Method for covalently modifying magnetic nanoparticles by metal ion-induced DNA (Deoxyribose Nucleic Acid) and application of magnetic nanoparticles in quantitative detection of ochratoxin A

Abstract

The invention discloses a method for covalently modifying magnetic nanoparticles by metal ion-induced DNA (Deoxyribose Nucleic Acid) and application of the magnetic nanoparticles in quantitative detection of ochratoxin A, and belongs to the technical field of fluorescent biosensing analysis. The method for covalently modifying the magnetic nanoparticles by metal ion-induced DNA provided by the invention comprises the following steps: adsorbing DNA molecules on the surfaces of polydopamine-coated magnetic nanoparticles under the induction of metal ions, and carrying out covalent linkage to obtain the DNA molecule-modified magnetic nanoparticles. In the method provided by the invention, the electrostatic repulsion between the DNA molecules and the surface of the polydopamine is overcome through the induction effect of the metal ions, and the modification amount of the DNA molecules on the surfaces of the magnetic nanoparticles is greatly increased. The method is simple in steps, and the obtained DNA molecule modified magnetic nanoparticles have very high sequence selectivity and capture capability on uncombined OTA aptamers, and are beneficial to improving the sensitivity and accuracy of OTA detection.

Description

Technical Field

[0001] The present application belongs to the technical field of fluorescent biosensor analysis, and in particular relates to a method for metal ion-induced DNA covalent modification of magnetic nanoparticles and its application in quantitative detection of ochratoxin A. Background Art

[0002] Ochratoxin A (OTA) is a common fungal toxin that is widely found in foods such as cereals, dried fruits, coffee, wine, and beer. It is nephrotoxic, hepatotoxic, and carcinogenic to humans and animals, and has been identified as a possible human carcinogen by the International Agency for Research on Cancer. The main detection methods for OTA include chromatography and enzyme-linked immunosorbent assay (ELISA). Chromatography mainly includes liquid chromatography and liquid chromatography-mass spectrometry, which require expensive instruments and complex sample pretreatment. The ELISA method uses the specific interaction between antigen and antibody to improve the sensitivity of detection by amplifying the enzymatic reaction signal. However, the use of antibodies has defects such as poor reproducibility and short service life.

[0003] Nucleic acid aptamers are single-stranded oligonucleotide sequences obtained by in vitro SELEX technology screening. They have high specificity and affinity for the target and are called artificial antibodies. Compared with antibodies, aptamers have the advantages of easy synthesis and modification, low cost, small batch differences and good stability. In 2008, Cruz-Aguado et al. (J. Agric. Food Chem, 2008, 56, 10456) screened out a 36-base OTA aptamer. This aptamer showed excellent binding ability and specificity to OTA and is one of the most studied and used aptamers. At present, a variety of OTA detection methods have been developed based on this aptamer, including colorimetry, fluorescence, electrochemistry, Raman, etc. Among them, the fluorescence method is widely used due to its simple operation.

[0004] Dopamine is an important neurotransmitter. In a weakly alkaline environment, it can spontaneously polymerize to form polydopamine, which is deposited on the surface of magnetic nanoparticles to obtain polydopamine-coated magnetic nanoparticles. The polydopamine coating can be covalently linked to molecules containing amino or sulfhydryl groups, such as DNA molecules, through Michael addition or Schiffer base reaction. However, since both DNA molecules and polydopamine are negatively charged under weakly alkaline reaction conditions, electrostatic repulsion makes it difficult for DNA molecules to approach the polydopamine surface to react, so the reaction efficiency is generally not high. At present, there are few reports on how to induce DNA molecules to efficiently modify polydopamine-coated magnetic particles and how to apply them to the fluorescent quantitative detection of OTA content. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a method for metal ion-induced DNA covalent modification of magnetic nanoparticles and its application in the quantitative detection of ochratoxin A.

[0006] In order to achieve the above-mentioned invention object, this application provides the following technical solutions:

[0007] In one aspect, the present application provides a method for metal ion-induced DNA covalent modification of magnetic nanoparticles, comprising the following steps:

[0008] DNA molecules are adsorbed on the surface of magnetic nanoparticles under the induction of metal ions and covalently linked to obtain magnetic nanoparticles modified with DNA molecules.

