A SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe

Abstract

The present invention relates to an SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe in immunological analysis and detection technology. In the first step, the surface of Co3O4 magnetic nanoparticles is modified with carboxyl groups and activated, and then connected with a secondary antibody Ab2 to obtain Co3O4 magnetic nanoparticles. 4 NPs-Ab2 nano signal amplification probe; In the second step, the SPA protein was fixed on the surface plasmon resonance immunosensor SPR substrate, and then connected to the capture antibody Ab1 and blocked with bovine serum albumin, and then the antigen solution and Co3O 4 NPs-Ab2 signal amplification probe, incubated to form a sandwich SPR immunosensor. The carboxylated Co3O4 nanoparticles of the present invention, with their high refractive index and high molecular weight, can effectively enhance the SPR signal. At the same time, by separating and enriching targets in complex samples in an external magnetic field, the background interference of unknown compounds can be greatly reduced. It has good biocompatibility, prepares signal amplification probes, and combines the sandwich method to enhance the SPR signal for high-sensitivity detection of protein molecules.

Description

Technical Field

[0001] The invention relates to the technical field of immunological detection and analysis, and in particular to an SPR immunosensor of a Co3O4 magnetic nanoparticle signal amplification probe for detecting protein molecules. Background Art

[0002] In the 1960s, Otto and Kretschmann invented the method of exciting surface plasmon with visible light. In 1982, Nylander et al. first applied the surface plasmon resonance (SPR) technology to the field of immunosensors. In 1983, Liedberg et al. successfully applied SPR to the reaction between IgG protein and its antigen. SPR can be used for real-time analysis, and can monitor the interaction between DNA and protein, protein and protein, drug and protein, antigen and antibody, receptor and ligand, etc. in a simple and fast manner. It has a wide range of applications in biological research, medical testing, food testing, environmental monitoring and other fields.

[0003] SPR technology can be combined with immunosensors to achieve high-sensitivity detection of different types of proteins. The sandwich method is an important method for SPR immunosensors to determine antigenic substances. By introducing secondary antibodies, the mass of the SPR sensing surface is increased, and the refractive index of the sensing surface is changed, thereby improving the performance of the SPR sensor and improving the sensitivity and specificity.

[0004] Co3O4 magnetic nanoparticles can be used as signal amplification probes to improve the performance of SPR sensors. However, in actual SPR immunoassays, they are easily interfered by unknown compounds, which affects the accuracy, sensitivity and detection limit of the detection. Summary of the invention

[0005] The purpose of the present invention is to provide an SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe, which increases the SPR sensor signal intensity by improving the amplification probe of Co3O4 magnetic nanoparticles, thereby improving the accuracy, sensitivity and detection limit of SPR detection.

[0006] The object of the present invention is achieved by providing an SPR immunosensor based on a Co3O4 magnetic nanoparticle signal amplification probe, characterized in that it is prepared by the following steps:

[0007] Step 1: Carboxyl groups are modified on the surface of Co3O4 magnetic nanoparticles, and activated, and then connected to the secondary antibody Ab2 to prepare the Co3O4NPs-Ab2 nano signal amplification probe;

[0008] In the second step, the SPA protein was fixed on the surface plasmon resonance immunosensor SPR substrate, and then connected to the capture antibody Ab1 and blocked with bovine serum albumin. Then, the antigen solution and the Co3O4NPs signal amplification probe were introduced in sequence and incubated to form a sandwich SPR immunosensor.

[0009] The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe of the present invention synthesizes carboxylated Co3O4 nanoparticles, whose high refractive index and high molecular weight can effectively enhance the SPR signal, and can greatly reduce the background interference of unknown compounds by separating and enriching targets in complex samples in an external magnetic field. At the same time, it has good biocompatibility, utilizes its high specific surface area to fix antibody molecules, prepares signal amplification probes, and combines with the sandwich method to promote SPR signal enhancement for high-sensitivity detection of protein molecules.

