Immune ratio electrochemical sensing platform based on steric hindrance response and preparation method

Abstract

The invention discloses an immune ratio electrochemical sensing platform based on steric hindrance response and a preparation method, and relates to the technical field of biosensors, and the preparation method mainly comprises the following steps: taking cerium dioxide and a ferrous sulfate heptahydrate solution to react to prepare a ferroferric oxide nano material; the preparation method comprises the following steps: dispersing a ferroferric oxide nano material into a molybdenum disulfide solution to prepare magnetic nano particles, heating a chloroauric acid aqueous solution and a trisodium citrate solution to react until the solution becomes red to obtain a gold nano particle solution, and uniformly dispersing the magnetic nano particles into a phosphate buffer solution to obtain a gold nano particle solution; the preparation method comprises the following steps: preparing a magnetic nano-particle layer, depositing the magnetic nano-particle layer on the surface of a pencil lead electrode to obtain a magnetic nano-particle layer, coating the obtained pencil lead electrode with a gold nano-particle layer, and coating the gold nano-particle layer of the pencil lead electrode with a C reactive protein antibody by using an EDC / NHS cross-linking agent; according to the sensor disclosed by the invention, the C-reactive protein antibody is fixed on the sensor, so that high-accuracy, sensitive and high-selectivity detection on the CRP is effectively realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, in particular to an immunoratio electrochemical sensing platform based on steric hindrance response and a preparation method thereof. Background Art

[0002] C-reactive protein (CRP) is a reactive protein that rises sharply when the body is damaged or infected. The normal concentration range of CRP in healthy human plasma is 1 to 3 mg / L -1 If the concentration of CRP in plasma is too high, it will cause cardiovascular disease and inflammatory diseases. Therefore, sensitive detection of CRP has certain value in diagnosing systemic inflammation and infection and evaluating the efficacy of antibacterial drugs. At present, the clinical methods used for CRP detection mainly include chemiluminescence, enzyme-linked immunosorbent assay and immunoturbidimetry, etc. However, the equipment used in chemiluminescence and enzyme-linked immunosorbent assay is expensive and the operation process is complicated; the sensitivity of immunoturbidimetry is not high and the linear range is narrow. Therefore, it is necessary to establish a detection method with simple operation, fast detection response speed, accurate quantification and high sensitivity for analyzing CRP.

[0003] In recent years, electrochemical detection technology has gradually become a research hotspot due to its advantages such as high sensitivity, accurate detection, low cost, good controllability, and simple and easy operation of equipment. Electrochemical immunosensor is an important branch of electrochemical biosensor, which combines three technologies: immunoassay technology for specific recognition, high-sensitivity sensing technology, and electrochemical analysis technology. Using antibodies to antigens as recognition elements, the specific immune reaction occurring on the sensor surface can cause the electrical signal to change, thereby detecting a biosensor of the target object. It has the advantages of high efficiency and rapidity of electrochemical sensing and specific anti-interference of biorecognition.

[0004] However, due to the interference of problems such as electrode area deviation and electrode load density, the stability, reliability and reproducibility of traditional electrochemical aptamer sensors are affected to a certain extent. There is an urgent need to develop an electrochemical biosensor with high anti-interference and high selectivity. Summary of the invention

[0005] In view of this, the present invention proposes an immune ratiometric electrochemical sensing platform and preparation method based on steric hindrance response, in which gold nanoparticle-magnetic nanomaterial nanocomposite materials are used as materials for modifying lead core electrodes to form a signal amplification sensor. For the first time, a homemade lead core electrode is used as a working electrode, and a ratio strategy is adopted to detect and analyze CRP. Combined with the immune response strategy, an immune ratiometric electrochemical CRP aptamer sensor is constructed.

