Porous carbon material with luminol encapsulated in aptamer, preparation method of porous carbon material and application of porous carbon material in specific detection of enrofloxacin

Abstract

The invention relates to the technical field of chemiluminescence analysis, in particular to a porous carbon material with luminol encapsulated in an aptamer, a preparation method of the porous carbon material and application of the porous carbon material in specific detection of enrofloxacin. ZIF-8 is prepared and heated in a tubular furnace to obtain Z-800 with a porous carbon framework; carrying out carboxylation treatment on the Z-800 with the porous carbon framework; the preparation method comprises the following steps: carrying out activation treatment on carboxylated Z-800 by using a phosphate buffer solution, adding amino-modified cDNA into the carboxylated Z-800 to obtain cDNA functionalized Z-800, transferring the cDNA functionalized Z-800 into the phosphate buffer solution, adding luminol and an aptamer, and incubating to obtain the porous carbon material with luminol molecules encapsulated by the aptamer. According to the invention, the detection of the residual level of enrofloxacin in the sample is successfully realized, a CL signal is successfully amplified by the concept of enhancing CL by liquid drops, and the sensitivity of CL detection is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescence analysis, and in particular to a porous carbon material encapsulating luminol with aptamer, a preparation method thereof, and an application in the specific detection of enrofloxacin. Background Art

[0002] Metal-organic frameworks (MOFs) are a class of novel crystalline porous materials assembled from metal ions / clusters and organic linkers. MOFs have the advantages of high specific surface area, multiple pore structures, and adjustable pore size, and are ideal precursors for preparing porous carbon materials. As a kind of MOF, ZIF-8 not only has the above characteristics, but also has the advantages of high carbon content, stable structure, and strong loading capacity. Compared with other traditional carriers, ZIF-8 and its derivative porous carbon materials have a larger specific surface area and a more uniform pore structure, and have been widely used in the loading of small molecules such as dyes and drugs in recent years.

[0003] In recent years, gating technology has developed rapidly and has been widely used in the stimulus response mechanism to control the transport and release of guest molecules under specific conditions. In the gating system, different kinds of "locks" such as polymers, antibodies, aptamers, etc. are used to encapsulate guest molecules in the "cage" to ensure that the guest molecules can be released when the corresponding pH, temperature, biomolecules, and other predetermined stimulus conditions are reached. As single-stranded DNA or RNA nucleotides, aptamers have high specificity and affinity for target substances and are very suitable as "locks" in the gating system. When the target appears, the aptamer can specifically bind to the target and successfully open the "lock", thereby realizing the release of the guest molecule.

[0004] As an analytical method with high sensitivity and wide linear range, chemiluminescence has been widely used in the fields of chemical detection, life analysis, biosensors, etc. It is generally believed that the sensitivity of biosensors is directly related to the signal intensity. Therefore, various signal amplification strategies have been adopted in the CL signal amplification strategy. For example, relying on the catalytic action of nanomaterials, nucleic acid signal amplification technology can effectively achieve CL signal amplification, but these methods usually require complex synthesis processes and long nucleic acid incubation times. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a porous carbon material encapsulating luminol with aptamer, a preparation method thereof, and an application in the specific detection of enrofloxacin. The present invention prepares a MOF precursor, uses carbon framework Z-800 as a carrier, encapsulates luminol with enrofloxacin aptamer and cDNA, and successfully realizes the detection of the residual level of enrofloxacin in the sample. The concept of droplet-enhanced CL successfully amplifies the CL signal and improves the sensitivity of CL detection.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions: A preparation method of a porous carbon material encapsulating aptamer-luminol, comprising the following steps: Dissolve soluble zinc salt and 2-methylimidazole in water respectively to obtain a soluble zinc salt solution and a 2-methylimidazole solution. Add the soluble zinc salt solution to the 2-methylimidazole solution, stir at room temperature, then stand, and obtain ZIF-8 after post-treatment. Heat-treat the ZIF-8 in a tubular furnace to obtain Z-800 with a porous carbon framework.

[0007] Add the Z-800 with a porous carbon framework to an acidic solution for carboxylation to obtain carboxylated Z-800.

[0008] Activated the carboxylated Z-800 with phosphate buffer, then add amino-modified cDNA to the carboxylated Z-800. The carboxylated Z-800 binds to the amino-modified cDNA through an amide reaction to obtain cDNA-functionalized Z-800. Then transfer the cDNA-functionalized Z-800 to phosphate buffer, add luminol and aptamer, and incubate to obtain a porous carbon material encapsulating aptamer-luminol molecules.

