Magnetic bead and method for detecting glycosylated tumor markers

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a magnetic bead and a method for detecting glycosylated tumor markers. The magnetic bead for detecting the glycosylated tumor marker is a boric acid-based magnetic bead, the surface of the magnetic bead contains a boric acid group, the size of the magnetic bead is 300-500 nm, and the boron element content on the surface of the magnetic bead is greater than or equal to 1000 mg / g; specifically, boric acid-based magnetic beads are taken as a solid-phase extracting agent, the method comprises the steps of combining the magnetic beads with glycosylated tumor markers, closing the magnetic beads and coupling corresponding antibodies marked by signal molecules, and after magnetic separation, the magnetic beads are detected on a corresponding signal molecule detection instrument. The method is used for detecting the glycosylated tumor marker, and has the advantages of simple route, rapidness and convenience in operation, low cost and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to magnetic beads and methods for detecting glycosylated tumor markers. Background Art

[0002] Glycosylated tumor markers, such as alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, etc., play an important role in the diagnosis, treatment and monitoring of cancer, and specific glycosylation forms may provide higher diagnostic and prognostic value. At present, magnetic bead-based immunoassay technology has been widely used in clinical practice. Its detection process involves signal response mechanisms such as enzymatic reaction, chemiluminescence, electrochemiluminescence, and fluorescence. The double antibody sandwich method is often used for immunoassay strategy, which usually requires the first antibody to be coupled to the surface of the magnetic sphere first. After specific binding to the antigen to be detected, the enzyme-labeled second antibody is further coupled to respond to the signal. This detection strategy uses two different specific antibodies to sandwich different epitopes of the target antigen, which has high detection specificity and sensitivity. However, the double antibody sandwich method requires that the antigen has at least two sites that can bind to the antibody, so it is not suitable for small molecule antigen detection such as haptens with a molecular weight of less than 5000. In addition, the use of double antibodies is often accompanied by multiple steps such as coating, incubation, and washing. Not only is the detection cost high, but there is also a limitation of long operation time. Based on this, achieving low-cost, high-efficiency, and high-sensitivity detection of glycosylated tumor markers in serum still faces challenges.

[0003] The application of boric acid affinity chromatography has proved that boric acid molecules have a strong affinity for biomacromolecules containing cis-diol structures, and the two can achieve stable binding in weakly alkaline or neutral aqueous solutions. Glycosylated tumor markers contain cis-diol groups in their structural domains. These characteristics indicate that materials functionalized with borate can be used to detect glycosylated tumor markers. Summary of the invention

[0004] The purpose of the present invention is to provide a magnetic bead and method for rapid, efficient, low-cost and universal detection of glycosylated tumor markers in response to the current situation that the cost of existing magnetic bead detection of tumor markers is high and the operation time is long.

[0005] The present invention first provides a magnetic bead for detecting glycosylated tumor markers, which is a boric acid-based magnetic bead, specifically a magnetic microsphere containing a boric acid group on the surface, with a size of 300 to 500 nm, and a boron content of ≥1000 mg / g on the surface of the magnetic microsphere. The magnetic microsphere is prepared by the following method:

[0006] By using click chemistry reaction, boric acid molecules are reacted with magnetic microspheres containing epoxy groups on the surface, so that the boric acid groups are fixed on the surface of the microspheres; wherein the boric acid molecules are selected from molecules containing thiol groups and boric acid groups in their structures, and the amount of the boric acid molecules used satisfies the molar ratio of the thiol groups in their structures to the epoxy groups on the surface of the microspheres of (1-2):1.

[0007] The magnetic microspheres containing epoxy groups on the surface include but are not limited to one or more of glycidyl methacrylate modified magnetic microspheres, γ-glycidyloxypropyltrimethoxysilane modified magnetic microspheres, and epoxy-polyethylene glycol-carboxyl activated agarose magnetic microspheres. The boronic acid molecules are selected from 3-mercaptophenylboronic acid, 4-mercaptophenylboronic acid, phenylboronic acid-polyethylene glycol-mercapto, preferably 4-mercaptophenylboronic acid.

