An electrochemical detection method for detecting recombinant glycoprotein

Abstract

An electrochemical detection method for detecting recombinant glycoprotein, comprising the following steps: A: preparing a phenylboronic acid-copper-based metal organic framework composite, comprising the following sub-steps: A1: synthesis of a copper-based metal organic framework; A2: preparation of a phenylboronic acid-copper-based metal organic framework composite; B: detection of the recombinant glycoprotein, comprising the following sub-steps: B1: resuspending nickel ion affinity magnetic beads in a phosphate buffer solution; B2: moving the dispersion prepared in step B1 into a microcentrifuge tube; B3: adding a to-be-detected substance containing the recombinant glycoprotein into the microcentrifuge tube; B4: dispersing the phenylboronic acid-copper-based metal organic framework composite obtained in step A2 in water and adding the solution obtained in step B3 to obtain a magnetic bead composite; B5: transferring the composite prepared in step B4 to the surface of a glassy carbon electrode; B6: soaking the electrode obtained in B5 in a Tris-HCl buffer solution, and the present application has low cost.

Description

Technical Field

[0001] This invention relates to electrochemical detection methods, and particularly to an electrochemical method for detecting recombinant human erythropoietin, belonging to the field of chemistry. Background Technology

[0002] With the development of recombinant DNA technology, a large number of recombinant proteins have been synthesized and used as drugs to regulate various physiological processes. Notably, over 60% of recombinant proteins are glycosylated, such as interferon-β1, recombinant human erythropoietin (rHuEPO), glucocerebrosidase, and the SARS-CoV-2 recombinant spike protein nanoparticle vaccine. rHuEPO is a recombinant protein that can increase the oxygen-carrying capacity of blood and is used to improve the quality of life of patients with chronic kidney disease; however, it is accompanied by increased side effects such as increased blood pressure, headache, and thrombosis. Simultaneously, rHuEPO has also been illegally used as a banned substance to enhance athletic performance. Therefore, its detection is of great significance in fields such as disease diagnosis. Although various methods exist for the detection of protein-based disease biomarkers, "sandwich" sensors (such as enzyme-linked immunosorbent assay) remain the most popular and effective method. In this detection system, the target protein can be captured by antibodies (primary antibodies) on the sensor surface, and then the captured protein is recognized and signaled by antibodies (secondary antibodies) labeled with signal molecules or materials. However, antibodies or specially labeled antibodies often suffer from drawbacks such as high cost, difficulty in preparation and purification, and susceptibility to denaturation. Therefore, it is essential to develop an electrochemical sensor for the detection of recombinant glycoproteins that does not rely on biorecognition elements. Summary of the Invention

[0003] The purpose of this invention is to provide an electrochemical detection method for the detection of recombinant glycoproteins.

[0004] To achieve the objective of this invention, the following technical solution is adopted: an electrochemical detection method for recombinant glycoprotein detection, comprising the following steps:

[0005] A: Preparation of phenylboronic acid-copper-based metal-organic framework complex includes the following sub-steps:

[0006] A1: Synthesis of copper-based metal-organic frameworks: Copper nitrate, 2-aminoterephthalic acid and polyvinylpyrrolidone were used as precursors. Copper nitrate and 2-aminoterephthalic acid were dissolved in DMF, and polyvinylpyrrolidone was dissolved in a mixture of DMF and ethanol. The mixture was then transferred to a polytetrafluoroethylene reactor and reacted at 100°C for 8 hours. The solution was then cooled to room temperature, and the reaction product was centrifuged. The obtained solid was washed three times with DMF and ethanol respectively, and then dried under vacuum at 60°C for 12 hours to obtain the copper-based metal-organic framework.

[0007] A2: Preparation of phenylboronic acid-copper-based metal-organic framework complex: The solid obtained in step A1 was ultrasonically dispersed in ethanol, and then 4-formylphenylboronic acid was added. The mixture was refluxed at 80 °C for 12 hours. After cooling to room temperature, the mixture was centrifuged to remove the unreacted 4-formylphenylboronic acid in the upper layer. The resulting precipitate was washed with ethanol. The purified phenylboronic acid-copper-based metal-organic framework complex was vacuum dried at 60 °C for 12 hours and stored under dry conditions for later use.