[0006] The preparation method of the immunoratio electrochemical sensing platform based on steric hindrance response disclosed in the present invention comprises the following steps:

[0007] Step S1: adding cerium dioxide to a sodium hydroxide solution and stirring at 70-80° C. for 30 min, then adding a hydrochloric acid solution of ferrous sulfate heptahydrate and stirring for 30 min, collecting a black precipitate, washing it, and drying it for 24 h to obtain a ferrosoferric oxide nanomaterial;

[0008] Step S2: dispersing the ferroferric oxide nanomaterial into the molybdenum disulfide solution, stirring at room temperature for 30 minutes and then drying for 24 hours to obtain magnetic nanoparticles;

[0009] Step S3: heating the chloroauric acid aqueous solution to boiling, adding the trisodium citrate solution with stirring, and continuing to heat until the solution turns red, and then cooling to room temperature to obtain a gold nanoparticle solution;

[0010] Step S4: uniformly dispersing the magnetic nanoparticles in a 0.5 mol / L phosphate buffer solution with a pH of 7.3 by ultrasonication, depositing the resulting solution on the surface of a pencil core electrode to obtain a magnetic nanoparticle layer, and then modifying the surface of the magnetic nanoparticle layer of the pencil core electrode with a gold nanoparticle solution by a drop coating method to obtain a pencil core electrode coated with a gold nanoparticle layer and drying the pencil core electrode;

[0011] Step S5: dripping EDC / NHS crosslinking agent on the gold nanoparticle layer of the pencil core electrode, drying at room temperature, and then coating with C-reactive protein antibody, washing and drying to obtain the electrochemical sensing platform.

[0012] One embodiment of the present invention is that, in parts by weight, the ratio of cerium dioxide to ferrous sulfate heptahydrate is 1:5-8.

[0013] An embodiment of the present invention is that, in parts by weight, the ratio of molybdenum disulfide to ferrosoferric oxide is 1:1-10.

[0014] One implementation mode of the present invention is that the mass fraction of the chloroauric acid aqueous solution is 0.01%, the mass fraction of the trisodium citrate solution is 1%, and the ratio of the trisodium citrate solution to the chloroauric acid aqueous solution is 1:25-30 in volume ratio.

[0015] One embodiment of the present invention is that the amount of the magnetic nanoparticles dispersed into the phosphate buffer is 0.5 g of the magnetic nanoparticles per 100 mL of the phosphate buffer.

[0016] In one embodiment of the present invention, the method for depositing the magnetic nanoparticle layer on the surface of the pencil core electrode in step S4 is cyclic voltammetry.

[0017] An embodiment of the present invention is that the concentration of EDC in the EDC / NHS cross-linking agent is 0.4 mol / L, and the concentration of NHS is 0.1 mol / L.

[0018] One embodiment of the present invention is that the concentration of the C-reactive protein antibody is 1 mg / L, and the ratio of the C-reactive protein antibody to the phosphate buffer is 1:1000 in terms of volume.

[0019] In addition, the present invention also discloses an immunoratio electrochemical sensing platform based on steric hindrance response prepared by the above method.

[0020] The technical effects of the present invention are:

[0021] The sensor of the present invention uses the cross-linking effect of EDC / NHS to fix C-reactive protein antibody (anti-CRP) on the sensor to form an immune biosensor. In the presence of CRP, the antibody and CRP specifically bind to each other, hindering the electron transfer at the sensor interface, eliminating the influence of the environment on the detection, improving the detection sensitivity and accuracy, and thus effectively achieving high accuracy, sensitivity and high selectivity detection of CRP. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A is a physical picture of the gold nanoparticles in Example 1 of the present invention;

[0023] Figure 1 B is a TEM characterization image of the gold nanoparticles in Example 1 of the present invention;

[0024] Figure 2 A is the TEM and electron diffraction pattern of the magnetic nanoparticles in Example 1 of the present invention;

[0025] Figure 2 B is the hysteresis loop spectrum of the magnetic nanoparticles in Example 1 of the present invention;

[0026] Figure 2 C is the XRD spectrum of the magnetic nanoparticles in Example 1 of the present invention;

[0027] Figure 2 D is the XPS spectrum of the magnetic nanoparticles in Example 1 of the present invention;

[0028] Figure 3 A is the CV response curve of the electrode modification process in Example 1 of the present invention;