[0010] Furthermore, the Co3O4NPs-Ab2 nanometer signal amplification probe in the first step of the present invention includes the following step-by-step process:

[0011] 1.1 First, dissolve Co(NO3)2·6H2O and NH3·H2O in H2O, then add H2O2, heat in a water bath and stir to react to obtain [Co(NH3)6] 3+ Solution;

[0012] 1.2 To [Co(NH3)6] 3+ Co(NO3)2·6H2O solution was added to the solution, and after the reaction was completed by stirring in a water bath, the reactants were cooled to room temperature and collected to obtain a black Co3O4 solid precipitate. After washing, the solid phase was separated by centrifugation and dispersed in PBS buffer to obtain a suspension of Co3O4 magnetic nanoparticles.

[0013] 1.3 Add a mixed solution of dimercaptosuccinic acid (DMSA) and dimethyl sulfoxide (DMSO) to the suspension of Co3O4 magnetic nanoparticles and stir to modify the carboxyl groups on the surface of Co3O4NPs. Then activate the carboxyl groups with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Then add the secondary antibody (Ab2) and stir at 0-4°C. Purify the probe molecules by low-temperature centrifugation and washing to obtain the Co3O4NPs-Ab2 nano signal amplification probe, which is dispersed in PBS buffer and stored at low temperature.

[0014] Further, in step 1.1, the molar ratio of Co(NO3)2·6H2O, NH3·H2O and H2O2 is 5:50:3, the mass fraction of NH3·H2O is 25%, and the water bath temperature is 50-70°C.

[0015] Furthermore, in step 1.2, the amount of Co(NO3)2·6H2O solution added was 20 mL 0.025 mol / L.

[0016] Furthermore, in step 1.3, the mixing ratio of Co3O4 magnetic nanoparticles, dimercaptosuccinic acid (DMSA), and dimethyl sulfoxide (DMSO) is 4:5:280, and the usage ratio of EDC / NHS and secondary antibody (Ab2) is 20:14:1.

[0017] The second step of the present invention comprises the following step-by-step process:

[0018] 2.1 Heat a mixed solution of H2O2, ammonia and ultrapure water in a molar ratio of 2:3:56 to 95-100°C, soak the SPR chip in it, then rinse the chip alternately with ethanol and ultrapure water, and then blow dry with nitrogen;

[0019] 2.2 Place the dried chip in step 2.1 into the detection cell of the surface plasmon resonance instrument, and pass PBS buffer into the detection cell until a stable baseline is reached;

[0020] 2.3 Add Staphylococcus aureus protein A (SPA) solution to the detection cell and incubate until the signal is stable, then add PBS buffer to rinse until the SPR signal is stable;

[0021] 2.4 Inject the primary antibody (Ab1) into the detection pool and incubate with the SPA reaction until the signal is stable, then rinse with PBS buffer until the SPR signal is stable;

[0022] 2.5 Inject bovine serum albumin (BSA) solution into the surface of the SPR chip in the detection pool for blocking and incubation until the signal becomes stable again, and then rinse with PBS until the SPR signal is stable;

[0023] 2.6 Inject antigen and primary antibody (Ab1) into the surface of the SPR chip in the detection pool and incubate until the signal is stable, then rinse with PBS until the SPR signal is stable;

[0024] 2.7 The Co3O4NPs-Ab2 nano signal amplification probe was introduced into the detection pool to react with the antigen and incubated until the signal was stable, and then PBS was introduced to rinse until the SPR signal was stable to obtain an SPR immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe.

[0025] Furthermore, in step 2.3, the concentration of the SPA solution was 100 μg / mL, and in step 2.4, the concentration of Ab1 was 100 μg / mL.

[0026] Furthermore, in step 2.5, the concentration of the BSA solution was 200 μg / mL.

[0027] Furthermore, in step 2.7, the concentration of Co3O4NPs-Ab2 is 10 μg / mL.

[0028] Furthermore, in steps 2.2 to 2.7, the flow rate of the solution into the detection cell is 5 μL / min; and the incubation time of each step is 30 to 40 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the surface plasmon resonance immunosensor and immunoassay based on the Co3O4 magnetic nanoparticle signal amplification probe of the present invention.

[0030] Figure 2 Scanning and transmission electron microscopy images of Co3O4NPs, Co3O4NPs-COOH, and Co3O4NPs-Ab2 prepared in the present invention. Figure 3 This is a feasibility study diagram of the surface plasmon resonance immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe of the present invention.