[0008] The method for detecting glycosylated tumor markers using the boric acid-based magnetic beads prepared above comprises the following specific steps:

[0009] Step 1, binding of magnetic beads with glycosylated tumor markers: boric acid-based magnetic beads and the sample to be tested are mixed, the amount of boric acid-based magnetic beads is 0.1-1 mg, the amount of sample is 10-60 μL, 50-200 μL of alkaloid buffer is added, and the mixed solution is shaken at 700-1000 rpm in a constant temperature mixer at 25-37° C. for 15-120 min to complete the adsorption between the glycosylated tumor marker and the magnetic beads, and then magnetic separation is performed, and the solution is placed on a magnetic rack until the magnetic beads are completely adsorbed;

[0010] Step 2, washing the magnetic beads: discard the supernatant obtained in step (1), add PBS buffer and let it stand for 1 to 2 minutes, then magnetically separate and discard the supernatant, add PBS buffer and let it stand for 1 to 2 minutes, then magnetically separate and discard the supernatant to ensure that there are no obvious droplets;

[0011] Step 3, blocking of magnetic beads: add 200-1000 μL of blocking buffer containing 5-20 wt% bovine serum albumin, vortex mix it, and shake it at 700-1000 rpm in a constant temperature mixer at 25-37° C. for 1-3 h to complete the blocking of bovine serum albumin on the nonspecific adsorption sites on the surface of the magnetic beads;

[0012] Step 4, washing of magnetic beads: after the blocking experiment is completed, the supernatant of step (3) is discarded, PBS buffer is added and allowed to stand for 1 to 2 minutes, and the supernatant is discarded by magnetic separation again, PBS buffer is added and allowed to stand for 1 to 2 minutes, and the supernatant is discarded by magnetic separation again to ensure that there are no obvious droplets;

[0013] Step 5, coupling of magnetic beads and antibodies: add 50-200 μL of the antibody solution labeled with the signal molecule, and shake and mix the mixed solution in a constant temperature mixer at 25-37° C. at 700-1000 rpm for 10-30 min to fully couple the magnetic beads with the labeled antibody;

[0014] Step 6, washing of magnetic beads: add ultrapure water and let stand for 1 to 2 minutes, then magnetically separate and discard the supernatant, add ultrapure water and let stand for 1 to 2 minutes, then magnetically separate and discard the supernatant to ensure that there are no obvious droplets;

[0015] Step 7, detection: disperse it in 20-100 μL ultrapure water and test it on the corresponding signal molecule detection instrument.

[0016] The sample to be tested in step 1 of the present invention preferably comprises a serum sample containing various glycosylated tumor markers (including alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, and procalcitonin).

[0017] The alkaloid in step 1 of the present invention is preferably Tris base or Tris-HCl base.

[0018] The alkaloid buffer in step 1 of the present invention is a phosphate buffer containing Tris base or Tris-HCl base, and the phosphate concentration is 0.3mM to 3mM.

[0019] The pH value of the alkaloid buffer in step 1 of the present invention is in the range of 7.5 to 9.0.

[0020] The pH value of the PBS buffer in step 2 and step 4 of the present invention is 7.5-9.0.

[0021] The signal molecule in step 5 of the present invention is preferably one or more of acridinium ester, fluorescein isothiocyanate, horseradish peroxidase, and the like.

[0022] The antibody in step 5 of the present invention is a monoclonal antibody corresponding to the target molecule to be detected, and the concentration of the antibody solution is 0.1-2.0 mg / mL, and the pH range is 7.5-9.0.

[0023] The corresponding signal molecule detection instrument in step 7 of the present invention is preferably one or more of a chemiluminescence immunoassay analyzer, a fluorescence analyzer, and an enzyme marker.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The boric acid-based magnetic beads used have a strong binding ability with the catechol group and have a high detection sensitivity for glycosylated tumor markers;

[0026] (2) The present invention has universal applicability for the detection of glycosylated tumor markers and a wide range of applications. By specifically binding to antibodies labeled with different signal molecules (such as acridinium ester, fluorescein isothiocyanate, horseradish peroxidase, etc.), it can achieve qualitative or quantitative detection of multiple types of glycosylated tumor markers under different response modes (chemiluminescence, fluorescence, colorimetric reaction);

[0027] (3) The present invention can achieve direct interaction between magnetic beads and target glycosylated tumor markers. The magnetic bead surface does not need to be coupled with antibodies to construct a complex double antibody sandwich structure, and the process is simpler.