[0008] B: Detection of recombinant glycoproteins includes the following sub-steps:

[0009] B1: Wash the nickel ion affinity magnetic beads twice and resuspend them in phosphate buffer solution to obtain a nickel ion affinity magnetic bead dispersion;

[0010] B2: Take the nickel ion affinity magnetic bead dispersion prepared in step B1 and transfer it into a centrifuge tube;

[0011] B3: The analyte containing recombinant glycoprotein is added to a centrifuge tube containing a magnetic bead dispersion. Under the action of an external magnetic field, the magnetic beads that capture the recombinant glycoprotein are separated and washed twice with phosphate buffer solution before being dispersed in phosphate buffer solution.

[0012] B4: The phenylboronic acid-copper-based metal-organic framework complex obtained in step A2 was ultrasonically dispersed in water and then added to the solution obtained in step B3; after incubation for 30 minutes, magnetic separation and washing were performed to obtain the magnetic bead complex;

[0013] B5: Transfer the magnetic bead composite obtained in step B4 to the surface of the magnetic glassy carbon electrode;

[0014] B6: Immerse the electrode obtained in B5 in Tris-HCl buffer solution;

[0015] C: Electrochemical testing: The electrode prepared in step B6 was used as the working electrode for electrochemical performance testing.

[0016] Furthermore, the phosphate buffer solution described in step B1 is 50 mM, pH 7.4, 300 mM NaCl, and 30 mM Mimidazole.

[0017] Furthermore, the Tris-HCl buffer solution mentioned in step B6 is 50 mM, pH 7.4, 100 mM NaCl.

[0018] Furthermore, in step C, the electrochemical test uses a three-electrode system, with a magnetic glassy carbon electrode as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a Pt electrode as the auxiliary electrode.

[0019] The beneficial technical effect of the electrochemical method for detecting recombinant glycoproteins provided by this invention is as follows: recombinant glycoproteins contain hexahistidine (His6) and glycosyl groups, and hexahistidine can bind to NTA-Ni on nickel ion affinity magnetic beads. 2+ Complex interactions: the sugar group can interact with phenylboronic acid to form a boronic ester bond, hence NTA-Ni 2+ Complexes and phenylboronic acid can effectively replace antibodies to specifically recognize recombinant glycoproteins. This invention utilizes NTA-Ni... 2+ Magnetic beads modified with complexes capture recombinant glycoproteins; the recombinant glycoproteins are recognized by a phenylboronic acid-copper-based metal-organic framework complex through borate ester bonds, which facilitates the fixation of the copper-based metal-organic framework onto the electrode surface through magnetic separation and chemical action. The invention has high sensitivity, does not require expensive and easily denatured antibodies, has low cost, and good stability. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the testing principle of the recombinant glycoprotein rHuEPO of this invention.

[0021] Figure 2 This is a scanning electron microscope (SEM) characterization of a copper-based metal-organic framework.

[0022] Figure 3 This is a scanning electron microscope (SEM) characterization of the phenylboronic acid-copper-based metal-organic framework complex.

[0023] Figure 4 This is a Zeta potential characterization diagram of copper-based metal-organic frameworks and phenylboronic acid-copper-based metal-organic framework complexes.

[0024] Figure 5 These are the infrared absorption spectra of copper-based metal-organic frameworks and phenylboronic acid-copper-based metal-organic framework complexes (the lower curve represents the phenylboronic acid-copper-based metal-organic framework).

[0025] Figure 6 The graph shows the test results of the working electrode under conditions containing and without rHuEPO.

[0026] Figure 7 shows the test results of rHuEPO at different concentrations.

[0027] Figure 8 shows the linear relationship between peak current and rHuEPO concentration.

