An igf-2 electrochemical biosensor based on boron affinity and its preparation method and application

Abstract

The application provides an IGF-2 electrochemical biosensor based on boron affinity and a preparation method and application thereof, the application uses amino carbon nanotubes as a carrier, then 4-formylphenylboronic acid (4-FPBA) is covalently anchored to the surface of the MWCNTs to synthesize nanomaterial (NH2-MWCNTs@4-FPBA); the NH2-MWCNTs@4-MPBA is modified on an electrode, then IGF-2 templates are captured through boron affinity, and then a molecular imprinting layer MIP generated by electro-polymerization of aniline and meta-aminobenzenesulfonic acid is plated; after the IGF-2 templates are dissociated and removed in an acidic solution, three-dimensional cavities complementary to the IGF-2 templates are formed in the molecular imprinting layer MIP, and the molecular imprinting layer MIP can be used for detection of IGF-2. The application realizes detection of the concentration of IGF-2 in a sample through the way of boron affinity and molecular imprinting instead of an antibody, so that the IGF-2 detection is more rapid and simple, has high sensitivity and low cost.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis technology, specifically relating to an insulin growth factor-2 (IGF-2) electrochemical biosensor based on boric acid affinity, its preparation method, and its application. Background Technology

[0002] Insulin-like growth factor (IGF) is a group of growth-promoting polypeptides widely distributed in human tissues such as the liver, kidneys, lungs, heart, brain, and intestines. IGF in milk is highly homologous to insulin; 45-50% of the amino acid sequence of the IGF molecule is identical to that of insulin. Both have a certain affinity for each other's specific receptors, and IGF also possesses some of the functions of insulin. Therefore, IGF can effectively promote glucose transport, promote the synthesis of glycogen, fat, and protein, and significantly lower blood glucose levels, which is closely related to the prevention and treatment of diabetes. Developing dairy products that regulate blood glucose is of great significance for preventing the occurrence of carbohydrate metabolism diseases in humans.

[0003] Several methods for detecting IGF-2 levels have been established, including enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and optical immunoassay. These strategies have shown advantages such as high sensitivity and accuracy; however, each analytical technique has one or more drawbacks, such as time consumption, excessive reagent consumption, high cost, and narrow dynamic range. For example, ELISA is widely used as a sandwich-based method for detecting biomarkers. However, its limitations include poor sensitivity and a long pretreatment process. Summary of the Invention

[0004] The purpose of this invention is to solve the problems in the prior art and provide an IGF-2 electrochemical biosensor based on boric acid affinity, its preparation method and application. This invention uses boric acid affinity and molecular imprinting to replace antibodies to detect the concentration of IGF-2 in samples, making IGF-2 detection faster, simpler, more sensitive and lower in cost.

[0005] This invention is achieved through the following technical solution:

[0006] A method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity includes the following steps:

[0007] Step 1: Using a thin film coated with a metal catalyst as a substrate, multi-walled carbon nanotubes are prepared by vapor-phase chemical deposition using a carbon source gas and a carrier gas.

[0008] Step 2: After purification, multi-walled carbon nanotubes are added to a mixture of concentrated sulfuric acid and nitric acid, sonicated, separated into solid and liquid, washed, and dried to obtain carboxylated MWCNTs;

[0009] Step 3: Disperse carboxylated MWCNTs in ethylenediamine by ultrasonication, add a coupling agent, and after the reaction, separate the solid and liquid, wash, and dry to obtain NH2-MWCNTs;

[0010] Step 4: Add NH2-MWCNTs and 4-formylphenylboronic acid to ethanol, stir, and intermittently add NaBH4 during stirring to prepare NH2-MWCNTs@4-FPBA;

[0011] Step 5: Modify the electrode with NH2-MWCNTs@4-FPBA, add IGF-2, and generate a molecularly imprinted layer on the electrode by electropolymerization of aniline and m-aminobenzenesulfonic acid;

[0012] Step 6: IGF-2 in the molecularly imprinted layer is dissociated and removed in an acidic solution to obtain an electrochemical biosensor.

[0013] Preferably, in step 1, the carbon source gas is CH4, the carrier gas is H2, and the metal catalyst is iron, cobalt, or nickel.

[0014] Preferably, in step 3, the mass-to-volume ratio of the carboxylated MWCNTs to ethylenediamine is (1-3):1.

