A green preparation method for surface plasmon enhanced biomolecule detection without modification

Abstract

A method for green preparation of unmodified surface plasmon enhanced biomolecule detection, belonging to the field of biomolecule detection methods, comprises the following steps: adding a ferric chloride solution to a chloroplast solution; adjusting the pH; performing centrifugation to collect the precipitate; washing, centrifuging, and collecting the precipitate; adding a capture antibody; centrifuging and collecting the precipitate; washing away the unbound capture antibody; adding a blocking solution; washing, centrifuging, and collecting the precipitate; adding the biomolecule to be detected; centrifuging and collecting the precipitate; washing away the unreacted biomolecule to be detected; adding a detection antibody; centrifuging and collecting the precipitate; washing away the unreacted detection antibody; adding streptavidin labeled with an enzyme; centrifuging and collecting the precipitate; washing away the uncrosslinked streptavidin; adding a chemical fluorescence substrate; adding a termination solution, and detecting the absorbance of the solution. The present invention solves the problem that the gold nanoparticles prepared by the existing method cannot be directly applied to ELISA and need further biochemical surface modification.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecule detection methods, and particularly relates to a method for green preparation of a surface plasmon enhanced biomolecule detection method without modification. Background Art

[0002] In today's society, cancers, AIDS and various diseases seriously threaten the health and life rights of human beings. Early diagnosis and early treatment are the most effective ways to prevent and control tumors and reduce mortality. Therefore, ultrasensitive detection of tumor biomarkers has become an urgent requirement for cancer clinical diagnosis and treatment. The concentrations of relevant biomarkers for diseases such as cancers and AIDS in blood reach levels lower than femtogram per milliliter (fg / ml), reaching the attogram per milliliter (ag / ml) level. Therefore, how to ultrasensitively detect low-concentration biomolecules plays a crucial role in the early, rapid, accurate diagnosis and treatment of diseases.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive detection technique for detecting antibodies or antigens, which is based on immunological reactions and combines the specific reactions of antigens and antibodies with the efficient catalytic action of enzymes on substrates.

[0004] In order to detect lower-concentration biomolecules, the fluorescence signal intensity of ELISA fluorescence products can be enhanced to increase the detection sensitivity. The studies by Yu Huakang et al. (Acta Physica Sinica, 2019, 68: 149101) and Zhu Xupeng et al. (Acta Physica Sinica, 2019, 68: 147304) have both demonstrated that the surface plasmon effect of gold nanoparticles can enhance the fluorescence intensity. Jia, Min et al. in Food Chemistry, 2021, 344: 128602 used the surface plasmon effect of gold nanoparticles to improve the detection sensitivity of BPA to 6.20 pg / mL, proving the feasibility of the surface plasmon effect of gold nanoparticles in enhancing the detection sensitivity in ELISA.

[0005] However, there are currently two main problems with gold nanoparticle ELISA: (1) Some strongly corrosive chemical reagents such as hydroxylamine, borohydride, organic solvents, etc. are used in the traditional preparation process of gold nanoparticles, and surface modification is usually required later to reduce their biological toxicity. Therefore, these toxic reagents cause serious environmental pollution and high production costs. (2) The surface of gold nanoparticles prepared by traditional methods lacks active groups such as amino groups and carboxyl groups, and biochemical surface modification is still required when applying them to ELISA.

[0006] Based on this, it is necessary to invent a method for enhancing biomolecule detection by the surface plasmon effect of green-prepared gold nanoparticles without surface modification, so as to solve the problems of low sensitivity in existing traditional ELISA biomolecule detection methods and the need for further biochemical surface modification due to biological toxicity and lack of active groups such as amino and carboxyl groups on the surface of gold nanoparticles prepared by traditional methods in existing gold nanoparticle ELISA biomolecule detection methods. Summary of the Invention

[0007] In order to solve the problem that the surface of gold nanoparticles prepared by traditional methods in existing gold nanoparticle ELISA biomolecule detection methods needs further biochemical surface modification due to biological toxicity and lack of active groups such as amino and carboxyl groups, the present invention provides a method for enhancing biomolecule detection by surface plasmon without surface modification prepared by green method.