[0031] Figure 4 This is the linear curve of the surface plasmon resonance immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe of the present invention for detecting HIgG standard samples.

[0032] In order to better illustrate the technical process and purpose of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods. DETAILED DESCRIPTION

[0033] This embodiment is as follows Figure 1 The process shown specifically prepares an SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe.

[0034] First, synthesize Co3O4 magnetic nanoparticles:

[0035] Dissolve 0.291 g Co(NO3)2·6H2O and 810 μL ammonia in 50 mL water, transfer to a three-necked flask, and add 60 μL H2O2 while stirring to obtain [Co(NH3)6] 3+ The solution was heated in a water bath at 60 °C for 15 min, and then 20 mL of 0.025 MCo (NO3) 2·6H2O solution was added and heated and stirred at 60 °C for 3 h. After the reaction was completed, it was cooled to room temperature, washed by centrifugation, and dispersed in water to obtain a suspension of Co3O4 magnetic nanoparticles with a dispersion concentration of 1 mg / mL.

[0036] Then, a surface plasmon resonance immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe is prepared, which specifically includes the following steps:

[0037] (1) Take 50 mL of the above Co3O4 suspension, add 0.045 g DMSA and 1 mL DMSO, stir and react for 5 h to modify the surface of Co3O4 with carboxyl groups, and obtain a Co3O4NPs-COOH stable solution; then add a mixed solution of 100 μL EDC and 70 μL NHS at a concentration of 10 mg / mL to the above stable solution to activate the carboxyl groups, and then add 50 μL of secondary antibody (Ab2) at a concentration of 1 mg / mL, stir and react at low temperature at 4°C for 30 min, continue to centrifuge at 10000 rpm for 30 min at low temperature, and the obtained solid is dispersed in PBS buffer at a concentration of 0.01 mol / L to obtain a Co3O4NPs-Ab2 suspension with a dispersion concentration of 10 μg / mL;

[0038] (2) Heat a mixed solution of ultrapure water, H2O2 and ammonia (mass fraction 25%) in a volume ratio of 1:1:5 to a slight boil, soak the SPR chip in it, then rinse the chip alternately with ethanol and ultrapure water, and then blow dry with nitrogen;

[0039] (3) Place the treated chip into the detection cell of the surface plasmon resonance instrument and pass PBS buffer (0.01 mol / L) at a flow rate of 5 μL / min until the detection cell reaches a stable baseline;

[0040] (4) A 100 μg / mL Staphylococcus aureus protein A (SPA) solution was passed into the detection cell at a flow rate of 5 μL / min until the signal was stable, and then PBS (0.01 mol / L) was passed through the cell for flushing until the SPR signal was stable;

[0041] (5) A primary antibody (Ab1) with a concentration of 100 μg / mL was introduced into the detection cell at a flow rate of 5 μL / min to react with SPA until the signal was stable, and then PBS (0.01 mol / L) was introduced to rinse until the SPR signal was stable;

[0042] (6) Pass 200 μg / mL bovine serum albumin (BSA) solution into the detection cell at a flow rate of 5 μL / min to block until the signal becomes stable again, and then pass PBS (0.01 mol / L) to rinse until the SPR signal is stable;

[0043] (7) The HIgG antigen solution was passed into the detection cell at a flow rate of 5 μL / min to react with the primary antibody (Ab1) until the signal was stable, and then PBS (0.01 mol / L) was passed to rinse until the SPR signal was stable;

[0044] (8) After the SPR signal stabilizes, the Co3O4NPs-Ab2 suspension prepared in step (1) is introduced to react with the antigen until the signal stabilizes, and then PBS (0.01 mol / L) is introduced to rinse until the SPR signal stabilizes to obtain an SPR immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe.

