Sandwich immunomagnetic relaxation switch sensor based on magnetic separation and Cu < 2 + > mediation as well as preparation method and application of sandwich immunomagnetic relaxation switch sensor

Abstract

The invention discloses a sandwich immunomagnetic relaxation switch sensor based on magnetic separation and Cu < 2 + > mediation as well as a preparation method and application thereof. The preparation method comprises the following steps: reacting dopamine under an alkaline condition to obtain polydopamine nanoparticles; covalently binding active groups on the surfaces of the polydopamine nanoparticles with amino groups of an antibody to obtain a polydopamine-antibody conjugate; mixing the aminated ferroferric oxide magnetic beads with an activated antibody solution to carry out a coupling reaction, and then carrying out cleaning, magnetic separation and closing to obtain a magnetic nanoparticle-antibody conjugate; and mixing the magnetic nanoparticle-antibody conjugate, the antigen solution and the polydopamine-antibody conjugate, and continuously reacting to obtain the sandwich immune complex. The sandwich immune complex prepared by the invention can be used as a sensor to realize efficient antibody fixation and chelating adsorption of paramagnetic Cu < 2 + > to regulate and control a magnetic relaxation signal, is combined with a magnetic separation technology to be used for quantitative determination of protein in a complex matrix, and is sensitive in detection and short in detection time.

Description

Technical Field

[0001] The present invention relates to the field of sensor technology, in particular to a method based on magnetic separation and Cu 2+ Mediated sandwich immunomagnetic relaxation switch sensor and its preparation method and application. Background Art

[0002] In the development of biosensor technology, the sensitive detection of trace biomarkers in complex sample environments has always been the core direction and key bottleneck. Immunoglobulins (Ig) can be divided into five categories based on molecular structure and function. Among them, serum IgG has the highest abundance and has become the key detection target. It has important application value in disease diagnosis, drug development and biological product monitoring. At the same time, it can provide a technical reference for the detection of other biomarkers as a methodological model. Although traditional detection systems such as enzyme-linked immunosorbent assay and electrochemical sensors have nanogram-level sensitivity, they still require multiple pretreatment steps in complex sample applications such as whole blood. In addition, the free components in the sample matrix are prone to cause nonspecific interference, resulting in limited direct detection capabilities.

[0003] The detection signal of the magnetic relaxation switch sensor (MRS) is not affected by the turbidity or color of the sample, and has been successfully used in the rapid analysis of pathogens and protein markers. This technology achieves the quantification of the target by monitoring the state or concentration changes of magnetic nanoparticles in aqueous solution, or by detecting the concentration changes or valence changes of paramagnetic substances, which cause changes in the relaxation time (T1 or T2) of water protons. However, the spontaneous aggregation of magnetic nanoparticles in traditional MRS systems is prone to produce false positive signals, which seriously affects the accuracy of detection. Therefore, the construction of a new pre-treatment-free detection method suitable for complex matrices has become an important breakthrough in improving the efficiency of rapid detection of clinical samples. Summary of the Invention

[0004] The purpose of the present invention is to provide a method based on magnetic separation and Cu 2+ The sandwich immunomagnetic relaxation switch sensor and its preparation method and application are designed to solve the problem that the spontaneous aggregation of magnetic nanoparticles in the above-mentioned MRS system easily produces false positive signals, which seriously affects the detection accuracy. The magnetic separation-magnetic relaxation switch sensor prepared by the present invention utilizes the rich catechol and imine structures on the surface of polydopamine to achieve efficient antibody immobilization and chelation adsorption of paramagnetic Cu 2+ Modulate magnetic relaxation signals and combine them with magnetic separation technology for quantitative protein determination in complex matrices.

[0005] To achieve the above object, the first aspect of the present invention provides a method based on magnetic separation and Cu 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor comprises the following steps:

[0006] (1) Polydopamine nanoparticles are obtained by spontaneous oxidative polymerization of dopamine under alkaline conditions;

[0007] (2) The active groups on the surface of polydopamine nanoparticles are covalently bonded to the amino groups of the antibody through Michael addition reaction to obtain polydopamine-antibody conjugates;

[0008] (3) mixing the amino-ferroferric oxide magnetic beads with the activated antibody solution to undergo a coupling reaction, followed by washing, magnetic separation, and blocking to obtain a magnetic nanoparticle-antibody conjugate;

[0009] (4) The magnetic nanoparticle-antibody conjugate and the antigen solution are mixed to undergo a binding reaction to obtain a mixture, polydopamine-antibody conjugate is added to the mixture, and the reaction is continued to obtain a sandwich immune complex, which is a sandwich immune magnetic relaxation switch sensor.

[0010] Preferably, in step (1), the specific preparation process of polydopamine nanoparticles is:

[0011] Ethanol and ultrapure water are mixed, and then ammonia water is added. After mixing evenly, dopamine solution is quickly injected and stirred at room temperature for reaction. The precipitate is then collected by centrifugation and washed. The product obtained by washing is polydopamine nanoparticles; the polydopamine nanoparticles are redispersed in ultrapure water to obtain a polydopamine nanoparticle suspension for later use.

