Electrochemical biosensor for detecting amyloid protein oligomer and preparation method of electrochemical biosensor

Abstract

The invention discloses an electrochemical biosensor for detecting an amyloid protein oligomer. According to the sensor, methylene blue (MB) is packaged in a ZIF-67 metal organic framework to synthesize a ZIF-67 (at) MB composite material, and the ZIF-67 (at) MB composite material is modified on the surface of a nuclear track membrane nanopore channel to form a complete electrochemical detection system with an electrode. The detection mechanism is based on the competitive coordination effect of the amyloid protein oligomer and cobalt ions, so that ZIF-67 is damaged to release methylene blue, and quantitative analysis of the A beta oligomer is realized by detecting an electrochemical signal. The sensor disclosed by the invention has the advantages of high sensitivity, strong specificity, good stability, simplicity, convenience and rapidness in operation and the like, and a powerful tool is provided for early diagnosis of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical analysis, and in particular to an electrochemical biosensor for detecting amyloid protein oligomers and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease. One of its main pathological characteristics is the abnormal aggregation of amyloid protein (Aβ) in the brain to form amyloid plaques. In the process of Aβ aggregation, Aβ oligomers are considered to be the key factors leading to neurotoxicity and cognitive dysfunction. They can appear in the early stages of AD and are closely related to the progression of the disease. Therefore, accurate detection of Aβ amyloid protein, especially its oligomers, is extremely critical for the early diagnosis and disease monitoring of Alzheimer's disease. Existing detection methods, such as immunoblotting and nuclear magnetic resonance technology, have shortcomings such as complicated operation, time-consuming detection, expensive equipment, and poor sensitivity, which make it difficult to meet the needs of clinical rapid and accurate detection. Therefore, a new detection technology is urgently needed to overcome these problems.

[0003] Electrochemical biosensors have become a research hotspot in the field of biomolecule detection due to their high sensitivity, rapid response and low cost. Metal organic frameworks (MOFs), as a new type of porous material, have a large specific surface area, adjustable pore structure and abundant active sites, showing great potential in the field of biosensing. By combining electrochemical detection technology with MOF materials, highly sensitive detection of biomolecules can be achieved. Summary of the invention

[0004] The present invention aims to construct an electrochemical biosensor for detecting amyloid oligomers. ZIF-67 is a kind of electrochemical biosensor composed of cobalt ions (Co 2+ ) and 2-methylimidazole through coordination to form a metal organic framework (MOF) material, whose highly ordered porous structure and large specific surface area are conducive to the loading and transmission of substances. The present invention utilizes these characteristics of ZIF-67 and uses it as a nanocontainer to encapsulate electrochemically active methylene blue (MB) to form a ZIF-67@MB composite material. When the target molecule Aβ amyloid protein oligomer is present in the system, the ZIF-67 structure will be broken, thereby releasing the MB encapsulated inside it into the solution. Since MB has good electrochemical activity and can undergo redox reactions on the electrode surface to generate detectable electrochemical signals, it is possible to detect changes in current to achieve highly sensitive and specific detection of Aβ oligomers, providing a powerful tool for early diagnosis of Alzheimer's disease.

[0005] An electrochemical biosensor for detecting amyloid oligomers, comprising: a ZIF-67@MB nanocomposite and a nuclear pore membrane, wherein the ZIF-67@MB composite is modified in the nanopores of the nuclear pore membrane as a signal amplification medium for the construction of an electrochemical biosensor. When the sensor contacts a sample solution containing Aβ oligomers, it reacts with Co in ZIF-67. 2+ The coordination reaction occurs, which destroys the structure of ZIF-67 and releases the MB encapsulated inside ZIF-67. Since MB is electrochemically active, its release amount is positively correlated with the concentration of Aβ oligomers in the sample. By detecting the electrochemical signals in the system, quantitative analysis of Aβ oligomers can be achieved.

[0006] Application of the sensor in early diagnosis of Alzheimer's disease.