[0029] Figure 3 B is the EIS spectrum of the electrode modification process in Example 1 of the present invention;

[0030] Figure 3 C is a peak current response curve of different concentrations of anti-CRP in Example 1 of the present invention;

[0031] Figure 3 D is a curve diagram showing the effect of immune response time on peak current in Example 1 of the present invention;

[0032] Figure 4 A is different Fe[(CN)6] in Example 1 of the present invention 3- / 4- Cyclic voltammetry of the solution;

[0033] Figure 4 B is a differential pulse voltammetric response diagram and voltammetric spectra of different methylene blue concentrations in Example 1 of the present invention;

[0034] Figure 4 C is the cyclic voltammetry spectrum in Example 1 of the present invention;

[0035] Figure 4 D is the differential pulse voltammetric response diagram in Example 1 of the present invention;

[0036] Figure 5 A is the cyclic voltammogram at different pH values ​​in Example 1 of the present invention;

[0037] Figure 5 B is a differential pulse voltammetric response diagram at different pH values ​​in Example 1 of the present invention;

[0038] Figure 5 C is the CV graph at different scanning rates in Example 1 of the present invention;

[0039] Figure 5 D is a correlation curve between the redox peak potential and the scan rate 1 / 2 power in Example 1 of the present invention;

[0040] Figure 6 A is the 2mM [Fe(CN)6] 3- / 4- DPV response curves of different MB concentrations;

[0041] Figure 6 B is the different [Fe(CN)6] 3- / 4- DPV response curve;

[0042] Figure 7 A is the DPV response curve of different concentrations of C-reactive protein in Example 1 of the present invention;

[0043] Figure 7 B is the CRP concentration in Example 1 of the present invention and I Fe / I MB The standard curve of

[0044] Figure 7 C is the linear relationship between the CRP concentration and the MB oxidation peak current change in Example 1 of the present invention;

[0045] Figure 7 D is the CRP concentration and [Fe(CN)6] in Example 1 of the present invention 3- / 4- The linear relationship between the oxidation peak current change and

[0046] Figure 8 A is the sensor in Example 1 of the present invention at 200 ng·mL -1 DPV response curve under CRP;

[0047] Figure 8 B is a histogram of the CRP selectivity experiment in Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used therein are all commercially available unless otherwise specified.

[0049] Embodiment 1:

[0050] (1) Synthesis of magnetic nanomaterials:

[0051] 5.0g CeO 2 Add to a solution containing 1 mol·L -1 The suspension was stirred at 80°C for 30 min in a three-necked flask containing 250 mL of NaOH solution, and then 125 mL of 0.87 mol·L -1 FeSO 4 7H2O solution (0.1 mol·L -1 HCl) was slowly dripped into the mixture, and the black precipitate obtained was collected by magnetic field after 30 minutes, washed with pure water and ethanol until neutral, and dried in vacuum at 50°C for 24 hours. 3 O 4 Nanomaterials were ultrasonically dispersed in 50 mL of 0.1 mol·L -1 MoS 2 The solution was dried for 24 h after 30 min to collect the black solid FeNPs@MoS 2 .

[0052] (2) Synthesis of gold nanomaterials:

[0053] Place 100 mL of 0.01% chloroauric acid aqueous solution in a round-bottom flask and heat to boiling. Add 4 mL of 1% trisodium citrate solution accurately while stirring. Continue heating until the solution changes color to red. Stop heating, wait for the solution to return to room temperature, and store in a 4°C refrigerator.

[0054] (3) Pretreatment of lead core electrodes:

[0055] Preparation method of PGE: Select a syringe with a smaller caliber and squeeze the insulating glue into the needle-free syringe. Align the caliber with one end of the pen core tube and push the insulating glue in. When the amount of insulating glue reaches 2 / 3 of the pen core tube, hold the interface and insert the pencil core from the other end of the pen core tube. The length exposed at the interface after insertion is defined as h. Gently wipe off the excess insulating glue with paper, and place the prepared product in a ventilated place for 3-5 days to wait for the insulating glue to solidify. Before modifying the electrode, use a mixture of alumina powder of different particle sizes and deionized water to polish the h end of PGE on the antler skin, then ultrasonically treat in ethanol and deionized water for 5 minutes to further remove impurities on the electrode surface, and dry for use.

