An anti-pollution electrochemical biosensor with thorny vine-like micro-nano scale and its preparation method and application

Abstract

The present invention discloses an electrochemical biosensor with a thorny vine-like micro-nano scale anti-pollution property, and a preparation method and application thereof, and relates to the technical field of electrochemical biosensors. An electrochemical biosensor with a thorny vine-like micro-nano scale anti-pollution property, with a copper mesh as a substrate, on which copper hydroxide, a copper metal organic framework and gold nanoparticles are modified; and an anti-pollution polypeptide with a hydrophilic structure is coupled and modified on the copper metal organic framework. The present invention designs an electrochemical biosensor with a thorny vine-like micro-nano scale anti-pollution property. The electrochemical biosensor has a thorny vine structure with a micro-nano dual-scale structure, which improves the roughness of the biosensor sensing interface, and is combined with an anti-pollution polypeptide with a hydrophilic structure to improve the hydrophilic performance from multiple dimensions, so that the biosensor designed by the present invention has excellent anti-pollution properties, and can achieve accurate detection in complex serum.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical biosensors, and in particular to a thorny vine-shaped micro-nanoscale anti-pollution electrochemical biosensor and a preparation method and application thereof. Background Art

[0002] A biosensor is a device that can detect biological molecules or biological processes. It uses the interaction between biomolecules in the body and biological elements in the sensor to produce a measurable signal; these sensors are widely used in fields such as medicine, environmental monitoring, food safety and biological research.

[0003] The main challenge that affects the accurate detection of biosensors during use lies in the biological molecules in complex biological fluids, such as cells, DNA, proteins, etc. These biomolecules tend to non-specifically adsorb on the sensor surface, thereby contaminating the sensing interface of the biosensor, causing the sensor to generate false positive signals during use or reducing the sensitivity of the biosensor system.

[0004] At present, relevant research has shown that constructing anti-fouling biosensors is an effective way to overcome the challenges of biological contamination in complex biological media without sample pretreatment. It can achieve reliable detection of complex biological fluids and is of great significance for disease diagnosis and monitoring.

[0005] Therefore, the inventors believe that how to design a biosensor with better anti-pollution properties is a technical problem that technicians in this field urgently need to solve.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In view of the above technical problems, the embodiments of the present invention provide a thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor and a preparation method and application thereof to solve the problems raised in the above background technology.

[0008] The present invention provides the following technical solutions:

[0009] A thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor is based on a copper mesh, which is modified with copper hydroxide, a copper metal organic framework and gold nanoparticles; the copper metal organic framework is coupled with an anti-pollution polypeptide modified with a hydrophilic structure.

[0010] Preferably, the amino acid sequence of the anti-pollution polypeptide is CEKEKEKEK

[0011] Preferably, the copper mesh has a micron-scale grid structure, the copper hydroxide has a micron-scale thorny vine-like structure grown on the surface of the copper mesh, and the copper metal organic framework has a nanoscale structure grown on the surface of the thorny vine-like structure of the copper hydroxide.

[0012] A method for preparing the above-mentioned thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor comprises the following steps:

[0013] S1: Sodium hydroxide and (NH4)2S2O8 are dissolved in ultrapure water to prepare solution A, a copper mesh is immersed in solution A for reaction and then washed with ultrapure water to obtain a composition A of copper hydroxide and copper mesh, and the composition A is named CuOH / Cu Net;

[0014] S2: 2,3,6,7,10,11-hexahydroxytriphenyl is dissolved in N,N-dimethylformamide, and ultrapure water is added to prepare solution B, CuOH / Cu Net is immersed in solution B and heated to 70°C for incubation, and then rinsed with acetone and ultrapure water, and dried in air to obtain a composition B of a copper metal organic framework, copper hydroxide, and copper net, and the composition B is named CuMOF / CuOH / Cu Net;

[0015] The Cu MOF / CuOH / Cu Net was immersed in APTES and stirred overnight to obtain APTES-modified Cu MOF / CuOH / Cu Net; wherein APTES is 3-aminopropyltriethoxysilane;

[0016] S3: Add ultrapure water and 1% HAuCl4 to a flask and heat to 100°C in an oil bath with stirring to prepare solution C, add 50 mg / mL sodium citrate solution to solution C, react at 100°C and cool to room temperature to prepare solution D;

[0017] A certain amount of solution D was added into water to mix and prepare solution E, and APTES-modified Cu MOF / CuOH / Cu Net was immersed in solution E and stirred at room temperature to prepare a composition C of gold nanoparticles, copper metal organic framework, copper hydroxide and copper net, and the composition C was named AuNPs / Cu MOF / CuOH / Cu Net;