[0007] The method for preparing an electrochemical biosensor for detecting amyloid oligomers comprises the following steps: S1: Preparation of ZIF-67@MB composite material: Co(NO3)2·6H2O was dissolved in methanol solution, and then the mixed solution and MB solution were fully stirred in methanol, 2-methylimidazole was added dropwise, and stirred for 20-40 min; the precipitate was washed with methanol several times, and the ZIF-67@MB composite material was obtained after drying; S2: Construction of biosensor: The synthesized ZIF-67@MB is placed in the nanopores of the nuclear pore membrane and dried to obtain the ZIF-67@MB functionalized nuclear pore membrane; then the bovine serum albumin solution is dripped on the surface of the ZIF-67@MB functionalized nuclear pore membrane to seal it and obtain an electrochemical biosensor.

[0008] The concentration of the Co(NO3)2·6H2O is 0.05-0.5 mol / L, and the concentration of the 2-methylimidazole is 0.5-1.0 mol / L.

[0009] The application is the use of the sensor in preparing a kit for detecting amyloid oligomers.

[0010] Technical principle of the present invention:

[0011] Co 2+ The coordination effect between ZIF-67 and Aβ oligomers is stronger than that between ZIF-67 and methylimidazole. 2+ The coordination reaction occurs, which causes the ZIF-67 structure to break, thereby releasing the electrochemically active MB encapsulated inside it. The amount of MB released is positively correlated with the concentration of Aβ oligomers in the sample. By detecting the electrochemical signals in the system, quantitative analysis of Aβ oligomers can be achieved.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] 1. The high specific surface area and rich pore structure of ZIF-67 enable it to encapsulate a large number of MBs, significantly enhancing the signal output capability of the sensor. At the same time, the specific interaction between Aβ oligomers and ZIF-67 greatly improves the detection sensitivity of the sensor to Aβ oligomers.

[0014] 2. The nuclear pore membrane provides a solid support for the growth of ZIF-67, enhances the stability and reproducibility of the sensor, and ensures the reliability of the detection results.

[0015] 3. The preparation process of the sensor is relatively simple, does not require complex and sophisticated instruments and equipment, and the detection process is rapid, making it easy to achieve on-site instant detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 Schematic diagram of the detection principle of an electrochemical biosensor for detecting amyloid oligomers

[0018] Figure 2 Optimization of (A) MB concentration and (B) incubation time during electrochemical biosensor construction

[0019] Figure 3 DPV curves of the response to different concentrations of Aβ oligomers

[0020] Figure 4 Standard curve of the current change rate curve in response to different concentrations of Aβ oligomers

[0021] Figure 5 Evaluation of the repeatability of electrochemical biosensor for Aβ oligomer detection

[0022] Figure 6 Evaluation of the reproducibility of electrochemical biosensor for the detection of Aβ oligomers

[0023] Figure 7 Evaluation of the stability of electrochemical biosensor for detection of Aβ oligomers

[0024] Figure 8 Evaluation of the specificity of electrochemical biosensors for the detection of Aβ oligomers DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following embodiments.

[0026] Example 1

[0027] A method for preparing an electrochemical biosensor for detecting amyloid oligomers, wherein the method is as follows Figure 1 As shown, the following steps are included: S1: Preparation of ZIF-67@MB composite material: Co(NO3)2·6H2O was dissolved in methanol solution, and then the mixed solution was thoroughly stirred with 3 mol / L MB solution in methanol ( Figure 2 A), the concentration of Co(NO3)2·6H2O was adjusted to 0.15 mol / L, 2-methylimidazole was added dropwise to make its concentration 0.8 mol / L, and then stirred for 30 min; the precipitate was washed with methanol several times and dried to obtain the ZIF-67@MB composite material; S2: Construction of biosensor: The synthesized ZIF-67@MB is placed in the nanopores of the nuclear pore membrane and dried to obtain the ZIF-67@MB functionalized nuclear pore membrane; then the bovine serum albumin solution is dripped on the surface of the ZIF-67@MB functionalized nuclear pore membrane to seal it and obtain an electrochemical biosensor.

