C;

[0014] Step 2: Binding of gold nanoparticles to antibodies:

[0015] Mix 1-2 mol NHS and 1-2 mol EDC, and add hydrochloric acid solution to adjust the pH to 5-7;

[0016] Add MES buffer, use 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the surface carboxyl group of the purified marker C to NHS Ester, use the amino group of the antibody to couple with the nano-gold NHSester, the nano-gold ball is combined with the thiol group of the marker to form a gold-sulfur bond, and then use the bridge material as a bridge, the thiol group on one side is combined with the nano-gold to form a gold-sulfur bond, and the carboxyl group on one side is connected with the amino group at the constant end of the antibody by dehydration condensation, and the antibody is connected with the labeled nano-gold ball, and coupled at 37°C for more than 3 hours to obtain the conjugate D. After the coupling is completed, the pH is adjusted to 7.5 with a sodium hydroxide solution;

[0017] Step 4: The antibody with Raman marker binds to the antigen to be tested:

[0018] The tissue frozen sections to be labeled and the conjugate D are sliced ​​and labeled to obtain the labeled sample E;

[0019] Step 5: Perform Raman spectroscopy on the final sample:

[0020] The Raman spectrometer collects the Raman spectrum signal of sample E to obtain a Raman spectrum graph, and determines whether the region is the desired antigen based on the result of the Raman spectrum.

[0021] Optionally, the preparation of the gold nanoparticles:

[0022] Dissolve chloroauric acid in water to obtain a chloroauric acid solution with a concentration of 0.01 g / ml;

[0023] Dissolve sodium citrate in water to obtain a sodium citrate solution with a concentration of 0.01 g / ml;

[0024] Add water, chloroauric acid solution and sodium citrate solution into a container in a volume ratio of 100:1:0.7, put in a rotor, rotate and heat to boiling, keep aging for 15 minutes, and then naturally cool to room temperature to obtain nano gold sol.

[0025] Optionally, pre-centrifuge at 2000 rpm for 10 minutes, rotate the centrifuge tube 180°, centrifuge twice at 12000 rpm for 20 minutes, rotate the centrifuge tube 180°, and centrifuge at 15000 rpm for 20 minutes to obtain a pink-brown turbid liquid purified product C.

[0026] Optionally, the bridge substance is any one of 4-mercaptobenzoic acid and 5,5'-dithiobis(2-nitrobenzoic acid).

[0027] Optionally, the labeling substance may be but is not limited to any one of naphthylthiol, 2-chlorobenzenethiol, diphenylthiol, 4-ethylthiophenol, 2-methylthiophenol, and 4,4'-thiodiphenylthiol.

[0028] In summary, this application at least includes the following beneficial effects:

[0029] 1. The present invention adopts Raman spectroscopy labeling method to replace fluorescent labeling, and utilizes the characteristics of narrow Raman spectroscopy peak and good spectral signal specificity to achieve the simultaneous expression and presentation of more types of tissue components.

[0030] 2. The present invention relates to a Raman labeling method to replace fluorescent labeling to achieve more colorful immune labeling. The self-assembly of thiol and nano-gold is used to modify the nano-gold, and the substance containing thiol and hydroxyl is used as a bridge to connect the nano-gold and the antibody. The specific recognition of the antibody and the specific spectrum of the modified nano-gold ball are used to identify the substance. The distribution recognition of the antigen in the tissue section can be achieved.

[0031] 3. The specific technical principle utilizes Raman surface enhancement technology. The gold nanospheres are combined with the thiol groups of naphthalenethiol (or 2-chlorobenzenethiol, diphenylthiol, etc.) to form a gold-sulfur bond to achieve the effect of fixed Raman enhancement. Then 4-mercaptobenzoic acid is used as a bridge, and the thiol group on one side is combined with the gold nanosphere to form a gold-sulfur bond. The carboxyl group on one side is connected to the amino group at the constant end of the antibody by dehydration condensation. The antibody and the labeled gold nanosphere are connected. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0033] Figure 1 It is the Raman spectrum. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The following is combined with Figure 1 This application is described in further detail.

[0036] The embodiment of the present application discloses a multicolor immunolabeling method based on Raman spectroscopy technology.

[0037] Embodiment 1

[0038] A multicolor immunolabeling method based on Raman spectroscopy technology, including a method for preparing multicolor Raman microspheres, and a method for using the overall process;

[0039] Step 1: First, prepare a suitable nano-gold sol:

[0040] Dissolve 1 g of chloroauric acid (Chinese medicine) in 100 ml of pure water to obtain a chloroauric acid solution;

[0041] Dissolve 1 g of sodium citrate (Chinese medicine) in 100 ml of pure water to obtain a sodium citrate solution;

[0042] Add 100 ml pure water to the flask, put in a rotor, add 1 ml chloroauric acid solution, add 0.65 ml sodium citrate solution. Rotate and heat until boiling, keep aging for 15 minutes, and then cool naturally to room temperature to obtain nano-gold sol (72nm and 41nm are the best particle sizes).

