Molecularly imprinted sensing electrode for protein detection and preparation method thereof

Abstract

The application discloses a molecular imprinting sensing electrode for protein detection, which comprises a gold electrode, and a ternary NTA-Ni complex is modified on the surface of the gold electrode 2+ A template protein complex is formed on the NTA-Ni 2+ A layer of polydopamine imprinting film is formed around the template protein ternary complex, and the template protein and Ni are removed by washing the electrode 2+ An imprinting cavity capable of specifically recognizing a target protein is formed, and the molecular imprinting sensing electrode for protein detection is obtained, wherein the template protein is a commercially available recombinant protein containing a His6 label and corresponds to the target protein to be detected. Compared with a traditional imprinting polymer film, the imprinting technology has the following advantages: organic solvent polymerization can be avoided, the template removal condition is mild, and the mass transfer performance of the protein in and out of the imprinting cavity is excellent.

Description

Technical Field

[0001] This invention relates to sensors for protein detection, and particularly to a molecularly imprinted sensing electrode for protein detection and its preparation method, belonging to the field of chemical technology. Background Technology

[0002] Protein biomarkers reflect complex physiological changes in organisms under disease states, and their concentration changes are often closely related to the onset and development of certain diseases. Currently, methods for detecting protein biomarkers mainly include immunoassay, mass spectrometry, electrophoresis, and high-performance liquid chromatography (HPLC). However, while these methods are relatively sensitive, they require expensive reagents and instruments, as well as skilled operators. Therefore, developing simple, economical, and reliable methods for the selective determination of protein biomarkers is of great significance.

[0003] Electrochemical sensors, as point-of-care detection devices, can convert biorecognition events into various electrochemical signals, demonstrating great potential for sensitive detection of biomarkers. The specific recognition of biomolecules plays a decisive role in the development of electrochemical sensors. Molecularly imprinted polymeric membranes (MIPs), as a class of artificially synthesized receptors, hold promise as a replacement for natural biological receptors. Compared to biological receptors such as nucleic acids and antibodies, MIPs offer advantages such as long-term durability, low cost, and good robustness in the detection of low molecular weight targets. However, traditional MIPs prepared by polymerizing functional monomers with template molecules have many limitations in the recognition and detection of large molecular biomarkers such as proteins due to their large size, complex and variable structure, and poor environmental stability. For example, proteins undergo irreversible conformational changes during polymerization, the large size of MIPs makes it difficult to remove template proteins from the imprinted cavity, and the rebinding ability of natural proteins to the imprinted cavity is weak.

[0004] Biomolecules contain a rich variety of epitope groups, including thiol, amino, and glycosyl groups, which makes directional or site-specific surface imprinting of proteins feasible. These epitope groups facilitate the immobilization of proteins on substrate surfaces functionalized with aldehydes, boric acids, and other specific molecules through covalent coupling reactions, boric acid affinity, and electrostatic adsorption. This orientation-based immobilization method can, to some extent, hinder conformational changes in proteins. However, small molecules containing epitope groups similar to those in the template protein can also be captured by the imprinted membrane, resulting in significant non-specific adsorption. For example, polysaccharide molecules present in complex biological samples can compete with target glycoproteins for binding to boric acid-containing MIPs, thereby reducing the specificity of analytical methods. Furthermore, the practical application of directional immobilization methods is limited by inherent limitations of proteins and imprinted surfaces, including the need for polymerization with organic solvents, precise control of film thickness, and stringent template removal conditions. These factors affect the spatial structure of the template protein and cause degradation, deformation, or collapse of the MIP cavity, thus limiting the rebinding of the target protein to the MIP in its native conformation. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems in existing protein detection methods and to provide a molecularly imprinted sensing electrode for protein detection and its preparation method.

[0006] To achieve the objectives of this invention, the following technical solution is adopted: a molecularly imprinted sensing electrode for protein detection, comprising a gold electrode, wherein a ternary NTA-Ni structure is modified on the surface of the gold electrode. 2+ - Template protein complex, via electropolymerization on NTA-Ni 2+ A polyscopolamine-imprinted membrane forms around the template protein ternary complex. The template protein and Ni are removed by rinsing the electrode. 2+ Then, an imprinted cavity is formed that can specifically recognize the target protein, resulting in a molecularly imprinted sensing electrode that can be used for protein detection. The template protein is a commercially available recombinant protein containing His6 label, corresponding to the target protein to be detected.