[0018] S4: The step of coupling and modifying the anti-pollution polypeptide with a hydrophilic structure onto AuNPs / Cu MOF / CuOH / Cu Net comprises: dissolving the anti-pollution polypeptide with a hydrophilic structure in PBS and adding a 20% TCEP solution to obtain an anti-pollution polypeptide solution with a hydrophilic structure, immersing AuNPs / Cu MOF / CuOH / Cu Net in the anti-pollution polypeptide solution with a hydrophilic structure and incubating at room temperature to obtain a composition D of an anti-pollution polypeptide with a hydrophilic structure, gold nanoparticles, a copper metal organic framework, copper hydroxide, and a copper net, wherein the composition D is named Pep / AuNPs / Cu MOF / CuOH / Cu Net;

[0019] S5: Pep / AuNPs / Cu MOF / CuOH / Cu Net was immersed in a 2 μM aptamer solution containing 20% ​​TCEP and incubated at room temperature for 2 h to prepare a thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor. This thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor was named Apt / Pep / AuNPs / Cu MOF / CuOH / Cu Net.

[0020] An application of the above-mentioned thorny vine-shaped micro-nanoscale anti-pollution electrochemical biosensor in disease diagnosis and monitoring.

[0021] An electrochemical biosensor with thorny vine-like micro-nano scale anti-pollution properties, a preparation method and application thereof provided in an embodiment of the present invention have the following beneficial effects: the present invention designs an electrochemical biosensor with thorny vine-like micro-nano scale anti-pollution properties. This electrochemical biosensor has a thorny vine structure with a micro-nano dual-scale structure, which improves the roughness of the biosensor sensing interface, and is combined with an anti-pollution polypeptide with a hydrophilic structure to improve the hydrophilic performance from multiple dimensions, so that the biosensor designed in the present invention has excellent anti-pollution properties and can achieve accurate detection in complex serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram of a method for constructing an anti-pollution electrochemical biosensor according to the present invention;

[0023] Figure 2 It is the scanning electron microscope image of each structure of the present invention;

[0024] Figure 2 A is the SEM image of Cu Net; Figure 2 B is a scanned magnified image of Cu Net; Figure 2 C is the SEM image of CuOH / Cu Net; Figure 2 D is the SEM image of AuNPs / Cu MOF / CuOH / Cu Net; Figure 2E is a scanned magnified image of AuNPs / Cu MOF / CuOH / Cu Net.

[0025] Figure 3 This is a laser confocal image of the anti-pollution performance using a laser confocal instrument;

[0026] Figure 3 A is a laser confocal image of the copper mesh soaked in fluorescently modified bovine serum albumin; Figure 3 B is the laser confocal image of Pep / AuNPs / Cu MOF / CuOH / Cu Net after being immersed in fluorescently modified bovine serum albumin;

[0027] Figure 4 This is a response curve diagram of the present invention to the concentration of carcinoembryonic antigen protein. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments made by those skilled in the art without creative work are within the scope of protection of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments and drawings. The reagents and biological materials described in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0031] 1. Design principle of the solution of the present invention:

[0032] (1) Research has found that introducing anti-fouling materials into the sensing interface of biosensors is an important way to construct anti-fouling biosensors. Common anti-fouling materials include polyethylene glycol (PEG)-based materials, zwitterionic materials, peptide-based biomaterials, etc. Among them, peptide-based biomaterials have become important anti-fouling materials for constructing biosensors due to their physical and chemical properties, biocompatibility, and strong hydration ability.

[0033] Studies have found that polypeptides composed of specific amino acids such as glutamic acid (E), lysine (K), histidine (H), aspartic acid (D), arginine (R) and cysteine ​​(C) are usually used as anti-fouling materials for electrode modification; based on this, the present invention designs a hydrophilic anti-fouling polypeptide with a hydrophilic structure, and the amino acid sequence of the hydrophilic anti-fouling polypeptide is: CEKEKEKEK;

[0034] (2) Further studies have found that the roughness of a surface can affect its hydrophilicity or hydrophobicity; for example, increasing the roughness usually increases the hydrophilicity or hydrophobicity of a material, depending on the intrinsic properties of the material; this is because the roughness increases the surface area in contact with air or water, thus affecting its overall wettability;

[0035] It can be seen from the Wenzel equation that the hydrophilicity or hydrophobicity of the solid surface is affected by the roughness of the material itself; by appropriately changing the roughness of the solid surface within a certain range, the hydrophobic surface can be further improved to a super-hydrophobic surface, while the hydrophilic surface can be further improved to a super-hydrophilic surface; therefore, increasing the roughness of the biosensor sensing interface can improve the hydrophilicity and anti-pollution ability of the electrode;