[0012] Preferably, in step (2), the specific preparation process of the polydopamine-antibody conjugate is:

[0013] The polydopamine nanoparticle suspension was mixed with the antibody solution and incubated to promote full binding of the antibody to the surface of the polydopamine nanoparticles. Subsequently, a bovine serum albumin solution was added to block excess sites and the incubation was continued. After the reaction was completed, the polydopamine-antibody conjugate was obtained by centrifugation and washing. The polydopamine-antibody conjugate was dispersed in PBS buffer to obtain a polydopamine-antibody conjugate dispersion, which was stored at 4°C for future use.

[0014] Preferably, in step (3), the specific preparation process of the magnetic nanoparticle-antibody conjugate is:

[0015] (31) Pretreatment of magnetic nanoparticles: Take aminated ferroferric oxide magnetic beads, add them to ultrapure water for washing, and then dilute to ultrapure water to obtain an aminated ferroferric oxide magnetic bead dispersion;

[0016] (32) Antibody activation: dilute the antibody with PBS buffer, then add EDC and NHS solution to obtain activated antibody solution;

[0017] (33) Coupling reaction: Add the amino-containing ferroferric oxide magnetic bead dispersion to the activated antibody solution to undergo coupling reaction;

[0018] (34) Cleaning and purification: After the coupling reaction, magnetic separation was performed and the mixture was washed several times with PBST buffer;

[0019] (35) Site blocking: After washing, add bovine serum albumin solution for blocking reaction;

[0020] (36) Product storage: After blocking, the product was magnetically separated, washed with PBS buffer, and then resuspended in PBS buffer to obtain a magnetic nanoparticle-antibody conjugate dispersion, which was stored at 4°C for future use.

[0021] Preferably, in step (4), the specific preparation process of the sandwich immune complex is:

[0022] (41) Antigen-antibody binding reaction: Mix the magnetic nanoparticle-antibody conjugate dispersion, antigen solution, and PBS buffer, and shake for reaction;

[0023] (42) Addition of signal probe: Then, polydopamine nanoparticle suspension was added to the system after the oscillation reaction, PBS buffer was added, and the oscillation reaction was continued;

[0024] (43) After the reaction, the cells were placed on a magnetic separation rack to remove the unbound free components in the supernatant and then washed repeatedly with PBST buffer to obtain the sandwich immune complex.

[0025] Preferably, the antibody solution is an IgG antibody solution, and the antigen solution is an IgG antigen solution.

[0026] Preferably, in step (42), after supplementing with PBS buffer, the final concentration of the polydopamine-antibody conjugate dispersion is 0.8 mg / mL; the final concentration of the magnetic nanoparticle-antibody conjugate dispersion is 0.4 mg / mL.

[0027] The second aspect of the present invention provides a method based on magnetic separation and Cu 2+ The sandwich immunomagnetic relaxation switch sensor is prepared by the above preparation method.

[0028] The third aspect of the present invention provides a method based on magnetic separation and Cu 2+ Application of sandwich immunomagnetic relaxation switch sensor for immunoglobulin detection.

[0029] Preferably, the application process includes the following steps:

[0030] S1: Resuspend the sandwich immune complex in MES buffer and then add copper chloride solution for reaction;

[0031] S2: After the reaction is completed, the supernatant is collected by magnetic separation and the transverse relaxation time T2 sample of the water molecules in the supernatant is measured using a CPMG pulse sequence. The blank sample T2 blank is used as a reference and the change value △T2 is calculated according to △T2 = T2 sample - T2 blank;

[0032] S3: Calculate the concentration of immunoglobulin based on the transverse relaxation time change value △T2.

[0033] Preferably, in step S1, after the sandwich immune complex, MES buffer and copper chloride solution are mixed, the final concentration of the copper chloride solution is 0.5 mM.

[0034] Therefore, the present invention adopts the above-mentioned one based on magnetic separation and Cu 2+ The sandwich immunomagnetic relaxation switch sensor, preparation method and application thereof have the following beneficial effects:

[0035] (1) The present invention utilizes paramagnetic ions as the output of magnetic relaxation signals, which not only overcomes the problems of nonspecific aggregation and false positives in traditional MRS, but also has the advantages of simple preparation, long shelf life, and easy operation.

[0036] (2) The magnetic separation sandwich immunomagnetic relaxation switch sensor constructed by the present invention in combination with magnetic separation technology can achieve rapid separation and enrichment of target analytes in complex matrices, and has good specificity (based on antibody-antigen immune recognition), fast separation speed and short detection time.

[0037] (3) The present invention utilizes the abundant catechol and imine groups on the surface of polydopamine to achieve dual functions: it can provide abundant active sites for covalent coupling of antibodies, thus achieving efficient and stable fixation of antibodies; it can also strongly chelate and adsorb paramagnetic ions Cu 2+ , to achieve precise control of the T2 signal of water molecules in the solution and establish a sensor signal readout system. In addition, Cu 2+ Also, because of its superior nuclear relaxation enhancement performance (superior to most paramagnetic metal ions), it can more sensitively regulate the change of T2 value and achieve high-sensitivity detection.