[0028] Figure 9 This is a selective test diagram of the sensor of the present invention. Detailed Implementation

[0029] To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the process and do not limit the scope of the present invention. The present invention is illustrated by the following embodiments, but is not limited to the following embodiments. Any variations are included within the technical scope of the present invention.

[0030] In this invention, the various abbreviations represent the following substances: NH2-BDC: 2-aminoterephthalic acid, PVP: polyvinylpyrrolidone, FPBA: 4-formylphenylboronic acid, FPBA-Cu-MOFs: phenylboronic acid-copper-based metal-organic framework complex, rHuEPO: recombinant human erythropoietin, MBs-NTA-Ni 2+ :NTA-Ni 2+ Complex-modified magnetic beads (i.e., the nickel ion affinity magnetic beads of this invention) are commercially available, such as those offered by Thermo Fisher Scientific; MGCE: magnetic glassy carbon electrode; HSA: human serum albumin; IgG: human immunoglobulin; carcinoembryonic antigen: carcinoembryonic antigen; mM, μM, and pM are concentration units, representing 10⁻⁶ m / s², respectively. -3 mol / L, 10 -6 mol / L, 10 -12 mol / L, µL: microliter, Tris: English name: Tris(hydroxymethyl)aminomethane, Chinese aliases: tris(hydroxymethyl)aminomethane; aminobutanetriol; tris(hydroxymethyl)aminomethane.

[0031] The following examples use recombinant human erythropoietin as the glycoprotein to be tested for detailed explanation. Further explanation of the accompanying drawings follows. Figure 1 middle, Represents MBs-NTA-Ni 2+ , Representing rhuEPO, Represents a phenylboronic acid-copper-based metal-organic framework complex. Representing MGCE, The numbers represent centrifuge tubes. For clarity, the molecules in the diagram are not shown to actual scale. Figure 6 The concentration of rhuEPO was 50 ng / mL, the scan rate was 0.05 V / s, and the supporting electrolyte was Tris-HCl buffer solution (50 mM, pH 7.4, 100 mM NaCl). Figure 7 The concentrations of rHuEPO were 0, 0.01, 0.05, 0.1, 1, 10, 25, 50, 100, and 300 ng / mL, respectively, with the arrows indicating the scanning direction. Figure 8The concentrations of rHuEPO in the medium were 0.01, 0.05, 0.1, 1, 10, 25, and 50, respectively. Figure 9 The table shows the response of the electrodes to CEA (1), thrombin (2), HSA (3), IgG (4), rHuEPO (5), and a mixture of 1-5 (6). The concentration of rHuEPO was 50 ng / mL, and the concentrations of 1-4 were all 500 ng / mL.

[0032] Example 1:

[0033] (A): Synthesis of phenylboronic acid-copper-based metal-organic framework complex:

[0034] A1: First, 5.4 mg of NH2-BDC and 24.2 mg of copper nitrate were dissolved in 4 mL of DMF solution. Then, 0.20 g of PVP was dissolved in a mixture of 4 mL of DMF and 4 mL of ethanol under stirring. Next, the two solutions were mixed and sonicated for 30 minutes. The mixture was then transferred to a polytetrafluoroethylene reactor and reacted at 100 °C for 8 hours. After the solution cooled to room temperature, the product was centrifuged, washed three times with DMF and ethanol respectively, and then vacuum dried at 60 °C for 12 hours to obtain the copper-based metal-organic framework.

[0035] A2: Weigh 6 mg of the copper-based metal-organic framework prepared in step A1 into a 15 mL round-bottom flask, add 6 mL of ethanol, and sonicate for 10 minutes to disperse it evenly; add 6 mg of FPBA to the prepared complex dispersion, and reflux at 80 °C for 12 hours; then centrifuge and wash to obtain the phenylboronic acid-copper-based metal-organic framework complex, which is vacuum dried at 60 °C for 12 hours and stored under dry conditions for later use; the synthesized phenylboronic acid-copper-based metal-organic framework complex was analyzed by scanning electron microscopy, Zeta potential, and infrared absorption spectroscopy, and the results are shown in the figure. Figure 2 , Figure 3 , Figure 4 , Figure 5 ;