[0045] At the same time, the Co3O4NPs, Co3O4NPs-COOH, and Co3O4NPs-Ab2 prepared above were analyzed by electron microscopy. Figure 2 As shown, Figure 2 Figures A and D are scanning and transmission electron microscopy images of Co3O4NPs, respectively. It can be seen that the particle size of Co3O4NPs is about 50 nm, showing a relatively regular spherical shape; Figure 2 Figures B and E are scanning and transmission electron microscopy images of Co3O4NPs-COOH, respectively. It can be seen that the size and morphology of Co3O4NPs remain basically unchanged after carboxylation. Figure 2 Figures C and F are scanning and transmission electron microscopy images of Co3O4NPs-Ab2, respectively. It can be seen from the figures that after binding with the antibody, Ab2 is wrapped outside the Co3O4NPs, and Co3O4NPs-Ab2 is successfully prepared.

[0046] Figure 3 This is a feasibility study diagram of the surface plasmon resonance immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe in this embodiment. Figure 3 As can be seen in A, when HIgG is present, the SPR resonance angle shifts by 0.092° after the introduction of Co3O4NPs-Ab2. Under the same conditions, when HIgG is replaced with 0.01 M PBS and Co3O4NPs-Ab2 is introduced for incubation, the SPR resonance angle shifts by only 0.016°. Since Co3O4NPs-Ab2 is slightly larger in size and magnetic, it will aggregate, causing nonspecific adsorption. However, this nonspecific adsorption only increases the SPR signal by 0.016°, which is a smaller signal increase than the basic specific binding. Figure 3 B is the SPR resonance angle shifted by 0.092° after the introduction of Co3O4NPs-Ab2 in the presence of HIgG. Under the same conditions, the SPR resonance angle shifted by only 0.034° when 10 μg / mLAb2 was used instead of Co3O4NPs-Ab2. Based on the above data, it can be proved that the prepared surface plasmon resonance immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe is feasible.

[0047] like Figure 4 The SPR signal curves of the above sensor detecting HIgG standard samples of different concentrations are shown in the figure. It can be clearly seen from the figure that the SPR signal increases with the increase of HIgG concentration. Figure 4 A is the sensor diagram of the surface plasmon resonance SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe to detect 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 30 μg / mL, and 50 μg / mL HIgG standard samples. Figure 4 B is the linear curve of concentration and SPR signal. It can be clearly seen from the figure that the detection linear range is 0.01-50 μg / mL. The linear equation is ∆θ(mDeg)=4.1709C HIgG (μg / mL) + 41.4895, where ∆θ is the SPR angle offset, C is the HIgG concentration, and the linear correlation coefficient is 0.9929. Based on the above data, the surface plasmon resonance immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe has high sensitivity and low detection limit for detecting HIgG.

[0048] Example 2

[0049] This example is to investigate the reliability of the actual application of the SPR immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe. After adding the standard concentration of HIgG antibody to the untreated actual serum sample, the SPR immunoassay was performed according to the process of Example 1. The difference is that in step (7), the samples with the spiked concentrations as described in Table 1 were respectively introduced for SPR immunoassay, and the detection depth and recovery rate were obtained.

[0050] Table 1. Determination of HIgG actual samples

[0051] sample Spike concentration (μg / mL) Detection concentration (μg / mL) Recovery rate (%) 1 1.00 0.973 97.3 2 5.00 5.27 105.4 3 10.00 10.32 103.2 4 15.00 14.73 98.2 5 30.00 30.48 101.6

[0052] It can be seen from Table 1 that the recovery rate ranges from 97.3% to 105.4%, indicating that the SPR sensor can accurately detect HIgG in actual serum samples.