[0056] (4) Preparation of immunomodified electrodes:

[0057] Take 50 mg of magnetic nanoparticles (FeNPs@MoS 2 ) were dispersed in 10 mL 0.5 M phosphate buffer (PBS) and uniformly dispersed by ultrasonic. Then, FeNPs@MoS were deposited on the surface of pencil graphite electrode (PGE) by cyclic voltammetry (CV). 2 , FeNPs@MoS 2 -PGE. Gold nanomaterials (AuNPs) were modified on FeNPs@MoS using drop coating method. 2 -PGE, and AuNPs / FeNPs@MoS was prepared after drying. 2 -PGE. Then the prepared 0.4M EDC / 0.1M NHS was drop-coated on the AuNPs / FeNPs@MoS 2 -PGE surface, after the electrode is dried, 10 μL of 1 mg / L anti-CRP is applied to the electrode surface, dried at low temperature, rinsed with 0.05 M pH = 7.3 PBS to remove the unfixed antibody, and dried to obtain anti-CRP / AuNPs / FeNPs@MoS 2 -PGE. When not in use, the electrodes were immersed in PBS and stored at 4°C.

[0058] In order to better illustrate the technical effects of the present invention, performance evaluations are provided below for relevant embodiments or comparative examples.

[0059] 1. Morphological characterization of gold nanoparticles:

[0060] Gold nanoparticles (AuNPs) are used as an amplifier of electrical signals to improve the sensitivity of the developed immunosensor. The physical image and TEM characterization image of the AuNPs prepared in Example 1 are shown in FIG. Figure 1 As shown, the solution exhibits a clear pink color, which is similar to the color of gold nanoparticles prepared by other researchers. They are visualized using a transmission electron microscope (TEM) and their actual size distribution is determined based on the TEM image. It can be observed that the morphology of AuNPs is mostly spherical. From the enlarged TEM image in the upper right corner, it can be seen that the average diameter of AuNPs is 25±5nm, which shows that gold nanoparticles have been successfully prepared in this experiment.

[0061] 2. Characterization of the physical and chemical properties of magnetic nanoparticles:

[0062] The magnetic nanoparticles (FeNPs@MoS 2 ) to verify the synthesis process. Figure 2 As shown in A, after hydrothermal treatment, the product presents a huge bulk structure, and the stripes with a lattice spacing of 0.60 nm correspond to MoS 2 The (002) crystal plane of the nanoparticles. The 0.32 nm lattice fringes in the figure can be attributed to the (111) crystal plane of the cerium oxide nanoparticles. The lattice fringes at 0.30 nm in the figure can be attributed to the Fe 3 O 4 (220) crystal plane.

[0063] FeNPs@MoS 2 The X-ray diffraction spectrum of Figure 2 As shown in Figure B, according to the standard card (JCPDS-19-0629), the characteristic diffraction peaks at 2θ = 30.1°, 35.4°, and 57.3° correspond to the (220), (311), and (511) crystal planes of ferroferric oxide, respectively. 2 The diffraction peaks are shown at 28.5°, 33.1°, 47.4° and 56.3°, corresponding to (111), (200), (202), (311) crystal planes, which are attributed to CeO 2 The fluorite cubic structure (JCPDS card number 81-0792). And when the synthesized magnetic nanomaterials are mixed with MoS 2 After the composite, obvious MoS 2 (002) crystal plane characteristic diffraction peak.

[0064] The room temperature magnetization curve of FeNPs is shown in Figure 2As shown in C, it can be seen from the hysteresis curve (VSM) of FeNPs nanoparticles that FeNPs@MoS 2 The hysteresis loop curve of the nanoparticles presents an "S" shape and passes through the origin, which indicates that the synthesized nanomaterial has good superparamagnetism and its magnetic saturation intensity is 12.5emu / g.

