Au NPs@Co-LDH@ZIF-67 composite, and preparation method and application thereof

Abstract

The application relates to the technical field of tumor detection, in particular to an AuNPs@Co-LDH@ZIF-67 composite and a preparation method and application thereof; the biosensor comprises a working electrode and an anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate, the working electrode comprises a screen-printed electrode (SPCE), and the detection end surface of the screen-printed electrode (SPCE) is sequentially connected with chitosan (Chi) and AuNPs@Co-LDH@ZIF-67, namely the biosensor is AuNPs@Co-LDH@ZIF-67 / Chi / SPCE. The biosensor has good oxidase simulation activity, the chitosan is combined to increase the loading of the antibody, the detection performance of the electrode is greatly improved, and the tumor cells can be efficiently detected.

Description

Technical Field

[0001] This invention relates to the field of tumor detection technology, and in particular to an Au NPs@Co-LDH@ZIF-67 complex, its preparation method, and its application. Background Technology

[0002] Early screening for tumors is crucial for diagnosis, treatment, and prognosis. Circulating tumor cells (CTCs) originate from primary or metastatic tumors and contain abundant tumor-related information. CTCs can enter the bloodstream via the blood or lymphatic system, leading to distant metastases. Previous studies have shown that CTCs can be used to predict disease progression and survival in early-stage cancer patients, predict treatment response and recurrence risk, better investigate treatment resistance mechanisms, and identify new therapeutic targets. Therefore, CTCs are of great significance in assisting early tumor screening and diagnosis, and in predicting treatment response and recurrence risk.

[0003] Conventional methods for detecting circulating tumor cells (CTCs) mainly include flow cytometry, immunoassay microarrays, and polymerase chain reaction (PCR). These methods are expensive, time-consuming, and require sophisticated equipment. Therefore, there is a need to develop more economical, rapid, and sensitive early tumor cell detection platforms. In recent years, biosensors have attracted widespread attention due to their advantages such as fast response speed, simple operation, high specificity, low cost, high sensitivity, and ease of miniaturization. Among them, colorimetric biosensors are widely used due to their simplicity, practicality, direct readout, low cost, and ease of operation. Standard colorimetric assays typically use biological enzymes, such as horseradish peroxidase, glucose oxidase, alkaline phosphatase, and other proteases, to catalyze the colorimetric reaction. However, the high cost, difficult purification, high temperature sensitivity, and susceptibility to denaturation of biological enzymes greatly limit their practical application. In contrast, artificial nanozymes with natural enzyme activity are an effective alternative to natural enzymes. Compared with traditional biological enzymes, artificial nanozymes have advantages such as simple preparation, low cost, and good stability. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention solves the above-mentioned technical problems through the following technical solution:

[0005] The first aspect of this invention discloses an Au NPs@Co-LDH@ZIF-67 complex, which is synthesized by in-situ etching of ZIF-67 with chloroauric acid to form a heterostructure Co-LDH@ZIF-67, and then reducing chloroauric acid with sodium borohydride to form the Au NPs@Co-LDH@ZIF-67 complex.

[0006] The second invention discloses a method for preparing the above-mentioned Au NPs@Co-LDH@ZIF-67 complex:

[0007] Synthesis of ZIF-67: 90-110 mM cobalt nitrate and 3-5 mM hexadecyltrimethylammonium bromide were dissolved in deionized water; simultaneously, 780-800 mM 2-methylimidazole was dissolved in deionized water; then, the 2-methylimidazole aqueous solution was added to the mixed solution of cobalt nitrate and hexadecyltrimethylammonium bromide, and the mixture was vigorously stirred under magnetic force to obtain a purple solution; the purple solution was centrifuged and the precipitate was collected, washed with ethanol, and the obtained purple precipitate was dried to obtain ZIF-67 powder;

[0008] Synthesis of AuNPs@Co-LDH@ZIF-67: ZIF-67 powder was dispersed in a methanol solution; then, 0.05-0.15 g / ml chloroauric acid methanol solution was added to the ZIF-67 methanol solution and magnetically stirred at room temperature; then, 0.01-0.02 M sodium borohydride methanol solution was added dropwise to the above mixture and magnetically stirred at room temperature; the mixture was centrifuged and the precipitate was collected, washed with methanol, and dried to obtain the AuNPs@Co-LDH@ZIF-67 complex.