[0028] (4) No secondary antibody is required, so the detection cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 (a) is a morphology of the magnetic microspheres modified with γ-glycidyloxypropyltrimethoxysilane described in Example 1 of the present invention; Figure 1 (b), (c) and (d) are morphological images of the boric acid-based magnetic beads prepared in Example 1, Example 2 and Example 3 of the present invention, respectively.

[0030] Figure 2 The chemiluminescent signal intensity when different amounts of boric acid-based magnetic beads are used in the operations of Example 4, Example 5 and Example 6 respectively. DETAILED DESCRIPTION

[0031] Example 1: Preparation of boric acid-based magnetic beads

[0032] Boric acid-based magnetic beads were prepared by modifying the surface of magnetic microspheres modified with γ-glycidyloxypropyltrimethoxysilane with boric acid groups using the click chemistry reaction between thiol and epoxy. The specific method is as follows: weigh 100 mg of 4-mercaptophenylboronic acid in a three-necked flask, add 50 mL of isopropanol, and sonicate for 2 minutes to fully dissolve it. Then, 50 mg of epoxy-based magnetic microspheres were added, and the magnetic microspheres were evenly dispersed by sonication. The flask was then placed in a mechanical stirring device, and 10 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene was added to the mixed solution under stirring. The reaction was continued at room temperature for 24 hours. After the reaction was completed, the product was separated by magnetic adsorption, and washed with ethanol and water several times. Finally, the product was dispersed in a certain amount of deionized water, and freeze-dried for use. The morphology of the epoxy-based magnetic microspheres used and the morphology of the boric acid-based magnetic beads prepared after the reaction are shown as follows: Figure 1 As shown in (a) and (b).

[0033] Example 2: The experimental operation of this example is the same as that of Example 1, except that the monomer containing a boronic acid group is replaced by 3-mercaptophenylboronic acid.

[0034] Example 3: The experimental operation of this example is the same as that of Example 1, except that the monomer containing the boronic acid group is replaced by phenylboronic acid-polyethylene glycol-mercapto from 4-mercaptophenylboronic acid, and the reaction addition amount is changed from 50 mg to 100 mg.

[0035] Example 4: Chemiluminescent immunoassay detection of alpha-fetoprotein (AFP) using the boric acid-based magnetic beads prepared in Example 1

[0036] (1) 2.5 mg, 5 mg, 50 mg, 200 mg, and 300 mg of prepared magnetic beads were dispersed in 1.5 mL centrifuge tubes, respectively. 200 μL of Tris base buffer (pH = 7.5) containing 0.5 mM phosphate and 15 μL of serum solution containing AFP (concentration of 300 ng / mL) were added thereto. The mixture was vortexed for 3 min. The mixture was placed on a constant temperature mixer at 37°C and shaken at 1000 rpm for 120 min. The supernatant was removed by magnetic separation and washed twice with PBS buffer at pH 7.5, each time with 500 μL.

[0037] (2) After washing and removing the supernatant, the boric acid-based magnetic beads were dispersed in 500 μL of blocking buffer (a 10 wt% BSA aqueous solution), vortexed for 3 min, and placed on a constant temperature mixer at 37°C. The blocking reaction was shaken at 1000 rpm for 3 h. The product was collected by magnetic separation and washed twice with PBS buffer at pH 7.5, with an amount of 500 μL each time.

[0038] (3) Add 100 μL of 300 ng / mL acridinium ester-labeled AFP antibody solution to the above magnetic beads, and shake the mixed solution at 1000 rpm in a constant temperature mixer at 37°C for 15 min; remove the supernatant by magnetic separation, and wash twice with ultrapure water, each time with 500 μL. Finally, disperse the above magnetic beads in 1 mL of ultrapure water to prepare an immunomagnetic bead solution for use.

[0039] (4) Add the immunomagnetic bead solution to the sampling slot of the chemiluminescent immunoassay analyzer, and add the pre-excitation solution (strong sodium oxide solution) and the excitation solution (hydrogen peroxide solution) to the injection slots A and B, respectively. Set the injection volumes of the sample, pre-excitation solution, and excitation solution to 15 μL / time, 30 μL / time, and 30 μL / time, respectively. In the instrument parameter interface, set the incubation time of substrate excitation to 10 min, set the sample to be tested in parallel 3 times, and finally start the chemiluminescent immunoassay analyzer for detection. Figure 2The results of the luminescence intensity test of serum samples with AFP concentrations of 0 ng / mL and 300 ng / mL when different concentrations of boric acid-based microspheres were used for injection. As can be seen from the figure, when the sample does not contain AFP, no luminescence value was detected at different concentrations of microspheres. In the serum sample containing 300 ng / mL of AFP, with the increase of the concentration of boric acid-based microspheres injected, that is, with the increase of the number of injected microspheres at the same magnetic bead injection volume, the chemiluminescence signal gradually increased, indicating its feasibility in actual sample detection.