[0065] FeNPs@MoS 2 The XPS scan spectrum is as follows Figure 2 D, it can be seen from the figure that the main elements contained in the composite nanomaterial are Ce, Fe and C. The small figure in the upper right corner shows two strong peaks of Mo 3d, which confirms that Mo 4+ There is no peak at 226.4 eV that can be assigned to S2s, which indicates the presence of S 2- Therefore, the XPS results confirm the interaction between FeNPs and MoS 2 Assembled. Among them, Fe 2p 3 / 2 There are two peaks at 710.47 eV and 712.48 eV, which represent Fe 2+ and Fe 3+ .Fe 2p 1 / 2 The peak is also divided into 724eV (Fe 2+ ) and 726eV(Fe 3+ ) peaks, it can be seen that Fe 2+ The content is higher than Fe 3+ The photoelectron peak between 870-920 eV can be attributed to the +4 valence Ce element. In summary, the synthesized FeNPs@MoS 2 The composite material contains Fe and Ce elements stably, which indicates that the composite material has been successfully synthesized.

[0066] 3. Electrochemical behavior test of immunosensor:

[0067] The CV response curve of the immunosensor electrode modification process in Example 1 is as follows: Figure 3 As shown in A, in the presence of 2 mM [Fe(CN)6] 3- / 4- The test was carried out in PBS buffer solution containing 0.05M KCl (pH=7.0), with a scanning rate of 100mV / s and a scanning potential of -0.2~0.6V. As the scanning proceeded, a pair of obvious reversible redox peak currents appeared in the unmodified PGE, indicating that the prepared bare PGE had good conductivity. 2When deposited on the surface of PGE, the sensor's response current increased significantly due to its excellent conductivity as a metal nanoparticle. At the same time, AuNPs have excellent catalytic properties, conductivity and biocompatibility. 2 When the PGE surface is formed, the current response further increases, indicating that AuNPs / FeNPs@MoS 2 The composite nanomaterials have good electron transfer ability and have been successfully modified on PGE. When anti-CRP was fixed to AuNPs / FeNPs@MoS by EDC / NHS crosslinker 2 -PGE surface, due to the certain insulating properties of the antibody, the electron transfer on the electrode surface is hindered and the redox peak current is significantly reduced.

[0068] The EIS spectrum of the electrode modification process is shown in Figure 3 As shown in Figure B, the electron transport on the surface is mainly investigated. 2 -PGE has the smallest electron transfer resistance Ret value and the highest slope of the straight line. This shows again that AuNPs and FeNPs@MoS 2 The synergistic effect of nanomaterials can greatly increase the electron transfer rate. When anti-CRP was fixed on the electrode through a crosslinker, the impedance increased significantly, indicating that the antibody was successfully fixed to the electrode surface, blocking the electron transfer of the electrochemical probe. This EIS test result is consistent with the above CV results. The results show that the CRP immunosensor probe was successfully prepared by the layer-by-layer modification method and can be used for the detection of CRP.

[0069] The electrical signal response of different anti-CRP concentrations is as follows Figure 3 As shown in C, in the range of 0.2 to 1.0 μM, as the concentration of the fixed antibody increases, [Fe(CN)6] 3- / 4- The peak current signal gradually decreased. When the anti-CRP mass concentration reached 1 μM, the antibody concentration continued to increase, and the peak current response decreased slowly and showed a platform, indicating that the binding sites on the electrode surface had reached saturation.

[0070] Figure 3 D is the curve of the effect of immune response time on peak current. The immune sensor anti-CRP / AuNPs / FeNPs@MoS prepared in Example 1 2 -PGE was immersed in a solution containing 500 ng·mL -1 The cells were incubated in CRP solution for 10-80 min and then rinsed with PBS buffer to remove antigens not bound to the antibody. Figure 3As can be seen in D, the detection signal decreases significantly with the increase of incubation time. This is because the amount of immune complexes generated on the electrode surface increases with the increase of incubation time, thereby blocking electron transfer. When the incubation time reaches 60 minutes, the detection signal no longer decreases if the incubation time is continued to increase. At this time, the binding of antigen and antibody is close to saturation.

