Preparation method and application of colloidal gold magnetic beads

Abstract

The invention relates to the technical field of medical detection, in particular to a preparation method and application of colloidal gold magnetic beads. Amino magnetic beads capable of expanding the number of groups, reducing the steric effect and improving the capture efficiency are obtained; washing the treated amino magnetic beads, adding an EGS coupling agent, washing after reaction, adding small molecules with one end containing amino and the other end containing sulfydryl, enabling the microspheres and the amino of the small molecules to form covalent bonds, then washing, and modifying the carboxyl magnetic beads into sulfydryl magnetic beads; adding a chloroauric acid solution into the sulfydryl magnetic beads, fully mixing, and then adding a reducing agent; and then heating to 40-90 DEG C, and reacting for 1-6 hours to prepare the colloidal gold magnetic beads. Compared with the prior art, the preparation method and the application of the colloidal gold magnetic bead provided by the invention not only meet multiple detection requirements of flow fluorescence, but also can adsorb natural allergen antigens, realize high throughput and reduce the omission ratio of allergen detection.

Description

[Technical field]

[0001] The invention relates to the technical field of medical detection, and in particular to a preparation method and application of colloidal gold magnetic beads. [Background technology]

[0002] Colloidal gold material, due to its ability to adsorb antigens and antibodies through electrostatic action, can be used as a carrier, so it is one of the most widely used and mature materials in the field of in vitro diagnosis. It has the characteristics of easy operation, no damage to antigens or antibodies, and low cost.

[0003] Flow cytometry is an emerging diagnostic technology based on flow analysis and magnetic fluorescence coded microsphere system. Compared with traditional detection methods such as colloidal gold test strips and chemiluminescence, it has the advantages of high sensitivity, fast detection speed, and multiplex detection.

[0004] As people pay more and more attention to health and the number of people with allergies increases, allergen detection has gradually attracted attention in the field of medical diagnosis. However, allergens have certain particularities, that is, allergens are a mixture and there are many types. A single allergen contains one or more allergenic fragments, and different people may be allergic to different fragments of the same allergen, including one or several of them. However, these allergenic fragments have different structures and properties. Therefore, when manufacturing allergen diagnostic reagents, there will be problems such as being unable to uniformly coat the surface of the carrier material, resulting in missed detection; some test strips can only detect a single allergen, have low throughput, and patients need to draw blood multiple times.

[0005] Therefore, it is necessary to construct a method that can detect allergens with high throughput and accuracy. [Summary of the invention]

[0006] In order to overcome the above problems, the present invention proposes a preparation method and application of colloidal gold magnetic beads that can effectively solve the above problems.

[0007] The present invention provides a technical solution to solve the above technical problems: providing a preparation method and application of colloidal gold magnetic beads, comprising the following steps:

[0008] Step S1, obtaining amino magnetic beads that expand the number of groups and reduce steric hindrance to improve capture efficiency, activating carboxyl magnetic beads with EDC, adding long-chain polyamino polymers, and coupling them with microspheres;

[0009] Step S2, washing the amino magnetic beads treated in step S1, adding EGS coupling agent, washing after reaction, adding a small molecule containing an amino group at one end and a thiol group at the other end, so that the microspheres form a covalent bond with the amino group of the small molecule, and then washing to modify the carboxyl magnetic beads into thiol magnetic beads;

[0010] Step S3, adding the chloroauric acid solution to the thiol magnetic beads, mixing thoroughly, and then adding a reducing agent; then heating to 40-90° C., reacting for 1-6 hours, to obtain colloidal gold magnetic beads.

[0011] Preferably, the long-chain polyamino polymer includes one or more of polyethyleneimine, polylysine, and polyacrylamine, and has a molecular weight between 1,000 and 1,000,000.

[0012] Preferably, the small molecules containing an amino group at one end and a thiol group at the other end include β-mercaptoethylamine and 2-amino-5-mercapto-1,3,4-thiadiazole.

[0013] Preferably, the reducing agent includes one or more of sodium citrate, tetrakishydroxymethylphosphonium chloride, tannic acid, tannic acid, and vitamin C.

[0014] Preferably, in step S3, the reaction temperature is 60°C to 90°C.

[0015] Preferably, the ratio of chloroauric acid to reducing agent is 100:1 to 1:100.

[0016] Preferably, the ratio of chloroauric acid to reducing agent is 2.5:1 to 10:1.

