A magnetic bead and method for detecting glycosylated tumor marker

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a magnetic bead and a method for detecting glycosylation tumor marker. The magnetic bead for detecting the glycosylation tumor marker is a boronic acid-based magnetic bead, and the magnetic microspheres on the surface of the magnetic bead contain boronic acid groups, the size of the magnetic microspheres is 300-500 nm, and the content of boron elements on the surface of the magnetic microspheres is greater than or equal to 1000 mg / g. Specifically, the boronic acid-based magnetic bead is used as a solid-phase extraction agent, and the method comprises the following steps: the combination of the magnetic bead and the glycosylation tumor marker, the blocking of the magnetic bead, and the coupling of the corresponding antibody of a signal molecule label, and the detection of the magnetic bead on a corresponding signal molecule detection instrument after magnetic separation. The application has the advantages of simple route, rapid and convenient operation, low cost and the like for detecting the glycosylation tumor marker.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a magnetic bead and a method for detecting glycosylated tumor markers. BACKGROUND

[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 can provide higher diagnostic and prognostic value. At present, the magnetic bead-based immunoassay technology is widely used in clinical practice, and its detection process involves enzymatic reaction, chemiluminescence, electrochemiluminescence, fluorescence, etc. The immunoassay strategy usually adopts a double-antibody sandwich method, which requires the coupling of the first antibody on the surface of the magnetic bead, and further coupling of the enzyme-labeled second antibody after specific binding to the antigen to respond to the signal. This detection strategy uses two different specific antibodies to sandwich the target antigen at different epitopes, and has high detection specificity and sensitivity. However, the double-antibody sandwich method requires at least two sites on the antigen that can bind to the antibody, and therefore is not suitable for the detection of small molecule antigens such as hapten with a molecular weight less than 5000. The use of double antibodies often involves multiple coating, incubation, washing and other steps, which not only increases the detection cost, but also limits the operation time. Therefore, it is still a challenge to realize low-cost, high-efficiency and high-sensitivity detection of glycosylated tumor markers in serum.

[0003] The application of boronic acid affinity chromatography has proved that boronic acid molecules have strong affinity for biological macromolecules containing cis-diol structures, and the two can be stably combined in weakly alkaline or neutral aqueous solution. The glycosylated tumor marker domain contains cis-diol groups. These characteristics show that borate functionalized materials can be used for the detection of glycosylated tumor markers. SUMMARY

[0004] The present application aims to provide a magnetic bead and a method for detecting glycosylated tumor markers, which can be used for rapid and efficient, low-cost and universal detection of glycosylated tumor markers, in view of the current situation of high cost and long operation time of existing magnetic bead detection of tumor markers.

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

[0006] The boronic acid molecules are reacted with the magnetic microspheres containing epoxy groups on the surface by using the click chemistry reaction, so that the boronic acid groups are fixed on the surface of the microspheres; wherein the boronic acid molecules are selected from molecules containing thiol and boronic acid groups in the structure, and the amount of the boronic acid molecules satisfies that the molar ratio of thiol in the structure to the epoxy group on the surface of the microspheres is (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, gamma-glycidoxypropyltrimethoxysilane modified magnetic microspheres, and epoxy-polyethylene glycol-carboxyl activated agarose magnetic microspheres. The boronic acid molecules are selected from 3-mercapto phenyl boronic acid, 4-mercapto phenyl boronic acid, and phenyl boronic acid-polyethylene glycol-mercapto, preferably 4-mercapto phenyl boronic acid.

[0008] The boronic acid-based magnetic beads prepared by the above method are used for detecting glycosylated tumor marker, and the specific steps are as follows:

[0009] Step 1, binding of magnetic beads and glycosylated tumor marker: the boronic acid-based magnetic beads and the sample to be detected are mixed, the amount of the boronic acid-based magnetic beads is 0.1-1 mg, the amount of the sample is 10-60 μL, 50-200 μL of alkaline buffer is added, and the mixed solution is shaken in a constant temperature shaker at a speed of 700-1000 rpm for 15-120 min to complete the adsorption between the glycosylated tumor marker and the magnetic beads, and then the magnetic separation is used to stand on the magnetic stand until the adsorption of the magnetic beads is completed.