[0071] 4. Characterization of electroactive reference substance concentration:

[0072] The optimal concentration of electroactive substances was determined by comparing the concentrations of MB and [Fe(CN)6] 3- / 4- The CV and the corresponding DPV are determined, and the test results are as follows Figure 4 As shown, the experimental results show that 2mM [Fe(CN)6] 3- / 4- The signal response intensity of [Fe(CN)6] is comparable to that of 16 μM MB. 3- / 4- and MB at fixed concentrations of 2 mM and 16 μM.

[0073] 5. Evaluation of pH and scanning rate selection:

[0074] Since CRP is not an electroactive substance, [Fe(CN)6] is selected 3- / 4- The current response indirectly reflects the concentration change of CRP. MB is selected as the reference substance. In the electrolyte solution containing a two-component mixture, the change of the electrical signal shows a certain regularity. In the electrolyte solution, 2mM [Fe(CN)6] 3- / 4- and 16 μM MB, and 500 ng·mL -1 The pH of the electrolyte solution was optimized by adjusting the pH of the CRP using 0.05 M PBS buffer. The obtained electrolyte solutions were measured by DPV. The results are shown in Figure 5 As shown in Figures A and 5B, it can be seen that with the gradual increase in pH, the peak potential of the oxidation current of MB moves to the left / more negative potential direction and the intensity gradually increases, while Fe[(CN)6] 3- / 4- The peak intensity of Fe[(CN)6] decreases with increasing pH. It is worth noting that when pH is 7.3, 3- / 4- and MB have relatively high oxidation current peak intensities, which are comparable to those of 2 mM [Fe(CN)6] 3- / 4-The peak intensity of the oxidation current is almost the same as that of 16μM MB, the peak potential of the oxidation is not offset and the peak shape is good, which is very suitable for the effective construction of the ratio electrochemical sensing system proposed in this work. The pH of the human physiological environment or tissue microenvironment is about 7.3, and CRP is a protein in the human body, so pH = 7.3 can be selected as the detection solution pH of this work.

[0075] In order to better understand the electrochemical behavior of the anti-CRP / AuNPs / FeNPs-PGE immunosensor, the probe was investigated in the presence of 2 mM [Fe(CN)6] 3- / 4- and 16 μM MB, and contained 500 ng·mL -1 In the PBS electrolyte solution with CRP and pH = 7.3, the scanning rate range was changed to a~i: 10~120mV·s -1 , every 10mV·s -1 Test a set of data and observe how the peak current changes with the scan rate. The test results are as follows: Figure 5 As shown in C and D, with the increase of scanning rate, the peak current increases and the change of peak potential difference becomes larger and larger. -1 In the range, the redox peak current of MB has a linear relationship with the 1 / 2 power of the scan rate, oxidation peak: IMB(OP) = 9.39v1 / 2-32.06 (R2 = 0.9979); reduction peak: IMB(RP) = -29.73v1 / 2+23.63 (R2 = 0.9988), [Fe(CN)6] 3- / 4- The redox peak current has a linear relationship with the 1 / 2 power of the scan rate, oxidation peak: IOP = 26.91v1 / 2-4.86 (R2 = 9992); reduction peak: IRP = -23.84v1 / 2-21.94 (R2 = 0.9981). The results indicate that the chemical reaction process on the prepared immunosensor is controlled by the surface.

[0076] 6. Evaluation of the mutual impact between different components:

[0077] MB (12 μM) + [Fe(CN)6] 3- / 4- (1~10mΜ) and [Fe(CN)6] 3- / 4- (2 mM) + MB (10-20 μM) PBS electrolyte solution (pH = 7.3) and add 500 ng·mL -1 CRP, a series of DPV curves were tested and the experimental results were analyzed. The test results are as follows Figure 6 As shown in A, when the MB concentration is fixed, it is found that [Fe(CN)6] 3- / 4- The gradual increase in concentration caused [Fe(CN)6] 3- / 4-The peak intensity of the oxidation current of 2.5%-6.3% increased gradually, but it had only a negligible effect on MB. 3- / 4- Concentration of electrolyte solution was tested. Figure 6 B shows that the increase of MB concentration only affects [Fe(CN)6] 3- / 4- These results demonstrate that increasing the concentration of a single electroactive substance has little effect on the oxidation current peak of another substance whose concentration remains unchanged. This indicates that there is no interaction between the reference substance and the analyte, further confirming the feasibility of constructing the scheme in an electrolyte solution.