[0009] Optionally, the metal ions include Mg 2+ , Ca 2+ and Zn 2+ At least one of .

[0010] Optionally, the molar concentration of the metal ions is 2-20 mM.

[0011] Optionally, the molar concentration of the metal ion is independently selected from any value among 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, or any range therebetween.

[0012] Optionally, the DNA molecule comprises a complementary sequence to an OTA aptamer.

[0013] Optionally, the nucleotide sequence of the OTA aptamer is shown in SEQ ID NO.1.

[0014] Optionally, the OTA aptamer is labeled with a fluorophore or a luminescent nanomaterial.

[0015] Optionally, the fluorophore comprises FAM, Alexa Fluor 488, Cy3, Cy5 or TAMRA.

[0016] Optionally, the luminescent nanomaterial includes quantum dots or carbon dots.

[0017] Optionally, the complementary sequence of the OTA aptamer has a length of 10 to 36 bases.

[0018] Preferably, the complementary sequence of the OTA aptamer is shown as SEQ ID NO.2.

[0019] Optionally, one end of the complementary sequence of the OTA aptamer is modified with an amino group or a thiol group.

[0020] Optionally, the magnetic nanoparticles include Fe3O4 magnetic nanoparticles.

[0021] Optionally, the surface of the Fe3O4 magnetic nanoparticles is coated with polydopamine.

[0022] Optionally, the coating thickness of the polydopamine is 20 to 40 nm.

[0023] Optionally, the coating thickness of the polydopamine is independently selected from any value among 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, or any range value therebetween.

[0024] Optionally, the covalent attachment is performed via a Michael addition reaction or a Schiff base reaction.

[0025] Optionally, the reaction time of the covalent bonding is 2 to 24 hours.

[0026] Optionally, the reaction time of the covalent linkage is independently selected from any value among 2h, 5h, 10h, 15h, 20h, 24h, or any range between two of them.

[0027] In a second aspect, the present application provides the application of the above-mentioned method of metal ion-induced DNA covalent modification of magnetic nanoparticles in the quantitative detection of ochratoxin A.

[0028] In a third aspect, the present application provides a method for quantitatively detecting ochratoxin A based on fluorescently labeled aptamers and magnetic nanoparticles modified with DNA molecules, comprising the following steps:

[0029] (1) mixing the OTA aptamer with the sample to be tested, using the OTA aptamer to specifically bind to OTA in the sample to be tested, adding and using the DNA molecule-modified magnetic nanoparticles prepared by the above method to recognize and capture the unbound OTA aptamer, and then removing the unbound OTA aptamer by magnetic separation;

[0030] (2) The content of ochratoxin A in the sample to be tested is obtained by measuring the fluorescence spectrum intensity of the system in which the unbound OTA aptamer is removed in step (1).

[0031] Optionally, the nucleotide sequence of the OTA aptamer in step (1) is as shown in SEQ ID NO.1.

[0032] Optionally, the OTA aptamer in step (1) is labeled with a fluorophore or a luminescent nanomaterial.

[0033] Optionally, the fluorophore comprises FAM, Alexa Fluor 488, Cy3, Cy5 or TAMRA.

[0034] Optionally, the luminescent nanomaterial includes quantum dots or carbon dots.

[0035] Optionally, the DNA molecule in step (1) is a complementary sequence of the OTA aptamer.

[0036] Optionally, the length of the DNA molecule in step (1) is 10 to 36 bases.

[0037] Preferably, the nucleotide sequence of the DNA molecule in step (1) is as shown in SEQ ID NO.2.

[0038] Optionally, one end of the DNA molecule in step (1) is modified with an amino group or a thiol group.

[0039] Optionally, the magnetic nanoparticles in step (1) include Fe3O4 magnetic nanoparticles.

[0040] Optionally, the surface of the Fe3O4 magnetic nanoparticles is coated with polydopamine.

[0041] Optionally, the coating thickness of the polydopamine is 20 to 40 nm.