[0009] The third aspect of this invention discloses the application of the above-mentioned AuNPs@Co-LDH@ZIF-67 complex in the quantitative detection of biomarkers, wherein the AuNPs@Co-LDH@ZIF-67 complex is used as an oxidase-like enzyme to oxidize and discolor the chromogenic substrate.

[0010] A fourth aspect of the present invention discloses a bioconjugate formed by bioconjugation of an anti-EGFR antibody with the above-mentioned Au NPs@Co-LDH@ZIF-67 complex.

[0011] The fifth aspect of this invention discloses a method for preparing the above-mentioned bioconjugate: the synthesized Au NPs@Co-LDH@ZIF-67 complex is dispersed in PBS buffer, anti-EGFR polyclonal antibody Ab2 is added, and the mixture is stirred at low temperature overnight; the obtained mixed solution is then centrifuged and the supernatant is discarded, and the precipitate is washed with PBS to obtain the anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate.

[0012] The sixth aspect of this invention discloses the application of the above-mentioned bioconjugate in a biosensor, wherein the screen-printed electrode of the biosensor is surface-modified using an anti-EGFR antibody.

[0013] Preferably, the specific method for the above surface modification is as follows: dissolve chitosan in 1% acetic acid solution to prepare a 0.5-1.5% chitosan solution; drop the chitosan solution onto the surface of a screen-printed carbon electrode; after the electrode has been completely dried at room temperature, drop a 2-3% glutaraldehyde solution onto the electrode and incubate at room temperature; after rinsing the electrode with PBS, drop 0.5-1.5 mg / mL anti-EGFR antibody Ab1 onto the surface of the chitosan-modified electrode and incubate overnight in a humidified chamber; wash the electrode with PBS to remove antibodies that have not been cross-linked with chitosan; drop a 0.5-1.5% bovine serum albumin solution onto the electrode surface and incubate to block the antibody, thus obtaining the surface-modified screen-printed electrode.

[0014] Preferably, the above-mentioned bioconjugate is used in conjunction with a screen-printed electrode with an anti-EGFR antibody surface modified to capture tumor cells.

[0015] Preferably, the logarithm of the absorbance signal detected by the biosensor to the tumor cell concentration is between 20 and 10. 4 The results showed a good linear relationship within the cell / mL range. The linear regression equation was A = 0.4975lgc - 0.1197, with a correlation coefficient of 0.9928, where A is the absorbance value of the solution in au and c is the concentration of tumor cells in cells / mL.

[0016] This invention synthesizes a heterostructure Co-LDH@ZIF-67 by in-situ etching ZIF-67 with chloroauric acid, and then reduces chloroauric acid with sodium borohydride to form an AuNPs@Co-LDH@ZIF-67 complex. This complex exhibits excellent oxidase properties, capable of directly oxidizing 3,3',5,5'-tetramethylbenzidine to blue without the use of hydrogen peroxide, and turning it yellow under acidic conditions. Furthermore, this invention reveals that the gold nanoparticles in the AuNPs@Co-LDH@ZIF-67 complex can effectively bind antibodies, making it an excellent carrier for antibody immobilization.