[0078] VII. Evaluation of the detection effect of immunosensor on CRP:

[0079] like Figure 7 As shown in the figure, the immunosensor responds to signals of different concentrations of CRP. As the concentration of CRP increases, the antigen-antibody undergoes a specific immune reaction. At the same time, the amount of immune complexes generated on the electrode surface also increases, blocking the electron transfer channels on the modified electrode surface, thereby causing the electron transfer performance of the electrode surface to decrease and the peak current value to decrease. Figure 7 A is the DPV response curve of the immunosensor. It can be clearly observed that as the CRP concentration increases, the response of the internal reference electroactive substance oxidation peak current is inversely proportional to it, which indicates that the macromolecular protein attaches to the electrode to form a steric hindrance, which reduces the peak current detected. When the concentration of CRP is between 0.01 and 800 ng·mL -1 When the range changes, MB and [Fe(CN)6] 3- / 4- By comparing the change trend of the oxidation peak current of Figure 7 B linear curve, it can be seen that the ratio of the two can form a good linear relationship, and the regression equation is: y1 = -1.4 × 10 -4 x+1.06, the correlation coefficient is 0.9859. The detection limit can be calculated as: 0.455fg·mL -1 The results show that there is a good ratio relationship between the two, which can be used to detect different concentrations of CRP.

[0080] like Figure 7 As shown in C, when the concentration of CRP is between 0.01 and 800 ng·mL -1 When the oxidation peak current of MB changes within the range of , the concentration of CRP antigen is negatively correlated, and the linear regression equations are: y = -0.09x + 181.34 (R 2 =0.9810); at the same time [Fe(CN)6] 3- / 4- The oxidation peak current also decreases with the increase of CRP concentration. The test results are as follows Figure 7D, which shows a good linear relationship: y = -0.11x + 191.74 (R 2 =0.9982). The above data show that the detection limits of the prepared immunosensors are: 0.147 fg·mL -1 and 0.088 fg·mL -1 .

[0081] Based on the above results, the performance of the immunosensor prepared in Example 1 was compared with other CRP immunosensors, and the results are shown in Table 1:

[0082] Table 1 Comparison of CRP determination using different modified electrodes

[0083]

[0084] From the results in Table 1, it can be seen that the immunosensor in this embodiment has a wide linear range and a low detection limit, which is due to the AuNPs / FeNPs@MoS 2 It has good electron transfer ability and biocompatibility, providing a good immunosensing platform for detection reactions.

[0085] 8. Immunosensor stability and anti-interference test:

[0086] For a concentration of 200 ng·mL -1 The CRP sensor was measured 8 times in parallel, and its DPV curve is as follows Figure 8 As shown in A, the relative standard deviation is 2.531%. The results show that the immunosensor proposed in this experiment has excellent repeatability, and the dual signal ratio strategy effectively enhances the stability of detection and reduces the error caused by human factors.

[0087] Figure 8 B shows the addition of 10 μM K + , Ca 2+ , Cl - 、SO4 2- , BSA, AA, L-try, L-tyr and 500ng·mL -1 Histogram of different DPV responses of the constructed sensing system after CRP. Figure 8 B shows that the immunosensor is sensitive to 500 ng·mL - 1 The response current of CRP changed the most, while that of MB and [Fe(CN)6] 3- / 4-The △I of the aptamer sensor was 45.25±1.22μA and 42.32±1.26μA respectively. The interference of other interfering factors was minimal, indicating that the aptamer sensor had high selectivity for CRP. Since the current change of the immunosensor was caused by the exposure of the negatively charged phosphate skeleton, due to its specific recognition ability, the prepared sensor had good anti-interference ability.

