Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor

An immunosensor and probe technology, applied in the field of molecular recognition probes, can solve problems such as difficulty in overcoming the influence of non-specific adsorption, limited enrichment effect of secondary antibodies, and inability to update electrodes, thereby eliminating the need for centrifugation and washing processes. , not easy to inactivate, and the effect of low detection limit

Active Publication Date: 2011-11-23
SOUTHERN MEDICAL UNIVERSITY
11 Cites 10 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, the reported electrochemical amperometric sensors also have the following deficiencies in use: ① more "one-step method" is used, that is, the immune complex formed after the combination of antigen and antibody is used to cause the electrode surface current to drop, and the quantification is carried out according to the falling current, so that it is impossible It is difficult to avoid the influence of non-specific adsorption; ②When the antigen/antibody on the surface...
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Abstract

The invention relates to the field of biological monitoring, and specifically relates to a secondary antibody probe for a sandwich immune reaction by using an amperometric immunosensor, wherein, the secondary antibody probe is composed of calf thymus DNA molecular chains and nanospheres adhering to the calf thymus DNA molecular chains, and the surfaces of the nanospheres are covered by enzyme labeled antibody proteins while magnetic nanometer particles, which are formed by depositing a layer of amorphous zirconia on the surfaces of nanometer ferriferrous oxide particles, are filled in the nanospheres. The secondary antibody probe provided in the invention is suitable for measurring concentration of antigens by a sandwich immune reaction employing an amperometric immunosensor, and has the advantages of wide linear range of detection and low detection limits.

Application Domain

Material analysis by electric/magnetic meansBiological testing

Technology Topic

AntigenFerriferrous Oxide +7

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  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor
  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor
  • Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor

Examples

  • Experimental program(5)

Example Embodiment

[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.

Example Embodiment

[0058] Example 2 (Comparative Experiment 1)
[0059] 1. Experimental materials
[0060] 1. Antigen and antibody: AFP antigen standard, alpha-fetoprotein monoclonal primary antibody (anti-AFP), horseradish peroxidase-labeled alpha-fetoprotein polyclonal antibody (HRP-anti-AFP) and AFP ELISA kit All purchased Zhengzhou Bosai Biotechnology Co., Ltd.
[0061] 2. Amperometric immunosensor: prepared according to the method described in Example 1.
[0062] 3. Secondary antibody probe
[0063] 3.1. Sample: The secondary antibody probe prepared by the method described in Example 1.
[0064] 3.2. Reference substance
[0065] Reference substance 1:
[0066] (1) Preparation of magnetic nanoparticles
[0067] Preparation of magnetic nanoparticles: For specific preparation methods, please refer to the invention patent application with publication number CN 101302361A.
[0068] (2) Preparation and characterization of antibody-loaded nanospheres
[0069] 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 , The unbound antibody is separated by applying a magnetic field, and the antibody-loaded nanospheres are obtained by magnetic separation under the applied magnetic condition.
[0070] (3) Preparation of magnetic probe
[0071] 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, and the magnetic probes obtained are expressed as HRP-anti-AFP-ZMPs probes.
[0072] Reference substance 2: Prepared by referring to the method reported in the following literature: Tang J, Su B, Tang D, et al. Conductive carbon nanoparticles-based electrochemical immunosensor with enhanced sensitivity for [alpha]-fetoprotein using irregular-shaped gold nanoparticles-labeled enzyme-linked antibodies as signal improvement[J]. Biosensors and Bioelectronics, 2010, 25(12): 2657-2662. The resulting secondary antibody probes are represented by the code HRP-anti-AFP-GNGs.
[0073] Reference substance 3: Prepared by referring to the method reported in the following literature: Zhuo Y, Yi WJ, Lian WB, et al. Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification[J].Biosens Bioelectron.2011 , 26(5): 2188-2193. The resulting secondary antibody probe is codenamed AuNCs-HRP-Ab 2 Said.
[0074] Control 4: AFP polyclonal antibody labeled with horseradish peroxidase in the AFP ELISA kit (purchased from Zhengzhou Bosai Biotechnology Co., Ltd.) was used as control 4. Expressed as HRP-anti-AFP.
[0075] 2. Experimental method
[0076] 1. The experiment is divided into 5 groups. In each group, except for the added secondary antibody probe, the other operations are the same.
[0077] 2. Experimental process
[0078] 2.1 Preparation of quantitative standard curve:
[0079] (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, the prepared amperometric immunosensors were divided into 5 groups, each containing 10 sensors, 10 μL of AFP antigen solution standards of different concentrations were respectively added to the reaction chamber of the amperometric immunosensors, and incubated at 37°C for 30 min. After the reaction, the unreacted antigen was carefully washed away with PBS.
[0080] (b) The sample prepared in this example and the reference substance are combined to form a 1mg/ml suspension, and 10μL of the sample is added dropwise to the reaction chamber of the ampere immunosensor in group 1, and the reaction chambers of the sensors in groups 2 to 5 are respectively Add 10 μL of the reference substance 1 to 4 probe suspension, and incubate at 37°C for 30 min.
[0081] (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).
[0082] (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 of the corresponding relationship between the concentration of the standard solution, that is, the standard curve; 10 blank samples are used for testing, and the average value is calculated And the standard deviation s to As the detection limit.
[0083] Table 1 is a comparison of the effects of different secondary antibody probes in detecting AFP antigen. From Table 1, it can be concluded that the secondary antibody probe prepared in this application has linearity compared with the secondary antibody probe prepared by other methods for electrochemical detection. The advantages of wide range and high sensitivity.
[0084] Table 1 Comparison of the effects of different secondary antibody probes in detecting AFP antigen
[0085]