[0017] Based on this, a colorimetric biosensor was developed using the lung cancer cell line A549 as a model for the sensitive detection of lung cancer tumor cells. Chitosan was modified onto the surface of a screen-printed electrode, and an anti-EGFR antibody was cross-linked with chitosan using glutaraldehyde. After tumor cells were added to the surface of the antibody-modified screen-printed electrode, the antibody on the electrode specifically captured the tumor cells. The AuNPs@Co-LDH@ZIF-67 antibody complex specifically binds to the tumor cells on the electrode surface, forming a sandwich-type immune complex. The highly efficient oxidation properties of this complex catalyze the oxidation of TMB (3,3',5,5'-tetramethylbenzidine), thereby quantitatively detecting A549 cells. This cell sensor exhibits good sensitivity and stability and has broad application prospects in tumor cell detection.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) Using ZIF-67 nanocubes as templates, ZIF-67 was etched in situ with chloroauric acid to synthesize the Co-LDH@ZIF-67 complex. Then, sodium borohydride was used to reduce the chloroauric acid to form the AuNPs@Co-LDH@ZIF-67 complex. This complex exhibits excellent oxidase properties, capable of directly oxidizing 3,3',5,5'-tetramethylbenzidine to blue without the use of hydrogen peroxide, and turning it yellow under acidic conditions. Furthermore, the gold nanoparticles in the AuNPs@Co-LDH@ZIF-67 complex can effectively bind antibodies, making it an excellent carrier for antibody immobilization.

[0020] (2) This invention verifies that AuNPs@Co-LDH@ZIF-67 possesses oxidase-like activity, capable of directly oxidizing TMB to oxTMB without the use of hydrogen peroxide. Simultaneously, the calculated affinity constant of AuNPs@Co-LDH@ZIF-67 is 0.0414 mM, lower than that of the commonly used horseradish peroxidase, indicating its high affinity for the substrate 3,3',5,5'-tetramethylbenzidine.

[0021] (3) This invention also optimized the experimental conditions, determining the linear relationship between cell concentration and absorbance value under optimal capture antibody incubation time and secondary antibody complex incubation time. The constructed sensor detected tumor cells within a linear range of 20-10. 4 The sensor's detection limit is 5 cells / mL. The constructed sensor exhibits good specificity, repeatability, and reproducibility. In clinical sample testing, the recoveries of spiked cells at different concentrations ranged from 94% to 103.48%, with sample standard deviations all less than 5.73%. These results indicate that this cell sensor has great potential for application in clinical diagnostics. Attached Figure Description

[0022] Figure 1 The transmission electron microscope (TEM) image of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0023] Figure 2 The scanning electron microscope image of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0024] Figure 3 The X-ray diffraction pattern of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0025] Figure 4 Fourier transform infrared spectrum of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0026] Figure 5 X-ray photoelectron spectroscopy characterization of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0027] Figure 6 These are the cyclic voltammetry curves of different electrodes involved in this invention;

[0028] Figure 7 The ultraviolet absorption spectra of different electrodes involved in this invention are shown below;

[0029] Figure 8 The absorbance of different volumes of AuNPs@Co-LDH@ZIF-67 catalyzing TMB color development according to the present invention;

[0030] Figure 9 The time-kinetic curves of AuNPs@Co-LDH@ZIF-67 of this invention;

[0031] Figure 10 The absorbance of AuNPs@Co-LDH@ZIF-67 of the present invention for catalyzing TMB color development in the presence and absence of hydrogen peroxide;

[0032] Figure 11 The absorbance of AuNPs@Co-LDH@ZIF-67 of the present invention catalyzing TMB color development at different temperatures;

[0033] Figure 12 Enzyme kinetic analysis of AuNPs@Co-LDH@ZIF-67 of the present invention;

[0034] Figure 13 The chromatogram shows the oxidase activity of AuNPs@Co-LDH@ZIF-67 against o-phenylenediamine according to the present invention.

[0035] Figure 14 The image shows the oxidase activity of AuNPs@Co-LDH@ZIF-67 for 2,2'-nitro-bis(3-ethylbenzothiazoline-6-sulfonic acid) according to the present invention.

[0036] Figure 15 The chromatogram shows the oxidase activity of AuNPs@Co-LDH@ZIF-67 against 5-aminosalicylic acid according to the present invention.

[0037] Figure 16 To optimize the incubation time of tumor cells using the biosensor of the present invention;

[0038] Figure 17 To optimize the incubation time of the biosensor of the present invention for the secondary antibody complex;

[0039] Figure 18 The present invention provides absorbance curves for detecting tumor cells at different concentrations using the biosensor.