[0017] Preferably, the absorption peak of the colloidal gold is between 510nm and 560nm.

[0018] Preferably, the absorption peak of the colloidal gold is between 520 and 550 nm.

[0019] An application of colloidal gold magnetic beads obtained by the preparation method in allergen detection.

[0020] Compared with the prior art, the colloidal gold magnetic beads prepared by the preparation method of the colloidal gold magnetic beads of the present invention have both the high-throughput detection characteristics of the flow fluorescence method and the advantages of the high adsorption of the colloidal gold material, which is conducive to improving the detection efficiency and accuracy. It not only meets the multiple detection requirements of flow fluorescence, but also can adsorb natural allergen antigens, achieve high throughput, and reduce the missed detection rate of allergen detection.

Brief Description of the Drawings

[0021] Figure 1 This is a distribution diagram of colloidal gold encoded microspheres of colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0022] Figure 2 This is the e1 standard curve of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0023] Figure 3 This is a standard curve of e5 of colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0024] Figure 4 This is a d1 standard curve diagram of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0025] Figure 5 The d2 standard curve of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0026] Figure 6 This is a h1 standard curve diagram of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0027] Figure 7 This is the i6 standard curve of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0028] Figure 8 This is the e1 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0029] Fig. 9 This is the e5 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0030] Fig.10 This is a d1 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0031] Fig.11 This is a d2 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0032] Fig.12 This is the h1 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention;

[0033] Fig.13 This is the i6 linear evaluation graph of the colloidal gold magnetic beads obtained by the preparation method of the present invention. [Specific implementation method]

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] It should be noted that in the embodiments of the present invention, all directional indications (such as up, down, left, right, front, back, etc.) are limited to relative positions on the specified view, rather than absolute positions.

[0036] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0037] See also Figures 1 to 13 The preparation method of colloidal gold magnetic beads of the present invention comprises the following steps:

[0038] Step S1, a certain amount of carboxyl magnetic beads are activated by EDC, and a long-chain polyamino polymer is added to couple the polymer with the microspheres. The microspheres are carboxyl magnetic or carboxyl magnetic fluorescent coded microspheres.

[0039] The long-chain polyamino polymer includes but is not limited to polyethyleneimine, polylysine, polyacrylamine, etc., and the molecular weight should be between 1000 and 1000000.

[0040] After this operation, the number of surface active groups of the original microspheres can be greatly increased, thereby effectively increasing the loading capacity; in addition, the long-chain structure of the polymer can effectively reduce the steric effect, more efficiently capture the substances to be coated and detected in the solution, and greatly improve the detection sensitivity and accuracy.

[0041] Step S2, wash the amino microspheres treated in step S1, add a certain amount of EGS coupling agent (ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester)), react for 30 minutes and then wash, add a small molecule containing an amino group at one end and a thiol group at the other end to form a covalent bond between the microspheres and the amino group of the small molecule, then wash three times to modify the carboxyl magnetic beads into thiol magnetic beads.

[0042] The small molecules containing an amino group at one end and a thiol group at the other end include, but are not limited to, β-mercaptoethylamine and 2-amino-5-mercapto-1,3,4-thiadiazole.

[0043] Step S3, add a certain concentration of chloroauric acid solution to the thiol magnetic beads, mix thoroughly for 1 hour, and then add a certain amount of reducing agent. Then raise the temperature to 40-90°C and react for 1-6 hours. The particle size and quantity of the coupled colloidal gold can be adjusted by adjusting the dosage, compatibility, temperature and reaction time of the reducing agent. This will achieve the effect of not affecting the entry of the excitation light inside and outside the microspheres and the release of the emitted light.

[0044] The reducing agent includes but is not limited to one or more of sodium citrate, tetrakishydroxymethylphosphonium chloride, tannic acid, tannic acid, vitamin C, etc.

[0045] In step S3, the reaction temperature is 40°C to 95°C, and the optimal temperature is 60°C to 90°C.

[0046] The ratio of chloroauric acid to reducing agent is 100:1 to 1:100; optimally, 2.5:1 to 10:1.