[0015] Preferably, in step 3, the coupling agent is benzotriazole-tetramethylurea hexafluorophosphate.

[0016] Preferably, in step 4, the mass ratio of NH2-MWCNTs to 4-formylphenylboronic acid is 1:(1-3).

[0017] Preferably, in step 5, the electrode is a glassy carbon electrode.

[0018] Preferably, in step 5, the electropolymerization potential range is -0.6V to 0.6V, the scanning rate is 50 to 100mV / s, and the number of scanning cycles is 5 to 25.

[0019] Preferably, in step 6, the acidic solution is concentrated hydrochloric acid.

[0020] The IGF-2 electrochemical biosensor based on boric acid affinity was obtained using the preparation method described above.

[0021] The application of the IGF-2 electrochemical biosensor based on boric acid affinity in the detection of IGF-2.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention uses amino-carbon nanotubes (NH2-MWCNTs) as a carrier and covalently anchors 4-formylphenylboronic acid (4-FPBA) to the surface of MWCNTs to synthesize nanomaterials (NH2-MWCNTs@4-FPBA). NH2-MWCNTs@4-FPBA are then modified onto an electrode, and an IGF-2 template is captured via boric acid affinity. A molecularly imprinted layer (MIP) generated by the electropolymerization of aniline and m-aminobenzenesulfonic acid is then deposited. After dissociation and removal of the IGF-2 template in an acidic solution, a three-dimensional cavity complementary to the IGF-2 template is formed within the MIP, which can be used for IGF-2 detection. The nanomaterial NH2-MWCNTs@4-FPBA not only successfully binds IGF-2 but also exhibits excellent conductivity, significantly improving the electrical performance of electrochemical biosensors. Furthermore, this material is low in cost and suitable for large-scale production. This invention utilizes boric acid affinity and molecular imprinting to detect IGF-2 concentration in samples, replacing antibodies. This makes IGF-2 detection faster, simpler, and less costly. The electrochemical biosensor exhibits a wide linear range of 0.5–500 pg / ml and a low detection limit of 0.21 pg / ml, demonstrating high selectivity, repeatability, and stability. Its practical application in bovine milk samples has been proven. This method achieves simple, rapid, highly sensitive, highly specific, and antibody-free detection of IGF-2. Attached Figure Description

[0024] Figure 1 This diagram illustrates the fabrication of an IGF-2 electrochemical biosensor based on the nanocomposite material NH2-MWCNTs@4-MPBA and the IGF-2 detection method.

[0025] Figure 2 These are scanning electron microscope (SEM) images of the various stages of NH2-MWCNTs@4-FPBA formation in this invention: Figure 2 A represents the NH2-MWCNTs synthesized in step 3 of Example 1; Figure 2 B is the NH2-MWCNTs@4-FPBA synthesized in step 4.

[0026] Figure 3 Here are the cyclic voltammetry curves of the electrodes at each stage of this invention: Figure 3 In A, curve a represents the bare glassy carbon electrode (GCE), curve b represents the glassy carbon electrode modified with NH2-MWCNTs@4-FPBA (GCE / NH2-MWCNTs@4-FPBA), and curve c represents the electrode incubated with IGF-2 (GCE / NH2).

[0027] -MWCNTs@4-FPBA / IGF-2), curve d is the electrode with aniline and m-aminobenzenesulfonic acid molecular imprinted layer electropolymerized (GCE / NH2-MWCNTs@4-FPBA / IGF-2 / MIP), curve e is the electrode with template protein IGF-2 washed off (GCE / NH2-MWCNTs@4-FPBA / MIP), curve f is the electrode with IGF-2 rebound (GCE / NH2-MWCNTs@4-FPBA / IGF-2 / MIP); Figure 3 In B, curve g represents the electrode that washes away the template protein IGF-2 (GCE / NH2-MWCNTs@4-FPBA / MIP), and curve f represents the electrode that rebinds IGF-2 (GCE / NH2-MWCNTs@4-FPBA / IGF-2 / MIP). Figure 3 In C, curve g represents the blank control electrode (GCE / NH2-MWCNTs@4-FPBA / NIP) that did not bind template molecules in step 4, and curve f represents the control electrode (GCE / NH2) that contains IGF-2.

[0028] -MWCNTs@4-FPBA / IGF-2 / NIP).