[0040] Figure 19 This invention provides experimental verification of the anti-interference capability of the AuNPs@Co-LDH@ZIF-67 sensor.

[0041] Figure 20 This invention provides repeatability testing verification for the AuNPs@Co-LDH@ZIF-67 sensor.

[0042] Figure 21 This invention provides experimental verification of the reproducibility of the AuNPs@Co-LDH@ZIF-67 sensor. Detailed Implementation

[0043] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0044] Example 1

[0045] A method for fabricating a biosensor based on AuNPs@Co-LDH@ZIF-67, comprising the following steps: an anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate and an anti-EGFR antibody / chitosan modified screen-printed electrode:

[0046] Synthesis of ZIF-67

[0047] 99.5 mM cobalt nitrate and 4.12 mM hexadecyltrimethylammonium bromide were dissolved in 2 mL of deionized water. Simultaneously, 789 mM 2-methylimidazole was dissolved in 14 mL of deionized water. Then, the 2-methylimidazole aqueous solution was added to the mixed solution of cobalt nitrate and hexadecyltrimethylammonium bromide, and the mixture was vigorously stirred magnetically for 20 min. The purple solution was centrifuged, and the precipitate was collected, washed five times with ethanol, and dried at 60 °C to obtain ZIF-67 powder.

[0048] Synthesis of AuNPs@Co-LDH@ZIF-67

[0049] 50 mg of ZIF-67 powder was dispersed in 10 mL of methanol solution. Then, 0.8 mL of chloroauric acid methanol solution (0.1 g / mL) was added to the ZIF-67 methanol solution, and the mixture was magnetically stirred at room temperature for 20 min. 2 mL of 0.01 M sodium borohydride methanol solution was added dropwise to the mixture, and the mixture was magnetically stirred at room temperature for 20 min. The mixture was centrifuged, and the precipitate was collected, washed five times with methanol, and then dried at 60 °C.

[0050] Synthesis of anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate

[0051] The synthesized AuNPs@Co-LDH@ZIF-67 complex was dispersed in 2 mL PBS, and 20 μL of anti-EGFR polyclonal antibody (Ab2) was added. The mixture was stirred overnight at 4 °C. The resulting mixture was centrifuged and the supernatant was discarded. The precipitate was washed three times with PBS to obtain the anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate.

[0052] Preparation of anti-EGFR antibody / chitosan modified electrode

[0053] Dissolve 1 g of chitosan in 100 mL of 1% acetic acid solution to prepare a 1% chitosan solution. Dilute 50% glutaraldehyde solution with deionized water to a 2.5% glutaraldehyde solution. Add 4 μL of the 1% chitosan solution dropwise to the surface of a screen-printed carbon electrode. After the electrode has completely dried at room temperature, add 6 μL of 2.5% glutaraldehyde solution dropwise to the electrode and incubate at room temperature for 4 h. After rinsing the electrode with PBS, add 4 μL of 1 mg / mL anti-EGFR antibody dropwise to the surface of the chitosan-modified electrode and incubate overnight at 4°C in a humidified chamber. Wash the electrode with PBS to remove antibodies that are not cross-linked with chitosan. Add 1% bovine serum albumin solution dropwise to the electrode surface and incubate for 1 h to block the antibody. After washing the electrode with PBS, store the prepared electrode at 4°C.

[0054] 1. Morphological characterization of AuNPs@Co-LDH@ZIF-67

[0055] The morphological characterization of AuNPs@Co-LDH@ZIF-67 synthesized in Example 1 was performed, and the results are as follows: Figure 1 As shown, the AuNPs@Co-LDH@ZIF-67 composite has a rough surface with layered structures. Figure 2 As shown, corrosion marks appear on the Au NPs@Co-LDH@ZIF-67 composite.