[0047] In step S3, colloidal gold may be synthesized first, and the particle size of the coupled colloidal gold may be adjusted by adjusting the dosage of the reducing agent, the compatibility, the temperature and the reaction time. The absorption peak of the synthesized colloidal gold is required to be between 510nm and 560nm, and then the colloidal gold and the thiol magnetic beads are mixed in a certain proportion, and the amount of the coupled colloidal gold on the surface of the microspheres is adjusted to achieve the purpose of not affecting the detection. Stir at 25°C for 3 hours.

[0048] The absorption peak of the colloidal gold should be between 510nm and 560nm, and the optimal absorption peak is between 520nm and 550nm.

[0049] The colloidal gold magnetic beads prepared by the preparation method of the colloidal gold magnetic beads of the present invention have both the high-throughput detection characteristics of the flow fluorescence method and the advantages of the high adsorption of the colloidal gold material, which is conducive to improving the detection efficiency and accuracy. It not only meets the multiple detection requirements of the flow fluorescence method, but also can adsorb natural allergen antigens, achieve high throughput, and reduce the missed detection rate of allergen detection.

[0050] The preparation method of the colloidal gold magnetic beads of the present invention is described below with reference to specific examples.

[0051] Example 1

[0052] Preparation of colloidal gold magnetic beads

[0053] Step S11, weigh 4.5 mg EDC, add it to 1 ml of MES solution (ph ~ 4.75) containing 100 mg of encoded or unencoded carboxyl magnetic beads, vertically mix for 30 minutes, remove the supernatant by magnetic separation, wash three times with the same MES buffer, then add 20 ul of polyethyleneimine with a molecular weight of 100,000, shake and mix thoroughly, vertically mix and react for 3 hours to couple it with the microspheres; wash three times with deionized water, and the resulting amino magnetic beads have expanded the number of groups, reduced steric effect and improved capture efficiency.

[0054] Step S21, wash the amino microspheres treated in step S11, place them in 1 ml of 10 mM PBS solution, add 100 microliters of 50 mg of EGS coupling agent (ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)) DMSO solution, react for 30 minutes, wash with PBS, add 0.5 ml of PBS to resuspend, add 10 mg of β-mercaptoethylamine, fully dissolve and mix, vertically mix for 6 hours, so that the microspheres form a covalent bond with the amino group of the small molecule, and then wash three times, and the resulting thiol magnetic beads. The magnetic beads are placed in a three-necked flask, 50 ml of deionized water is added, and argon is filled for storage.

[0055] Step S31, first prepare the reducing agent: mix 12.9 mg of tetrakis hydroxymethyl phosphonium chloride with 30 mg of tannic acid, add 2 ml of deionized water and mix well for use; prepare 100 ml of 2% chloroauric acid solution, take 25 ml of the solution, add it to the solution obtained in S2, stir mechanically, mix well for 1 hour, and then add 2 ml of the reducing agent solution. Then heat to 75°C and react for 3 hours. Wash three times with deionized water, and finally prepare a microsphere solution with a concentration of 10 mg / ml and store it at 4°C away from light.

[0056] Application of Colloidal Gold Magnetic Beads

[0057] This embodiment provides a liquid phase chip based on 6 types of colloidal gold magnetic beads, but is not limited to the 6 types of this embodiment. The encoding method is the difference in fluorescence intensity of the APC / APC-A750 channels. The differently coded microspheres of the liquid phase chip are respectively coupled to 6 antigens, namely cat hair (e1), dog hair (e5), dust mites (d2), dust mites (d1), house dust (h1), and cockroaches (i6), but are not limited to the 6 antigens in this case. At the same time, the liquid phase chip also includes 1 biotinylated detection antibody and streptavidin-labeled phycoerythrin. The following briefly describes the preparation method of the liquid phase chip:

[0058] 1. Preparation of captured microspheres

[0059] Take 1.25 mL of microspheres, separate by magnetic separation, discard the supernatant, add 100-500 μL of 0.2 M potassium carbonate solution, and mix well;

[0060] Add 10-100 μg of natural allergen antigen, mix and incubate at room temperature for 3h-12h;

[0061] Microsphere magnetic separation, discard the supernatant, add 0.1-1mL blocking solution, preferably 1% BSA, and block for 3h-12h;

[0062] Magnetic separation of microspheres, discard the supernatant, add 5-10mL preservation solution to resuspend, and store at 4℃;

[0063] Mix the 6 coated microspheres together, add 5-10 mL of preservation solution to resuspend, and store at 4°C.

[0064] Test on a flow cytometer or flow fluorimeter, refer to Figure 1 .