[0007] Furthermore, the electrode was cleaned and rinsed with ethanol and hydrochloric acid to remove template proteins and Ni. 2+ This then forms an imprinted cavity capable of specifically recognizing the target protein.

[0008] A method for preparing a molecularly imprinted sensing electrode for protein detection includes the following steps: S1: Incubate the gold electrode with 50 μL of 50 mM (2-morpholinoethanesulfonic acid) (MES) buffer containing 0.1 mM mercapto-NTA and 1 mM tris(2-carboxyethyl)phosphine (TCEP) for 12 hours; S2: Connect the electrode from step S1 to 10 μM Ni2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complexes; S3: After rinsing the electrode from step S2 with water, incubate the electrode with 10 μg / mL of recombinant template protein with a His6 tag for 30 minutes to form a ternary NTA-Ni matrix on the electrode surface. 2+ - Template protein complex, yielding NTA-Ni 2+ - The template protein complex modifies the electrode, and the recombinant template protein corresponds to the target protein to be detected; for example, if the target protein is PSA, the recombinant template protein is recombinant PSA (represented as rPSA in this application), and if the target protein is CEA, the recombinant template protein is recombinant CEA (represented as rCEA in this application). S4: Electropolymerization of hyoscyamine on the electrode surface obtained in step S3 is performed by electrodeposition on NTA-Ni. 2+ - A polyhyoscyamine-imprinted membrane is formed around the template protein complex. The specific polymerization steps and conditions are as follows: The modified electrode obtained in step S3 is subjected to multi-step amperometric scanning for 15-20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with the applied potentials being 0 V for 5 s and 0.7 V for 1 s; then the electrode is rinsed with 20% ethanol and cleaned with 0.1 M HCl under ultrasonication to remove the template protein, thus obtaining a molecularly imprinted sensing electrode for protein detection.

[0009] An application of a molecularly imprinted sensing electrode for protein detection is disclosed. This electrode is prepared using the aforementioned method, with the recombinant template protein being recombinant PSA or recombinant CEA protein. The molecularly imprinted sensing electrode is used for the detection of either PSA or CEA.

[0010] The positive and beneficial technical effects of this invention are as follows: compared with traditional imprinted polymer membranes, this imprinting technology has significant advantages, such as: avoiding the use of organic solvents for polymerization, mild template removal conditions, and excellent mass transfer performance of proteins inside and outside the imprint cavity. Since many proteins in commercial kits are His6-tagged recombinant proteins, this invention is of great value for the development of imprinted materials and biosensors. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the preparation process and detection principle of the molecularly imprinted sensing electrode according to an embodiment of the present invention.

[0012] Figure 2 This is a feasibility analysis diagram for testing PSA.

[0013] Figure 3 It is an SPR curve that measures the binding ability of molecularly imprinted membranes to PSA.

[0014] Figure 4 This is an EIS curve of PSA at different concentrations measured by the sensing electrode (PSA concentrations are 0, 0.1, 1, 3.3, 6.7, 10, in ng / mL).

[0015] Figure 5 It is the impedance value R et Linear curves of PSA concentrations (PSA concentrations are 0, 0.1, 1, 3.3, 6.7, 10, in ng / mL).

[0016] Figure 6 This is a comparison chart of the selectivity of rPSA imprinted sensing electrodes for PSA detection.

[0017] Figure 7 This is a comparison chart of the selectivity of rCEA imprinted sensing electrodes for CEA detection. Detailed Implementation

[0018] To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0019] The present invention provides a molecularly imprinted sensing electrode for protein detection, comprising a gold electrode on which a ternary NTA-Ni structure is modified. 2+ - Template protein complex, via electropolymerization on NTA-Ni 2+ A polyscopolamine-imprinted membrane forms around the template protein ternary complex. The template protein and Ni are removed by rinsing the electrode. 2+ Then, an imprinted cavity is formed that can specifically recognize the target protein, forming a molecularly imprinted sensing electrode that can be used for protein detection. The template protein is a commercially available recombinant protein containing a His6 tag, corresponding to the target protein to be detected.