[0010] Step 2, washing of the magnetic beads: the supernatant obtained in step (1) is sucked and discarded, PBS buffer is added and stood for 1-2 min, then the supernatant is again separated and discarded by magnetic separation, PBS buffer is added and stood for 1-2 min, the supernatant is separated and discarded by magnetic separation, and no obvious droplets are ensured.

[0011] Step 3, blocking of the magnetic beads: 200-1000 μL of blocking buffer containing 5-20 wt% bovine serum albumin is added, and the mixture is vortexed and shaken in a constant temperature shaker at a speed of 700-1000 rpm for 1-3 h to complete the blocking of the non-specific adsorption sites on the surface of the magnetic beads by bovine serum albumin.

[0012] Step 4, washing of the magnetic beads: after the completion of the blocking experiment, the supernatant of step (3) is sucked and discarded, PBS buffer is added and stood for 1-2 min, then the supernatant is again separated and discarded by magnetic separation, PBS buffer is added and stood for 1-2 min, the supernatant is separated and discarded by magnetic separation, and no obvious droplets are ensured.

[0013] Step 5, coupling of magnetic beads and antibodies: 50-200 μL of signal molecule-labeled antibody solution is added, and the mixed solution is mixed at a constant temperature shaker at a speed of 700-1000 rpm for 10-30 min to fully couple the magnetic beads and the labeled antibody;

[0014] Step 6, washing of magnetic beads: after adding ultrapure water and standing for 1-2 min, the supernatant is removed by magnetic separation, ultrapure water is added and stood for 1-2 min, and the supernatant is removed by magnetic separation to ensure that there is no obvious liquid drop;

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

[0016] The sample to be detected in step 1 of the present application preferably comprises serum samples of various glycosylated tumor markers (including alpha-fetoprotein, carcinoembryonic antigen, prostate specific antigen, and calcitonin).

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

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

[0019] The pH value of the alkaloid buffer in step 1 of the present application is 7.5-9.0.

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

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

[0022] The antibody in step 5 of the present application 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 is 7.5-9.0,

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

[0024] Compared with the prior art, the present application has the following advantages:

[0025] (1) The boronic acid-based magnetic beads have strong binding capacity with catechol groups, and have high detection sensitivity for glycosylated tumor markers;

[0026] (2) The application has universality in detection of glycosylated tumor markers, wide application range, and can realize qualitative or quantitative detection of multiple types of glycosylated tumor markers in different response modes (chemiluminescence, fluorescence, color reaction) through antibodies labeled with different signal molecules (such as acridinium ester, fluorescein isothiocyanate, horseradish peroxidase, etc.);

[0027] (3) The application can realize direct action of magnetic beads and target glycosylated tumor markers, and the surface of the magnetic beads does not need to be coupled with antibodies to construct a complex double antibody sandwich structure, so that the process is more simple;

[0028] (4) The use of a second antibody is not required, and the detection cost is lower. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 (a) is a morphology diagram of the γ-glycidyl ether oxypropyl trimethoxysilane modified magnetic microspheres described in Example 1 of the application; Figure 1 (b), (c) and (d) are morphology diagrams of the boronic acid based magnetic beads prepared in Example 1, Example 2 and Example 3 of the application, respectively.

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

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

[0032] The boronic acid group is modified on the surface of the γ-glycidyl ether oxypropyl trimethoxysilane modified magnetic microspheres by using the click chemistry reaction of thiol and epoxy to prepare the boronic acid based magnetic beads. The specific method is as follows: 100 mg of 4-mercaptophenylboronic acid is weighed in a three-necked flask, 50 mL of isopropyl alcohol is added, and ultrasonic is used for 2 min to make it fully dissolved, then 50 mg of epoxy magnetic microspheres is added, and ultrasonic is used to disperse the magnetic microspheres uniformly, then the flask is placed in a mechanical stirring device, 10 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene is added to the mixed solution under stirring, and the reaction is continued to stir at room temperature for 24 h, after the reaction is completed, the product is separated by a magnet, and is washed with ethanol and water for multiple times, finally the product is dispersed in a certain amount of deionized water, and is freeze-dried for standby. The morphology of the used epoxy magnetic microspheres and the prepared boronic acid based magnetic beads after the reaction are shown in Figure 1 (a) and (b), respectively.