[0028] Example 2

[0029] An application of an electrochemical biosensor for detecting amyloid oligomers as described above comprises the following steps:

[0030] The functionalized nuclear pore membrane in Example 1 is fixed in the middle of the H-type electrolytic cell as a diaphragm, an electrolyte solution is placed in the electrolytic cell, and electrodes are placed in two chambers respectively, so as to construct a complete electrochemical detection system. It is connected to an electrochemical workstation, and the system current is detected by DPV detection means. At this time, the response current is recorded as I0;

[0031] The sample solutions containing different concentrations of amyloid oligomers were placed in the electrolytic cell and incubated for 15 min ( Figure 2 B), and electrochemical detection is performed. The current is recorded as I ( Figure 3 ); With amyloid concentration as the horizontal axis and current change I-I0 as the vertical axis, draw a standard curve ( Figure 4 ).

[0032] Example 3 In order to explore the performance of the sensor in the present invention, its reproducibility, repeatability and stability were evaluated, and experiments were carried out according to the detection method of Example 2. Figure 5As shown, the repeatability of the sensor was evaluated, and 6 sensors built in the same batch were selected to detect their response currents. The test results showed that the response current values ​​generated by the 6 electrodes to the target were basically the same, which was 64.81±0.67μA, and the relative standard deviation (RSD) was 1.04%. This result confirmed that the sensor has good repeatability.

[0033] like Figure 6 As shown in the figure, under the same conditions, the sensor was tested six times in a row, and its current change rate was 65.12±0.77μA, with an RSD of 1.18%. At the same time, the stability of the sensor was evaluated, as shown in the figure. Figure 7 As shown, after storage at 4°C for 5 weeks, 89.99% of the response current value was retained compared to the first week. The experimental results intuitively show that the sensor constructed by the present invention has good reproducibility, repeatability and stability, providing a powerful tool for the early diagnosis of Alzheimer's disease.

[0034] In order to explore the specificity of this detection method for Aβ oligomers, potential interfering substances including Aβ monomers, Aβ fibrils and C-reactive protein, a representative interfering substance in serum, were detected. When the interfering substances were present, the response current values ​​of other interfering substances were very low compared with the target Aβ oligomers ( Figure 8 ). The experimental results show that the electrochemical biosensor of the present invention has high specificity for Aβ oligomers and can effectively identify the target in a complex matrix, providing a strong guarantee for the detection of Aβ oligomers.

[0035] The above description is only the best embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent substitutions, improvements, etc. made should be included in the protection scope of the present invention.

Claims

1. An electrochemical biosensor for detecting amyloid oligomers, characterized in that The invention comprises: a ZIF-67@MB nanocomposite material and a nuclear pore membrane. The ZIF-67@MB composite material is modified in the nanopore of the nuclear pore membrane as a signal amplification medium for the construction of an electrochemical biosensor. When the sensor contacts a sample solution containing Aβ oligomers, it reacts with Co in ZIF-67. 2+ A coordination reaction occurs, which destroys the structure of ZIF-67 and releases the MB encapsulated inside ZIF-67.

2. An electrochemical biosensor for detecting amyloid oligomers according to claim 1, characterized in that: Application of the sensor in early diagnosis of Alzheimer's disease.

3. The method for preparing an electrochemical biosensor for detecting amyloid oligomers according to claim 1, characterized in that: The following steps are involved: S1: Preparation of ZIF-67@MB composite material: Co(NO3)2·6H2O was dissolved in methanol solution, and then the mixed solution and MB solution were fully stirred in methanol, 2-methylimidazole was added dropwise, and stirred for 20-40 min; the precipitate was washed with methanol several times, and the ZIF-67@MB composite material was obtained after drying; S2: Construction of biosensor: The synthesized ZIF-67@MB is placed in the nanopores of the nuclear pore membrane and dried to obtain the ZIF-67@MB functionalized nuclear pore membrane; then the bovine serum albumin solution is dripped on the surface of the ZIF-67@MB functionalized nuclear pore membrane to seal it and obtain an electrochemical biosensor.

4. The method for preparing an electrochemical biosensor for detecting amyloid oligomers according to claim 3, characterized in that: The concentration of the Co(NO3)2·6H2O is 0.05-0.5 mol / L, and the concentration of the 2-methylimidazole is 0.5-1.0 mol / L.

5. The use of the electrochemical biosensor for detecting amyloid oligomers according to claim 1, characterized in that: The application is the use of the sensor in preparing a kit for detecting Aβ oligomers.

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