A green preparation method for magnetic particle enhanced biomolecule detection without surface modification

Abstract

A method for green preparation of magnetic particle enhanced biomolecule detection without surface modification belongs to the field of biomolecule detection methods, and includes the following steps: adding metal iron salt and metal copper salt into a chloroplast solution; adding NaOH to adjust the pH value and stirring for reaction; centrifuging to collect the precipitate; washing; calcining; adding copper ferrite particles and a capture antibody corresponding to the biomolecule to be detected into a centrifuge tube; washing away the uncaptured capture antibody; adding a blocking solution; washing; adding the biomolecule to be detected; washing away the unreacted biomolecule to be detected; adding a detection antibody; washing away the unreacted detection antibody; adding streptavidin labeled with alkaline phosphatase; washing away the uncrosslinked streptavidin; adding a chemiluminescent substrate; adding a termination solution and detecting the absorbance. The present invention solves the problem that the magnetic beads prepared by the existing method cannot be directly applied to ELISA due to biological toxicity and lack of active groups on the surface 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 magnetic particle enhanced biomolecule detection method without surface modification. Background Art

[0002] Currently, malignant diseases such as malignant tumors and AIDS seriously endanger human health and survival. Early detection and early treatment of these malignant diseases are one of the most effective methods to reduce the mortality rate of patients and improve the quality of life. Biomarker detection is one of the important means for early detection of related diseases. Therefore, ultrasensitive detection of biomolecules such as biomarkers has become an urgent requirement for clinical diagnosis and treatment. However, the concentration of biomarkers for malignant diseases in blood is generally relatively low, even reaching the attomolar (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 based on immunological reactions, which combines the specific reaction 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 the ELISA fluorescent product can be enhanced to increase the detection sensitivity. Micro-nano particles have a larger specific surface area compared with the surface of solid-phase carriers and can directly or indirectly adsorb more biomolecules to be detected, thus playing a role in enriching the biomolecules to be detected and improving the detection sensitivity.

[0005] Through the retrieval of the prior art, it is found that Wang, Yuan-Kai et al. in Food Control, 2014, 40, 41-45 used magnetic bead ELISA to detect the biomolecule fumonisin B1 at a concentration of 0.24 ng / mL. Compared with traditional ELISA, the detection time was shortened and the detection sensitivity was improved, demonstrating the feasibility of magnetic bead ELISA in enhancing detection sensitivity. However, there are currently two main problems with magnetic bead ELISA: (1) Some strongly corrosive chemical reagents such as hydroxylamine, borohydride, and organic solvents are used in the traditional magnetic bead preparation process, and surface modification is usually required later to reduce its biological toxicity. Therefore, these toxic reagents cause serious environmental pollution and high production costs. (2) The surface of magnetic beads prepared by traditional methods lacks active groups such as amino 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 enhanced biomolecule detection by green preparation of surface-modification-free magnetic particles to solve the problems of low sensitivity in existing traditional ELISA biomolecule detection methods and the need for further biochemical surface modification of the surface of magnetic beads prepared by traditional methods in existing magnetic bead ELISA biomolecule detection methods due to biological toxicity and lack of active groups such as amino and carboxyl groups. Summary of the Invention

[0007] In order to solve the problem that the surface of magnetic beads prepared by traditional methods in existing magnetic bead 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 enhanced biomolecule detection by green preparation of surface-modification-free magnetic particles.

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

[0009] A method for enhanced biomolecule detection by green preparation of surface-modification-free magnetic particles, comprising the following steps:

[0010] Step S1: Add metal iron salt and metal copper salt into the chloroplast solution and stir until evenly mixed;

[0011] Step S2: Add NaOH solution, adjust the pH value of the solution to 8 - 10, and stir for reaction;

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

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

[0014] Step S5: Calcine the precipitate in a vacuum oven to obtain powdery copper ferrite particles;

[0015] Step S6: Put the copper ferrite particles into a centrifuge tube containing ultrapure water, add the capture antibody corresponding to the biomolecule to be detected, and stir until the copper ferrite particles and the capture antibody corresponding to the biomolecule to be detected react fully;

[0016] Step S7: Fix the copper ferrite particles by the magnetic field generated by an external magnet, and wash away the capture antibody not captured by the copper ferrite particles with a washing solution;

[0017] Step S8: Add a blocking solution and perform incubation;

[0018] Step S9: Fix the copper ferrite particles by the magnetic field generated by an external magnet and wash with a washing solution;