Example Embodiment

[0086] Example 3
[0087] 1. Preparation and characterization of the secondary antibody probe
[0088] (1) Preparation and characterization of magnetic nanoparticles
[0089] Same as in Example 1 step 1.
[0090] (2) Preparation and characterization of antibody-loaded nanospheres
[0091] Preparation of antibody-loaded nanospheres: Disperse 10 mg of magnetic nanoparticles in 5 mL of pH 7.0 phosphate buffer, and add 1 mg of horseradish peroxidase-labeled HIV p24 antibody (HRP-anti-HIV p24). After stirring for 6 hours, a magnetic field was applied to separate the unbound antibody to obtain antibody-loaded nanospheres.
[0092] (3) Preparation of nanospheres
[0093] Same as Example 1 step 3 shown.
[0094] (4) Preparation of secondary antibody probe
[0095] Same as Example 1 step 4 shown.
[0096] 2. Using secondary antibody probe to detect antigen concentration
[0097] (1) Preparation of amperometric immunosensor
[0098] (a) Graphene/chitosan (GS/CS) is modified into a film on the surface of the screen printed electrode
[0099] Same as step 2 (a) in Example 1.
[0100] (b) Electroplating gold
[0101] Same as step 2 (b) in Example 1
[0102] (c) After adding 10 μL of HIV p24 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 low temperature for 12 hours, and then the unbound and weakly bound antibodies are washed thoroughly with PBS.
[0103] (d) BSA closed
[0104] Same as step 2 (d) in Example 1
[0105] (2) Preparation of quantitative standard curve:
[0106] (a) Dilute the HIV antigen solution standard to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml , Add 10μL of HIV antigen solution standards of different concentrations into the reaction chamber of the Ampere immunosensor, and incubate at 37°C for 30min. After the reaction, the unreacted antigen was carefully washed away with PBS.
[0107] (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, and incubate at 37°C for 30 minutes;
[0108] (c) Add a 0.1mol/L phosphate buffer solution containing a concentration of 5mmol/L peroxyurine solution and 1mmol/L catechol to the reaction chamber of the ampere immunosensor for 3 minutes; on the electrochemical workstation, use the differential Pulse voltammetry (DPV) records the reduction peak current I, (scanning range from 0.2V to -0.5V).
[0109] (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. DPV reduction peak current and HIV antigen concentration value see Figure 5 Inset, the linear equation is: I=15+0.97Log[C p24 ].
[0110] Add the sample to be tested into the reaction chamber of the amperometric immunosensor, use DPV 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 step (a). The concentration of antigen in the sample to be tested can be calculated from the linear equation. Use 10 blank samples to test and calculate the average And standard deviation s to As the detection limit.
[0111] In this example, the detection range of AFP is 0.01-200ng/mL, and the detection limit is 4pg/mL.

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