[0020] Furthermore, the electrode was cleaned and rinsed with ethanol and hydrochloric acid to remove template proteins and Ni. 2+ This then forms an imprinted cavity capable of specifically recognizing the target protein.

[0021] A method for preparing a molecularly imprinted sensing electrode for protein detection includes the following steps: S1: Incubate the gold electrode with 50 μL of 50 mM (2-morpholinoethanesulfonic acid) (MES) buffer containing 0.1 mM mercapto-NTA and 1 mM TCEP for 12 hours; S2: Connect the electrode from step S1 to 10 μM Ni2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complexes; S3: After rinsing the electrode from step S2 with water, incubate the electrode with 10 μg / mL of recombinant template protein with a His6 tag for 30 minutes to form a ternary NTA-Ni structure on the electrode surface. 2+ - Template protein complex, yielding NTA-Ni 2+ - The template protein complex modifies the electrode, and the recombinant template protein corresponds to the target protein to be detected; for example, if the target protein is PSA, then the recombinant template protein is recombinant PSA (represented as rPSA in this application), and if the target protein is CEA, then the recombinant template protein is recombinant CEA (represented as rCEA in this application). S4: Electropolymerization of hyoscyamine on the electrode surface obtained in step S3 is performed by electrodeposition on NTA-Ni. 2+ - A polyhyoscyamine-imprinted membrane is formed around the template protein complex. The specific polymerization steps and conditions are as follows: The modified electrode obtained in step S3 is subjected to multi-step amperometric scanning for 15-20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with the applied potentials being 0 V for 5 s and 0.7 V for 1 s; then the electrode is rinsed with 20% ethanol and cleaned with 0.1 M HCl under ultrasonication to remove the template protein, thus obtaining a molecularly imprinted sensing electrode for protein detection.

[0022] The present invention also provides an application of a molecularly imprinted sensing electrode for protein detection. The molecularly imprinted sensing electrode for protein detection adopts the preparation method of the above-mentioned molecularly imprinted sensing electrode for protein detection. The recombinant template protein is recombinant PSA (rPSA) or recombinant CEA (rCEA). The molecularly imprinted sensing electrode for protein detection is used for the detection of PSA or applied to the detection of CEA.

[0023] The present invention will be described below using an imprint sensor for detecting PSA as an example.

[0024] I. Fabrication of Molecularly Imprinted Sensing Electrodes: S1: Incubate the gold electrode with 50 μL of 50 mM (2-morpholinoethanesulfonic acid) (MES) buffer (pH = 6.5) containing 0.1 mM mercapto-NTA and 1 mM TCEP for 12 hours; S2: Then the electrode is connected to 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complexes; S3: After rinsing the modified electrode with water, incubate the electrode with 10 μg / mL of His6-tagged recombinant PSA protein (rPSA) or recombinant CEA protein (rCEA) for 30 minutes to form a ternary NTA-Ni structure on the electrode surface. 2+ -rPSA or NTA-Ni 2+ -rCEA complex; S4: Then, hyoscyamine is electropolymerized on the electrode surface by electrodeposition in NTA-Ni 2+ -rPSA or NTA-Ni 2+ A polyhyoscyamine-imprinted film is formed around the -rCEA complex. The specific polymerization steps and conditions are as follows: NTA-Ni 2+ -rPSA or NTA-Ni 2+ The rCEA complex-modified electrode was subjected to multi-step amperometric scanning for 15–20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with applied potentials of 0 V (5 s) and 0.7 V (1 s). The electrode was then rinsed several times with 20% ethanol, followed by cleaning with 0.1 M HCl under gentle sonication to remove the template proteins rPSA and Ni. 2+ The sensing electrode is obtained.