[0008] The present invention adopts the following technical solutions:

[0009] A method for enhancing biomolecule detection by surface plasmon without modification prepared by green method, comprising the following steps:

[0010] Step S1: Add chloroauric acid solution to chloroplast solution and stir until evenly mixed;

[0011] Step S2: Add NaOH solution, adjust the pH value of the solution to 7.5 - 9, stir, and wait for the reaction between chloroauric acid and chloroplast;

[0012] Step S3: Inject the solution into a centrifuge tube, perform centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0013] Step S4: Inject ultrapure water into the centrifuge tube to wash the precipitate, then perform centrifugation using a centrifuge, remove the supernatant, and collect the precipitate. This washing process is repeated 1 - 5 times to obtain gold nanoparticles;

[0014] Step S5: After washing is completed, add the capture antibody corresponding to the biomolecule to be detected to the precipitate in Step S4, and stir the gold nanoparticles and the capture antibody solution corresponding to the biomolecule to be detected to react fully;

[0015] Step S6: Perform centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0016] Step S7: Add the washing solution to the centrifuge tube, stir, perform centrifugation using a centrifuge, remove the supernatant, wash away the capture antibody not captured by the gold nanoparticles, and collect the precipitate;

[0017] Step S8: Add a blocking solution to the precipitate in Step S7 and incubate;

[0018] Step S9: Add the washing solution into the centrifuge tube, stir for washing, after centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0019] Step S10: Add the biomolecule to be detected to the precipitate in Step S9, and wait until the capture antibody and the biomolecule to be detected react fully;

[0020] Step S11: After centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0021] Step S12: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the unreacted biomolecule to be detected, and collect the precipitate;

[0022] Step S13: Add the detection antibody to the precipitate in Step S12, and wait until the biomolecule to be detected and the detection antibody react fully;

[0023] Step S14: After centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0024] Step S15: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the unreacted detection antibody, and collect the precipitate;

[0025] Step S16: Add streptavidin labeled with an enzyme to the precipitate in Step S15, and wait until the detection antibody and streptavidin crosslink;

[0026] Step S17: After centrifugation using a centrifuge, remove the supernatant, and collect the precipitate;

[0027] Step S18: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the uncrosslinked streptavidin, and collect the precipitate;

[0028] Step S19: Add a chemiluminescent substrate to the precipitate in Step S16, and wait until the enzyme chemiluminescent substrate reacts to generate a fluorescent product;

[0029] Step S20: Add a termination solution, and measure the absorbance of the solution to calculate the concentration of the biomolecule to be detected.

[0030] Further, the temperature for the reaction of chloroauric acid and chloroplast is 25 °C, and the reaction time is 24 - 36 h.

[0031] Further, the rotation speed for the centrifugation is 10000 - 14000 rpm / min, and the centrifugation time is 10 - 30 min.

[0032] Further, the temperature for the full stirring reaction of the gold nanoparticles and the capture antibody solution corresponding to the biomolecule to be detected is 37°C, and the time for the full stirring reaction is 10 - 30 min.

[0033] Further, the temperature for the reaction of the capture antibody and the biomolecule to be detected is 37°C, and the reaction time is 10 - 30 min.

[0034] Further, the temperature for the reaction of the biomolecule to be detected and the detection antibody is 37°C, and the reaction time is 10 - 30 min.

[0035] Further, the temperature for the cross-linking of the detection antibody and streptavidin is 37°C, and the cross-linking time is 10 - 30 min.

[0036] Further, the temperature for the reaction of the enzyme and the chemiluminescent substrate to generate a fluorescent product is 37°C, and the time is 10 - 30 min.

[0037] The beneficial effects of the present invention are as follows:

[0038] Compared with the existing traditional ELISA biomolecule detection method, the green preparation of the surface plasmon enhanced biomolecule detection method without modification described in the present invention has the following advantages: First, the gold nanoparticles have a larger specific surface area and can adsorb more biomolecules, thus improving the detection sensitivity; Second, the gold nanoparticles can move freely in the liquid and can adsorb more biomolecules, thus improving the detection sensitivity; Third, the gold nanoparticles can use their own surface plasmon effect to enhance the fluorescence intensity of the fluorescent product, thereby further improving the detection sensitivity.

[0039] Compared with the existing gold nanoparticle ELISA biomolecule detection method, the green preparation of the surface plasmon enhanced biomolecule detection method without modification described in the present invention has the following advantages: First, the preparation of gold nanoparticles using chloroplasts avoids the use of some strongly corrosive chemical reagents in the traditional gold nanoparticle preparation process, thus avoiding the biological toxicity on the surface of gold nanoparticles and the environmental pollution caused by the preparation process; Second, the surface of the gold nanoparticles prepared using chloroplasts has active groups such as amino groups and carboxyl groups, which can be directly applied to ELISA detection, avoiding biochemical surface modification.