[0038] (4) As a validation of a novel strategy for quantitative analysis of trace proteins in complex matrices, the magnetic separation-magnetic relaxation switch sensor constructed in the present invention exhibited excellent performance in the quantitative detection of mouse immunoglobulin IgG. The constructed sensor exhibited good linearity in the range of 50-1000 ng / mL (Y=112.50lgC-160.16, r=0.9937), with a detection limit of 13.5 ng / mL. The actual serum sample test results showed good consistency compared with the classic protein quantitative analysis method enzyme-linked immunosorbent assay (ELISA). Moreover, the detection process of this method was greatly simplified, and the total analysis time was shortened to within 90 minutes. While maintaining high specificity, the detection efficiency was significantly improved compared with ELISA.

[0039] (5) The present invention is based on magnetic separation and Cu 2+ The sandwich immunomagnetic relaxation switch sensing platform mediated by enrichment synergy achieved highly sensitive and specific detection of IgG in mouse serum samples, providing a new strategy for the quantitative detection of trace functional proteins in complex matrices.

[0040] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A diagram of the sensing mechanism of the present invention;

[0042] Figure 2 Transmission electron microscopy image of polydopamine nanoparticles;

[0043] Figure 3 This is a transmission electron microscopy image of polydopamine-antibody conjugate;

[0044] Figure 4 is a transmission electron microscopy image of magnetic nanoparticle-antibody conjugate;

[0045] Figure 5 DLS size comparison of polydopamine nanoparticles and polydopamine-antibody conjugates;

[0046] Figure 6 is the zeta potential of polydopamine nanoparticles and polydopamine-antibody conjugates;

[0047] Figure 7 IR spectra of polydopamine nanoparticles and polydopamine-antibody conjugates;

[0048] Figure 8 is the zeta potential of amino-containing ferroferric oxide magnetic beads and magnetic nanoparticle-antibody conjugates;

[0049] Figure 9 Free Cu 2+ Evaluation results of solution magnetic relaxation response;

[0050] Figure 10 Adsorption of Cu by polydopamine-antibody conjugates 2+ Performance evaluation results;

[0051] Figure 11 Cu 2+ Concentration optimization results;

[0052] Figure 12 Optimize the concentration of magnetic nanoparticle-antibody conjugates;

[0053] Figure 13 Results for polydopamine-antibody conjugate concentration optimization;

[0054] Figure 14 Standard curve for IgG detection in immunosensor analysis;

[0055] Figure 15 The linear range of IgG detection for immunosensor analysis;

[0056] Figure 16 Evaluation of the specificity of the immunosensor for detecting IgG. DETAILED DESCRIPTION

[0057] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the present invention is not limited to this embodiment.

[0058] Example 1

[0059] A method based on magnetic separation and Cu 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor comprises the following steps:

[0060] (1) Preparation of polydopamine nanoparticles (PDANPs)

[0061] The preparation of polydopamine nanoparticles (PDANPs) is based on the spontaneous oxidative polymerization of dopamine (DA) under alkaline conditions.

[0062] The specific experimental steps are as follows: First, 40 mL of ethanol (purity ≥99.5%) was mixed with 90 mL of ultrapure water, followed by the addition of 2 mL of 26% ammonia water and magnetic stirring for 40 minutes. Subsequently, 10 mL of dopamine (DA) solution (50 mg / mL) was rapidly injected, and the color of the solution was observed to gradually darken from light yellow to dark brown, indicating that nanoparticles began to form. After the reaction system was stirred continuously at room temperature for 24 hours, it was centrifuged at 12,000 rpm for 10 minutes to remove unreacted DA monomers. After collecting the precipitate, it was repeatedly washed with ultrapure water three times and finally redispersed in 40 mL of ultrapure water to obtain a dispersed and stable polydopamine nanoparticle suspension (PDANPs suspension) for later use.

[0063] (2) Preparation of polydopamine-antibody conjugate (PDA@Ab)

[0064] Active groups such as hydroxyl groups on the surface of polydopamine nanoparticles (PDANPs) can covalently bind to the amino groups of antibodies through Michael addition reaction, thereby achieving antibody immobilization.

[0065] The specific operation process is as follows: First, 250 μL of PDANPs suspension is mixed with 25 μL of goat anti-mouse IgG antibody solution (1 mg / mL) in an aqueous solution and placed in a 37°C rotary mixer for 2 hours to promote the full binding of the antibody to the nanoparticle surface. Subsequently, 1% bovine serum albumin (BSA) solution is added to block excess sites and incubated for another 1 hour. After the reaction is completed, the solid and liquid phases are separated by centrifugation at 12,000 rpm for 10 minutes. After removing the supernatant, the mixture is washed three times with phosphate buffered saline containing 0.05% Tween 20 (PBST, 0.01 M, pH 7.4). Finally, the obtained polydopamine-antibody conjugate (PDA@Ab) is dispersed in 250 μL of PBS buffer to obtain a PDA@Ab dispersion, which is stored at 4°C for future use.