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

[0020] Step S11: Use the magnetic field generated by an external magnet to fix the copper ferrite particles, and wash away the unreacted biomolecule to be detected with a washing solution;

[0021] Step S12: Add the detection antibody and wait until the biomolecule to be detected and the detection antibody fully react;

[0022] Step S13: Use the magnetic field generated by an external magnet to fix the copper ferrite particles, and wash away the unreacted detection antibody with a washing solution;

[0023] Step S14: Add streptavidin labeled with alkaline phosphatase and wait until the detection antibody and streptavidin crosslink;

[0024] Step S15: Use the magnetic field generated by an external magnet to fix the copper ferrite particles, and wash away the uncrosslinked secondary antibody with a washing solution;

[0025] Step S16: Add a chemiluminescent substrate and wait until alkaline phosphatase reacts with the chemiluminescent substrate to generate a fluorescent product;

[0026] Step S17: Add a termination solution and measure the absorbance of the solution to calculate the concentration of the biomolecule to be detected.

[0027] Further, the metal iron salt includes one of ferric chloride or ferric nitrate, the metal copper salt includes one of copper chloride or copper nitrate, and the molar ratio of the metal iron salt to the metal copper salt is 2:1.

[0028] Further, the stirring reaction time in Step S2 is 12 - 24 h.

[0029] Further, the rotation speed of the centrifugation is 8000 - 12000 rpm / min, and the centrifugation time is 10 - 30 min.

[0030] Further, the temperature of the precipitation calcination in Step S5 is 200 °C, and the precipitation calcination time is 8 - 12 h.

[0031] Further, the incubation temperature in Step S8 is 37 °C, and the incubation time is 1 - 2 h.

[0032] Further, the temperature for the reaction of the solution containing copper ferrite particles and the capture antibody corresponding to the biomolecule to be detected is 37 °C, and the reaction time 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] Furthermore, 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] Furthermore, 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] Furthermore, the temperature for the reaction of alkaline phosphatase and 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 enhanced biomolecule detection method using green-prepared surface-modification-free magnetic particles of the present invention has the following advantages: First, the magnetic beads have a larger specific surface area, can adsorb more biomolecules, thus improving the detection sensitivity; Second, the magnetic beads can move freely in the liquid, can adsorb more biomolecules, thus improving the detection sensitivity; Third, the magnetic beads can be manipulated by applying an external magnetic field, which is beneficial for detection.

[0039] Compared with the existing magnetic bead ELISA biomolecule detection method, the enhanced biomolecule detection method using green-prepared surface-modification-free magnetic particles of the present invention has the following advantages: First, using chloroplasts to prepare magnetic beads avoids the use of some strongly corrosive chemical reagents in the traditional magnetic bead preparation process, thus avoiding the biological toxicity on the surface of the magnetic beads and environmental pollution caused by the preparation process; Second, the surface of the magnetic beads 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 problem of low sensitivity in the existing traditional ELISA biomolecule detection method and the problem that the magnetic beads prepared by the traditional method in the existing magnetic bead ELISA biomolecule detection method need further biochemical surface modification due to biological toxicity and lack of active groups such as amino groups and carboxyl groups on the surface. Specific Embodiments

[0041] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0042] Embodiment

[0043] Taking the detection of human interleukin 6 (IL-6) biomolecules as an example, the enhanced biomolecule detection method using green-prepared surface-modification-free magnetic particles includes the following steps:

[0044] Step S1: Add metal iron salt and metal copper salt into the chloroplast solution and stir until evenly mixed;

[0045] Step S2: Add NaOH solution and adjust the pH value of the solution to 8 - 10, then stir and react;

[0046] Step S3: Put the solution into a centrifuge tube, perform centrifugation using a centrifuge, and collect the precipitate;

[0047] Step S4: Inject ultrapure water into the centrifuge tube to wash the precipitate, then perform centrifugation using a centrifuge and collect the precipitate. This washing process is repeated 1 - 5 times;

[0048] Step S5: Calcinate the precipitate in a vacuum oven to obtain powdery copper ferrite particles;

[0049] Step S6: Put the copper ferrite particles into a centrifuge tube containing ultrapure water, add the capture antibody mouse anti - human IL - 6 capture antibody corresponding to the biomolecule to be detected, and stir until the copper ferrite particles and the capture antibody mouse anti - human IL - 6 capture antibody corresponding to the biomolecule to be detected react fully;

[0050] Step S7: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash away the un - captured capture antibody mouse anti - human IL - 6 capture antibody with a washing solution;

[0051] Step S8: Add a blocking solution, 1% bovine serum albumin PBS solution, and perform incubation.