[0029] Figure 4 The detection results of IGF-2 by the electrochemical biosensor obtained in Example 1 of this invention are as follows: Figure 4 A represents the CV curves of electrodes obtained by incubating IGF-2 solutions of different concentrations (curves a~l: 0, 0.5, 5, 10, 25, 50, 75, 100, 200, 300, 400 and 500 pg / ml); Figure 4 B is the calibration curve between the current difference and the IGF-2 concentration.

[0030] Figure 5 This is the specific detection of IGF-2 by the electrochemical biosensor obtained in Example 1 of this invention. Detailed Implementation

[0031] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0032] The preparation method of the insulin growth factor-2 electrochemical biosensor based on boric acid affinity according to the present invention includes the following steps:

[0033] Step 1: Using a thin film coated with a metal catalyst as a substrate, multi-walled carbon nanotubes (MWCNTs) are prepared by chemical vapor deposition using a carbon source gas and a carrier gas.

[0034] Step 2: The purified MWCNTs are added to a mixture of concentrated sulfuric acid and nitric acid, and sonicated to introduce carboxyl groups on the surface of MWCNTs. After solid-liquid separation, washing and drying, carboxylated MWCNTs are obtained.

[0035] Step 3: Disperse carboxylated MWCNTs in ethylenediamine by ultrasonication, add a coupling agent, and after the reaction, separate the solid and liquid, wash, and dry to obtain NH2-MWCNTs;

[0036] Step 4: Disperse NH2-MWCNTs and 4-formylphenylboronic acid (4-FPBA) in ethanol, stir at room temperature, and add NaBH4 at intervals to prepare NH2-MWCNTs@4-FPBA;

[0037] Step 5: Modify NH2-MWCNTs@4-FPBA onto a glassy carbon electrode, add template protein IGF-2, and generate a molecularly imprinted layer (MIP) on the glassy carbon electrode by electropolymerization of aniline and m-aminobenzenesulfonic acid.

[0038] Step 6: IGF-2 in the molecularly imprinted layer on the glassy carbon electrode is dissociated and removed in an acidic solution to obtain an electrochemical biosensor.

[0039] In step 1 above, the preferred carbon source gas is CH4, the preferred carrier gas is H2, and the preferred metal catalyst is iron, cobalt, or nickel.

[0040] In step 2 above, the preferred volume ratio of sulfuric acid to nitric acid is 3:1.

[0041] In step 3 above, the mass-to-volume ratio of carboxylated MWCNTs to ethylenediamine is (1-3):1, and the coupling agent is benzotriazole-tetramethylurea hexafluorophosphate (HBTU).

[0042] In step 4 above, the mass ratio of NH2–MWCNTs to 4-formylphenylboronic acid is 1:(1-3).

[0043] In step 5 above, the electropolymerization potential range is -0.6V to 0.6V, the scan rate is 50 to 100mV / s, and the number of scans is 5 to 25.

[0044] In step 6 above, the acidic solution is 0.2M concentrated hydrochloric acid.

[0045] The electrochemical biosensor prepared by the above method of the present invention can be used to detect IGF-2. By immersing the above electrochemical biosensor in a solution containing IGF-2, quantitative or qualitative electrochemical detection of IGF-2 and sensor performance research can be achieved.

[0046] The present invention forms a three-dimensional cavity in the molecularly imprinted layer (MIP) that is complementary to the IGF-2 template. The modified electrode is incubated with IGF-2 solutions of different concentrations at room temperature, and the DPV signal intensity is detected. A calibration curve is plotted with the IGF-2 concentration as the abscissa and the change value of the DPV signal as the ordinate, so as to realize the quantitative detection of IGF-2.

[0047] Example 1

[0048] Please see Figure 1 This invention discloses a fabrication and detection process for an electrochemical biosensor based on boric acid affinity for insulin-like growth factor-2 (IGF-2). The process involves preparing nanomaterials NH2-MWCNTs@4-FPBA and modifying them onto a glassy carbon electrode. Boric acid affinity is used to capture IGF-2 template molecules. An aniline and m-aminobenzenesulfonic acid molecularly imprinted layer is then formed via electropolymerization. The IGF-2 template molecules are then dissociated and removed in an acidic solution to obtain the electrochemical biosensor. In samples containing IGF-2, the electrochemical biosensor specifically recognizes IGF-2, and the generated electrochemical signal shows a significant positive correlation with the IGF-2 concentration, thus enabling sensitive detection of IGF-2.

