[0028] Example 1
[0029] 1. Preparation and characterization of the secondary antibody probe
[0030] (1) Preparation of magnetic nanoparticles
[0031] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.
[0032] Characterization of magnetic nanoparticles: X-ray fluorescence spectroscopy (XRF) was used to analyze Fe 3 O 4 /ZrO 2 For characterization, Zr-K appeared β (17.8keV), Zr-K α (15.8keV), Zr-L β (2.1ke), Zr-L α (2.0keV) peak and Fe-K β (7.1KeV), Fe-K α (6.4keV) peak, indicating the presence of Zr and Fe elements in the magnetic particles.
[0033] (2) Preparation and characterization of antibody-loaded nanospheres
[0034] Preparation of antibody-loaded nanospheres: Disperse 10 mg of magnetic nanoparticles in 5 mL pH 7.0 phosphate buffer, add 1 mg horseradish peroxide-labeled alpha-fetoprotein secondary antibody (HRP-anti-AFP), and stir for 6 hours , Magnetic separation under external magnetic conditions to obtain antibody-loaded nanospheres.
[0035] (3) Preparation of nanospheres
[0036] Disperse 10 mg of antibody-loaded nanospheres in 5 mL of pH 7.0 phosphate buffer, add 5 mg horseradish peroxidase and 10 mg bovine serum albumin (BSA) in sequence, stir at 4°C for 6 hours, and separate the magnetic nanospheres using an external magnetic field The particles are washed to obtain nanospheres.
[0037] (4) Preparation of secondary antibody probe
[0038] Disperse 10 mg of nanospheres in 5 mL of pH 7.0 phosphate buffer, add 12 mg of calf thymus DNA, stir at room temperature for 6 hours, and magnetically separate to obtain the secondary antibody probe.
[0039] Characterization of the secondary antibody probe: The preparation process of the probe was characterized by transmission electron microscopy ( image 3 ). by image 3 It can be seen that the secondary antibody probe has a one-dimensional "bead-like" linear structure.
[0040] Using X-ray fluorescence spectroscopy (the range of measuring elements is 9 F~ 92 U) The secondary antibody probe was characterized, showing that the Zr-kα peak is 2.1keV, Fe-kα peak is 6.4keV, P-kα peak is 1.13keV, and S-kα peak is 2.3keV. DNA contains a large number of phosphate groups. The appearance of the P peak further illustrates that the surface of the magnetic nanoparticles is coated with DNA.
[0041] The UV spectrum after the interaction of the magnetic nanoparticles and DNA was measured, and it was found that the characteristic absorption peak of DNA was red-shifted from 520nm to 524nm, indicating that the DNA and the magnetic nanoparticles had interacted and the absorption peak was red-shifted. by figure 2 It can be seen that the magnetic probe of the secondary probe has good superparamagnetism under the action of an external magnetic field of 0.3mTde.
[0042] 2. Preparation of Amperometric Immunosensor
[0043] Modification of commercial screen-printed electrodes:
[0044] (a) Graphene/chitosan (GS/CS) is modified into a film on the surface of the screen printed electrode
[0045] Add 2 mL of 0.2% chitosan (CS) acetic acid solution with pH 5.0 and 1 mg of GS to a 5 mL centrifuge tube, and mix to obtain a suspension; drop 5 mL of the suspension on the surface of the working electrode in the screen-printed electrode. Dry naturally at room temperature, and then wash with pH 7.0 phosphate buffer solution to remove loosely bound nanoparticles.
[0046] (b) Using the current-time curve method to directly electroplate gold on the working electrode surface of the screen-printed electrode: add 20 μL of 1% HAuCl 4 Drop on the surface of the working electrode, and apply a constant potential of -0.2V for 30S. Wash thoroughly with PBS after plating.
[0047] (c) After adding 10 μL of AFP monoclonal primary antibody to the reaction chamber of the screen-printed electrode, the screen-printed electrode is placed in a 4°C refrigerator and incubated at a low temperature for 12 hours, and then the unbound and weakly bound antibodies are fully washed with PBS.
[0048] (d) Block the unbound active sites on the surface of the unbound electrode with 3 mg/mL bovine serum albumin (BSA) to obtain an amperometric immunosensor.
[0049] 3. Detection of antigen concentration
[0050] The antigen detection process is to use the sensor with the immobilized primary antibody to sequentially incubate with the sample and the prepared secondary antibody probe. If the sample contains the antigen to be tested, the prepared secondary antibody probe is immobilized in the form of an immune complex On the surface of the electrode, after adding the substrate, it can catalyze the oxidation-reduction reaction of the substrate and generate electrical signals. The detection principle is as attached figure 1.
[0051] (1) Preparation of quantitative standard curve:
[0052] (a) Dilute the standard AFP antigen solution to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, respectively add 10μL of AFP antigen solution standards of different concentrations into the reaction chamber of the amperometric immunosensor, and incubate at 37°C for 30min. After the reaction, the unreacted antigen was carefully washed away with PBS.
[0053] (b) Prepare a 1mg/ml suspension of the secondary antibody probe synthesized in step 1, and add 10 mL of the secondary antibody probe suspension dropwise to the reaction chamber of the amperometric immunosensor, incubate at 37°C for 30 minutes, and carefully wash with PBS;
[0054] (c) Add a 0.1 mol/L phosphate buffer solution containing a concentration of 5 mmol/L of urine peroxide solution and 1 mmol/L of catechol to the reaction chamber of the amperometric immunosensor for 3 minutes; Voltammetry (CV) records the reduction peak current I (scanning range from -0.3V to -0.8V).
[0055] (d) A set of data of the reduction peak current obtained in step (C) and the concentration of the corresponding antigen standard solution. The average slope method is used to perform linear regression analysis on the concentration of the antigen standard solution by the reduction peak current to obtain the reduction peak current and antigen The linear equation corresponding to the concentration of the standard solution is the standard curve. CV reduction peak current and AFP antigen concentration value see Figure 4 Inset illustration. The linear equation is logI=0.66log[C AFP ]-5.15.
[0056] Add the sample to be tested into the reaction chamber of the amperometric immunosensor, use cyclic voltammetry (CV) to record the reduction peak current with the same method and reaction conditions as step (1), and then substitute the reduction peak current value obtained from the test sample into The concentration of the antigen in the sample to be tested can be calculated from the linear equation obtained in step (a). Use 10 blank samples to test and calculate the average And the standard deviation s to As the detection limit.
[0057] In this example, the detection range for AFP is 0.01-200ng/mL, and the detection limit is 4pg/mL.