[0066] (3) Preparation of magnetic nanoparticle-antibody conjugates (MNP@Ab)

[0067] (a) Magnetic nanoparticle pretreatment: 200 μL of amino-Fe3O4 magnetic beads (10 mg / mL) were added to 0.2 mL of ultrapure water and washed three times, and then the volume was adjusted to 0.2 mL of ultrapure water to obtain an amino-ferroferric oxide magnetic bead dispersion. The amino-Fe3O4 magnetic beads were purchased from Xi'an Huirui Biotechnology Co., Ltd., model R-GGDB41, and named amino magnetic beads 200 nm.

[0068] (b) Antibody activation: A 10 mg / mL goat anti-mouse IgG solution was diluted to 1 mg / mL with phosphate-buffered saline (PBS). 36 μL of the diluted goat anti-mouse IgG solution was mixed with 164 μL of PBS buffer. Then, 200 μL of EDC (1 mg / mL) solution and 200 μL of NHS (1 mg / mL) solution were added. The mixture was vortexed and activated at 37°C for 30 min to obtain an activated antibody solution.

[0069] (c) Coupling reaction: Add the amino-containing ferroferric oxide magnetic bead dispersion (10 mg / mL) to the activated antibody solution and incubate at 37°C with shaking for 4 h.

[0070] (d) Cleaning and purification: After the coupling reaction, the suspension was separated by magnetic separation and washed twice with PBST buffer (0.01 M, pH 7.4) containing 0.05% Tween 20;

[0071] (e) Site blocking: After washing, add 1 mL of 5% BSA solution (prepared in PBS) and block at 37°C with shaking for 60 min;

[0072] (f) Product storage: After blocking, the product was subjected to magnetic separation, washed once with PBST, and resuspended in 1 mL of PBS to obtain a magnetic nanoparticle-antibody conjugate dispersion (MNP@Ab dispersion, final concentration 2 mg / mL), which was stored at 4°C until use.

[0073] (4) Construction of magnetic separation and Cu 2+ Sandwich immunomagnetic relaxation switch sensor

[0074] (a) Antigen-antibody binding reaction: 100 μL of MNP@Ab dispersion (2 mg / mL) was mixed with 20 μL of gradient concentration mouse IgG antigen solution (50-4000 ng / mL) and 280 μL of PBS buffer, and the mixture was incubated at 37°C with constant shaking for 30 min.

[0075] (b) Addition of signal probe: 63 μL of PDA@Ab dispersion (6.4 mg / mL) was then added to the above reaction system, and PBS buffer was supplemented to a final volume of 500 μL. The mixture was shaken at 37°C for 30 min to form a mixture containing the PDA@Ab-Ag-MNP@Ab sandwich structure.

[0076] (c) After the reaction, the mixture was placed on magnetic separation for 3 minutes to remove unbound free components in the supernatant, and then washed three times with 1 mL of PBST.

[0077] (d) Finally, the washed sandwich immune complex was resuspended in 450 μL MES buffer (0.001 M, pH 5.5), and 50 μL CuCl2·2H2O solution (5 mM) was added and reacted at 37°C for 10 min;

[0078] (e) Data acquisition and analysis: After the reaction, the supernatant was collected by magnetic separation and the change in T2 value was measured (ΔT2 = T2 sample - T2 blank). The transverse relaxation time (T2) of water molecules in the solution was measured using a CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence. The key parameters were set as follows: 1 The H nuclear resonance frequency was 600.13 MHz, the 90° pulse width (P1) was 9.5 μs, the pulse interval (τ) was 2 ms, the repeat delay time (D1) was 6 s, the number of scans (ns) was 4, and the total measurement time was 17 min. Data acquisition was performed using TopSpin 4.1 software for data processing and analysis. T2 values ​​were calculated using an exponential fitting algorithm. Each concentration gradient was measured in triplicate.

[0079] (5) Copper ion magnetic relaxation response test process

[0080] A series of CuCl2·2H2O standard solutions (0.05, 0.1, 0.3, 0.5, 1, 2, 5, 10 mmol / L) were prepared. After vortex mixing, 400 μL was dispensed into a 5 mm NMR tube and a capillary tube encapsulated with heavy water was added for field lock. The T2 value was determined. The parameter setting (CPMG pulse sequence) in step (4) of Example 1 was referred to. Each concentration gradient was measured in parallel 3 times.

[0081] like Figure 1 As shown, the detection principle of the present invention is:

[0082] A. Construction of the magnetic signal probe: Polydopamine nanoparticles (PDA NPs) were prepared based on the self-polymerization reaction of dopamine. Target IgG antibodies were immobilized on the PDA surface via Michael addition reaction. After blocking nonspecific sites with bovine serum albumin, a polydopamine-antibody conjugate (PDA@Ab) was obtained.