[0036] (B): Detection of recombinant human erythropoietin, including the following sub-steps:

[0037] B1: Under the influence of an external magnetic field, 100 mL of 2 mg NTA-Ni 2+ Complex-modified magnetic beads (MBs-NTA-Ni) 2+ Wash twice and resuspend in 200 mL of phosphate buffer solution;

[0038] B2: Take 10 mL of 1 mg / mL MBs-NTA-Ni 2+ The dispersion was added to the centrifuge tube;

[0039] B3: Add 10 mL of 50 ng / mL recombinant human erythropoietin sample solution to the solution obtained in B2. After incubation at room temperature for 30 minutes, under the action of an external magnetic field, capture the MBs-NTA-Ni of recombinant human erythropoietin. 2+ After being separated and washed twice with phosphate buffer solution, it was dispersed in phosphate buffer solution;

[0040] B4: Sonicate 1 mg of the phenylboronic acid-copper-based metal-organic framework complex obtained in step A2 into 2 mL of water, then add 5 mL of the 0.5 mg / mL phenylboronic acid-copper-based metal-organic framework complex solution to the MBs-NTA-Ni obtained in step B3. 2+ In solution; incubate for 30 minutes, then perform magnetic separation and washing;

[0041] B5: Transfer the magnetic bead composite obtained in step B4 to the surface of the magnetic glassy carbon electrode;

[0042] B6: Immerse the electrode obtained in step B5 in Tris-HCl buffer solution and perform differential pulse voltammetry. The Tris-HCl buffer solution contains 100 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane (Tris), and has a pH of 7.4.

[0043] C: Electrochemical testing:

[0044] Electrochemical detection employs a three-electrode system, with a magnetic glassy carbon electrode as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a Pt electrode as the auxiliary electrode for electrochemical testing.

[0045] Examples and test results of magnetic bead modification of the working electrode with and without rhuEPO:

[0046] 66. Working electrodes modified with magnetic bead composites were prepared using all the steps in Example 1. Working electrodes modified with magnetic bead composites without rhuEPO were prepared using the steps in Example 1 except for step B3. The test results are shown in the figure. Figure 6 Curve a represents the differential pulse voltammetry result of the working electrode, curve b represents the differential pulse voltammetry result of the working electrode modified with the magnetic bead complex containing rhuEPO, and curve c represents the differential pulse voltammetry result of the working electrode modified with the magnetic bead complex without rhuEPO. Comparing the curves, it can be seen that this method can be used for the detection of recombinant glycoproteins.

[0047] Examples using different rhuEPO concentrations:

[0048] 8. Using the same steps as before, change the concentration of rhuEPO in step B3, while keeping other conditions unchanged. The test results are as follows. Figure 7 , Figure 8 As shown, Figure 7 The rhuEPO concentrations (ng / mL) used for each curve (a to j) from left to right are 0, 0.01, 0.05, 0.1, 1, 10, 25, 50, 100, and 300, respectively. Figure 7 As can be seen, the reduction current increases with increasing rhuEPO concentration, indicating that the amount of copper-based metal-organic framework immobilized on the electrode surface depends on the concentration of recombinant human erythropoietin. From... Figure 8 It can be seen that the current intensity is linearly related to the rhuEPO concentration in the range of 0.01~50 ng / mL, indicating that this method can be used for the quantitative detection of recombinant glycoproteins.

[0049] Comparative examples of responses to other proteins:

[0050] In step B3, replace rHuEPO with the proteins described in steps 1, 2, 3, and 4 below; step 5 still uses rHuEPO; and step 6 uses a mixture of steps 1-5. The other steps remain the same. Prepare the working electrodes for testing. The testing method in step B6 is differential pulse voltammetry. The concentration of rHuEPO is 50 ng / mL, and the concentration of the other proteins is 500 ng / mL. The other conditions remain unchanged. The test results for each sample are as follows: Figure 6 As shown in the figure. The proteins used in 1, 2, 3, 4, and 5 are: 1: CEA, 2: thrombin, 3: HSA, 4: IgG, 5: rHuEPO, 6: a mixture of 1-5. The concentration of rHuEPO is 50 ng / mL, and the concentrations of 1-4 are all 500 ng / mL. Figure 9 As can be seen, the electrochemical signals generated by proteins 1-4 are negligible. Furthermore, a comparison... Figure 9 As can be seen from points 5 and 6, proteins 1-4 do not interfere with the determination of recombinant human erythropoietin, indicating that the method of the present invention can selectively detect recombinant glycoproteins.