[0049] The detailed steps and results are as follows:

[0050] (1) Carbon source gas (CH4) and carrier gas (H2) are introduced into the CVD reaction system. The substrate is a thin film coated with metal catalyst (Fe). The reaction is carried out at 800℃ for 24h. Finally, the carbon source gas (CH4) and carrier gas (H2) are washed alternately and centrifuged to obtain the final multi-walled carbon nanotubes (MWCNTs).

[0051] (2) The purified MWCNTs were first sonicated at 40°C for 3 hours with a mixture of concentrated sulfuric acid and nitric acid (volume ratio of concentrated sulfuric acid to nitric acid 3:1) to introduce carboxyl groups onto the surface of the MWCNTs, thus obtaining carboxylated MWCNTs. The carboxylated MWCNTs were washed with distilled water using filter paper with a pore size of 10 μm, and then dried in a vacuum oven at 80°C for 4 hours.

[0052] (3) 20 mg of carboxylated MWCNTs were ultrasonically dispersed in 10 mL of ethylenediamine, and 2 mg of coupling agent HBTU was added. The mixture was ultrasonically dispersed for 4 h. After dilution with 200 mL of methanol, the mixture was filtered through 10 μm filter paper. The filtrate was washed with excess methanol to obtain NH2-MWCNTs. Then, the NH2-MWCNTs were dried in a vacuum oven at 80 °C for 4 h. The morphology of the resulting material is shown in [reference]. Figure 2 A;

[0053] (4) Disperse 0.2 g of NH2-MWCNTs and 0.4 g of 4-formylphenylboronic acid (4-FPBA) in 20 ml of ethanol, stir at room temperature for 12 hours, add 50 mg of NaBH4 every 4 hours, centrifuge the resulting NH2-NWCNTs@4-FPBA, wash alternately with anhydrous ethanol and deionized water, and finally dry in a vacuum oven at 45 °C. The morphology of the resulting material is shown in [reference]. Figure 2 B;

[0054] (5) NH2-MWCNTs@4-FPBA was dropped onto a glassy carbon electrode and immersed in a solution of the template protein IGF-2 for 12 hours. Then, an imprinted layer generated by electropolymerization of aniline and m-aminobenzenesulfonic acid was deposited. The electropolymerization potential range was -0.6V to 0.6V, the scan rate was 50mV / s, and the number of scans was 25. The IGF-2 template was removed by dissociation in 0.2M concentrated hydrochloric acid to obtain an electrochemical biosensor. Meanwhile, the control material NIP-MWCNTs@4-FPBA was synthesized without the addition of the template protein IGF-2.

[0055] (6) IGF-2 detection: The modified electrode was incubated with IGF-2 solutions of different concentrations (0, 0.5, 5, 10, 25, 50, 75, 100, 200, 300, 400 and 500 pg / ml) at room temperature for 10 min and the DPV signal was recorded.

[0056] Please see Figure 4 As can be seen from A, from 0 pg / ml to 500 pg / ml (al), the generated current signal continuously weakens as the IGF-2 concentration increases. Within the range of 0 pg / ml to 500 pg / ml, ΔI exhibits a linear relationship with the IGF-2 concentration, as shown in Figure A. Figure 4 As shown in B, the formula corresponding to the linear relationship is ΔI = 0.184C. IGF-2 +8.23(R 2 =0.995), and the detection limit was 0.21 pg / ml.

[0057] The electrochemical biosensor of the present invention was used to detect milk samples: the milk samples were centrifuged at 3000 r / min for 15 min at 10 °C, and the resulting supernatant was diluted 100 times with PBS buffer. The electrochemical biosensor was then used to detect IGF-2.

[0058] Table 1. Detection results of IGF-2 in milk samples by the electrochemical biosensor of the present invention.

[0059]

[0060] Using the linear equation ΔI = 0.184C IGF-2The IGF-2 concentration was calculated using +8.23 and compared with the results calculated by the existing ELISA method, as shown in Table 1. It can be seen that the data detected by the electrochemical biosensor of the present invention is not much different from the data detected by the ELISA method, indicating that the electrochemical biosensor of the present invention has high reliability in detecting IGF-2 concentration.

