An immobilized electrochemical sensor enzyme element and its preparation method and application

Abstract

The present invention provides an immobilized electrochemical sensing enzyme element, a preparation method and an application thereof. The immobilized electrochemical sensing enzyme element includes a working electrode and a mixture of chitosan and a fusion protein ChiBD-LGOX attached to the surface of the working electrode. The present invention constructs a fusion recombinant protein through genetic engineering technology; while using a chitosan matrix to embed the electrochemical enzyme element, a biological affinity tag is coupled for composite directional immobilization; further, a prussian blue is used as a base material to build an electrochemical sensor. The sensitivity of the constructed L-glutamic acid biosensor based on ChiBD-LGOX is increased by 3.4 times to reach 53.4 µA·mM-1·cm-2, and the stability of the detection sensitivity is increased to 95% within 14 days. The immobilization method for the electrochemical sensing enzyme element proposed by the present invention has the advantages of improving the sensitivity and service life of the sensor, and provides a general immobilization method for the preparation of electrochemical enzyme biosensors.

Description

Technical Field

[0001] The invention relates to the technical field of protein engineering and electrochemical sensor, and in particular to an immobilization method of an electrochemical sensor enzyme element and application thereof. Background Art

[0002] Immobilization of enzyme elements is one of the necessary and key steps in the design of enzyme biosensors. The immobilization method can affect the performance parameters (sensitivity, selectivity and stability) of the biosensor by affecting the orientation, loading, mobility, stability, structure and biological activity of the enzyme. Commonly used methods for immobilization of enzyme elements include adsorption, cross-linking, covalent bonding and embedding.

[0003] Adsorption is the process of adsorbing the enzyme onto an insoluble carrier through the action of polar bonds, hydrogen bonds, and hydrophobic bonds of enzyme molecules. However, due to the weak interaction between the enzyme and the carrier, there are disadvantages such as easy falling off and leakage. Cross-linking with functional reagents such as carbodiimide and glutaraldehyde is also a commonly used immobilization method. However, these methods are prone to cause changes in the higher-order structure of the enzyme protein, destroying some active centers, thereby causing loss of enzyme activity. Covalent bonding is an immobilization method in which the enzyme and the carrier are covalently bonded, and a coupling reaction occurs with enzyme-related groups after the carrier is activated. The advantage of the bonding method is that the enzyme and the carrier are firmly bound and the problem of enzyme leakage is not easy to occur, but this method is complicated to operate, the reaction conditions are harsh, and even the properties of the enzyme such as the specificity of the substrate will change.

[0004] The encapsulation method is to fix the enzyme in a three-dimensional matrix, such as an amphiphilic network such as electropolymerized membrane, photopolymer, silica gel, polysaccharide or carbon paste. It is simple to operate and has a mild reaction. Chitosan molecules are rich in hydroxyl and amino groups, have a very high affinity with various proteins, have good biocompatibility, provide a natural microenvironment for enzymes, and can provide sufficient accessibility for electrons to shuttle between enzymes and electrodes. It has been widely used as an immobilized enzyme carrier. However, since there is no chemical reaction between chitosan and enzymes, this encapsulation fixation has good biocompatibility but is also accompanied by the problem of enzyme leakage. Summary of the invention

[0005] In view of the above problems of the prior art, the present invention provides an immobilized electrochemical sensor enzyme element and its preparation method and application. The present invention simultaneously utilizes the affinity between chitosan and protein tags and the embedding effect of chitosan itself to immobilize the enzyme to obtain the electrochemical enzyme element, thereby achieving directional and orderly immobilization of the enzyme element, and the immobilization efficiency is high. The prepared electrochemical sensor enzyme element has high sensitivity and long service life.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] An immobilized electrochemical sensor enzyme element comprises a working electrode and a mixture of chitosan and fusion protein ChiBD-LGOX attached to the surface of the working electrode;

[0008] The fusion protein ChiBD-LGOX is prepared by the following method:

[0009] (1) Insert the protein tag ChiBD-AB into the multiple cloning site of the vector plasmid through a one-step cloning method;

[0010] (2) Fusion of the target protein gene LGOX after the protein tag;

[0011] (3) Introduce the plasmid connecting the protein tag ChiBD-AB and the target protein gene LGOX into the host strain for expression to obtain a crude enzyme solution;

[0012] (4) After purification, the fusion protein ChiBD-LGOX is obtained.

[0013] Steps (1), (2) and (3) are the same as the corresponding steps in the prior art CN 109321539A.

[0014] The present invention also provides a method for preparing the above-mentioned immobilized electrochemical sensor enzyme element, comprising:

[0015] The solution of the fusion protein ChiBD-LGOX and chitosan is mixed evenly and then drop-coated on the surface of the working electrode and dried to obtain the result.

[0016] Preferably, the chitosan solution is obtained by dissolving chitosan in a 1%wt acetic acid solution and adjusting the pH to 6.0 with NaOH.

[0017] Preferably, the ratio of the fusion protein ChiBD-LGOX to the chitosan is 20 nmol: 5-15 g.