[0056] 2. X-ray diffraction characterization of AuNPs@Co-LDH@ZIF-67

[0057] X-ray diffraction characterization was performed on AuNPs@Co-LDH@ZIF-67 synthesized in Example 1, and the results are as follows: Figure 3 As shown, the typical diffraction peaks at 7.3°, 10.4°, 12.7°, and 16.4° correspond to the (011), (002), (112), and (022) planes, respectively. This result is consistent with the simulation results of ZIF-67, indicating the successful synthesis of ZIF-67 nanocubes. The X-ray diffraction results of the Au NPs@Co-LDH@ZIF-67 composite retain the (011), (002), (112), and (222) plane diffraction peaks in the ZIF-67 crystal structure. Furthermore, with the increase of chloroauric acid content during the reaction, the spectrum of the Au NPs@Co-LDH@ZIF-67-3 composite shows a diffraction peak at 20.2°, which is attributed to the characteristic peak of LDH, indicating the successful synthesis of the AuNPs@Co-LDH@ZIF-67 composite.

[0058] 3. Fourier transform infrared spectroscopy characterization of AuNPs@Co-LDH@ZIF-67

[0059] Fourier transform infrared spectroscopy was performed on AuNPs@Co-LDH@ZIF-67 synthesized in Example 1, and the results are as follows: Figure 4 As shown, in the infrared spectrum of AuNPs@Co-LDH@ZIF-67, the absorption peak intensity is at 470 cm⁻¹. -1 The corresponding increase in concentration indicates that this absorption peak is a typical -OH characteristic peak in Co-LDH, confirming the presence of Co-LDH.

[0060] 4. X-ray photoelectron spectroscopy characterization of AuNPs@Co-LDH@ZIF-67

[0061] X-ray photoelectron spectroscopy characterization was performed on AuNPs@Co-LDH@ZIF-67 synthesized in Example 1, and the results are as follows: Figure 5As shown, the O1s fine spectrum of AuNPs@Co-LDH@ZIF-67 has two peaks at 531.7 eV and 532.94 eV, which are attributed to C=O / -OH and CO, respectively, indicating that ZIF-67 is partially converted to Co-LDH during the etching process.

[0062] 5. Characterization by cyclic voltammetry curves for different electrodes

[0063] Cyclic voltammetry was used to characterize the different electrodes involved in this invention. For example... Figure 6 As shown, the current intensity of the bare screen-printed electrode (curve a) significantly increased after chitosan modification, due to the enhanced conductivity of chitosan (curve b). Further modification of the chitosan-modified electrode with anti-EGFR antibody after glutaraldehyde treatment resulted in a decrease in peak current, due to the poor conductivity of proteins (curve c). Similarly, due to the poor conductivity of tumor cells, the peak current of the electrode further decreased when A549 cells bound to the anti-EGFR antibody / chitosan-modified electrode surface (curve d). This result indicates that the antibody and tumor cells gradually bound to the chitosan-modified screen-printed electrode surface. When the anti-EGFR antibody (Ab2)-AuNPs@Co-LDH@ZIF-67 bioconjugate was modified onto the A549 cell / anti-EGFR antibody / chitosan-modified screen-printed electrode surface, the peak current of the electrode further decreased, indicating that the bioconjugate successfully bound to A549 cells, thereby immobilizing them on the tumor cell / Ab1 / chitosan-modified electrode surface (curve e).

[0064] 6. Ultraviolet absorption spectral characterization of different electrodes

[0065] The different electrodes involved in this invention were characterized using ultraviolet absorption spectroscopy. For example... Figure 7 As shown, after adding substrate solutions to the surfaces of chitosan-modified electrodes (curve a), bare screen-printed electrodes (curve b), anti-EGFR antibody / chitosan-modified electrodes (curve c), and tumor cell / anti-EGFR antibody / chitosan (curve d) modified electrodes, the solutions remained colorless with almost no detectable absorbance. In contrast, the solution on the electrode surface modified with the anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate (curve e) turned blue, and a strong absorbance could be detected by a microplate reader. These results indicate that the color change of the substrate is attributed to the oxidase catalytic activity of the anti-EGFR antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate, while chitosan and antibodies do not catalyze the substrate color change.