[0040] The present invention effectively solves the problems of low sensitivity in the existing traditional ELISA biomolecule detection method and the problem that the gold nanoparticles prepared by the traditional method in the existing gold nanoparticle ELISA biomolecule detection method need further biochemical surface modification due to biological toxicity and the lack of active groups such as amino groups and carboxyl groups on the surface. Specific embodiments

[0041] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0042] Example

[0043] A green preparation and modification-free surface plasmon enhanced biomolecule detection method, taking human interleukin 6 (IL-6) biomolecule detection as an example, comprises the following steps:

[0044] Step S1: add 1 mL of 10 mM chloroauric acid solution to 10 mL of chloroplast solution prepared from 1 g of plant green leaves, and stir until well mixed;

[0045] Step S2: Add NaOH solution, adjust the pH value of the solution to 7.5, stir, and wait for the chloroauric acid to react with the chloroplasts;

[0046] Step S3: injecting the solution into a centrifuge tube, centrifuging it in a centrifuge, removing the supernatant, and collecting the precipitate;

[0047] Step S4: After ultrapure water is injected into the centrifuge tube to wash the precipitate, the precipitate is centrifuged in a centrifuge, the supernatant is removed, and the precipitate is collected. This washing process is repeated 3 times;

[0048] Step S5: after washing, add the capture antibody mouse anti-human IL-6 capture antibody corresponding to the biological molecule to be detected IL-6, and fully stir the gold nanoparticles and the capture antibody mouse anti-human IL-6 capture antibody solution corresponding to the biological molecule to be detected IL-6 to react;

[0049] Step S6: After centrifugation using a centrifuge, remove the supernatant and collect the precipitate;

[0050] Step S7: adding the washing solution into the centrifuge tube, stirring, centrifuging using a centrifuge, removing the supernatant, washing away the capture antibody mouse anti-human IL-6 capture antibody that is not captured by the gold nanoparticles, and collecting the precipitate;

[0051] Step S8: adding a blocking solution containing 1% bovine serum albumin PBS solution for incubation;

[0052] Step S9: adding washing liquid into the centrifuge tube, stirring and washing, centrifuging in a centrifuge, removing the supernatant, and collecting the precipitate;

[0053] Step S10: adding the biomolecule to be detected IL-6, and waiting for the capture antibody mouse anti-human IL-6 capture antibody to react with the biomolecule to be detected IL-6;

[0054] Step S11: After centrifugation using a centrifuge, remove the supernatant and collect the precipitate.

[0055] Step S12: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the unreacted biomolecule IL-6 to be detected, and collect the precipitate.

[0056] Step S13: Add the detection antibody, biotinylated goat anti-human IL-6 detection antibody, and wait until the biomolecule IL-6 to be detected and the detection antibody, biotinylated goat anti-human IL-6 detection antibody, fully react.

[0057] Step S14: After centrifugation using a centrifuge, remove the supernatant and collect the precipitate.

[0058] Step S15: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the unreacted detection antibody, biotinylated goat anti-human IL-6 detection antibody, and collect the precipitate.

[0059] Step S16: Add streptavidin labeled with alkaline phosphatase and wait until the detection antibody and streptavidin crosslink.

[0060] Step S17: After centrifugation using a centrifuge, remove the supernatant and collect the precipitate.

[0061] Step S18: Add the washing solution into the centrifuge tube, stir, after centrifugation using a centrifuge, remove the supernatant, wash away the uncrosslinked streptavidin, and collect the precipitate.

[0062] Step S19: Add the chemiluminescent substrate p-nitrophenyl phosphate disodium salt and wait until alkaline phosphatase and the chemiluminescent substrate p-nitrophenyl phosphate disodium salt react to generate a fluorescent product.

[0063] Step S20: Add the termination solution, NaOH solution, and detect the absorbance of the solution to calculate the concentration of the biomolecule to be detected.

[0064] The temperature of the reaction between chloroauric acid and chloroplast is 25 °C.

[0065] The reaction time of chloroauric acid and chloroplast is 24 h.

[0066] The rotation speed of the centrifugation is 10000 rpm / min.

[0067] The time of the centrifugation is 30 min.

[0068] The sufficient stirring reaction time of the gold nanoparticles and the capture antibody solution of mouse anti-human IL-6 capture antibody corresponding to the biomolecule IL-6 to be detected is 10 min.

[0069] The gold nanoparticles and the capture antibody mouse anti-human IL-6 corresponding to the biomolecule IL-6 to be detected are fully stirred, and the reaction temperature is 37°C.

[0070] The reaction temperature between the capture antibody mouse anti-human IL-6 capture antibody and the biomolecule IL-6 to be detected is 37°C.

[0071] The reaction time between the capture antibody mouse anti-human IL-6 capture antibody and the biomolecule IL-6 to be detected is 10 min.

[0072] The reaction temperature between the biomolecule IL-6 to be detected and the detection antibody biotinylated goat anti-human IL-6 detection antibody is 37°C.

[0073] The reaction time between the biomolecule IL-6 to be detected and the detection antibody biotinylated goat anti-human IL-6 detection antibody is 10 min.

[0074] The reaction temperature between the detection antibody biotinylated goat anti-human IL-6 detection antibody and streptavidin is 37°C.

[0075] The reaction time between the detection antibody biotinylated goat anti-human IL-6 detection antibody and streptavidin is 10 min.