[0036] (3) Further research found that there is a synergistic effect between microstructure and nanostructure in improving surface roughness; micro-nanostructures are widely present in nature, and flame-treated bionic shark skin surfaces have excellent low sliding angle superhydrophobicity; micro-casting (shark skin) and nano-spraying (lotus structure) are used to obtain dual-functional surfaces; and in 2002, Academician Jiang Lei of the Institute of Chemistry of the Chinese Academy of Sciences found that the superhydrophobicity of the lotus leaf surface is highly correlated with the complex structure of its surface. In the microstructure of the lotus leaf surface, a hierarchical structure composed of nanoparticles was also found, and the interaction between these hierarchical structures led to the superhydrophobicity of its surface;

[0037] In addition, geckos have the ability to climb walls and run on rooftops, water striders can walk lightly on water, and turtles can recognize the way over thousands of miles. These are all given by the special micro-nano structure (layered roughness) of their body structure and the low surface energy of their surface; the small size effect and large specific surface area brought about by the micro-nano structuring of materials can further increase the roughness of the materials; applying micro-nano structure technology to biosensors can enhance the hydrophilicity and anti-pollution ability of electrodes.

[0038] 2. Preparation route of the anti-pollution electrochemical biosensor of the present invention:

[0039] Figure 1 The figure shows a schematic diagram of the process of constructing a thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor, which includes a multi-step layer-by-layer prepared AuNPs / Cu MOF / CuOH / Cu Net multidimensional micro-nanoscale thorny vine-like material, wherein the CuOH / Cu Net is micron-sized, coupled with the AuNPs / Cu MOF nanostructure and the modified hydrophilic anti-pollution polypeptide, so that the sensor has good hydrophilicity and anti-pollution properties, and can realize the accurate detection function of CEA protein in complex serum.

[0040] 3. Preparation methods and process conditions of each composition in the present invention:

[0041] 1. Materials and reagents:

[0042] The anti-fouling peptides (Pep) with hydrophilic structure are CEKEKEKEK, (NH4)2S2O8, N,N-dimethylformamide, 2,3,6,7,10,11-hexahydroxytriphenyl, HAuCl4, fluorescently modified bovine serum albumin (FITC-BSA), carcinoembryonic antigen (CEA) and aptamer chain (Apt).

[0043] (II) Material preparation process:

[0044] Preparation of CuOH / Cu Net: 3.0 g of sodium hydroxide and 0.7 g of (NH4)2S2O8 were dissolved in 30 mL of ultrapure water, the copper net was immersed in the solution for 5 min, and then washed several times with ultrapure water;

[0045] Preparation of Cu MOF / CuOH / Cu Net: 30 mg of 2,3,6,7,10,11-hexahydroxytriphenyl was dissolved in 3 mL of N,N-dimethylformamide, and 30 mL of ultrapure water was added. Then, CuOH / Cu Net was immersed in the solution, heated to 70 °C for 20 min, rinsed thoroughly with acetone and ultrapure water, and dried in air;

[0046] 150 μL 3-aminopropyltriethoxysilane (APTES) was dissolved in 10 mL methanol solution, and then the prepared Cu MOF / CuOH / Cu Net was immersed in the above solution and stirred overnight to obtain APTES-modified Cu MOF / CuOH / Cu Net;

[0047] Preparation of AuNPs / Cu MOF / CuOH / Cu Net: Add 2 mL HAuCl4 (1%) to 48 mL ultrapure water in a flask, stir vigorously and heat to 100 °C in an oil bath, add 2 mL sodium citrate solution (50 mg / mL) to the above solution, react at 100 °C for 20 min, and cool to room temperature; take 10 mL of the above solution and add 20 mL of water to mix; immerse the APTES-modified Cu MOF / CuOH / Cu Net in the solution and stir at room temperature for 2 h;

[0048] Preparation of Pep / AuNPs / Cu MOF / CuOH / Cu Net: Anti-pollution peptide powder with the amino acid sequence of CEKEKEKEK was dissolved in a certain amount of PBS, 20% TCEP solution was added to obtain a 2 μM peptide solution, and AuNPs / Cu MOF / CuOH / CuNet was immersed in the peptide solution and incubated at room temperature for 2 h;

[0049] Preparation of Apt / Pep / AuNPs / Cu MOF / CuOH / Cu Net: Pep / AuNPs / Cu MOF / CuOH / Cu Net was immersed in 2 μM aptamer solution (containing 20% ​​TCEP) and incubated at room temperature for 2 h.