[0065] 2. Prepare biotin-labeled detection antibodies

[0066] Dissolve 10 mg of activated biotin N-hydroxysuccinimide ester in 1 mL of anhydrous DMF, take 10 μL and add 200 μg of mouse anti-human IgE monoclonal antibody or polyclonal antibody (detection antibody) and incubate at room temperature for 1 hour. Dialyze overnight with 100 mL of 0.02 M PBS solution to remove unreacted biotin N-hydroxysuccinimide ester to obtain biotin-labeled detection antibody.

[0067] 3. Preparation of reporter molecules

[0068] The reporter molecule is SA-PE, which is a commercially available product and is diluted to 5 μg / mL with 0.01-0.05 M PBS solution for use.

[0069] 4. Preparation of Calibrators

[0070] The calibrator adds fetal bovine serum as a matrix to eliminate the matrix effect. The matrix is ​​fetal bovine serum (20% v / v), BSA (0.1% w / v) PBS (10mmol / L), proclin300 (0.2% v / v), add 6 kinds of humanized antibodies, mix well, and set aside. Used for the establishment of the kit calibration curve.

[0071] 5. Use of the kit

[0072] The method of using this kit is as follows:

[0073] Step 1: Preparation of calibrators: Take the calibrators, mix them, and perform gradient dilution with the calibrator matrix to obtain calibrators of different concentrations for the establishment of the standard curve.

[0074] Step 2: Take 50 μL of calibrator / serum sample to be tested, add 50 μL of capture antibody microspheres, mix well, and incubate at 37°C for 30 min;

[0075] Step 3, after magnetic separation of the capture microspheres, wash with washing solution three times, add 50 μL of biotin-labeled detection antibody, add 50 μL of SA-PE, mix well, and incubate at 37°C in the dark for 30 min;

[0076] Step 4, after magnetic separation, the captured microspheres were washed three times with PBS and detected by flow cytometry or flow fluorimetry;

[0077] Step 5, the signal of phycoerythrin (PE) is read in the fluorescence channel by a flow cytometer or a flow fluorometer, and the signals reported by the phycoerythrin (PE) of the six coded microspheres represent the concentrations of the six allergen IgEs.

[0078] Test results and analysis of colloidal gold microsphere applications

[0079] 1. Establishment of standard curve

[0080] Use the calibrator provided in this kit to dilute the calibrator to different concentrations to obtain calibrators of different concentrations. Figure 2-Figure 7 The standard curve of

[0081] 2. Linear Evaluation

[0082] Select a high-concentration sample at the upper limit of the linear range and dilute it into at least 6 samples of different concentrations (xi). Test each concentration 3 times and calculate the mean value (yi) of the test results. Use the dilution concentration (xi) as the independent variable and the mean value (yi) of the test results as the dependent variable to calculate the linear regression equation. Calculate the correlation coefficient (R) of the linear regression. The results are shown in Figure 8-Figure 13 According to the formula, the correlation coefficient R is greater than 0.99, which shows a good correlation.

[0083] 3. Blank Limit and Detection Limit Assessment

[0084] Blank limit: Use sample diluent, repeat the test 20 times, calculate the mean (x-) and standard deviation (SD), and calculate the signal value of x-+2SD. Substitute this value into the standard curve equation of the kit to obtain the corresponding sample concentration value, which is the blank limit of this kit.

[0085] Detection limit: Test 5 samples with low concentrations close to the detection limit (the approximate detection limit is estimated based on the blank limit value, which is slightly higher than the blank limit). Test each sample 5 times and sort the test results by size. The number of test results below the blank limit value should be less than or equal to 3.

[0086] See Table 1 below. The blank limit is ≤0.1IU / mL and the detection limit is ≤0.35IU / mL, which has a high sensitivity.

[0087] Table 1. Blank limit and detection limit (IU / mL)

[0088]

[0089] IV. Clinical Evaluation

[0090] Ten healthy samples and ten clinically confirmed allergen-positive samples were selected, and colloidal gold microsphere reagents and ordinary microsphere reagents were tested respectively for comparison and analysis with clinically confirmed cases. The results are shown in Table 2. The positive and negative coincidence rates of colloidal gold microsphere reagents were 100%, which was significantly better than ordinary microsphere reagents and reduced the missed detection rate.