[0025] II. PSA testing: To detect the target PSA, the sensing electrode was incubated with a given concentration of PSA for 30 minutes. After thoroughly rinsing the electrode with 10 mM phosphate buffer, the electrode was placed in a 5 mM solution of [Fe(CN)6] containing 0.1 M KCl. 3− / 4− Electrochemical impedance spectroscopy (EIS) was performed in a (1:1) solution, with a platinum wire as the auxiliary electrode and an Ag / AgCl electrode as the reference electrode.

[0026] Figure 2 In the middle, curve 1 is NTA-Ni 2+ EIS curves of the -rPSA modified electrode; Curve 2 is the EIS curve of the electrode of Curve 1 after electropolymerization of scopolamine; Curve 3 is the EIS curve of the electrode of Curve 2 after washing with HCl; Curve 4 is the EIS curve of the electrode of Curve 3 after incubation with 10 ng / mL PSA solution. From Figure 2 It can be seen from NTA-Ni 2+ The impedance of the -rPSA-modified electrode (curve 1) increases after electropolymerization of hyoscyamine (curve 2), indicating that the hyoscyamine imprinted film polymerized on the electrode surface hinders the polymerization of [Fe(CN)6]. 3− / 4−Electron transfer; after washing the electrode in curve 2 with 0.1 M HCl solution, the impedance decreased (curve 3), indicating that this condition can remove rPSA and is beneficial to [Fe(CN)6]. 3− / 4− Electron transfer; the impedance of the molecularly imprinted electrode increased after incubation with PSA solution (curve 4), indicating that PSA can enter the cavity of the imprint, thereby hindering [Fe(CN)6]. 3− / 4− Electron transfer. These results indicate that this method can be used to prepare molecularly imprinted sensing electrodes for PSA detection.

[0027] Determination of dissociation constant: A clean gold film was incubated with 200 μL of 0.1 mM mercapto-NTA for 12 hours, followed by incubation with 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold film surface. 2+ Complex. Then NTA-Ni 2+ The complex-modified gold film was incubated with 10 μg / mL rPSA for 30 min, followed by 15–20 cycles of multi-step amperometric scanning in 0.1 M NaCl solution containing 0.5 mM scopolamine, with applied potentials of 0 V (5 s) and 0.7 V (1 s). After rinsing the gold film with 20% ethanol, it was treated with 0.1 M HCl under gentle sonication to remove rPSA and Ni. 2+ The final gold film was fixed onto the SPR instrument, and three concentrations of PSA samples (10 mM phosphate buffer) were delivered into the gold film channels via a syringe pump. The dissociation constant was calculated using the SPR system's built-in kinetic software based on the signal changes.

[0028] Figure 3 This is a SPR curve graph measuring the binding affinity of PSA to the molecularly imprinted membrane. The solid line represents the SPR curves after injecting three different concentrations of PSA onto the surface of the molecularly imprinted membrane, while the dashed line represents the curve simulated using SPR software. Figure 3 As can be seen, the change in SPR angle caused by PSA increases with increasing concentration. Based on the experimental results, the dissociation constant of the interaction between PSA and the molecularly imprinted membrane can be obtained. K D The molecular imprinting concentration (PIM) was 2.7 μM, indicating that the molecularly imprinted membrane has a strong ability to recognize PSA.

[0029] To evaluate the sensitivity of the imprinted sensing electrode of the present invention, the effect of PSA concentration on impedance change was measured, such as... Figure 4 As shown. From Figure 4 As can be seen, the impedance gradually increases with increasing PSA concentration, indicating that this sensing electrode can be used for PSA detection. Figure 5As shown, within the concentration range of 0.1 ~ 10 ng / mL, R et It has a linear relationship with PSA concentration, and the linear equation is R. et = 2106[PSA] (ng / mL) + 23309. The minimum detectable concentration is lower than the PSA concentration in the blood of prostate patients (~4 ng / mL), indicating that this sensing electrode has great potential for practical sample detection.