[0083] B. Capture probe construction: The capture antibody was covalently modified onto the surface of amino-Fe3O4 magnetic nanoparticles (MNPs) using an EDC / NHS activation strategy to prepare antibody-functionalized magnetic nanoparticles (MNP@Ab) with magnetic separation properties.

[0084] C. Construction of sandwich detection system: When the target IgG is present, PDA@Ab and MNP@Ab form a sandwich immune complex through antigen-antibody specific binding. After magnetic separation and washing, Cu is added to the complex system. 2+Based on the chelation of catechol / imine groups on the PDA surface, Cu 2+ The concentration decreases, resulting in a significant increase in T2 relaxation time; when there is no target, the Cu 2+ The concentration remained stable, and the T2 value did not change significantly. By detecting the quantitative relationship between the change in T2 signal and the concentration of the target, a new universal method suitable for the detection of trace functional proteins in complex matrices was established.

[0085] Example 2

[0086] The product prepared in Example 1 was characterized.

[0087] Figures 2 to 4 Transmission electron microscopy images of polydopamine (PDANPs), polydopamine-antibody conjugate (PDA@Ab), and magnetic nanoparticle-antibody conjugate (MNP@Ab) respectively. Transmission electron microscopy shows that the synthesized PDANPs have good monodispersity and regular spherical morphology. Statistical results show that the average particle size is 180nm ( Figure 2 After antibody coupling, the resulting PDA@Ab complex exhibits core-shell structural characteristics, with a particle size increased to 200 nm and slight aggregation. This phenomenon is attributed to the fact that the antibody molecules are anchored to the o-phenol active sites on the surface of the nanoparticles through an amidation reaction, and the subsequent introduction of bovine serum albumin (BSA) can form a dense protein layer to achieve surface sealing ( Figure 3 The functionalized magnetic nanoparticles (MNP@Ab) maintained a spherical morphology of approximately 200 nm, but the sample exhibited slight aggregation in the dispersed system ( Figure 4 ).

[0088] The particle size distributions of PDANPs and PDA@Ab were compared by dynamic light scattering (DLS). Figure 5 It can be seen that compared with PDANPs, the particle size of PDA@Ab is larger and the trend is consistent with the transmission electron microscopy results, confirming that the antibody is successfully grafted to the surface of the nanoparticles.

[0089] Figure 6 The Zeta potential analysis results of PDANPs and PDA@Ab showed that after coupling with antibodies, the Zeta potential value of the PDA@Ab composite system increased from -16.6±0.8mV to -12.7±0.6mV, indicating that the antibody molecules covalently bound to the catechol groups on the PDA surface through the amino groups, effectively neutralizing part of the surface negative charge.

[0090] Figure 7 The Fourier transform infrared spectra of PDANPs and PDA@Ab are shown in Figure 2. From the figure, we can see that the infrared spectrum of PDANPs is at 3400 cm -1The broad peak of OH / NH stretching vibration in the catechol group is shown at 1616 cm -1 、1508cm -1 、1440cm -1 The peak at 3400 cm is the C=C skeleton vibration peak of the benzene ring. -1 New peaks appeared near the -1 ), indicating that the catechol group and the antibody were reacted by Michael addition to introduce new NH / OH (from the amino and carboxyl groups in the antibody); similarly, at 2924 cm -1 and 1700cm -1 New absorption peaks appeared, which were attributed to the stretching vibration of the alkyl group and the formation of -C=N bond in the antibody molecule.

[0091] Figure 8 Comparison of the Zeta potential of MNP and MNP@Ab shows that EDC / NHS-mediated antibody coupling leads to a significant negative shift in the potential of MNP@Ab. This is because the amino groups are consumed during the antibody modification process, resulting in a reduction in the surface positive charge, thus confirming that the antibody has been stably bound to the surface of the magnetic nanoparticles.

[0092] Example 3

[0093] Copper ion binding efficiency evaluation experiment:

[0094] A gradient concentration of PDA@Ab solution (0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 mg / mL) was mixed with a CuCl2·2H2O solution (final concentration of 2 mM). The mixture was shaken at 37°C for 10 minutes and then centrifuged at 12000 rpm for 10 minutes to collect the supernatant. 400 μL of the supernatant was transferred to a nuclear magnetic resonance tube containing a heavy water capillary. The parameters (CPMG pulse sequence) in step (4) of Example 1 were used to measure the transverse relaxation time T2 of the solution and detect the change in T2 value (ΔT2 = T2 sample - T2 blank). Each concentration gradient was measured in parallel three times.

[0095] Figure 9 Free Cu 2+ The magnetic relaxation response evaluation results of the solution are shown in the figure. As the concentration of copper chloride dihydrate (CuCl2·2H2O) solution increases, the water molecules 1 The transverse relaxation time (T2) of H decreases gradually, showing a significant concentration dependence. This phenomenon indicates that the paramagnetic Cu 2+ The ions have a good magnetic relaxation response to water protons.