[0051] After a detailed description of the embodiments of the present invention, those skilled in the art will clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent applications. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention fall within the scope of the present invention, and the present invention is not limited to the embodiments described in the specification. To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the process and do not limit the scope of the present invention. The present invention is illustrated by the following embodiments, but is not limited to the following embodiments. Any variations are included within the technical scope of the present invention.

Claims

1. An electrochemical detection method for the detection of recombinant glycoproteins, characterized in that Comprising the following steps: A: Preparation of phenylboronic acid-copper-based metal organic framework complex, comprising the following sub-steps: A1: Synthesis of copper-based metal organic framework: using copper nitrate, 2-amino terephthalic acid and polyvinylpyrrolidone as precursors, dissolving copper nitrate and 2-amino terephthalic acid in DMF, dissolving polyvinylpyrrolidone in a mixture of DMF and ethanol, mixing the two, then transferring to a polytetrafluoroethylene reactor, reacting at 100℃ for 8 hours, then cooling the solution to room temperature, centrifuging the reaction product, washing the obtained solid with DMF and ethanol three times respectively, then vacuum drying at 60℃ for 12 hours to obtain the copper-based metal organic framework; A2: Preparation of phenylboronic acid-copper-based metal organic framework complex: ultrasonic dispersion of the solid obtained in step A1 in ethanol, then adding 4-formylphenylboronic acid, refluxing at 80℃ for 12 hours; after cooling to room temperature, centrifuging and discarding the unreacted 4-formylphenylboronic acid on the top, washing the obtained precipitate with ethanol, vacuum drying the obtained purified phenylboronic acid-copper-based metal organic framework complex at 60℃ for 12 hours, and storing under dry conditions for later use; B: Detection of recombinant glycoprotein, comprising the following sub-steps: B1: Washing the nickel ion affinity magnetic beads twice and resuspending in a phosphate buffer solution; B2: Taking the nickel ion affinity magnetic bead dispersion prepared in step B1 and moving it to a centrifuge tube; B3: Adding the test containing the recombinant glycoprotein to the centrifuge tube containing the magnetic bead dispersion, under the action of an external magnetic field, the magnetic beads capturing the recombinant glycoprotein are separated, and after washing twice with a phosphate buffer solution, they are dispersed in a phosphate buffer solution; B4: Ultrasonic dispersion of the phenylboronic acid-copper-based metal organic framework complex obtained in step 2 of A in water, then adding to the solution obtained in step B3; incubating for 30 minutes, then performing magnetic separation and washing to obtain a magnetic bead complex; B5: Transferring the magnetic bead complex prepared in step B4 to the surface of a magnetic glassy carbon electrode; B6: Immersing the electrode obtained in B5 in a Tris-HCl buffer solution; C: Electrochemical test: using the electrode prepared in step B6 as the working electrode to test the electrical properties.

2. The electrochemical detection method for detecting a recombinant glycoprotein according to claim 1, characterized in that: The phosphate buffer solution in step B1 is 50 mM, pH 7.4, 300 mM NaCl, 30 mM imidazole.

3. The electrochemical detection method for detecting a recombinant glycoprotein according to claim 1, characterized in that: The Tris-HCl buffer solution in step B6 is 50 mM, pH 7.4, 100 mM NaCl.

4. The electrochemical detection method for detecting a recombinant glycoprotein according to claim 1, characterized in that: The electrochemical test in step C uses a three-electrode system, with the magnetic glassy carbon electrode as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a Pt electrode as the auxiliary electrode.

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