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

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

[0035] Example 4: Chemiluminescence immunoassay detection of alpha-fetoprotein (AFP) using the boronic acid group magnetic beads prepared in Example 1

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

[0037] (2) The boronic acid group magnetic beads after washing and removing the supernatant were dispersed in 500 μL of blocking buffer (10 wt% BSA aqueous solution), vortexed for 3 min, and then placed in a constant temperature shaker at 37°C, shaken at 1000 rpm for 3 h, the product was collected by magnetic separation, and washed twice with 500 μL of PBS buffer with pH of 7.5.

[0038] (3) 100 μL of acridinium ester-labeled AFP antibody solution with a concentration of 300 ng / mL was added to the above magnetic beads, and the mixed solution was shaken in a constant temperature shaker at 37°C at a speed of 1000 rpm for 15 min; the supernatant was removed by magnetic separation, and washed twice with 500 μL of ultrapure water. Finally, the above magnetic beads were dispersed in 1 mL of ultrapure water to prepare an immunomagnetic bead solution for use.

[0039] (4) The immunomagnetic bead solution was added to the sampling groove of the chemiluminescence immunoassay instrument, and pre-activation liquid (sodium hypochlorite solution) and activation liquid (hydrogen peroxide solution) were added to sampling grooves A and B, respectively. The sampling capacities of the sample, pre-activation liquid, and activation liquid were set to 15 μL / time, 30 μL / time, and 30 μL / time, respectively. In the instrument parameter interface, the incubation time for substrate activation was set to 10 min, the sample was tested in parallel for 3 times, and finally the chemiluminescence immunoassay instrument was started for detection. Figure 2The luminescence intensity detection results of AFP concentration of 0 ng / mL and 300 ng / mL serum samples when using different concentrations of boric acid-based microspheres for sample injection. As can be seen from the figure, when the sample does not contain AFP, no luminescence value is detected under different concentrations of microsphere dosage. In the 300 ng / mL AFP-containing serum sample, with the increase of the concentration of boric acid-based microspheres, that is, under the same magnetic bead injection volume, the number of injected microspheres increases, and the chemiluminescence signal gradually increases, indicating the feasibility of its detection in actual samples.

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

[0041] Example 6: Chemiluminescence immunoassay detection of alpha-fetoprotein (AFP) using boric acid-based magnetic beads prepared in Example 3. Specifically, the operation is basically the same as that of Example 4, except that the boric acid-based magnetic beads used are replaced by 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 detected 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 detected is a serum solution of prostate specific antigen PSA.

[0044] It should be noted that the above examples 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 boric acid-based magnetic beads prepared in Example 1

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

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

[0048] (4) The immunomagnetic bead solution was added to a glass dish and placed in the sample slot of the fluorescence detector. The excitation wavelength was set to 490 nm, and the intensity of the emitted light at 520 nm was collected and recorded.

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

[0050] This example was the same as the operations of (1)-(2) of Example 4, except for the subsequent operations of (3)-(4). The subsequent operations of (3) and (4) of this example are described as follows:

[0051] (3) 100 μL of horseradish peroxidase-labeled AFP antibody solution with a concentration of 300 ng / mL was added to the above magnetic beads, and the mixed solution was coupled in a constant-temperature shaker at 37°C at a speed of 1000 rpm for 15 min. The supernatant was removed by magnetic separation, and the magnetic beads were washed twice with ultrapure water, each time with a volume of 500 μL. Finally, the magnetic beads were dispersed in 1 mL of ultrapure water to prepare an immunomagnetic bead solution for use.

[0052] (4) The immunomagnetic bead solution was added to a colorimetric dish, and 50 uL of 3,3',5,5'-tetramethylbenzidine color developing solution was added thereto. The mixture was incubated at room temperature for 30 min in the dark, and qualitative detection of AFP was achieved by color change. Alternatively, the absorbance was directly measured at 370 nm (or 652 nm) for quantitative analysis.