[0061] Example 2

[0062] Please see Figure 1 This invention discloses a fabrication and detection process for an electrochemical biosensor based on boric acid affinity for insulin-like growth factor-2 (IGF-2). The process involves preparing nanomaterials NH2-MWCNTs@4-FPBA and modifying them onto a glassy carbon electrode. Boric acid affinity is used to capture IGF-2 template molecules. An aniline and m-aminobenzenesulfonic acid molecularly imprinted layer is then formed via electropolymerization. The IGF-2 template molecules are then dissociated and removed in an acidic solution to obtain the electrochemical biosensor. In samples containing IGF-2, the electrochemical biosensor specifically recognizes IGF-2, and the generated electrochemical signal shows a significant positive correlation with the IGF-2 concentration, thus enabling sensitive detection of IGF-2.

[0063] The detailed steps and results are as follows:

[0064] (1) Carbon source gas (CH4) and carrier gas (H2) are introduced into the CVD reaction system. The substrate is a thin film coated with metal catalyst (Fe). The reaction is carried out at 800℃ for 24h. Finally, the carbon source gas (CH4) and carrier gas (H2) are washed alternately and centrifuged to obtain the final multi-walled carbon nanotubes (MWCNTs).

[0065] (2) The purified MWCNTs were first sonicated at 40°C for 3 hours with a mixture of concentrated sulfuric acid and nitric acid (volume ratio of concentrated sulfuric acid to nitric acid 3:1) to introduce carboxyl groups onto the surface of the MWCNTs, thus obtaining carboxylated MWCNTs. The carboxylated MWCNTs were washed with distilled water using filter paper with a pore size of 10 μm, and then dried in a vacuum oven at 80°C for 4 hours.

[0066] (3) Disperse 20 mg of carboxylated MWCNTs in 20 mL of ethylenediamine by ultrasonication, add 2 mg of coupling agent HBTU, continue ultrasonication for 4 h, dilute with 200 mL of methanol, filter with filter paper with a pore size of 10 μm, wash the filtrate with excess methanol to obtain NH2-MWCNTs; then dry NH2-MWCNTs in a vacuum oven at 80 °C for 4 h;

[0067] (4) Take 0.2g NH2-MWCNTs and 0.2g 4-formylphenylboronic acid (4-FPBA) and disperse them in 20ml ethanol. Stir at room temperature for 12 hours, add 50mg NaBH4 every 4 hours, centrifuge the obtained NH2-NWCNTs@4-FPBA, wash with anhydrous ethanol and deionized water alternately, and finally dry in a vacuum oven at 45℃.

[0068] (5) NH2-MWCNTs@4-FPBA was dropped onto a glassy carbon electrode and immersed in a template protein IGF-2 solution for 12 hours. Then, an imprinted layer generated by electropolymerization of aniline and m-aminobenzenesulfonic acid was deposited. The electropolymerization potential range was -0.6V to 0.6V, the scan rate was 80mV / s, and the number of scans was 20. The IGF-2 template was removed by dissociation in 0.2M concentrated hydrochloric acid to obtain an electrochemical biosensor. Meanwhile, the template protein IGF-2 was not added during the synthesis of the control material NIP-MWCNTs@4-FPBA.

[0069] Example 3

[0070] Please see Figure 1 This invention discloses a fabrication and detection process for an electrochemical biosensor based on boric acid affinity for insulin-like growth factor-2 (IGF-2). The process involves preparing nanomaterials NH2-MWCNTs@4-FPBA and modifying them onto a glassy carbon electrode. Boric acid affinity is used to capture IGF-2 template molecules. An aniline and m-aminobenzenesulfonic acid molecularly imprinted layer is then formed via electropolymerization. The IGF-2 template molecules are then dissociated and removed in an acidic solution to obtain the electrochemical biosensor. In samples containing IGF-2, the electrochemical biosensor specifically recognizes IGF-2, and the generated electrochemical signal shows a significant positive correlation with the IGF-2 concentration, thus enabling sensitive detection of IGF-2.

[0071] The detailed steps and results are as follows:

[0072] (1) Carbon source gas (CH4) and carrier gas (H2) are introduced into the CVD reaction system. The substrate is a thin film coated with metal catalyst (Fe). The reaction is carried out at 800℃ for 24h. Finally, the carbon source gas (CH4) and carrier gas (H2) are washed alternately and centrifuged to obtain the final multi-walled carbon nanotubes (MWCNTs).