[0050] 4. Performance test of the present invention;

[0051] (A) Scanning electron microscopy of microstructure:

[0052] Figure 2 As shown, Figure 2 A is the microscopic morphology of the copper net (Cu Net). Figure 2 As can be seen from A, the copper mesh is a regular grid structure with a micron-scale size ( Figure 2 B); from Figure 2 In C, we can see the micron-scale columnar structure that has grown, and copper hydroxide (CuOH) can grow evenly on the surface of the copper mesh;

[0053] from Figure 2 As can be seen in D, the copper metal organic framework (Cu MOF) with nanoscale size can be uniformly grown on the surface of the micron-sized CuOH fiber column ( Figure 2 E), gold nanoparticles (AuNPs) can improve the conductivity of materials ( Figure 2 F), under the synergistic effect of micron- and nanoscale sizes, the structure of AuNPs / Cu MOF / CuOH / Cu Net can greatly increase the roughness of the prepared material.

[0054] (II) Anti-pollution performance evaluation:

[0055] The copper mesh ( Figure 3 A) and Pep / AuNPs / Cu MOF / CuOH / Cu Net( Figure 3 B) The cells were treated with 2 mM FITC-BSA for 0.5 h respectively, and then the anti-fouling performance was imaged using a laser confocal instrument. The confocal images after FITC-BSA treatment were recorded to evaluate the anti-fouling performance.

[0056] In this experiment, FITC-BSA was used as a protein mimic in biological samples. Figure 3 A and Figure 3 The results of B show that Figure 3 B has no obvious nonspecific adsorption of FITC-BSA, while Figure 3 A has a large amount of FITC-BSA adsorbed therein; thus, it can be seen that the biosensor designed in the present invention has good anti-pollution performance.

[0057] (III) Sensor test:

[0058] The flexible electrochemical biosensor was used to detect CEA protein at different concentrations using DPV technology, which showed a wide linear range and a low detection limit ( Figure 4 ).

[0059] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor, characterized in that: The following steps are involved: S1: Sodium hydroxide and (NH4)2S2O8 are dissolved in ultrapure water to prepare solution A, a copper mesh is immersed in solution A for reaction and washed with ultrapure water after the reaction to obtain a composition A of copper hydroxide and copper mesh, and the composition A is named Cu(OH)2 / Cu Net; S2: 2,3,6,7,10,11-hexahydroxytriphenyl was dissolved in N,N-dimethylformamide, and then ultrapure water was added to prepare solution B, Cu(OH)2 / Cu Net was immersed in solution B and heated to 70°C for incubation, then rinsed with acetone and ultrapure water, and dried in air to obtain a composition B of copper metal organic framework, copper hydroxide, and copper net, and the composition B was named Cu MOF / Cu(OH)2 / Cu Net; The Cu MOF / Cu(OH)2 / Cu Net was immersed in APTES and stirred overnight to obtain APTES-modified Cu MOF / Cu(OH)2 / CuNet; S3: ultrapure water and 1% HAuCl4 were added to a flask and heated to 100°C in an oil bath with stirring to prepare solution C, 50 mg / mL sodium citrate solution was added to solution C, the mixture was reacted at 100°C and cooled to room temperature to prepare solution D; A certain amount of solution D was added to water and mixed to prepare solution E. APTES-modified Cu MOF / Cu(OH)2 / Cu Net was immersed in solution E and stirred at room temperature to obtain a composition C of gold nanoparticles, copper metal organic framework, copper hydroxide and copper net. The composition C was named AuNPs / Cu MOF / Cu(OH)2 / Cu Net. S4: dissolving the anti-pollution polypeptide with a hydrophilic structure in PBS and adding 20% ​​TCEP solution to obtain an anti-pollution polypeptide solution with a hydrophilic structure, immersing AuNPs / Cu MOF / Cu(OH)2 / Cu Net in the anti-pollution polypeptide solution with a hydrophilic structure and incubating at room temperature to obtain a composition D of an anti-pollution polypeptide with a hydrophilic structure, gold nanoparticles, a copper metal organic framework, copper hydroxide and a copper net, and the composition D is named Pep / AuNPs / Cu MOF / Cu(OH)2 / Cu Net; S5: Pep / AuNPs / Cu MOF / Cu(OH)2 / Cu Net was immersed in a 2 μM aptamer solution containing 20% ​​TCEP and incubated at room temperature for 2 h to prepare a thorny vine-like micro-nanoscale anti-fouling electrochemical biosensor; The copper mesh is in a micron-scale grid structure, the copper hydroxide is in a micron-scale thorny vine-like structure growing on the surface of the copper mesh, and the copper metal organic framework is in a nano-scale structure growing on the surface of the thorny vine-like structure of copper hydroxide; the amino acid sequence of the anti-pollution polypeptide is CEKEKEKEK.

2. An application of an electrochemical biosensor prepared by the method for preparing a thorny vine-like micro-nanoscale anti-pollution electrochemical biosensor as claimed in claim 1 in the preparation of disease diagnosis and monitoring products.

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