[0043] Step 2: Preparation of multicolor Raman microspheres - surface modification of gold nanoparticles:

[0044] The bridge substance and the labeling substance are assembled onto the surface of the nano-gold through gold-sulfur bonds;

[0045] Here you need to use the organic solvent acetonitrile (Chinese medicine analytical grade or above), draw 5 ml of organic solvent into the glass test tube;

[0046] 1 mg of a bridge substance (the bridge substance may be any one of 4-mercaptobenzoic acid and 5,5'-dithiobis(2-nitrobenzoic acid)) is weighed and added to a test tube, and 1 mg of a marker substance 4,4'-thiodibenzenethiol (the marker substance may be and is not limited to any one of naphthalenethiol, 2-chlorobenzenethiol, dibenzenethiol, 4-ethylthiophenol, 2-methylthiophenol, and 4,4'-thiodibenzenethiol) is weighed and added to a test tube, and the mixture is vortexed at 500 rpm for 5 minutes to obtain substance A;

[0047] 5 ml of nano-gold was added to the test tube, 5 ml of substance A was added to the test tube, vortexed and tightly mixed, sealed at a constant temperature of 28°C, and provided with a shaker for self-assembly for 2 hours to obtain substance B;

[0048] Place substance B in a centrifuge and centrifuge it three times. Initially, centrifuge it at 2000 rpm for 10 minutes for pre-centrifugation. Rotate the centrifuge tube 180° and centrifuge it twice at 12000 rpm for 20 minutes. Rotate the centrifuge tube 180° and centrifuge it at 15000 rpm for 20 minutes to purify it. A pink-brown turbid liquid purified substance C is obtained.

[0049] Step 3: Binding of gold nanoparticles to antibodies:

[0050] Weigh 50 mg each of NHS and EDC and dissolve them in 10 ml of water;

[0051] Adjust the pH to 5 with 1 mol hydrochloric acid solution;

[0052] Use 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the surface carboxyl group of the purified marker C to NHS Ester, and use the amino group of the antibody to couple with the nanogold NHS ester. MES buffer is also required here. Couple at 37°C for more than 3 hours to obtain the conjugate D. After the coupling is completed, use 1 mol of sodium hydroxide solution to adjust the pH to 7.5.

[0053] Step 4: The antibody with Raman marker binds to the antigen to be tested:

[0054] The tissue cryosections to be labeled and the conjugate D are sliced ​​and labeled to obtain labeled samples E.

[0055] Step 5: Perform Raman spectroscopy on the final sample:

[0056] The Raman spectrometer collects the Raman spectrum signal of sample E and obtains the Raman spectrum diagram (such as Figure 1 As shown in the figure, the Raman spectrum results are used to determine whether the area is the desired antigen. The peaks pointed by the four arrows in the figure are used to determine that the marker at this time is 4,4'-thiodibenzene thiol, thereby determining the corresponding antibody and antigen.

[0057] Embodiment 2

[0058] like Figure 1 As shown, a multicolor immunolabeling method based on Raman spectroscopy technology, including a method for preparing multicolor Raman microspheres, and a method for using the overall process;

[0059] Step 1: First, prepare a suitable nano-gold sol:

[0060] Dissolve 1 g of chloroauric acid (Chinese medicine) in 100 ml of pure water to obtain a chloroauric acid solution;

[0061] Dissolve 1 g of sodium citrate (Chinese medicine) in 100 ml of pure water to obtain a sodium citrate solution;

[0062] Add 100 ml pure water to a conical flask, put in a rotor, add 1 ml chloroauric acid solution, and add 0.75 ml sodium citrate solution. Rotate and heat until boiling. Keep aging for 15 minutes. Then cool naturally to room temperature to obtain nano-gold sol (72nm and 41nm are the best particle sizes).

[0063] Step 2: Preparation of multicolor Raman microspheres - surface modification of gold nanoparticles:

[0064] The bridge substance and the labeling substance are assembled onto the surface of the nano-gold through gold-sulfur bonds;

[0065] Here you need to use the organic solvent acetonitrile (Chinese medicine analytical grade or above), draw 10 ml of organic solvent into the glass test tube;

[0066] 5 mg of a bridge substance (4-mercaptobenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid)) was weighed and added to a test tube, and 5 mg of a marker substance 4,4'-thiodibenzenethiol (the marker substance may be but is not limited to naphthylthiol, 2-chlorobenzenethiol, dibenzenethiol, 4-ethylthiophenol 2-methylthiophenol, 4,4'-thiodibenzenethiol, etc.) was weighed and added to a test tube, and vortexed at 500 rpm for 5 minutes to obtain substance A;

[0067] 10 ml of nano-gold was added to the test tube, 10 ml of substance A was added to the test tube, vortexed and tightly mixed, sealed at a constant temperature of 28°C, and provided with a shaker for self-assembly for 2 hours to obtain substance B;

[0068] Place substance B in a centrifuge and centrifuge it three times. Initially, centrifuge at 2000 rpm for 10 minutes for pre-centrifugation. Rotate the centrifuge tube at 180° for two times and centrifuge at 12000 rpm for 20 minutes. Rotate the centrifuge tube again at 180° and centrifuge at 15000 rpm for 20 minutes to purify the substance. A pink-brown turbid liquid purified substance C is obtained.