[0052] Step S9: Fix the copper ferrite particles using the magnetic field generated by an external magnet and wash with a washing solution.

[0053] Step S10: Add the biomolecule to be detected, IL - 6, and wait until the capture antibody mouse anti - human IL - 6 capture antibody and the biomolecule to be detected, IL - 6, react fully;

[0054] Step S11: Fix the copper ferrite particles using the magnetic field generated by an external magnet and wash away the unreacted biomolecule to be detected, IL - 6, with a washing solution;

[0055] Step S12: Add the detection antibody biotinylated goat anti - human IL - 6 detection antibody and wait until the biomolecule to be detected, IL - 6, and the detection antibody biotinylated goat anti - human IL - 6 detection antibody react fully;

[0056] Step S13: Fix the copper ferrite particles using the magnetic field generated by an external magnet and wash away the unreacted detection antibody biotinylated goat anti - human IL - 6 detection antibody with a washing solution;

[0057] Step S14: Add streptavidin labeled with alkaline phosphatase and wait until the biotinylated goat anti-human IL-6 detection antibody and streptavidin crosslink;

[0058] Step S15: Fix the copper ferrite particles using the magnetic field generated by an external magnet and wash away the uncrosslinked streptavidin with the washing solution;

[0059] Step S16: Add the chemiluminescent substrate disodium p-nitrophenyl phosphate and wait until the alkaline phosphatase reacts with the chemiluminescent substrate disodium p-nitrophenyl phosphate to generate a fluorescent product;

[0060] Step S17: Add the termination solution NaOH solution and measure the absorbance of the solution to calculate the concentration of the biomolecule IL-6 to be detected.

[0061] The metal iron salt is ferric chloride hexahydrate with a molar amount of 0.0001 mol.

[0062] The metal copper salt is copper(II) chloride dihydrate with a molar amount of 0.00005 mol.

[0063] The chloroplast solution is a 10 ml solution diluted from the chloroplasts extracted from 0.2 g of green plant leaves.

[0064] The time for the stirring reaction is 12 h.

[0065] The rotation speed for the centrifugation treatment is 10000 rpm / min.

[0066] The time for the centrifugation treatment is 20 min.

[0067] The temperature for the precipitation calcination is 200 °C.

[0068] The time for the precipitation calcination is 10 h.

[0069] The temperature for the reaction between the solution containing copper ferrite particles and the capture antibody mouse anti-human IL-6 capture antibody corresponding to the biomolecule IL-6 to be detected is 37 °C.

[0070] The temperature for the incubation is 37 °C.

[0071] The time for the incubation is 1 h.

[0072] The time for the reaction between the solution containing copper ferrite particles and the capture antibody mouse anti-human IL-6 capture antibody corresponding to the biomolecule IL-6 to be detected is 20 min.

[0073] The temperature for the reaction between the capture antibody mouse anti-human IL-6 capture antibody and the biomolecule IL-6 to be detected is 37 °C.

[0074] The reaction time between the capture antibody, mouse anti-human IL-6 capture antibody, and the biomolecule to be detected, IL-6, is 20 min.

[0075] The reaction temperature between the biomolecule to be detected, IL-6, and the detection antibody, biotinylated goat anti-human IL-6 detection antibody, is 37 °C.

[0076] The reaction time between the biomolecule to be detected, IL-6, and the detection antibody, biotinylated goat anti-human IL-6 detection antibody, is 20 min.

[0077] The reaction temperature between the detection antibody, biotinylated goat anti-human IL-6 detection antibody, and streptavidin is 37 °C.

[0078] The reaction time between the detection antibody, biotinylated goat anti-human IL-6 detection antibody, and streptavidin is 20 min.

[0079] The reaction temperature between alkaline phosphatase and the chemiluminescent substrate p-nitrophenyl phosphate disodium salt to produce a fluorescent product is 37 °C.

[0080] The reaction time between alkaline phosphatase and the chemiluminescent substrate p-nitrophenyl phosphate disodium salt to produce a fluorescent product is 20 min.