[0088] The modified immuno-PGE was used to detect CRP, and a spike recovery experiment was performed to reflect its applicability in water samples. The results are shown in Table 2:

[0089] Table 2 CRP spike recovery experiment

[0090]

[0091] Table 2 shows the results of the recovery experiment, indicating that the recovery is between 84.14% and 94.91%, and the RSD is relatively stable, which shows that this method meets the requirements of routine analysis. At the same time, it can be proved that it has good repeatability and reliable application value.

[0092] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the embodiments of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing an immunoratio electrochemical sensing platform based on steric hindrance response, characterized in that: The following steps are involved: Step S1: adding cerium dioxide to a sodium hydroxide solution and stirring at 70-80° C. for 30 min, then adding a hydrochloric acid solution of ferrous sulfate heptahydrate and stirring for 30 min, collecting a black precipitate, washing it, and drying it for 24 h to obtain a ferrosoferric oxide nanomaterial; Step S2: dispersing the ferroferric oxide nanomaterial into the molybdenum disulfide solution, stirring at room temperature for 30 minutes and then drying for 24 hours to obtain magnetic nanoparticles; Step S3: heating the chloroauric acid aqueous solution to boiling, adding the trisodium citrate solution with stirring, and continuing to heat until the solution turns red, and then cooling to room temperature to obtain a gold nanoparticle solution; Step S4: uniformly dispersing the magnetic nanoparticles in a 0.5 mol / L phosphate buffer solution with a pH of 7.3 by ultrasonication, depositing the resulting solution on the surface of a pencil core electrode to obtain a magnetic nanoparticle layer, and then modifying the surface of the magnetic nanoparticle layer of the pencil core electrode with a gold nanoparticle solution by a drop coating method to obtain a pencil core electrode coated with a gold nanoparticle layer and drying the pencil core electrode; Step S5: dripping EDC / NHS crosslinking agent on the gold nanoparticle layer of the pencil core electrode, drying at room temperature, and then coating with C-reactive protein antibody, washing and drying to obtain the electrochemical sensing platform.

2. The method for preparing an immunoratio electrochemical sensing platform based on steric hindrance response according to claim 1, characterized in that: In parts by weight, the ratio of cerium dioxide to ferrous sulfate heptahydrate is 1:5-8.

3. The method for preparing a steric hindrance response-based immunoratio electrochemical sensing platform according to claim 1, characterized in that: In parts by weight, the ratio of molybdenum disulfide to ferrosoferric oxide is 1:1-10.

4. The method for preparing a steric hindrance response-based immunoratio electrochemical sensing platform according to claim 1, characterized in that: The mass fraction of the chloroauric acid aqueous solution is 0.01%, the mass fraction of the trisodium citrate solution is 1%, and in terms of volume ratio, the ratio of the trisodium citrate solution to the chloroauric acid aqueous solution is 1:25-30.

5. The method for preparing a steric hindrance response-based immunoratio electrochemical sensing platform according to claim 1, characterized in that: The amount of the magnetic nanoparticles dispersed in the phosphate buffer is 0.5 g of the magnetic nanoparticles per 100 mL of the phosphate buffer.

6. The method for preparing a steric hindrance response-based immunoratio electrochemical sensing platform according to claim 1, characterized in that: The method for depositing the magnetic nanoparticle layer on the surface of the pencil core electrode in step S4 is cyclic voltammetry.

7. The method for preparing a steric hindrance response-based immunoratio electrochemical sensing platform according to claim 1, characterized in that: The concentration of EDC in the EDC / NHS cross-linking agent is 0.4 mol / L, and the concentration of NHS is 0.1 mol / L.

8. The method for preparing an immunoratio electrochemical sensing platform based on steric hindrance response according to claim 1, characterized in that: The concentration of the C-reactive protein antibody is 1 mg / L, and the ratio of the C-reactive protein antibody to the phosphate buffer is 1:1000 by volume.

9. An immunoratio electrochemical sensing platform based on steric hindrance response, characterized in that: The method is prepared by any one of claims 1 to 8.

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