[0073] (2) The purified MWCNTs were first sonicated at 40°C for 3 hours with a mixture of concentrated sulfuric acid and nitric acid (volume ratio of concentrated sulfuric acid to nitric acid 3:1) to introduce carboxyl groups onto the surface of the MWCNTs, thus obtaining carboxylated MWCNTs. The carboxylated MWCNTs were washed with distilled water using filter paper with a pore size of 10 μm, and then dried in a vacuum oven at 80°C for 4 hours.

[0074] (3) Disperse 20 mg of carboxylated MWCNTs in 60 mL of ethylenediamine by ultrasonication, add 2 mg of coupling agent HBTU, continue ultrasonication for 4 h, dilute with 200 mL of methanol, filter with filter paper with a pore size of 10 μm, wash the filtrate with excess methanol to obtain NH2-MWCNTs; then dry NH2-MWCNTs in a vacuum oven at 80 °C for 4 h;

[0075] (4) Take 0.2g NH2-MWCNTs and 0.6g 4-formylphenylboronic acid (4-FPBA) and disperse them in 20ml ethanol. Stir at room temperature for 12 hours, add 50mg NaBH4 every 4 hours, centrifuge the obtained NH2-NWCNTs@4-FPBA, wash with anhydrous ethanol and deionized water alternately, and finally dry in a vacuum oven at 45℃.

[0076] (5) NH2-MWCNTs@4-FPBA was dropped onto a glassy carbon electrode and immersed in a template protein IGF-2 solution for 12 hours. Then, an imprinted layer generated by electropolymerization of aniline and m-aminobenzenesulfonic acid was deposited. The electropolymerization potential range was -0.6V to 0.6V, the scan rate was 100mV / s, and the number of scans was 5. The IGF-2 template was removed by dissociation in 0.2M concentrated hydrochloric acid to obtain an electrochemical biosensor. Meanwhile, the control material NIP-MWCNTs@4-FPBA was synthesized without the addition of template protein IGF-2.

[0077] Example 4

[0078] Cyclic voltammetry tests were performed on the electrodes obtained at different stages of Example 1. A control electrode (GCE / NH2-MWCNTs@4-MPBA / NIP) was prepared according to the method in Example 1, but without the template protein IGF-2 in step 4. Cyclic voltammetry tests were then performed on the control electrode.

[0079] Please see Figure 3 The cyclic voltammetry test of this invention was performed in a K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 M KCl. Figure 3Curve a in section A shows that the peak current of the untreated glassy carbon electrode is 97.0 μA. Curve b shows that the peak current increased to 137.7 μA after NH2-MWCNTs@4-FPBA modification of the glassy carbon electrode, an increase of 41.96%, indicating that NH2-MWCNTs@4-FPBA has good conductivity and amplifies the electrochemical signal. The decrease in current value in curve c is due to the adsorption of IGF-2 onto the electrode by the affinity of boric acid. Since IGF-2 adsorption also hinders electron transfer, this confirms the success of the experimental principle. Next, after electropolymerization to form a molecularly imprinted layer, the current value further decreases (curve d). Under acidic conditions, IGF-2 is successfully washed away, as shown by the increase in current value in curve e. When IGF-2 is present on the electrode surface (curve f), it can bind to the vacancies left by the removal of IGF-2 in the molecularly imprinted layer, again hindering electron transfer and thus significantly reducing the peak current (a decrease of 40.8%). Conversely, in… Figure 3 In curve C, the control electrode also failed to exhibit specific recognition in the presence of IGF-2 (curve f). This indicates that the present invention has prepared an electrochemical biosensor through molecular imprinting, which can achieve the detection of IGF-2.

[0080] Example 5

[0081] This embodiment tests the specificity of the electrochemical biosensor obtained in Example 1 for IGF-2. EGF, GH, IL-10 and IL-6 are used as interfering agents. The concentration of IGF-2 is 300 pg / ml, and the concentration of other interfering agents is twice the concentration of IGF-2.

[0082] Please see Figure 5 The results showed that the ΔI obtained from detecting EGF, GH, IL-10 and IL-6 was much lower than that obtained from detecting IGF-2, indicating that the electrochemical biosensor of the present invention has high specificity for IGF-2.