[0042] Optionally, the coating thickness of the polydopamine is independently selected from any value among 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, or any range value therebetween.

[0043] Optionally, in step (1), the dosage ratio of the sample to be tested, the OTA aptamer, and the magnetic nanoparticles modified with DNA molecules is 50-100 μL: 0.01-0.05 nmol: 0.05-0.1 mg.

[0044] Optionally, the magnetic separation in step (1) is performed using a magnet.

[0045] Optionally, during the fluorescence spectrum intensity measurement process in step (2), the excitation wavelength is 450-500 nm, the emission wavelength is 500-600 nm, and the scanning speed is 1000-1500 nm / min.

[0046] Preferably, in the fluorescence spectrum intensity measurement process in step (2), the excitation wavelength is 480 nm, the emission wavelength is 520 nm, and the scanning speed is 1200 nm / min.

[0047] Compared with the prior art, this application has the following beneficial effects:

[0048] (1) In the present invention, a DNA molecule labeled with amino or thiol is placed in the presence of metal ions such as Mg 2+The DNA molecule is covalently linked to the surface of polydopamine-coated ferroferric oxide magnetic nanoparticles under induction, and the DNA molecule is the complementary sequence of the ochratoxin A (OTA) aptamer. Then, the magnetic nanoparticles modified with complementary DNA molecules are used for quantitative analysis of OTA. The fluorescently labeled aptamer is first mixed with the sample to be tested, a part of the aptamer binds to the OTA in the sample, and the unbound aptamer is captured by the complementary DNA molecule connected to the magnetic particles, and the unbound aptamer is separated from the bound OTA aptamer by magnetic separation. The higher the content of OTA in the sample to be tested, the more bound aptamers are retained in the solution after magnetic separation, and the stronger the fluorescence. Based on this, quantitative sensing analysis of OTA can be achieved.

[0049] (2) In the method provided in the present application, metal ions such as Mg 2+ The induction effect overcomes the electrostatic repulsion between DNA molecules and the surface of polydopamine, greatly increasing the reaction probability of DNA molecules and polydopamine, thereby increasing the amount of DNA molecules modified on the surface of Fe3O4@PDA particles. After the reaction, the DNA that is not covalently linked is modified by EDTA and Mg 2+ The method is simple, and the obtained Fe3O4@PDA@cDNA magnetic particles have high sequence selectivity and capture ability for unbound OTA aptamers. With the help of magnetic separation, bound OTA aptamers can be easily separated from unbound OTA aptamers, which helps to improve the sensitivity and accuracy of OTA detection. In addition, by replacing the aptamer and its corresponding complementary DNA, this method can be easily extended to the detection of other targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a TEM electron microscope image of ferroferric oxide (Fe3O4) magnetic nanoparticles in Example 1 of the present application;

[0051] Figure 2 This is a TEM electron microscope image of the polydopamine-coated ferroferric oxide (Fe3O4@PDA) magnetic nanoparticles in Example 1 of the present application;

[0052] Figure 3 Mg under different reaction conditions in Example 1 of the present application 2+ The amount of covalent modification of the induced complementary DNA molecules on the surface of Fe3O4@PDA particles (Note: A represents the amount of covalent modification when the amount of complementary DNA molecules added is 1-5 nmol, other reaction conditions: 1 mg of Fe3O4@PDA particles, 1 mL of Tris-HCl buffer (10 mM, pH 8.5, 150 mM NaCl, 10 mMMgCl2), reaction time 10 h; B represents Mg 2+The amount of covalent modification of complementary DNA molecules at a concentration of 0-20 mM. Other reaction conditions: 2 nmol of cDNA-NH2, 1 mg of Fe3O4@PDA particles, 1 mL of Tris-HCl buffer (10 mM, pH 8.5, 150 mM NaCl, 0-20 mM MgCl2) in the reaction solution, and 10 h of reaction time; C represents the calibration curve for quantifying cDNA-NH2 using Super Gold nucleic acid dye).