[0066] 7. Analysis of AuNPs@Co-LDH@ZIF-67 oxidase activity behavior

[0067] The oxidation behavior of AuNPs@Co-LDH@ZIF-67 synthesized in Example 1 was characterized. Figure 8 As shown, the absorbance of oxTMB at 630 nm increases with the increase of the volume of AuNPs@Co-LDH@ZIF-67 used. This result indicates that the content of oxidized TMB is closely related to the volume of AuNPs@Co-LDH@ZIF-67 used. Figure 9 As shown, the absorbance of the solution increases with time, indicating that AuNPs@Co-LDH@ZIF-67 can continuously oxidize TMB and cause it to change color over a period of time. Figure 10 As shown, there was no significant difference in absorbance change during TMB oxidation catalyzed by Au NPs@Co-LDH@ZIF-67 under conditions of added and unadded hydrogen peroxide, indicating that this nanozyme possesses oxidase activity and can directly oxidize TMB to oxTMB without the use of hydrogen peroxide. Figure 11 As shown, when the solution temperature increased from 20℃ to 60℃, the absorbance of AuNPs@Co-LDH@ZIF-67 oxidizing TMB showed no significant difference. However, previous studies have shown that when the temperature exceeds 37℃, the catalytic activity of horseradish peroxidase used in traditional enzymatic reactions decreases significantly or even disappears. This result indicates that the artificially synthesized nanozyme AuNPs@Co-LDH@ZIF-67 has better thermal stability compared to the widely used traditional horseradish peroxidase. Figure 12 As shown, apparent steady-state kinetics experiments were used to perform kinetic analysis on AuNPs@Co-LDH@ZIF-67 to evaluate its catalytic activity. The Kc of AuNPs@Co-LDH@ZIF-67 was... m The calculated value was 0.0414 mM, lower than that of the traditionally widely used horseradish peroxidase (0.434 mM). This is due to K... m The lower the value, the higher its affinity for the substrate. This result confirms that AuNPs@Co-LDH@ZIF-67 has a high affinity for TMB, meaning that AuNPs@Co-LDH@ZIF-67 has excellent oxidase mimicry activity.

[0068] 8. Analysis of oxidase activity of AuNPs@Co-LDH@ZIF-67 on different substrates

[0069] The catalytic activity of AuNPs@Co-LDH@ZIF-67 on several other common substrates was investigated, including o-phenylenediamine (OPD), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate, ABTS), and 5-aminosalicylic acid (5-ASA). Figure 13 As shown, with the increase of the volume of AuNPs@Co-LDH@ZIF-67 used, the absorbance of OPD increases accordingly, indicating that OPD can be directly oxidized by AuNPs@Co-LDH@ZIF-67. Meanwhile, as... Figure 14 As shown, with the addition of AuNPs@Co-LDH@ZIF-67, the ABTS solution turned green. With increasing volume of AuNPs@Co-LDH@ZIF-67, the absorbance of the green ABTS solution continuously increased, indicating that AuNPs@Co-LDH@ZIF-67 successfully oxidized ABTS to produce its color. Similarly, as... Figure 15 As shown, under the action of different volumes of Au NPs@Co-LDH@ZIF-67, 5-aminosalicylic acid can be oxidized to produce brown products of varying degrees. Furthermore, the absorbance and color increase with increasing volume of Au NPs@Co-LDH@ZIF-67, indicating that Au NPs@Co-LDH@ZIF-67 can also oxidize 5-aminosalicylic acid to produce a colored product.

[0070] 9. Sensor testing conditions optimization

[0071] The optimal incubation time for tumor cells and the capture antibody was investigated by testing the differences in absorbance after incubation of tumor cells with anti-EGFR antibody (Ab1) for different times. Figure 16 As shown, the absorbance of the TMB solution gradually increased with the extension of incubation time between tumor cells and the capture antibody. This may be due to the increasing number of tumor cells binding to the capture antibody, leading to a continuous increase in the AuNPs@Co-LDH@ZIF-67 complex that recognizes tumor cells. The absorbance of the TMB solution reached its maximum at 60 min of incubation, indicating that all tumor cells had bound to the capture antibody. Thereafter, further extension of the incubation time did not significantly increase the absorbance. Therefore, 60 min was considered the optimal incubation time for tumor cells and the capture antibody.