Claims

1. A method for green preparation of magnetic particle enhanced biomolecule detection without surface modification, characterized in that: It includes the following steps: Step S1: Add metal iron salt and metal copper salt into the chloroplast solution, and stir until evenly mixed; The metal iron salt is ferric chloride hexahydrate with a molar amount of 0.0001 mol; The metal copper salt is copper(II) chloride dihydrate with a molar amount of 0.00005 mol; The chloroplast solution is a 10 ml solution diluted from the chloroplast extracted from 0.2 g of plant green leaves; Step S2: Add NaOH solution, adjust the pH value of the solution to 8 - 10, and stir for reaction; Step S3: Put the solution into a centrifuge tube, perform centrifugation using a centrifuge, and collect the precipitate; Step S4: Inject ultrapure water into the centrifuge tube to wash the precipitate, then perform centrifugation using a centrifuge, and collect the precipitate. This washing process is repeated 1 - 5 times; Step S5: Calcine the precipitate in a vacuum oven to obtain powdery copper ferrite particles; Step S6: Put the copper ferrite particles into a centrifuge tube containing ultrapure water, add the mouse anti - human IL - 6 capture antibody corresponding to the biomolecule IL - 6 to be detected, and stir until the copper ferrite particles and the mouse anti - human IL - 6 capture antibody corresponding to the biomolecule IL - 6 to be detected react fully; Step S7: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash away the mouse anti - human IL - 6 capture antibody not captured by the copper ferrite particles with a washing solution; Step S8: Add a blocking solution and incubate; Step S9: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash with a washing solution; Step S10: Add the biomolecule IL - 6 to be detected, and wait until the mouse anti - human IL - 6 capture antibody and the biomolecule IL - 6 to be detected react fully; Step S11: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash away the unreacted biomolecule IL - 6 to be detected with a washing solution; Step S12: Add the biotinylated goat anti - human IL - 6 detection antibody, and wait until the biomolecule IL - 6 to be detected and the biotinylated goat anti - human IL - 6 detection antibody react fully; Step S13: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash away the unreacted biotinylated goat anti - human IL - 6 detection antibody with a washing solution; Step S14: Add streptavidin labeled with alkaline phosphatase, and wait until the biotinylated goat anti - human IL - 6 detection antibody and streptavidin cross - link; Step S15: Fix the copper ferrite particles using the magnetic field generated by an external magnet, and wash away the uncross - linked streptavidin with a washing solution; Step S16: Add a chemical fluorescence substrate, and wait until alkaline phosphatase and the chemical fluorescence substrate react to generate a fluorescent product; Step S17: Add a termination solution, and detect the absorbance of the solution to calculate the concentration of the biomolecule IL - 6 to be detected.

2. A method for detecting biomolecules enhanced by green-prepared magnetic particles without surface modification according to claim 1, characterized in that: The stirring reaction time in Step S2 is 12 - 24 h.

3. A method for detecting biomolecules enhanced by green-prepared surface-modification-free magnetic particles according to claim 1, characterized in that: The rotation speed of the centrifugation is 8000 - 12000 rpm / min, and the centrifugation time is 10 - 30 min.

4. A method for detecting biomolecules enhanced by green-prepared surface-modification-free magnetic particles according to claim 1, characterized in that: The calcination temperature of the precipitate in Step S5 is 200 °C, and the calcination time of the precipitate is 8 - 12 h.

5. A method for detecting biomolecules enhanced by green-prepared surface-modification-free magnetic particles according to claim 1, characterized in that: The incubation temperature in Step S8 is 37 °C, and the incubation time is 1 - 2 h.

6. A method for detecting biomolecules enhanced by green-prepared surface-modification-free magnetic particles according to claim 1, characterized in that: The temperature for the reaction of the solution containing cupric ferrite particles with the mouse anti-human IL-6 capture antibody corresponding to the biomolecule IL-6 to be detected is 37 °C, and the reaction time is 10 - 30 min.

7. A method for detecting biomolecules enhanced by green-prepared surface-modification-free magnetic particles according to claim 1, characterized in that: The temperature for the reaction of the mouse anti-human IL-6 capture antibody with the biomolecule IL-6 to be detected is 37 °C, and the reaction time is 10 - 30 min.

8. A method for detecting biomolecules enhanced by green-prepared magnetic particles without surface modification according to claim 1, characterized in that: The temperature for the reaction of the biomolecule IL-6 to be detected with the biotinylated goat anti-human IL-6 detection antibody is 37 °C, and the reaction time is 10 - 30 min.

9. A method for detecting biomolecules enhanced by green-prepared magnetic particles without surface modification according to claim 1, characterized in that: The temperature for the cross-linking of the biotinylated goat anti-human IL-6 detection antibody with streptavidin is 37 °C, and the cross-linking time is 10 - 30 min; The temperature for the reaction of alkaline phosphatase with the chemiluminescent substrate to generate a fluorescent product is 37 °C, and the time is 10 - 30 min.