[0009] In a preferred embodiment of the present invention, in the phosphate buffer, the dosage ratio of cDNA-functionalized Z-800, luminol and aptamer is 200 mg: 140 μL - 160 μL: 90 μL - 110 μL, wherein the concentration of luminol is 10 mM and the concentration of aptamer is 10 -6 M.

[0010] In a preferred embodiment of the present invention, the mass ratio of soluble zinc salt to 2-methylimidazole is 1.1 - 1.3: 21.8 - 23.6, the dosage ratio of soluble zinc salt to water is 1.1 - 1.3 g: 7.8 - 8.2 mL, and the dosage ratio of 2-methylimidazole to water is 21.8 - 23.6: 78 - 82 mL.

[0011] In a preferred embodiment of the present invention, the stirring time at room temperature is 4 - 6 h.

[0012] In a preferred embodiment of the present invention, the heat treatment method is to first treat at 145 - 155 °C for 3 - 5 hours to remove bound water, and then heat at 750 - 850 °C for 1.5 - 2.5 hours.

[0013] In a preferred embodiment of the present invention, the acidic solution is prepared by adding ammonium persulfate and sulfuric acid to water, and the dosage ratio of ammonium persulfate, sulfuric acid and water is 27.0 g - 27.8 g: 13.8 mL - 14.2 mL: 118 mL - 122 mL.

[0014] In a preferred embodiment of the present invention, the phosphate buffer solution is a mixed solution composed of EDC and NHS, wherein the molar ratio of EDC to NHS is 1.9 - 2.1:0.95 - 1.05.

[0015] In a preferred embodiment of the present invention, the incubation time condition is incubation at 37 °C for 4 - 6 hours.

[0016] Another object of the present invention is to provide a porous carbon material encapsulating luminol with an aptamer prepared by the preparation method described in any one of the above.

[0017] The third object of the present invention is to provide an application of the porous carbon material encapsulating luminol with an aptamer described above in the specific detection of enrofloxacin. After adding enrofloxacin to the porous carbon material encapsulating luminol with an aptamer and stirring, the supernatant is measured for CL signal by magnetic separation method, wherein the ratio of enrofloxacin to the porous carbon material is 1.9 - 2.1:0.95 - 1.05.

[0018] In a preferred embodiment of the present invention, the pH of the supernatant is adjusted to 9.0 - 12.5 and added to an H2O2 solution with immiscible Bnbzo droplets at the bottom for generating amplified CL signal, wherein the volume ratio of Bnbzo droplets to the H2O2 solution is 1:25.

[0019] In a preferred embodiment of the present invention, the detection limit of enrofloxacin is 0.03 nM.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. First, the ZIF-8 precursor is prepared in the present invention, and then heat treatment is carried out to obtain Z-800 with a porous carbon framework. The Z-800 is carboxylated, and the carboxylated Z-800 is combined with cDNA modified with amino groups through amidation reaction. Then, luminol and aptamer are added, and luminol is encapsulated with the aptamer and cDNA to obtain a porous carbon material encapsulating luminol with an aptamer. In the porous carbon material encapsulating luminol with an aptamer prepared in the present invention, in the absence of enrofloxacin, luminol is locked in a cage and cannot be released. The supernatant reacts with hydrogen peroxide to produce only a weak CL signal. Once enrofloxacin appears, the aptamer accurately recognizes it, and enrofloxacin specifically binds to the aptamer, opening the switch for encapsulating luminol, resulting in the release of luminol into the supernatant, enabling it to react with hydrogen peroxide to produce a strong CL signal. The porous carbon material generated with ZIF-8 as the precursor in the present invention has good chemiluminescent reagent loading performance due to its large specific surface area, uniform pore size distribution, and good pore volume ratio. Combining with high-quality aptamer technology, the encapsulation of luminol is successfully achieved, which has high specificity for enrofloxacin and realizes the detection of enrofloxacin.

[0021] 2. Droplet-enhanced CL is a simple and effective CL signal amplification method, which improves the collection efficiency of CL signals and makes the CL signals grow exponentially. Compared with the original traditional method, the droplet-enhanced CL of the present invention has successfully reduced the detection limit of enrofloxacin from 2.0 nM to 0.03 nM, and has great application potential for quantitative detection. This strategy is of great significance for the application of antibiotics such as enrofloxacin in on-site detection, and lays a foundation for the rapid and simple detection of enrofloxacin in the future.

[0022] 3. The present invention has developed a droplet-enhanced CL detection strategy for enrofloxacin, prepared a MOF precursor, used the carbon framework Z-800 as a carrier, and encapsulated luminol with aptamers. This strategy has successfully achieved the detection of enrofloxacin residue levels in real samples. The concept of droplet-enhanced CL has successfully amplified the CL signal and improved the sensitivity of CL detection. Brief Description of the Drawings

[0023] Figure 1 It is the enrofloxacin detection strategy based on the droplet-enhanced CL technology of the present invention.