[0018] The present invention also provides the use of the immobilized electrochemical sensor enzyme element in a biosensor.

[0019] Preferably, the working electrode is a Prussian blue modified electrode. More preferably, the Prussian blue modified electrode is prepared by the method of patent CN101532979A.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The electrochemical sensor enzyme element immobilization method proposed in the present invention not only immobilizes the enzyme element in the chitosan matrix, but also utilizes the bioaffinity tag ChiBD to bind to the chitosan specific site, thereby achieving directional and orderly immobilization of the enzyme element, further improving the immobilization efficiency;

[0022] (2) The L-glutamate biosensor constructed by the fusion protein ChiBD-LGOX in the present invention not only improves the service life, but also improves the detection sensitivity due to the directional fixation of the enzyme element. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a protein structure simulation and molecular docking diagram of the fusion protein ChiBD-LGOX used in the present invention.

[0024] Figure 2 Comparative graph of the biocompatibility of the immobilization agents chitosan and glutaraldehyde in Example 1.

[0025] Figure 3 Comparison of the immobilization efficiency of the enzyme elements LGOX and ChiBD-LGOX using the immobilization reagent chitosan.

[0026] Figure 4 Performance response diagram of L-glutamate biosensor detected by chronoamperometry, where (A) is L-glutamate biosensor based on LGOX and (B) is L-glutamate biosensor based on ChiBD-LGOX.

[0027] Figure 5 Sensitivity changes of L-glutamate biosensors based on LGOX and ChiBD-LGOX.

[0028] Figure 6 It is a schematic diagram of the structure and working process of the immobilized electrochemical sensor enzyme element of the present invention. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, which can help better understand the present invention. The contents described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0030] Example 1

[0031] 1. Preparation of fusion protein ChiBD-LGOX:

[0032] The method of case CN 109321539A is to insert the protein tag ChiBD-AB into the multiple cloning site of the vector plasmid by a one-step cloning method; fuse the target protein gene LGOX after the protein tag; introduce the plasmid connecting the protein tag ChiBD-AB and the target protein gene LGOX into the host strain for expression to obtain a crude enzyme solution;

[0033] After purification, the fusion protein ChiBD-LGOX was obtained.

[0034] 2. Preparation of immobilized electrochemical enzyme sensor element:

[0035] Chitosan was dissolved in 1% acetic acid solution, and the pH was adjusted to 6.0 with NaOH to obtain a 1% wt chitosan solution.

[0036] 20 nmol / L of fusion protein ChiBD-LGOX was premixed with an equal volume of chitosan solution, then drop-coated on a Prussian blue modified electrode and dried naturally to obtain an immobilized electrochemical sensor enzyme element.

[0037] 3. AlphaFold2 was used to simulate and predict the three-dimensional structure of the prepared fusion protein ChiBD-LGOX, and used for molecular docking with the substrate chitosan. AutoDock was used to molecularly dock the structure of ChiBD-LGOX with the substrate chitosan. PyMOL was used to visualize the molecular structure. Figure 1 As shown, the ChiBD binding domain can act as a bioaffinity tag to bind to chitosan-specific sites, allowing ChiBD-LGOX to be fixed on chitosan in a directional and orderly manner. This indicates that the immobilization strategy of the present invention can provide a more suitable and uniform orientation, expose more active domains to the electrode surface, enhance the interface exchange with the electrolyte, and help improve the performance of enzyme electrochemical sensors.

[0038] 4. Biocompatibility:

[0039] Pure enzyme LGOX and ChiBD-LGOX were prepared to obtain enzyme solutions with an enzyme concentration of 20 nmol / L.

[0040] The samples were mixed with glutaraldehyde solution and chitosan solution of different concentrations (0.5%, 1.0%, 1.5%, 2.0%) in an equal volume ratio of 1:1 and the enzyme activity was detected after being placed at room temperature for 1 h.

[0041] The relative enzyme activity without mixing with the immobilization reagent (glutaraldehyde, chitosan) is defined as 100%. Figure 2 As shown, the biocompatibility of chitosan is better than that of the traditional immobilization agent glutaraldehyde, and mixing with 1%wt chitosan solution in equal volumes is the best choice.

[0042] 5. Immobilization effect:

[0043] 20 nmol / L LGOX and ChiBD-LGOX enzyme solutions were mixed with an equal volume of 1%wt chitosan solution, and enzyme element LGOX and enzyme element ChiBD-LGOX were obtained according to the above-mentioned preparation method of immobilized electrochemical sensor enzyme element.

[0044] The encapsulation interaction between LGOX and chitosan is a disordered one, while the immobilization pattern between ChiBD-LGOX and chitosan is a directional and ordered one due to the presence of affinity tag.

[0045] The obtained different enzyme elements, i.e., modified electrodes, were immersed in 50 mL of electrode buffer, and the enzyme activity in the electrode buffer was measured to calculate its immobilization rate.