[0053] Figure 4 The capture capacity of Fe3O4@PDA@cDNA for OTA aptamer (i.e. Apt-F) obtained under different reaction conditions in Example 1 of the present application (Note: A represents the amount of cDNA-NH2 added of 1 to 5 nmol; B represents Mg 2+ concentration 0-20 mM, hybridization conditions: Apt-F 50 nM, Fe3O4@PDA@cDNA particles 0.1 mg / mL, hybridization buffer 10 mM Tris-HCl, pH 8.5, containing 1 M NaCl and 0.02% Tween 20, incubation time 20 min);

[0054] Figure 5 The fluorescence spectra of the supernatant at different OTA concentrations in Example 1 of the present application and the OTA calibration curve calculated therefrom (Note: A represents the fluorescence spectra of the supernatant after magnetic separation at different OTA concentrations; B represents the hybridization percentage of Apt-F and Fe3O4@PDA@cDNA magnetic particles at each OTA concentration calculated from the fluorescence spectra in A, and the hybridization percentage is linear in the OTA concentration range of 10 to 100 ng / mL (R 2 =0.996)). DETAILED DESCRIPTION

[0055] The present application is further described below in conjunction with specific embodiments. The following are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application discloses the following preferred embodiments, they are not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

[0056] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0057] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0058] Example 1

[0059] Mg 2+ Induced DNA covalently modified polydopamine coated magnetic nanoparticles for quantitative analysis of ochratoxin A:

[0060] (1) Preparation of Fe3O4@PDA magnetic nanoparticles coated with polydopamine:

[0061] Preparation of magnetic Fe3O4 nanoparticles: Dissolve ferric chloride hexahydrate (1.08 g), trisodium citrate dihydrate (0.40 g) and anhydrous sodium acetate (1.20 g) in 20 mL of ethylene glycol under vigorous stirring. Transfer the reaction mixture to a stainless steel reactor lined with polytetrafluoroethylene and heat at 200°C for 10 hours. After cooling to room temperature, the obtained Fe3O4 magnetic nanoparticles are washed several times with ethanol and water, respectively, and dried in vacuum. The TEM electron microscope image of Fe3O4 is shown in the figure below. Figure 1 As shown, the average particle size is about 122 nm.

[0062] Polydopamine coating: 3 mg Fe3O4 was ultrasonically dispersed in 24 mL Tris-HCl buffer (10 mM, pH 8.5), 7 mg dopamine hydrochloride was added, and the mixture was shaken for 5 h to obtain Fe3O4@PDA nanoparticles. Washed with water several times, the mixture was redispersed in 3 mL Tris-HCl (10 mM, pH 8.5) to obtain Fe3O4@PDA stock solution (recorded as 1 mg / mL). The TEM electron microscope image of Fe3O4@PDA is shown in the figure below. Figure 2 As shown, the thickness of the polydopamine coating is about 30 nm.

[0063] (2)Mg 2+ Inducing covalent modification of complementary DNA molecules:

[0064] Take 1 mL of Fe3O4@PDA stock solution, and redisperse it in 1 mL of Mg 2+ 1-5 nmol of amino-modified complementary DNA molecules (nucleotide sequence as shown in SEQ ID NO.2: (5'-3') NH2-C6-TGT CCG ATG CTC CCT) were added to a buffer (10 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0-20 mM MgCl2) and shaken for 10 h at room temperature. The magnetic particles were washed three times with EDTA solution to remove the non-covalently linked DNA molecules to obtain magnetic particles modified with complementary DNA molecules (Fe3O4@PDA@cDNA). The Fe3O4@PDA@cDNA particles were dispersed in 1 mL Tris-HCl buffer (10 mM, pH 8.5) (recorded as 1 mg / mL) and stored at 4°C for use.

[0065] (3) Measurement of the amount of modification of complementary DNA molecules on Fe3O4@PDA particles under different reaction conditions:

[0066] The supernatant and EDTA washing solution of the above DNA modification reaction were collected, and the remaining unreacted DNA molecules in the supernatant and the DNA molecules washed away by the EDTA solution were quantified using Super Gold nucleic acid dye to calculate the modification amount of the DNA molecules under each reaction condition.

[0067] The amount of modified DNA molecules = the amount added - the remaining DNA molecules in the reaction supernatant - the DNA molecules washed away by EDTA solution

[0068] The results are as follows Figure 3 shown.