[0083] Comparative Example

[0084] The performance of the electrochemical biosensor obtained in Example 1 of this invention was investigated and compared with the detection performance of existing IGF-2 biosensors. Existing IGF-2 biosensors are Comparative Examples 1-3. The electrochemical biosensor of Comparative Example 1 was prepared using the method described in “Rezaei, B, Majidi, N, Rahmani, H, et al. Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode[J]. Biosensors and Bioelectronics, 2011, 26(5):2130-2134. DOI:10.1016 / j.bios.2010.09.020.”; the electrochemical biosensor of Comparative Example 2 was prepared using the method described in “Serafín, V, Agüí, L, P, Pingarrón, JM, et al. Electrochemical immunosensor for the determination of insulin-like growth factor-1 using electrodes modified with carbon nanotubes–poly(pyrrole propionic acid) hybrids[J]. Biosensors and Bioelectronics, 2013, 52: 98-104. DOI: 10.1016 / j.bios.2013.08.021.”; The electrochemical biosensor of Comparative Example 3 was prepared using the method described in “Liu, LB, Gopinath, SCB, et al. Gold-enhanced current-volt dielectrodejunction for biosensing with an aptamer-insulin-like growth factor-1-antibodysandwich pattern[J]. MATERIALS EXPRESS, 2022, 12(3): 464-471. DOI: 10.1166 / mex.2022.2153.”.

[0085] Table 2 Comparison of the detection performance of the electrochemical biosensor of the present invention with existing products.

[0086]

[0087] A comparison of the data in Table 2 shows that, compared with existing detection technologies, the electrochemical biosensor prepared in this invention has a lower limit of detection. Furthermore, compared with commercially available IGF-2 enzyme-linked immunosorbent assay kits (with a limit of detection of 0.1 ng / ml), this invention also has the advantages of a low limit of detection and high sensitivity.

Claims

1. A method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity, characterized in that, Includes the following steps: Step 1: Using a thin film coated with a metal catalyst as a substrate, multi-walled carbon nanotubes are prepared by vapor-phase chemical deposition using a carbon source gas and a carrier gas. Step 2: After purification, multi-walled carbon nanotubes are added to a mixture of concentrated sulfuric acid and nitric acid, sonicated, separated into solid and liquid, washed, and dried to obtain carboxylated MWCNTs; Step 3: Disperse carboxylated MWCNTs in ethylenediamine by ultrasonication, add a coupling agent, and after the reaction, separate the solid and liquid, wash, and dry to obtain NH2-MWCNTs; Step 4: Add NH2-MWCNTs and 4-formylphenylboronic acid to ethanol, stir, and intermittently add NaBH4 during stirring to prepare NH2-MWCNTs@4-FPBA; the mass ratio of NH2-MWCNTs to 4-formylphenylboronic acid is 1:(1~3). Step 5: Modify the electrode with NH2-MWCNTs@4-FPBA, add IGF-2, and generate a molecularly imprinted layer on the electrode by electropolymerization of aniline and m-aminobenzenesulfonic acid; the electropolymerization potential range is -0.6 V to 0.6 V, the scan rate is 50 to 100 mV / s, and the number of scans is 5 to 25. Step 6: IGF-2 in the molecularly imprinted layer is dissociated and removed in an acidic solution to obtain an electrochemical biosensor.

2. The method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity according to claim 1, characterized in that, In step 1, the carbon source gas is CH4, the carrier gas is H2, and the metal catalyst is iron, cobalt, or nickel.

3. The method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity according to claim 1, characterized in that, In step 3, the mass-to-volume ratio of the carboxylated MWCNTs to ethylenediamine is (1~3):

1.

4. The method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity according to claim 1, characterized in that, In step 3, the coupling agent is benzotriazole-tetramethylurea hexafluorophosphate.

5. The method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity according to claim 1, characterized in that, In step 5, the electrode is a glassy carbon electrode.

6. The method for preparing an IGF-2 electrochemical biosensor based on boric acid affinity according to claim 1, characterized in that, In step 6, the acidic solution is concentrated hydrochloric acid.

7. An IGF-2 electrochemical biosensor based on boric acid affinity obtained by the preparation method according to any one of claims 1 to 6.

8. The application of the IGF-2 electrochemical biosensor based on boric acid affinity as described in claim 7 in the detection of IGF-2.

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