[0030] Figure 6 This is a comparison chart of the selectivity of the rPSA blot sensing electrode for PSA detection. Columns 1-7 represent, in order: blank control, CEA, AFP, CA125, 10% fetal bovine serum, PSA, and CEA+AFP+CA125+10% fetal bovine serum+PSA. The concentration of PSA was 10 ng / mL, and the concentration of other proteins was 50 ng / mL. To evaluate the selectivity of this method, samples including carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), antigen 125 (CA125), and 10% fetal bovine serum were analyzed. Figure 6 As shown, none of these samples caused R et Significant changes occurred, with corresponding impedance values ​​comparable to the blank control experiment (columns 1-5), indicating that the imprinted sensing electrode has excellent specificity for PSA detection. Furthermore, the presence of these test samples did not significantly affect PSA measurement (columns 6 and 7), indicating that the sensing electrode has good anti-interference capabilities.

[0031] In addition to detecting PSA, the applicant also detected CEA. For CEA detection, recombinant CEA protein (rCEA) was used, and other experimental procedures were the same as for PSA detection. The results for CEA detection were consistent with those for PSA detection, indicating the broad applicability of this blotting sensing method. Figure 7 This is a comparison graph showing the selectivity of the rCEA blot sensing electrode for CEA detection. Columns 1-7 represent, in order: blank control, PSA, AFP, CA125, 10% fetal bovine serum, CEA, and PSA+AFP+CA125+10% fetal bovine serum+CEA. The concentration of CEA is 10 ng / mL, and the concentration of other proteins is 50 ng / mL. Figure 7 As shown, compared with the blank control group, PSA, AFP, CA125, and 10% fetal bovine serum samples did not cause R in the rCEA-imprinted sensing electrode. et Significant changes occurred (columns 1-5), with CEA causing a significant increase in impedance, indicating that the imprinted sensing electrode has excellent specificity for CEA detection. The presence of these test samples did not significantly affect the CEA measurement (columns 6 and 7), further demonstrating that the sensing electrode has good anti-interference capabilities.

[0032] The reagents and samples used in this invention are all available for purchase.

[0033] 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 shall fall within the scope of the technical solution of the present invention, and the present invention is not limited to the embodiments of the examples given in the specification.

Claims

1. A molecularly imprinted sensing electrode for protein detection, comprising a gold electrode, characterized in that: A ternary NTA-Ni2+-template protein complex was modified on the surface of a gold electrode, and the NTA-Ni2+-template protein complex was further modified using electropolymerization. 2+ A polyscopolamine-imprinted membrane forms around the template protein ternary complex. The template protein and Ni are removed by rinsing the electrode. 2+ Then, an imprinted cavity is formed that can specifically recognize the target protein, forming a molecularly imprinted sensing electrode that can be used for protein detection. The template protein is a commercially available recombinant protein containing His6 label, corresponding to the target protein to be detected.

2. The molecularly imprinted sensing electrode for protein detection according to claim 1, characterized in that: The electrode was cleaned and rinsed with ethanol and hydrochloric acid to remove template protein and Ni. 2+ This then forms an imprinted cavity capable of specifically recognizing the target protein.

3. A method for preparing a molecularly imprinted sensing electrode for protein detection, characterized in that... Includes the following steps: S1: Incubate the gold electrode with 50 μL of 50 mM MES buffer containing 0.1 mM mercapto-NTA and 1 mM TCEP for 12 hours; S2: Connect the electrode from step S1 to 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complexes; S3: After rinsing the electrode from step S2 with water, incubate the electrode with 10 μg / mL of recombinant template protein with His6 tag for 30 minutes to form a ternary NTA-Ni2+-template protein complex on the electrode surface, thus obtaining a ternary NTA-Ni2+-template protein complex modified electrode, where the recombinant template protein corresponds to the target protein to be detected. S4: Electropolymerize hyoscyamine on the electrode surface obtained in step S3 by electrodeposition to form a polyhyoscyamine imprinted film around the NTA-Ni2+-template protein complex. The specific polymerization steps and conditions are as follows: The modified electrode obtained in step S3 is subjected to multi-step amperometric scanning for 15-20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with the applied potentials being 0 V for 5 s and 0.7 V for 1 s. The electrode was then rinsed with 20% ethanol and cleaned with 0.1 M HCl under ultrasonication to remove the template protein, thus obtaining a molecularly imprinted sensing electrode for protein detection.

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