[0069] Figure 3 A in is the amount of covalent modification of DNA molecules on Fe3O4@PDA particles calculated when 1-5 nmol complementary DNA is added. Figure 3 As can be seen from A in the figure, the more complementary DNA molecules are added during the reaction, the higher the concentration of complementary DNA molecules in the reaction solution, and the more complementary DNA molecules are covalently linked to the Fe3O4@PDA particles.

[0070] Figure 3 B in 0~20mM Mg 2+ The amount of modification of complementary DNA molecules under the conditions. Figure 3 As can be seen from B, Mg 2 + It can indeed increase the amount of modification of DNA molecules. 2+ The amount of DNA modification increased significantly after Mg 2+ As the concentration increases, the amount of modification on the DNA molecules gradually increases.

[0071] Figure 3 C in the figure is the calibration curve for quantifying complementary DNA using Super Gold nucleic acid dye.

[0072] (4) Comparison of the ability of Fe3O4@PDA@cDNA obtained under different reaction conditions to capture OTA aptamer (i.e. Apt-F):

[0073] In 400 μL hybridization buffer (10 mM Tris-HCl, pH 8.5, 1 M NaCl, 0.02% Tween 20), 50 μL Fe3O4@PDA@cDNA (final concentration 0.1 mg / mL) and 50 μL FAM-labeled OTA aptamer (Apt-F, final concentration 50 nM, nucleotide sequence as shown in SEQ ID NO.1: (5'-3') GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGGACA-FAM) were added and mixed and incubated for 20 min. After magnetic separation using a magnet (50×20×15 mm), the fluorescence of unhybridized Apt-F in the solution was measured, and the hybridization percentage of Apt-F and Fe3O4@PDA@cDNA obtained under different reaction conditions was calculated.

[0074] Hybridization percentage = (fluorescence intensity of 50 nM Apt-F at 520 nm - fluorescence intensity of unhybridized Apt-F at 520 nm in the supernatant after incubation with Fe3O4@PDA@cDNA) / fluorescence intensity of 50 nM Apt-F at 520 nm * 100%

[0075] The results are as follows Figure 4 shown.

[0076] Figure 4 A and Figure 4 B in (2) is respectively 1-5 nmol complementary DNA molecules and 0-20 mM Mg 2+ The hybridization percentage of Fe3O4@PDA@cDNA particles obtained under the same conditions and 50nM Apt-F. It can be seen that the ability of Fe3O4@PDA@cDNA to capture Apt-F is positively correlated with the modification amount of complementary DNA molecules. The more modified complementary DNA molecules, the stronger the ability to capture Apt-F.

[0077] (5) OTA quantitative analysis:

[0078] In 300 μL binding buffer (10 mM Tris-HCl, pH 8.5, 1 M NaCl, 6 mM CaCl2, 0.02% Tween 20), add 50 μL Apt-F (final concentration 50 nM) and 75 μL OTA of different concentrations. After incubation for 5 minutes, add 75 μL Fe3O4@PDA@cDNA (final concentration 0.15 mg / mL) and incubate for another 20 minutes. After magnetic separation, measure the fluorescence spectrum of the supernatant.

[0079] The results are as follows Figure 5 shown.

[0080] Figure 5A in the figure is the fluorescence spectrum of the supernatant measured at different OTA concentrations. It can be seen that the higher the OTA concentration, the stronger the fluorescence of the supernatant. This is because the higher the OTA concentration, the more aptamers bind to OTA, and these aptamers that bind to OTA cannot be captured by Fe3O4@PDA@cDNA and remain in the solution, resulting in stronger fluorescence of the supernatant.

[0081] Figure 5 B in the figure is the calculated relationship curve between the hybridization percentage of Apt-F and Fe3O4@PDA@cDNA and the OTA concentration, which is linear in the range of OTA concentration of 10-100 ng / mL (R 2 =0.996). The detection limit (3S / N) of this method was 2.77 ng / mL.

[0082] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for metal ion-induced DNA covalent modification of magnetic nanoparticles, characterized in that: The steps include: DNA molecules are adsorbed on the surface of magnetic nanoparticles under the induction of metal ions and covalently linked to obtain magnetic nanoparticles modified with DNA molecules.