[0096] Example 4

[0097] Copper ion binding efficiency evaluation experiment:

[0098] A gradient concentration of PDA@Ab solution (0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 mg / mL) was mixed with a CuCl2·2H2O solution (final concentration of 2 mM). The mixture was shaken at 37°C for 10 minutes and then centrifuged at 12000 rpm for 10 minutes to collect the supernatant. 400 μL of the supernatant was transferred to a nuclear magnetic resonance tube containing a heavy water capillary. The parameters of step (4) (CPMG pulse sequence) in Example 1 were used to determine the transverse relaxation time T2 of the solution and detect the change in T2 value (ΔT2 = T2 sample - T2 blank). Each concentration gradient was measured in parallel three times.

[0099] Figure 10 Adsorption of Cu by polydopamine-antibody conjugates 2+ The performance evaluation results are shown in the figure. As the concentration gradient of PDA@Ab complex increases, the adsorbed Cu 2+ Gradually increased, free Cu in the supernatant 2+ The concentration showed a decreasing trend, resulting in a gradual increase in the T2 change value. It is worth noting that when the PDA concentration exceeded 1.8 mg / mL, the unbound nanoparticles that remained after centrifugation at 12,000 rpm for 10 minutes caused a significant interference effect, resulting in a slight decrease in the T2 change value.

[0100] Example 5

[0101] (1) Copper ion concentration optimization experiment:

[0102] CuCl2·2H2O solutions with final concentrations of 0, 0.2, 0.5, 0.8, and 1 mg / mL were prepared and reacted with PDA@Ab (0.3 mg / mL) in a 37°C shaker for 10 min. The supernatant was centrifuged at 12,000 rpm for 10 min to obtain the supernatant. 400 μL of the supernatant was injected into an NMR tube and the parameters (CPMG pulse sequence) in step (4) of Example 1 were used to measure the transverse relaxation time T2 of the solution and detect changes in the T2 value (ΔT2 = T2 sample - T2 blank). Each concentration gradient was measured in parallel three times.

[0103] (2) Magnetic nanoparticle-antibody conjugate (MNP@Ab) concentration optimization experiment:

[0104] Specific binding was achieved by adding a gradient of MNP@Ab solutions (0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL) to 20 μL of mouse IgG antigen solution (1000 ng / mL). The volume was adjusted to 400 μL with PBS buffer and incubated at 37°C for 30 minutes. Subsequently, 63 μL of PDA@Ab probe (6.4 mg / mL) was added, and the volume was brought up to 500 μL with PBS, and the reaction continued for 30 minutes. After incubation, the supernatant was removed by magnetic separation for 3 minutes, and the complex was washed three times with 1 mL of PBST (containing 0.05% Tween 20). The complex was then resuspended in 450 μL of MES buffer (0.001 M, pH 5.5) and incubated with 50 μL of CuCl2·2H2O solution (5 mmol / L) at 37°C for 10 minutes. After magnetic separation, the change in T2 value of the supernatant was detected (ΔT2 = T2 sample - T2 blank). The parameters were set according to step (4) of Example 1. Each concentration gradient was measured in parallel three times to determine the optimal MNP@Ab concentration.

[0105] (3) Polydopamine-antibody conjugate (PDA@Ab) concentration optimization experiment:

[0106] The optimized MNP@Ab concentration system was used to specifically bind to 20 μL of mouse IgG antigen. The reaction system was adjusted to 400 μL with PBS and reacted at 37°C with constant temperature shaking for 30 minutes. Subsequently, gradient concentrations of PDA@Ab (0.4-1.2 mg / mL) were added to the above reaction system, and the total volume was made up to 500 μL with PBS buffer, and the reaction was continued at 37°C for 30 minutes. After incubation, the unbound components were removed by magnetic separation for 3 minutes, and after washing three times with PBST (containing 0.05% Tween 20), the complex was dispersed in 450 μL of MES buffer (0.001 M, pH 5.5), and 50 μL of CuCl2·2H2O (5 mmol / L) was added to react at 37°C for 10 minutes. After magnetic separation, the change in the supernatant T2 value was measured (ΔT2 = T2 sample - T2 blank). According to the detection parameters in step (4) of Example 1, each concentration gradient was measured in parallel three times to determine the optimal PDA@Ab concentration.

[0107] Figures 11 to 13 The key parameter optimization results of the sandwich immunomagnetic relaxation sensing system are presented, including Cu 2+ Working concentration ( Figure 11 ), MNP@Ab concentration ( Figure 12 ) and PDA@Ab concentration ( Figure 13In the presence of target IgG, MNP@Ab and PDA@Ab form a sandwich complex through specific binding. After washing with PBST buffer, the concentration of PDA@Ab bound to the sandwich complex in the system drops below 1.8 mg / mL. Therefore, it is necessary to investigate the effect of low concentration PDA@Ab (0.3 mg / mL) on Cu 2+ Response characteristics. Experimental data show that 0.5mM Cu 2+ It can produce the most significant T2 signal change; 0.4mg / mL MNP@Ab can achieve the best magnetic separation efficiency; and 0.8mg / mL PDA@Ab can effectively control the background signal while ensuring detection sensitivity.