[0024] Figure 2 In it, A is the TEM image of ZIF-8, and B is the TEM image of Z-800.

[0025] Figure 3 In it, A is the XRD pattern of ZIF-8 and Z-800, B is the N2 adsorption-desorption isotherm of Z-800, c is the micropore size distribution curve of Z-800, and D is the FTIR spectra of Z-800 and carboxyl-functionalized Z-800.

[0026] Figure 4 In it, A is the UV-visible spectra of carboxyl-functionalized Z-800 (orange) and the complex of carboxyl-functionalized Z-800 and cDNA (blue), B is the UV-visible spectra of luminol in the supernatant before encapsulation (blue) and luminol in the supernatant after encapsulation (green), C is the fluorescence spectra of luminol in the supernatant before encapsulation (brown) and luminol in the supernatant after encapsulation (purple), D is the polyacrylamide gel electrophoresis analysis of the cDNA-binding aptamer map, from left to right are markers, cDNA, enrofloxacin aptamer, cDNA and enrofloxacin aptamer (the fourth from the left), E is the CL intensity of luminol in the supernatant before encapsulation (orange) and luminol in the supernatant after encapsulation (blue), and F is the principle of droplet-enhanced CL.

[0027] Figure 5 In it, A and B are the optical microscope morphologies of Bnbzo in bright field and dark field in solution respectively, and C is the intensity map of droplet-enhanced CL.

[0028] Figure 6Among them, A is the CL intensity of enrofloxacin at concentrations of 5 nM (a), 10 nM (b), 30 nM (c), 50 nM (d), 100 nM (e), 200 nM (f), and 500 nM (g) in the absence of Bnbzo droplets, B is the linear relationship between the CL intensity and the enrofloxacin concentration in the absence of Bnbzo droplets, C is the CL intensity of enrofloxacin at 0.1 nM (a), 0.5 nM (b), 1 nM (c), 5 nM (d), 10 nM (e), 30 nM (f), 50 nM (g), 500 nM (h), 200 nM (i), and 500 nM (j) in the presence of Bnbzo droplets, and D is the linear relationship between the CL intensity and the enrofloxacin concentration in the presence of Bnbzo droplets.

[0029] Figure 7 In it, A is the CL reaction intensity of different antibiotics, and B is the result of repeating the CL signal intensity 6 times when the enrofloxacin concentration is 30 nM. Detailed implementation mode

[0030] The following combines the embodiments of the present invention, and uses preferred embodiments and accompanying drawings for detailed description. The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0031] It should be noted that all the professional terms used in the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the following embodiments of the present invention can be obtained through market purchase or prepared by existing methods.

[0032] Table 1 shows the relevant raw material information used in the present invention Example 1 A preparation method of an aptamer-encapsulated luminol porous carbon material includes the following steps: (1) Dissolve 1.2 g of Zn(NO3)2·6H2O in 8.0 mL of deionized water, and dissolve 22.7 g of 2-methylimidazole in 80.0 mL of deionized water. Then, under stirring conditions, dropwise add the Zn(NO3)2·6H2O solution into the 2-methylimidazole solution, stir at room temperature for 5 hours, let stand for 12 hours, wash several times with methanol and deionized water, centrifuge, and dry overnight under vacuum at 60°C to obtain ZIF-8.

[0033] (2) Place the ZIF-8 on a quartz boat and treat it at 150 °C for 4 hours in a tube furnace filled with argon to remove the bound water. Then raise the temperature to 800 °C and maintain it under this condition for 2 hours. After cooling in the tube furnace, remove the quartz boat to obtain a black powder, namely Z-800 with a porous carbon framework.

[0034] (3) Add 27.4 g of ammonium persulfate and 14.0 mL of sulfuric acid to 120 mL of deionized water to form an acidic solution. Then add 200 mg of Z-800 to the acid solution and stir at room temperature for 4 h to obtain carboxylated Z-800. After the reaction, wash the carboxylated Z-800 several times with deionized water until the pH value reaches 7, and then dry it in vacuo to obtain carboxylated Z-800.