Claims

1. A SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe, characterized in that: Prepared by the following steps: In the first step, the surface of Co3O4 magnetic nanoparticles is modified with carboxyl groups and activated, and then connected with the secondary antibody Ab2 to obtain the Co3O4NPs-Ab2 nano signal amplification probe; this step specifically includes the following step-by-step process: 1.1 First, dissolve Co(NO3)2·6H2O and NH3·H2O in H2O, then add H2O2, heat in a water bath and stir to react for 15-20 min to obtain a [Co(NH3)6]3+ solution; 1.2 Add Co(NO3)2·6H2O solution to [Co(NH3)6]3+ solution, continue to react under water bath with stirring for 2.5-3h, collect the obtained black Co3O4 solid phase precipitate after cooling to room temperature, wash and centrifuge to separate the solid phase to obtain Co3O4 magnetic nanoparticles, disperse in water to obtain a suspension of Co3O4 magnetic nanoparticles with a dispersion concentration of 0.5-1mg / mL; 1.3 Add a mixed solution of dithiosuccinic acid (DMSA) and dimethyl sulfoxide (DMSO) to the suspension of Co3O4 magnetic nanoparticles and stir to modify the carboxyl group on the surface of Co3O4 NPs, then activate the carboxyl group with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), add a secondary antibody (Ab2), stir at 0-4°C for 20-30 minutes, and then continue to centrifuge and wash at 0-4°C to purify the probe molecule to obtain Co3O4 NPs-Ab2 nano signal amplification probe, which is dispersed in PBS buffer and stored at low temperature; In the second step, the SPA protein was fixed on the surface plasmon resonance immunosensor SPR substrate, and then connected to the capture antibody Ab1 and blocked with bovine serum albumin. Then, the antigen solution and the Co3O4 NPs-Ab2 signal amplification probe were introduced in sequence and incubated to form a sandwich SPR immunosensor.

2. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 1, characterized in that: In step 1.1, the ratio of the amounts of Co(NO3)2·6H2O, NH3·H2O and H2O2 is 5:50:3, the mass fraction of NH3·H2O is 25%, and the water bath temperature is 50-70°C.

3. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 1, characterized in that: In step 1.2, the molar ratio of [Co(NH3)6]3+ to Co(NO3)2·6H2O is 2:1, and the concentration of Co(NO3)2·6H2O solution is 0.025 mol / L.

4. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 1, characterized in that: In step 1.3, the molar ratio of Co3O4 magnetic nanoparticles, dimercaptosuccinic acid (DMSA), and dimethyl sulfoxide (DMSO) is 4:5:280, and the molar ratio of EDC, NHS, and secondary antibody is 20:14:

1.

5. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 1, characterized in that: Step 2 includes the following step-by-step process: 2.1 Heat a mixed solution of H2O2, ammonia and ultrapure water in a molar ratio of 2:3:56 to 95-100°C, soak the SPR chip in it, then rinse the chip alternately with ethanol and ultrapure water, and then blow dry with nitrogen; 2.2 Place the dried chip in step 2.1 into the detection cell of the surface plasmon resonance instrument, and pass PBS buffer into the detection cell until a stable baseline is reached; 2.3 Add Staphylococcus aureus protein A (SPA) solution into the detection cell and incubate until the signal is stable, then add PBS buffer to rinse until the SPR signal is stable; 2.4 Inject the primary antibody (Ab1) into the detection pool and incubate with SPA reaction until the signal is stable, then pass PBS buffer to rinse until the SPR signal is stable; 2.5 Inject bovine serum albumin (BSA) solution into the surface of the SPR chip in the detection pool for blocking and incubation until the signal becomes stable again, and then rinse with PBS until the SPR signal becomes stable; 2.6 Inject antigen and primary antibody (Ab1) into the surface of the SPR chip in the detection pool and incubate until the signal is stable, then rinse with PBS until the SPR signal is stable; 2.7 The Co3O4 NPs-Ab2 nano signal amplification probe was introduced into the detection pool to react with the antigen and incubated until the signal was stable, and then PBS was introduced to rinse until the SPR signal was stable to obtain an SPR immunosensor based on the Co3O4 magnetic nanoparticle signal amplification probe.

6. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 5, characterized in that: In step 2.3, the concentration of SPA solution was 100 μg / mL, and in step 2.4, the concentration of Ab1 was 100 μg / mL.

7. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 5, characterized in that: In step 2.5, the concentration of the BSA solution was 200 μg / mL.

8. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 5, characterized in that: In step 2.7, the concentration of Co3O4 NPs-Ab2 was 10 μg / mL.

9. The SPR immunosensor based on Co3O4 magnetic nanoparticle signal amplification probe according to claim 5, characterized in that: In steps 2.2 to 2.7, the flow rate of the solution into the detection cell is 5 μL / min; the incubation time of each step is 30 to 40 min.

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