[0040] Example 5: Chemiluminescent immunoassay detection of alpha-fetoprotein (AFP) using the boric acid-based magnetic beads prepared in Example 2. Specifically, the operation is substantially the same as that in Example 4, except that the boric acid-based magnetic beads used are replaced with the boric acid-based magnetic beads prepared in Example 2.

[0041] Example 6: Chemiluminescent immunoassay detection of alpha-fetoprotein (AFP) using the boric acid-based magnetic beads prepared in Example 3. Specifically, the operation is substantially the same as that in Example 4, except that the boric acid-based magnetic beads used are replaced with the boric acid-based magnetic beads prepared in Example 3.

[0042] Example 7 This example is basically the same as Example 4, except that the sample to be tested is a serum solution of carcinoembryonic antigen CEA.

[0043] Example 8: This example is basically the same as Example 4, except that the sample to be tested is a serum solution of prostate specific antigen PSA.

[0044] It should be noted that the above embodiments only list the detection of certain glycosylated tumor markers, but are not limited to the above types.

[0045] Example 9: Fluorescence detection of AFP using the boric acid-based magnetic beads prepared in Example 1

[0046] This embodiment is the same as the operation of (1)-(2) of embodiment 4, except for the subsequent operation of (3)-(4). The subsequent operation of (3) and (4) of this embodiment is as follows:

[0047] (3) Add 100 μL of 300 ng / mL fluorescein isothiocyanate labeled AFP antibody solution to the above magnetic beads, and shake the mixed solution at 1000 rpm in a constant temperature mixer at 37°C for 15 min; remove the supernatant by magnetic separation, and wash twice with ultrapure water, each time with 500 μL. Finally, disperse the above magnetic beads in 1 mL of ultrapure water to prepare an immunomagnetic bead solution for use.

[0048] (4) Add the immunomagnetic bead solution to a glass dish and place it in the sample slot of a fluorescence detector. Set the excitation wavelength to 490 nm and collect and record the intensity of the emitted light at 520 nm.

[0049] Example 6: Colorimetric analysis of AFP using the boric acid-based magnetic beads prepared in Example 1

[0050] This embodiment is the same as the operation of (1)-(2) of embodiment 4, except for the subsequent operation of (3)-(4). The subsequent operation of (3) and (4) of this embodiment is as follows:

[0051] (3) Add 100 μL of 300 ng / mL horseradish peroxidase-labeled AFP antibody solution to the magnetic beads, and shake the mixed solution at 1000 rpm in a constant temperature mixer at 37°C for 15 min; remove the supernatant by magnetic separation, and wash twice with ultrapure water, each time with 500 μL. Finally, disperse the magnetic beads in 1 mL of ultrapure water to prepare an immunomagnetic bead solution for use.

[0052] (4) Add the immunomagnetic bead solution to a cuvette, and add 50uL of 3,3',5,5'-tetramethylbenzidine colorimetric solution to it, incubate at room temperature and away from light for 30 minutes, and detect AFP qualitatively by color change. Alternatively, directly measure the absorbance at 370nm (or 652nm) for quantitative analysis.

[0053] It should be noted that Example 2 and Examples 5-6 only list the detection strategies under the partial response mechanism of Example 1 for AFP, but are not limited to AFP, nor to the chemiluminescence, fluorescence or colorimetric reaction mechanisms mentioned above.

Claims

1. A magnetic bead for detecting glycosylated tumor markers, characterized in that: It is a boric acid-based magnetic bead, specifically a magnetic microsphere containing a boric acid group on the surface, the particle size of the microsphere is 300-500nm, and the boron content on the surface of the magnetic microsphere is ≥1000mg / g; it is prepared by the following method: By using click chemistry reaction, boric acid molecules are reacted with magnetic microspheres containing epoxy groups on the surface, so that the boric acid groups are fixed on the surface of the microspheres; wherein the boric acid molecules are selected from molecules containing thiol groups and boric acid groups in their structures, and the amount of the boric acid molecules used satisfies the molar ratio of the thiol groups in their structures to the epoxy groups on the surface of the microspheres of (1-2):

1.