[0108] Figures 14 and 15 Based on the optimized detection system parameters, the present invention established a standard curve and systematically evaluated the quantitative detection performance of the magnetic relaxation sensor by testing mouse IgG standards at different concentration gradients (50-4000 ng / mL). The change in T2 relaxation time (ΔT2) and the logarithm of the target concentration (log C) showed a linear correlation in the range of 50-1000 ng / mL. The linear regression equation was Y = 112.50log C - 160.16 (r = 0.9937, n = 3), and the limit of detection (LOD) was 13.5 ng / mL.

[0109] Example 6

[0110] The immunoassay anti-interference experimental process is as follows:

[0111] (a) Interference sample preparation: Five test systems were set up: a positive control (500 ng / mL IgG), a single interference group (500 ng / mL hemoglobin (Hb); 2500 ng / mL IgM; 2500 ng / mL IgA), a combined interference group (a mixture of 500 ng / mL IgG, 2500 ng / mL IgM, and 2500 ng / mL IgA), and a negative control (PBS buffer).

[0112] (b) Standard assay procedure: According to the optimized parameters, final concentrations of 0.4 mg / mL MNP@Ab and 0.8 mg / mL PDA@Ab were added, along with 0.5 mmol / L CuCl2·2H2O solution. The mixture was reacted at 37°C for 10 min, and the supernatant was obtained by magnetic separation.

[0113] (c) Signal detection analysis: The T2 value of the supernatant was measured and the change in T2 value was detected (ΔT2 = T2 sample - T2 blank). The test parameters were set as in step (4) of Example 1. Each concentration gradient was measured in parallel three times.

[0114] Test results see Figure 16 ,like Figure 16As shown, the T2 value of the single interference group (IgM / IgA / Hb) did not change significantly, indicating that non-target immunoglobulins and hemoglobin did not significantly interfere with the detection system. In the competitive binding group, even in the presence of 5 times the concentration of interfering substances (IgM / IgA), 500ng / mL IgG can still induce significant changes in T2 relaxation time, and its signal intensity is not significantly different from that of the single target group. This shows that the sensor prepared by the present invention has good anti-interference ability and can achieve specific detection of target immunoglobulin IgG in the presence of other non-target immunoglobulins and hemoglobin.

[0115] Example 7

[0116] The sandwich immunomagnetic relaxation switch sensor prepared in Example 1 was used to test actual samples. The test process was as follows:

[0117] Serum samples from 5 SPF-grade BALB / c female mice (8 weeks old) were tested and evaluated. The serum was prepared as follows: whole blood was collected into a centrifuge tube by the eyeball blood sampling method, and allowed to stand at room temperature for 1 hour to promote blood clot formation, and then centrifuged at 3000 rpm for 15 minutes at 4°C. The upper serum obtained after centrifugation was transferred to a new centrifuge tube. In order to completely remove residual cell debris, the initially separated serum was centrifuged again at 3000 rpm for 10 minutes. Finally, the clarified supernatant was collected and divided into sample tubes and placed in a -80°C refrigerator for long-term storage. Before testing, the original serum was diluted 1000 times to make the IgG concentration in the linear detection range of 50-1000 ng / mL for actual sample testing.

[0118] A single sample was selected for spike validation: the background IgG content was first determined, and then standard antigens at 50%, 100%, and 150% of the background value were added. According to the operating procedure of step (4) in Example 1, 0.4 mg / mL MNP@Ab, 0.8 mg / mL PDA@Ab, and 0.5 mM CuCl2·2H2O were added in sequence. After constant temperature oscillation reaction at 37°C for 10 minutes, the supernatant was collected by magnetic separation, and the T2 value was determined. The change in T2 value (ΔT2 = T2 sample - T2 blank) was detected. The test parameters were set as in step (4) in Example 1, and each concentration gradient was measured in parallel 3 times. The test results are shown in Tables 1 and 2.

[0119] Table 1 shows the recovery rates of spiked samples. In this example, Mouse Serum No. 1 was used as the real sample for spiked recovery experiments. The original concentration before spike addition was 239.10 μg / mL. As shown in Table 1, the magnetic relaxation switch sensor achieved recovery rates of 101.02% to 104.24% for mouse IgG, with relative standard deviations (RSDs) ranging from 1.33% to 5.28%, demonstrating the high accuracy of this experimental method.

[0120] Table 1 Actual sample spiked recovery test results

[0121]

[0122] Table 2 shows the detection of IgG content in serum samples of five healthy female BALB / c mice (8 weeks old). After testing, the results of the five mouse serum samples were 239.10, 208.02, 213.99, 219.67, and 221.64 μg / mL, respectively, which are consistent with the results detected by the traditional enzyme-linked immunosorbent assay commercial kit, fully confirming the accuracy of this sensing method in actual biological sample detection.