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

Claims

1. A magnetic bead for glycosylation of a tumor marker for detection, characterized in that, The application relates to a boronic acid-based magnetic bead, in particular to a magnetic microsphere containing a boronic acid group on the surface, wherein the particle size of the microsphere is 300-500 nm, and the content of boron on the surface of the magnetic microsphere is greater than or equal to 1000 mg / g; and the magnetic microsphere is prepared by the following method. The boronic acid molecule is reacted with the magnetic microsphere containing an epoxy group on the surface by using a click chemistry reaction, so that the boronic acid group is fixed on the surface of the microsphere; wherein the boronic acid molecule is selected from a molecule containing a mercapto group and a boronic acid group in the structure, and the amount of the boronic acid molecule satisfies that the molar ratio of the mercapto group in the structure to the epoxy group on the surface of the microsphere is (1-2):

1. The magnetic microsphere containing an epoxy group on the surface is selected from a glycidyl methacrylate modified magnetic microsphere, a gamma-glycidoxypropyltrimethoxysilane modified magnetic microsphere and a carboxyl-polyethylene glycol-epoxy activated agarose magnetic microsphere; and the boronic acid molecule is selected from 3-mercapto phenyl boronic acid, 4-mercapto phenyl boronic acid and phenyl boronic acid-polyethylene glycol-mercapto.

2. The method for detecting glycosylated tumor marker using the magnetic beads according to claim 1, characterized in that, The specific steps are as follows: Step 1, binding of the magnetic bead and a glycosylated tumor marker: the boronic acid-based magnetic bead and a sample to be detected are mixed, the amount of the boronic acid-based magnetic bead is 0.1-1 mg, the amount of the sample is 10-60 muL, 50-200 muL of a biological alkali buffer is added, the mixed solution is shaken in a constant-temperature mixing instrument at a speed of 700-1000 rpm for 15-120 min to complete the adsorption between the glycosylated tumor marker and the magnetic bead, and then the magnetic separation is adopted, and the magnetic bead is completely adsorbed after being placed on a magnetic frame. Step 2, washing of the magnetic bead: the supernatant obtained in step (1) is sucked and discarded, PBS buffer is added and placed for 1-2 min, then the supernatant is again separated and discarded by using the magnetic separation, PBS buffer is added and placed for 1-2 min, the supernatant is separated and discarded by using the magnetic separation, and no obvious liquid drops are ensured; Step 3, blocking of the magnetic bead: 200-1000 muL of a blocking buffer containing 5-20 wt% of bovine serum albumin is added, the solution is vortexed and mixed, and the mixed solution is shaken in a constant-temperature mixing instrument at a speed of 700-1000 rpm for 1-3 h to complete the blocking of the non-specific adsorption sites on the surface of the magnetic bead by the bovine serum albumin; Step 4, washing of the magnetic bead: after the blocking experiment is completed, the supernatant of step (3) is sucked and discarded, PBS buffer is added and placed for 1-2 min, then the supernatant is again separated and discarded by using the magnetic separation, PBS buffer is added and placed for 1-2 min, the supernatant is separated and discarded by using the magnetic separation, and no obvious liquid drops are ensured; Step 5, coupling of the magnetic bead and an antibody: 50-200 muL of an antibody solution labeled with a signal molecule is added, the mixed solution is shaken and mixed in a constant-temperature mixing instrument at a speed of 700-1000 rpm for 10-30 min to fully couple the magnetic bead and the labeled antibody; Step 6, washing of the magnetic bead: ultrapure water is added and placed for 1-2 min, then the supernatant is again separated and discarded by using the magnetic separation, ultrapure water is added and placed for 1-2 min, the supernatant is separated and discarded by using the magnetic separation, and no obvious liquid drops are ensured; Step 7, detection: the magnetic bead is dispersed in 20-100 muL of ultrapure water, and the magnetic bead is tested on a corresponding signal molecule detection instrument.

3. The method of claim 2, wherein, In step 1, The sample to be detected is a serum sample of various glycosylated tumor markers, specifically a serum sample of alpha-fetal protein, 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 0.3 mM to 3 mM. The pH value of the alkaloid buffer is 7.5 to 9.

0.

4. The method of claim 2, wherein, The pH value of the PBS buffer in steps 2 and 4 is 7.5 to 9.

0.

5. The method of claim 2, wherein, In step 5: The signal molecule is selected from acridinum 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 to 2.0 mg / mL, and the pH value is 7.5 to 9.

0.

6. The method of claim 2, wherein, The corresponding signal molecule detection instrument in step 7 is selected from a chemiluminescence immunoassay instrument, a fluorescence instrument and an enzyme label instrument.

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