[0072] like Figure 17As shown, the absorbance of the TMB solution increased with the extension of incubation time between tumor cells and the bioconjugate. This result indicates that the content of the labeled antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate bound to the electrode surface continuously increases with prolonged incubation time. The absorbance of the solution reached its maximum when the incubation time between the labeled antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate and tumor cells was 40 min. Afterward, there was no significant increase in the absorbance signal. This result indicates that when the incubation time between tumor cells and the antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate reaches 40 min, the antibody complex can completely capture tumor cells on the electrode surface. Therefore, to save detection time, 40 min is considered the optimal incubation time for tumor cells and the labeled antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate.

[0073] 10. Establishment of standard curve and determination of detection limit

[0074] A standard curve was established and the detection limit was determined for the biosensor synthesized in Example 1. The experimental results are as follows: Figure 18 As the number of tumor cells increases, the absorbance of the solution also increases accordingly. This is because the increased number of tumor cells allows for greater recognition by the AuNPs@Co-LDH@ZIF-67 complex, and the AuNPs@Co-LDH@ZIF-67 catalyzes the color development of TMB, leading to an increase in absorbance. (20-10) 4 Within the cell / mL range, the absorbance detected by the sensor showed a linear correlation with the logarithm of the cell concentration, with a standard curve of A = 0.4975lgc - 0.1197 and a correlation coefficient of 0.9928. Based on a signal-to-noise ratio of 3, the detection limit of this method was calculated to be 5 cells / mL.

[0075] 11. Verify the anti-interference capability of the biosensor

[0076] For the biosensor synthesized in Example 1, proteins that may coexist with EGFR in blood or interfere with its detection, including immunoglobulin G, carcinoembryonic antigen, and BSA, were used as interfering substances to verify its anti-interference ability. In addition, negative control experiments were conducted using a tumor cell-free group, an Ab1-free group, and an antibody-AuNPs@Co-LDH@ZIF-67 bioconjugate-free group. Figure 19As shown, when using this sensor to detect interfering substances such as albumin, immunoglobulin G, and carcinoembryonic antigen, the absorbance of the interference group is almost negligible. Meanwhile, in the control group, without the addition of capture antibodies, A549 cells, and labeled antibody complexes during sensor construction, the control group also failed to catalyze TMB color development. Strong absorbance was detected in the solutions containing A549 cells and mixed samples containing A549 cells and interfering substances. In both the interference and control groups, due to the absence of EGFR protein, the AuNPs@Co-LDH@ZIF-67 bioconjugate could not recognize EGFR and modify the electrode, thus failing to catalyze TMB color development. These results indicate that the constructed sensor has good selectivity.

[0077] 12. Verify the reproducibility of the biosensor

[0078] To evaluate the repeatability of the sensor's detection signal, cell sensors were independently fabricated using the same steps, with a detection concentration of 10. 3 Tumor cells / mL were compared to the difference in detection signals between the same batch and different batches of sensors. For example... Figure 20 As shown, the relative standard deviation of absorbance values ​​from the same batch of sensors was 1.4%, while the relative standard deviation of absorbance values ​​from different batches of sensors was 2.3%. These experimental results demonstrate that the cell sensor constructed in this experiment exhibits acceptable reproducibility.

[0079] 13. Verify the regenerative properties of biosensors

[0080] To investigate the regenerative properties of the sensor, chitosan was drop-coated onto the surface of a screen-printed carbon electrode. The chitosan was then treated with glutaraldehyde, followed by the addition of a capture antibody to cross-link the chitosan, forming a capture antibody / chitosan-modified screen-printed electrode. This electrode was stored in PBS buffer at 4°C for later use. Every four days after storage, the electrode was washed, and A549 cells and the AuNPs@Co-LDH@ZIF-67 complex were sequentially added to the electrode surface for specific binding. The absorbance of the sensor in detecting cells was then measured. Figure 21 The results showed that after 16 days of electrode storage, the absorbance of the sensor still maintained 92.5% of the initial signal, indicating that the sensor has good regeneration capability.