[0046] like Figure 3 As shown in the figure, the immobilization rates of enzyme elements LGOX and ChiBD-LGOX were 79.2% and 89.6%, respectively. The enzyme no longer fell off after repeated immersion, indicating that the immobilization efficiency of chitosan encapsulation was excellent. ChiBD-LGOX with chitosan affinity tag interacted more closely with the chitosan matrix, and the immobilization effect was improved.

[0047] 6. L-glutamate biosensor based on the immobilized electrochemical sensor enzyme element of the present invention:

[0048] (1) Using a three-electrode system, 3 μL of 20 nmol / L LGOX and ChiBD-LGOX enzyme solutions were mixed with 3 μL of 1% chitosan solution, respectively, and drop-coated on a Prussian blue modified electrode and dried at room temperature to construct L-glutamate biosensors based on LGOX and ChiBD-LGOX, respectively.

[0049] (2) Prepare 50 mM PBS phosphate buffer (pH 6.5) as the electrode buffer, immerse the electrode in 50 mL of electrode buffer, select an applied potential of -0.05 V, perform a chronoamperometric scan, and activate for 30 min until the current baseline is stable.

[0050] (3) Sodium L-glutamate solution with a final concentration of 0.025-0.2 mM was continuously injected into the electrode buffer, and the current change value was recorded. A linear relationship was established based on the sodium L-glutamate concentration and the current change value, and the sensitivity, linear range and detection limit of the L-glutamate biosensor were calculated.

[0051] (4) The electrode prepared in step (1) was stored at 4°C, and its sensitivity was tested every 24 h according to steps (2) and (3). The sensitivity change was continuously monitored for 14 days to reflect the stability of the prepared L-glutamate biosensor.

[0052] like Figure 4 As shown in the figure, adding a certain amount of sodium glutamate solution to the electrode buffer will cause a stable current step with a response time of 15 s. According to the linear relationship between the concentration of sodium L-glutamate and the current change value, the sensitivity of the L-glutamate biosensor based on GLOX is calculated to be 15.7 µA·mM -1 cm-2 , the linear range is 50-1000 µM, the detection limit is 35 µM, and the sensitivity of the L-glutamate biosensor based on ChiBD-LGOX is 53.4 µA·mM -1 cm -2 , linear range of 25-300 µM, and detection limit of 9 µM. The sensitivity of electrochemical sensors is directly related to the accessibility and activity of immobilized enzymes. Accessibility can be improved by directional and ordered immobilization to avoid unnecessary steric hindrance. The sensitivity of the improved ChiBD-LGOX-based L-glutamate biosensor increased by 3.4 times, which can be attributed to the affinity of the ChiBD tag, which enables ChiBD-LGOX to be fixed in the chitosan matrix in an orderly and site-specific manner, which is beneficial to the enzyme catalysis reaction.

[0053] like Figure 5 As shown in the figure, after 14 days, the response current of the L-glutamate biosensor based on LGOX was 81% of the original value. The L-glutamate biosensor based on ChiBD-LGOX showed stronger storage stability and maintained 95% of the initial sensitivity within 14 days. This is because the ChiBD tag can provide additional affinity, enhance the binding of the enzyme element to the chitosan matrix, and thus reduce the leakage of the enzyme element. The composite immobilization method of coupling chitosan embedding and bioaffinity tag proposed in the present invention is conducive to the development of highly sensitive and stable electrochemical enzyme biosensors, and has a wide range of practical application value.

Claims

1. An immobilized electrochemical sensor enzyme element, It is characterized in that It comprises a working electrode and a mixture of chitosan and fusion protein ChiBD-LGOX attached to the surface of the working electrode; the ratio of the fusion protein ChiBD-LGOX to the chitosan is 20 nmol: 5-15 g; The fusion protein ChiBD-LGOX is prepared by the following method: (1) Insert the protein tag ChiBD-AB into the multiple cloning site of the vector plasmid through a one-step cloning method; (2) Fusion of the target protein gene LGOX after the protein tag; (3) Introduce the plasmid connecting the protein tag ChiBD-AB and the target protein gene LGOX into the host strain for expression to obtain a crude enzyme solution; (4) After purification, the fusion protein ChiBD-LGOX is obtained.

2. The immobilized electrochemical sensor enzyme element according to claim 1, It is characterized in that The working electrode is a Prussian blue modified electrode.

3. A method for preparing the electrochemical sensor enzyme element according to any one of claims 1 to 2, It is characterized in that include: The solution of the fusion protein ChiBD-LGOX and chitosan is mixed evenly and then drop-coated on the surface of the working electrode and dried to obtain the result.

4. The preparation method according to claim 3, It is characterized in that The drying is natural drying.

5. The preparation method according to claim 3, It is characterized in that The chitosan solution is obtained by dissolving chitosan in a 1%wt acetic acid solution and adjusting the pH to 6.0 with NaOH.

6. Use of the immobilized electrochemical sensor enzyme element according to any one of claims 1 to 2 in a biosensor.

7. The use according to claim 6, It is characterized in that The biosensor adopts a three-electrode system.

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