[0076] The reaction temperature between alkaline phosphatase and the chemiluminescent substrate p-nitrophenyl phosphate disodium salt to generate a fluorescent product is 37°C.

[0077] The reaction time between alkaline phosphatase and the chemiluminescent substrate p-nitrophenyl phosphate disodium salt to generate a fluorescent product is 10 min.

Claims

1. A green preparation method for surface plasmon enhanced biomolecule detection without modification, characterized in that: It includes the following steps: Step S1: Add 1 mL of chloroauric acid solution with a concentration of 10 mM to 10 mL of chloroplast solution prepared from 1 g of green leaves of plants, and stir until evenly mixed; Step S2: Add NaOH solution to adjust the pH value of the solution to 7.5 - 9, stir, and wait for the reaction between chloroauric acid and chloroplast; Step S3: Inject the solution into a centrifuge tube, perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S4: Inject ultrapure water into the centrifuge tube to wash the precipitate, then perform centrifugation using a centrifuge, remove the supernatant, and collect the precipitate. This washing process is repeated 1 - 5 times to obtain gold nanoparticles; Step S5: After washing, add the mouse anti - human IL - 6 capture antibody corresponding to the biomolecule IL - 6 to be detected to the precipitate in Step S4, and stir the gold nanoparticles and the mouse anti - human IL - 6 capture antibody solution corresponding to the biomolecule IL - 6 to be detected for full reaction; Step S6: Perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S7: Add the washing solution to the centrifuge tube, stir, perform centrifugation using a centrifuge, then remove the supernatant, wash away the mouse anti - human IL - 6 capture antibody not captured by the gold nanoparticles, and collect the precipitate; Step S8: Add the blocking solution to the precipitate in Step S7 and incubate; Step S9: Add the washing solution to the centrifuge tube, stir and wash, perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S10: Add the biomolecule IL - 6 to be detected to the precipitate in Step S9 and wait until the mouse anti - human IL - 6 capture antibody and the biomolecule IL - 6 to be detected fully react; Step S11: Perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S12: Add the washing solution to the centrifuge tube, stir, perform centrifugation using a centrifuge, then remove the supernatant, wash away the unreacted biomolecule IL - 6 to be detected, and collect the precipitate; Step S13: Add the biotinylated goat anti - human IL - 6 detection antibody to the precipitate in Step S12 and wait until the biomolecule IL - 6 to be detected and the biotinylated goat anti - human IL - 6 detection antibody fully react; Step S14: Perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S15: Add the washing solution to the centrifuge tube, stir, perform centrifugation using a centrifuge, then remove the supernatant, wash away the unreacted biotinylated goat anti - human IL - 6 detection antibody, and collect the precipitate; Step S16: Add streptavidin labeled with an enzyme to the precipitate in Step S15 and wait until the detection antibody and streptavidin cross - link; Step S17: Perform centrifugation using a centrifuge, then remove the supernatant and collect the precipitate; Step S18: Add the washing solution to the centrifuge tube, stir, perform centrifugation using a centrifuge, then remove the supernatant, wash away the uncross - linked streptavidin, and collect the precipitate; Step S19: Add the chemiluminescent substrate to the precipitate in Step S18 and wait until the enzyme and the chemiluminescent substrate react to generate a fluorescent product; Step S20: Add a termination solution and measure the absorbance of the solution to calculate the concentration of the biomolecule to be measured.

2. The method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, wherein: The temperature of the reaction between chloroauric acid and chloroplasts is 25 °C, and the reaction time is 24 - 36 h.

3. A method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, characterized in that: The rotation speed of the centrifugation treatment is 10000 - 14000 rpm / min, and the centrifugation time is 10 - 30 min.

4. A method for green preparation of a surface plasmon enhanced biomolecule detection without modification according to claim 1, characterized in that: The temperature of the full stirring reaction between the gold nanoparticles and the mouse anti-human IL-6 capture antibody solution corresponding to the biomolecule IL-6 to be detected is 37 °C, and the full stirring reaction time is 10 - 30 min.

5. A method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, characterized in that: The temperature of the reaction between the mouse anti-human IL-6 capture antibody and the biomolecule IL-6 to be detected is 37 °C, and the reaction time is 10 - 30 min.

6. A method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, characterized in that: The temperature of the reaction between the biomolecule IL-6 to be detected and the biotinylated goat anti-human IL-6 detection antibody is 37 °C, and the reaction time is 10 - 30 min.

7. A method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, characterized in that: The temperature of the cross-linking between the biotinylated goat anti-human IL-6 detection antibody and streptavidin is 37 °C, and the cross-linking time is 10 - 30 min.

8. A method for green preparation of unmodified surface plasmon enhanced biomolecule detection according to claim 1, characterized in that: The temperature of the reaction between the enzyme and the chemiluminescent substrate to generate a fluorescent product is 37 °C, and the time is 10 - 30 min.