[0069] Step 3: Binding of gold nanoparticles to antibodies:

[0070] Weigh 50 mg each of NHS and EDC and dissolve them in 10 ml of water;

[0071] Adjust the pH to 7 with 1 mol hydrochloric acid solution;

[0072] Use 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the surface carboxyl group of the purified marker C to NHS Ester, and use the amino group of the antibody to couple with the nanogold NHS ester. MES buffer is also required here. Couple at 37°C for more than 3 hours to obtain the conjugate D. After the coupling is completed, use 1 mol of sodium hydroxide solution to adjust the pH to 7.5.

[0073] Step 4: The antibody with Raman marker binds to the antigen to be tested:

[0074] The tissue cryosections to be labeled and the conjugate D are sliced ​​and labeled to obtain labeled samples E.

[0075] Step 5: Perform Raman spectroscopy on the final sample:

[0076] The Raman spectrometer collects the Raman spectrum signal of sample E and obtains the Raman spectrum diagram (such as Figure 1 As shown in the figure, the Raman spectrum results are used to determine whether the area is the desired antigen. The peaks pointed by the four arrows in the figure are used to determine that the marker at this time is 4,4'-thiodibenzene thiol, thereby determining the corresponding antibody and antigen.

[0077] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multicolor immunolabeling method based on Raman spectroscopy, characterized in that: include: Step 1: Preparation of multicolor Raman microspheres - surface modification of gold nanoparticles Assemble the bridge substance and the labeling substance onto the surface of the gold nanoparticles through gold-sulfur bonds: dissolving the bridge material in an organic solvent to obtain a bridge material solution having a concentration of 1 mol; Dissolving the marker substance in an organic solvent to obtain a marker substance solution having a concentration of 1 mol; Pipette appropriate amount of bridge substance solution and marker substance solution with a concentration ratio of 0.5:1 into the test tube, and vortex to obtain substance A; Nanogold and substance A were added into the test tube in a volume ratio of 2:1, vortexed and tightly mixed, sealed at a constant temperature of 28°C, and provided with a shaker for self-assembly for 2 hours to obtain substance B; Place substance B in a centrifuge and centrifuge it three times to obtain a pink-brown turbid liquid purified substance C; Step 2: Binding of gold nanoparticles to antibodies: Mix 1-2 mol NHS and 1-2 mol EDC, add hydrochloric acid solution to adjust the pH to 5-7; Add MES buffer, use 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the surface carboxyl group of the purified marker C to NHS Ester, use the amino group of the antibody to couple with the nano-gold NHS ester, the nano-gold ball is combined with the thiol group of the marker to form a gold-sulfur bond, and then use the bridge material as a bridge, the thiol group on one side is combined with the nano-gold to form a gold-sulfur bond, and the carboxyl group on one side is connected with the amino group at the constant end of the antibody by dehydration condensation, and the antibody is connected with the labeled nano-gold ball, and coupled at 37°C for more than 3 hours to obtain the conjugate D. After the coupling is completed, the pH is adjusted to 7.5 with a sodium hydroxide solution; Step 4: The antibody with Raman marker binds to the antigen to be tested: The tissue frozen sections to be labeled and the conjugate D are sliced ​​and labeled to obtain the labeled sample E; Step 5: Perform Raman spectroscopy on the final sample: The Raman spectrometer collects the Raman spectrum signal of sample E to obtain a Raman spectrum graph, and determines whether the region is the desired antigen based on the result of the Raman spectrum.

2. The multicolor immunolabeling method based on Raman spectroscopy according to claim 1, characterized in that: Preparation of the nano gold: Dissolve chloroauric acid in water to obtain a chloroauric acid solution with a concentration of 0.01 g / ml; Dissolve sodium citrate in water to obtain a sodium citrate solution with a concentration of 0.01 g / ml; Add water, chloroauric acid solution and sodium citrate solution into a container in a volume ratio of 100:1:0.7, put in a rotor, rotate and heat to boiling, keep aging for 15 minutes, and then naturally cool to room temperature to obtain nano gold sol.

3. The multicolor immunolabeling method based on Raman spectroscopy according to claim 1, characterized in that: The initial centrifugation was performed at 2000 rpm for 10 minutes, the centrifuge tube was rotated 180°, and the mixture was centrifuged twice at 12000 rpm for 20 minutes. The centrifuge tube was rotated 180° and centrifuged at 15000 rpm for 20 minutes to obtain a pink-brown turbid liquid purified product C.

4. The multicolor immunolabeling method based on Raman spectroscopy according to claim 1, characterized in that: The bridge substance is any one of 4-mercaptobenzoic acid and 5,5'-dithiobis(2-nitrobenzoic acid).

5. The multicolor immunolabeling method based on Raman spectroscopy according to claim 1, characterized in that: The labeling substance may be, but is not limited to, any one of naphthylthiol, 2-chlorobenzenethiol, diphenylthiol, 4-ethylthiophenol, 2-methylthiophenol, and 4,4′-thiodiphenylthiol.

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