2. The magnetic beads for detecting glycosylated tumor markers according to claim 1, characterized in that: The magnetic microspheres containing epoxy groups on the surface are selected from magnetic microspheres modified with glycidyl methacrylate, magnetic microspheres modified with γ-glycidyloxypropyltrimethoxysilane, and carboxyl-polyethylene glycol-epoxy activated agarose magnetic microspheres; the boric acid molecules are selected from 3-mercaptophenylboronic acid, 4-mercaptophenylboronic acid, and phenylboronic acid-polyethylene glycol-mercapto.

3. A method for detecting glycosylated tumor markers using the magnetic beads according to claim 1 or 2, characterized in that: The specific steps are: Step 1, binding of magnetic beads with glycosylated tumor markers: boric acid-based magnetic beads and the sample to be tested are mixed, the amount of boric acid-based magnetic beads is 0.1-1 mg, the amount of sample is 10-60 μL, 50-200 μL of alkaloid buffer is added, and the mixed solution is shaken at 700-1000 rpm in a constant temperature mixer at 25-37° C. for 15-120 min to complete the adsorption between the glycosylated tumor marker and the magnetic beads, and then magnetic separation is performed, and the solution is placed on a magnetic rack until the magnetic beads are completely adsorbed; Step 2, washing the magnetic beads: discard the supernatant obtained in step (1), add PBS buffer and let it stand for 1 to 2 minutes, then magnetically separate and discard the supernatant, add PBS buffer and let it stand for 1 to 2 minutes, then magnetically separate and discard the supernatant to ensure that there are no obvious droplets; Step 3, blocking of magnetic beads: add 200-1000 μL of blocking buffer containing 5-20 wt% bovine serum albumin, vortex mix it, and shake it at 700-1000 rpm in a constant temperature mixer at 25-37° C. for 1-3 h to complete the blocking of bovine serum albumin on the nonspecific adsorption sites on the surface of the magnetic beads; Step 4, washing of magnetic beads: after the blocking experiment is completed, the supernatant of step (3) is discarded, PBS buffer is added and allowed to stand for 1 to 2 minutes, and the supernatant is discarded by magnetic separation again, PBS buffer is added and allowed to stand for 1 to 2 minutes, and the supernatant is discarded by magnetic separation again to ensure that there are no obvious droplets; Step 5, coupling of magnetic beads and antibodies: add 50-200 μL of the antibody solution labeled with the signal molecule, and shake and mix the mixed solution in a constant temperature mixer at 25-37° C. at 700-1000 rpm for 10-30 min to fully couple the magnetic beads with the labeled antibody; Step 6, washing of magnetic beads: add ultrapure water and let stand for 1 to 2 minutes, then magnetically separate and discard the supernatant, add ultrapure water and let stand for 1 to 2 minutes, then magnetically separate and discard the supernatant to ensure that there are no obvious droplets; Step 7, detection: disperse it in 20-100 μL ultrapure water and test it on the corresponding signal molecule detection instrument.

4. The method according to claim 3, characterized in that In step 1: The samples to be tested are serum samples of various glycosylated tumor markers, specifically serum samples of alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, and procalcitonin; The alkaloid is Tris base or Tris-HCl base; The alkaloid buffer is a phosphate buffer containing Tris base or Tris-HCl base, and the phosphate concentration is 0.3mM to 3mM; The pH value of the alkaloid buffer solution ranges from 7.5 to 9.

0.

5. The method according to claim 3, characterized in that: The pH value of the PBS buffer in step 2 and step 4 is 7.5-9.

0.

6. The method according to claim 3, characterized in that In step 5: The signal molecule is selected from acridinium ester, fluorescein isothiocyanate, and horseradish peroxidase; The antibody is a monoclonal antibody corresponding to the target molecule to be detected, and the concentration of the antibody solution is 0.1-2.0 mg / mL, and the pH value ranges from 7.5 to 9.

0.

7. The method according to claim 3, characterized in that The corresponding signal molecule detection instrument in step 7 is selected from a chemiluminescence immunoassay analyzer, a fluorescence analyzer, and an enzyme marker.

Images