[0091] Table 2. Clinical evaluation

[0092]

[0093] Example 2

[0094] Step S21, weigh 10 mg of EDC, add it to 1 ml of MES solution (ph ~ 4.75) containing 200 mg of encoded or unencoded carboxyl magnetic beads, vertically mix for 30 minutes, remove the supernatant by magnetic separation, wash three times with the same MES buffer, then add 100 ul of poly-lysine with a molecular weight of 50,000, shake and mix thoroughly, vertically mix and react for 5 hours to couple it with the microspheres; wash three times with deionized water, and the resulting amino magnetic beads have expanded the number of groups, reduced steric effect and improved capture efficiency.

[0095] Step S22, wash the amino microspheres treated in the first step, place them in 2 ml of 10 mM PBS solution, add 200 microliters of 50 mg of EGS coupling agent (ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)) DMSO solution, react for 30 minutes, wash with PBS, add 1 ml of PBS to resuspend, add 50 mg of 2-amino-5-mercapto-1,3,4-thiadiazole, fully dissolve and mix, vertically mix for 6 hours, so that the microspheres form a covalent bond with the amino group of the small molecule, and then wash three times, and the resulting thiol magnetic beads. Place the magnetic beads in a three-necked flask, add 100 ml of deionized water, and store them in argon.

[0096] Step S23, first prepare the reducing agent: mix 25.5 mg of tetrakis hydroxymethyl phosphonium chloride with 50 mg of vitamin C, add 3 ml of deionized water and mix well for use; prepare 100 ml of 2% chloroauric acid solution, take 25 ml of the solution, add it to the solution obtained in S2, stir mechanically, mix well for 1 hour, and then add 2 ml of the reducing agent solution. Then heat to 80°C and react for 5 hours. Wash three times with deionized water, and finally prepare a microsphere solution with a concentration of 10 mg / ml and store it at 4°C away from light.

[0097] Compared with the prior art, the colloidal gold magnetic beads prepared by the preparation method of the colloidal gold magnetic beads of the present invention have both the high-throughput detection characteristics of the flow fluorescence method and the advantages of the high adsorption of the colloidal gold material, which is conducive to improving the detection efficiency and accuracy. It not only meets the multiple detection requirements of flow fluorescence, but also can adsorb natural allergen antigens, achieve high throughput, and reduce the missed detection rate of allergen detection.

[0098] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any modifications, equivalent substitutions and improvements made within the concept of the present invention should be included in the patent protection scope of the present invention.

Claims

1. A method for preparing colloidal gold magnetic beads, characterized in that: The steps include: Step S1, obtaining amino magnetic beads that expand the number of groups and reduce steric hindrance to improve capture efficiency, activating carboxyl magnetic beads with EDC, adding long-chain polyamino polymers, and coupling them with microspheres; Step S2, washing the amino magnetic beads treated in step S1, adding EGS coupling agent, washing after reaction, adding a small molecule containing an amino group at one end and a thiol group at the other end, so that the microspheres form a covalent bond with the amino group of the small molecule, and then washing to modify the carboxyl magnetic beads into thiol magnetic beads; Step S3, adding the chloroauric acid solution to the thiol magnetic beads, mixing thoroughly, and then adding a reducing agent; then heating to 40-90° C., reacting for 1-6 hours, to obtain colloidal gold magnetic beads.

2. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The long-chain polyamino polymer includes one or more of polyethyleneimine, polylysine and polyacrylamine, and has a molecular weight between 1,000 and 1,000,000.

3. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The small molecules having an amino group at one end and a thiol group at the other end include β-mercaptoethylamine and 2-amino-5-mercapto-1,3,4-thiadiazole.

4. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The reducing agent includes one or more of sodium citrate, tetrakis(hydroxymethyl)phosphonium chloride, tannic acid, tannic acid, and vitamin C.

5. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: In step S3, the reaction temperature is 60°C to 90°C.

6. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The ratio of chloroauric acid to reducing agent is 100:1 to 1:

100.

7. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The ratio of chloroauric acid to reducing agent is 2.5:1 to 10:

1.

8. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The absorption peak of the colloidal gold is between 510nm and 560nm.

9. The method for preparing colloidal gold magnetic beads according to claim 1, characterized in that: The absorption peak of the colloidal gold is between 520 and 550 nm.

10. Use of colloidal gold magnetic beads obtained by the preparation method according to any one of claims 1 to 9 in allergen detection.

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