[0123] Table 2 Actual sample testing

[0124]

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method based on magnetic separation and Cu 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: The following steps are involved: (1) Polydopamine nanoparticles are obtained by spontaneous oxidative polymerization of dopamine under alkaline conditions; (2) The active groups on the surface of polydopamine nanoparticles are covalently bonded to the amino groups of the antibody through Michael addition reaction to obtain polydopamine-antibody conjugates; (3) mixing the amino-ferroferric oxide magnetic beads with the activated antibody solution to undergo a coupling reaction, followed by washing, magnetic separation, and blocking to obtain a magnetic nanoparticle-antibody conjugate; (4) The magnetic nanoparticle-antibody conjugate and the antigen solution are mixed to undergo a binding reaction to obtain a mixture, polydopamine-antibody conjugate is added to the mixture, and the reaction is continued to obtain a sandwich immune complex, which is a sandwich immune magnetic relaxation switch sensor.

2. A method based on magnetic separation and Cu according to claim 1 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: In step (1), the specific preparation process of polydopamine nanoparticles is: Ethanol and ultrapure water are mixed, and then ammonia water is added. After mixing evenly, dopamine solution is quickly injected and stirred at room temperature for reaction. The precipitate is then collected by centrifugation and washed. The product obtained by washing is polydopamine nanoparticles; the polydopamine nanoparticles are redispersed in ultrapure water to obtain a polydopamine nanoparticle suspension for later use.

3. A method based on magnetic separation and Cu according to claim 2 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: In step (2), the specific preparation process of the polydopamine-antibody conjugate is as follows: The polydopamine nanoparticle suspension was mixed with the antibody solution and incubated to promote full binding of the antibody to the surface of the polydopamine nanoparticles. Subsequently, a bovine serum albumin solution was added to block excess sites and the incubation was continued. After the reaction was completed, the polydopamine-antibody conjugate was obtained by centrifugation and washing. The polydopamine-antibody conjugate was dispersed in PBS buffer to obtain a polydopamine-antibody conjugate dispersion, which was stored at 4°C for future use.

4. A method based on magnetic separation and Cu according to claim 3 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: In step (3), the specific preparation process of the magnetic nanoparticle-antibody conjugate is as follows: (31) Pretreatment of magnetic nanoparticles: Take aminated ferroferric oxide magnetic beads, add them to ultrapure water for washing, and then dilute to ultrapure water to obtain an aminated ferroferric oxide magnetic bead dispersion; (32) Antibody activation: dilute the antibody with PBS buffer, then add EDC and NHS solution to obtain activated antibody solution; (33) Coupling reaction: Add the amino-containing ferroferric oxide magnetic bead dispersion to the activated antibody solution to undergo coupling reaction; (34) Cleaning and purification: After the coupling reaction, magnetic separation was performed and the mixture was washed several times with PBST buffer; (35) Site blocking: After washing, add bovine serum albumin solution for blocking reaction; (36) Product storage: After blocking, the product was magnetically separated, washed with PBS buffer, and then resuspended in PBS buffer to obtain a magnetic nanoparticle-antibody conjugate dispersion, which was stored at 4°C for future use.

5. A method based on magnetic separation and Cu according to claim 4. 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: In step (4), the specific preparation process of the sandwich immune complex is as follows: (41) Antigen-antibody binding reaction: Mix the magnetic nanoparticle-antibody conjugate dispersion, antigen solution, and PBS buffer, and shake for reaction; (42) Addition of signal probe: Then, polydopamine nanoparticle suspension was added to the system after the oscillation reaction, PBS buffer was added, and the oscillation reaction was continued; (43) After the reaction, the cells were placed on a magnetic separation rack to remove the unbound free components in the supernatant and then washed repeatedly with PBST buffer to obtain the sandwich immune complex.

6. A method based on magnetic separation and Cu according to claim 5. 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: The antibody solution is an IgG antibody solution, and the antigen solution is an IgG antigen solution.

7. A method based on magnetic separation and Cu according to claim 6. 2+ The preparation method of the sandwich immunomagnetic relaxation switch sensor is characterized by: In step (42), after supplementing with PBS buffer, the final concentration of the polydopamine-antibody conjugate dispersion is 0.8 mg / mL; the final concentration of the magnetic nanoparticle-antibody conjugate dispersion is 0.4 mg / mL.

8. A method based on magnetic separation and Cu 2+ A sandwich immunomagnetic relaxation switch sensor is characterized by: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. A method based on magnetic separation and Cu according to claim 8. 2+ Application of sandwich immunomagnetic relaxation switch sensor for immunoglobulin detection.

10. A method based on magnetic separation and Cu according to claim 9. 2+ Application of a sandwich immunomagnetic relaxation switch sensor, characterized by: The following steps are involved: S1: Resuspend the sandwich immune complex in MES buffer and then add copper chloride solution for reaction; S2: After the reaction is completed, the supernatant is collected by magnetic separation and the transverse relaxation time T2 sample of the water molecules in the supernatant is measured using a CPMG pulse sequence. The blank sample T2 blank is used as a reference and the change value △T2 is calculated according to △T2 = T2 sample - T2 blank; S3: Calculate the concentration of immunoglobulin based on the transverse relaxation time change value △T2.

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