2. The method for metal ion-induced DNA covalent modification of magnetic nanoparticles according to claim 1, characterized in that: The metal ions include Mg 2+ , Ca 2+ and Zn 2+ At least one of; Preferably, the molar concentration of the metal ions is 2-20 mM.

3. The method for metal ion-induced DNA covalent modification of magnetic nanoparticles according to claim 1, characterized in that: The DNA molecule includes a complementary sequence of the OTA aptamer; Preferably, the nucleotide sequence of the OTA aptamer is shown in SEQ ID NO.1; Preferably, the OTA aptamer is labeled with a fluorophore or a luminescent nanomaterial; Preferably, the complementary sequence of the OTA aptamer is 10 to 36 bases long; Preferably, one end of the complementary sequence of the OTA aptamer is modified with an amino group or a thiol group.

4. The method for metal ion-induced DNA covalent modification of magnetic nanoparticles according to claim 1, characterized in that: The magnetic nanoparticles include Fe3O4 magnetic nanoparticles; Preferably, the surface of the Fe3O4 magnetic nanoparticles is coated with polydopamine; Preferably, the coating thickness of the polydopamine is 20 to 40 nm.

5. The method for metal ion-induced DNA covalent modification of magnetic nanoparticles according to claim 1, characterized in that: The covalent bonding is carried out by Michael addition reaction or Schiffer base reaction; Preferably, the reaction time of the covalent bonding is 2 to 24 hours.

6. Use of the method for metal ion-induced DNA covalent modification of magnetic nanoparticles according to any one of claims 1 to 5 in the quantitative detection of ochratoxin A.

7. A method for quantitatively detecting ochratoxin A based on fluorescently labeled aptamers and magnetic nanoparticles modified with DNA molecules, characterized in that: The steps include: (1) mixing an OTA aptamer with a sample to be tested, using the OTA aptamer to specifically bind to OTA in the sample to be tested, adding and using the DNA molecule-modified magnetic nanoparticles prepared by the method according to any one of claims 1 to 5 to recognize and capture unbound OTA aptamers, and then removing the unbound OTA aptamers by magnetic separation; (2) The content of ochratoxin A in the sample to be tested is obtained by measuring the fluorescence spectrum intensity of the system in which the unbound OTA aptamer is removed in step (1).

8. The method for quantitatively detecting ochratoxin A based on fluorescently labeled aptamers and magnetic nanoparticles modified with DNA molecules according to claim 7, characterized in that: The nucleotide sequence of the OTA aptamer in step (1) is shown in SEQ ID NO.1; Preferably, the OTA aptamer in step (1) is labeled with a fluorophore or a luminescent nanomaterial; Preferably, the DNA molecule in step (1) is a complementary sequence of the OTA aptamer; Preferably, the length of the DNA molecule in step (1) is 10 to 36 bases; Preferably, one end of the DNA molecule in step (1) is modified with an amino group or a thiol group.

9. The method for quantitatively detecting ochratoxin A based on fluorescently labeled aptamers and magnetic nanoparticles modified with DNA molecules according to claim 7, characterized in that: The magnetic nanoparticles in step (1) include Fe3O4 magnetic nanoparticles; Preferably, the surface of the Fe3O4 magnetic nanoparticles is coated with polydopamine; Preferably, the coating thickness of the polydopamine is 20 to 40 nm.

10. The method for quantitatively detecting ochratoxin A based on fluorescently labeled aptamers and magnetic nanoparticles modified with DNA molecules according to claim 7, characterized in that: The dosage ratio of the sample to be tested, OTA aptamer, and DNA molecule-modified magnetic nanoparticles in step (1) is 50-100 μL: 0.01-0.05 nmol: 0.05-0.1 mg; Preferably, the magnetic separation in step (1) is performed using a magnet; Preferably, in the fluorescence spectrum intensity measurement process in step (2), the excitation wavelength is 450-500 nm, the emission wavelength is 500-600 nm, and the scanning speed is 1000-1500 nm / min.

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