[0081] 14. Verify the detection of biological samples by the biosensor synthesized in Example 1.

[0082] The feasibility of this sensor for detecting real samples was evaluated using a spiked recovery experiment. Different known concentrations (100, 500, 1000, and 5000 cells / mL) of A549 cells were added to plasma samples, and the sensor was used to detect the spiked samples. The obtained detection values ​​were compared with theoretical values. As shown in Table 1, the spiked recoveries for different cell concentrations ranged from 94% to 103.48%, and the standard deviations for all samples were less than 5.73%. These results indicate that this cell sensor has great potential for application in clinical diagnostics.

[0083] Table 1. Spike recovery experiment (n=3)

[0084]

[0085] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bioconjugate, characterized in that, The bioconjugate is formed by bioconjugation of an anti-EGFR antibody with an Au NPs@Co-LDH@ZIF-67 complex. The specific preparation method is as follows: the synthesized Au NPs@Co-LDH@ZIF-67 complex is dispersed in PBS buffer, anti-EGFR polyclonal antibody Ab2 is added, and the mixture is stirred at low temperature overnight. The resulting mixture is then centrifuged and the supernatant is discarded. The precipitate is washed with PBS to obtain the anti-EGFR antibody-Au NPs@Co-LDH@ZIF-67 bioconjugate. The preparation method of the Au NPs@Co-LDH@ZIF-67 complex is as follows: (1) Synthesis of ZIF-67: 90-110 mM cobalt nitrate and 3-5 mM hexadecyltrimethylammonium bromide were dissolved in deionized water; at the same time, 780-800 mM 2-methylimidazole was dissolved in deionized water; then the aqueous solution of 2-methylimidazole was added to the mixed solution of cobalt nitrate and hexadecyltrimethylammonium bromide, and the mixture was stirred vigorously under magnetic force to obtain a purple solution; the purple solution was centrifuged and the precipitate was collected, washed with ethanol, and the obtained purple precipitate was dried to obtain ZIF-67 powder; (2) Synthesis of Au NPs@Co-LDH@ZIF-67: ZIF-67 powder was dispersed in methanol solution; then 0.05-0.15 g / ml chloroauric acid methanol solution was added to ZIF-67 methanol solution and magnetically stirred at room temperature; then 0.01-0.02 M sodium borohydride methanol solution was added dropwise to the above mixed solution and magnetically stirred at room temperature; the mixed solution was centrifuged and the precipitate was collected, washed with methanol, and dried to obtain Au NPs@Co-LDH@ZIF-67 complex.

2. The application of the bioconjugate as described in claim 1 in a biosensor, characterized in that, The screen-printed electrodes of the biosensor are surface-modified using anti-EGFR antibodies.

3. The application according to claim 2, characterized in that, The specific method for surface modification is as follows: Chitosan is dissolved in 1% acetic acid solution to prepare a 0.5-1.5% chitosan solution; the chitosan solution is dropped onto the surface of a screen-printed carbon electrode; after the electrode is completely dried at room temperature, 2-3% glutaraldehyde solution is dropped onto the electrode and incubated at room temperature; after rinsing the electrode with PBS, 0.5-1.5 mg / mL anti-EGFR antibody Ab1 is dropped onto the surface of the chitosan-modified electrode and incubated overnight in a humidified chamber; the electrode is washed with PBS to remove antibodies that are not cross-linked with chitosan; 0.5-1.5% bovine serum albumin solution is dropped onto the electrode surface and incubated to block the antibody, thus obtaining the surface-modified screen-printed electrode.

4. The application according to claim 3, characterized in that, The absorbance signal detected by the biosensor corresponds to the logarithm of the tumor cell concentration in the range of 20-10. 4 The cells showed a good linear relationship within the range of cells / mL, with a linear regression equation of A = 0.4975 lg c - 0.1197 and a correlation coefficient of 0.9928, where A is the absorbance value of the solution in au and c is the concentration of tumor cells in cells / mL.

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