[0035] (4) Activate 3.0 mg of carboxylated Z-800 with 1.0 mL of 1.0×10 -3 M phosphate buffer solution containing 1.0 mM EDC and 0.5 mM NHS. Place the centrifuge tube in a shaker at 37 °C for 30 minutes. After 30 minutes, add an excess of 10 -6 M amino-modified cDNA to the centrifuge tube and place it in the shaker at 37 °C overnight again. Then, wash Z-800 with 10 mM phosphate buffer solution with a pH of 6.0 to remove the excess unbound cDNA to Z-800, and obtain cDNA-functionalized Z-800. Transfer the cDNA-functionalized Z-800 to 2.0 mL of 1 mM phosphate buffer solution with a pH of 7.4, add 150 μL of 10 mM luminol to the solution, and add 100 μL of 10 -6 M aptamer solution, and incubate at 37 °C for 5 hours to obtain a porous carbon material encapsulating luminol with aptamers.

[0036] Example 2 A preparation method of a porous carbon material encapsulating luminol with aptamers, comprising the following steps: (1) Dissolve 1.1 g of Zn(NO3)2·6H2O in 7.8 mL of deionized water, and dissolve 21.8 g of 2-methylimidazole in 78 mL of deionized water. Then, under stirring conditions, gradually add the Zn(NO3)2·6H2O solution dropwise to the 2-methylimidazole solution, stir at room temperature for 5 hours, let it stand for 12 hours, wash it several times with methanol and deionized water, centrifuge, and dry it overnight under vacuum at 60 °C to obtain ZIF-8.

[0037] (2) Place the ZIF-8 on a quartz boat and treat it at 145 °C for 3 hours in a tubular furnace filled with argon to remove the bound water. Then raise the temperature to 750 °C and maintain it under this condition for 1.5 hours. After cooling in the tubular furnace, remove the quartz boat to obtain a black powder, namely Z-800 with a porous carbon framework.

[0038] (3) Add 27.0 g of ammonium persulfate and 13.8 mL of sulfuric acid to 118 mL of deionized water to form an acidic solution. Then add 200 mg of Z-800 to the acid solution and stir at room temperature for 4 h to obtain carboxylated Z-800. After the reaction, wash the carboxylated Z-800 several times with deionized water until the pH value reaches 7, and then dry it in vacuo to obtain carboxylated Z-800.

[0039] (4) Activate 3.0 mg of the carboxylated Z-800 with 1.0 mL of 1.0×10 -3 M phosphate buffer solution containing 0.8 mM EDC and 0.45 mM NHS. Place the centrifuge tube in a shaker at 37 °C for 30 minutes. After 30 minutes, add 10 -6 M cDNA to the centrifuge tube and place it in the shaker at 37 °C overnight again. Then, wash the Z-800 with 10 mM phosphate buffer solution with a pH of 6.0 to remove the excess unbound cDNA to obtain cDNA-functionalized Z-800. Transfer the cDNA-functionalized Z-800 to 2.0 mL of 1 mM phosphate buffer solution with a pH of 7.4. Add 140 μL of 10 mM luminol to the solution, and add 90 μL of 10 -6 M aptamer solution, and incubate at 37 °C for 4 hours to obtain a porous carbon material encapsulating luminol with aptamers.

[0040] Example 3 A preparation method of a porous carbon material encapsulating luminol with aptamers, comprising the following steps: (1) Dissolve 1.3 g of Zn(NO3)2·6H2O in 8.2 mL of deionized water, and dissolve 23.6 g of 2-methylimidazole in 82 mL of deionized water. Then, under stirring conditions, slowly add the Zn(NO3)2·6H2O solution dropwise to the 2-methylimidazole solution, stir at room temperature for 5 hours, let it stand for 12 hours, wash it several times with methanol and deionized water, centrifuge, and dry it overnight under vacuum at 60 °C to obtain ZIF-8.

[0041] (2) Place the ZIF-8 on a quartz boat and treat it at 155 °C for 5 hours in a tube furnace filled with argon to remove the bound water. Then raise the temperature to 850 °C and maintain it under this condition for 2.5 hours. After cooling in the tube furnace, remove the quartz boat to obtain a black powder, namely Z-800 with a porous carbon framework.

[0042] (3) Add 27.8 g of ammonium persulfate and 14.2 mL of sulfuric acid to 122 mL of deionized water to form an acidic solution. Then add 200 mg of Z-800 to the acid solution and stir at room temperature for 4 h to obtain carboxylated Z-800. After the reaction, wash the carboxylated Z-800 several times with deionized water until the pH value reaches 7, and then dry it in vacuo to obtain carboxylated Z-800.

[0043] (4) Activate 3.0 mg of carboxylated Z-800 with 1.0 mL of 1.0×10 -3 M phosphate buffer solution containing 1.1 mM EDC and 0.52 mM NHS. Place the centrifuge tube in a shaker at 37 °C for 30 minutes. After 30 minutes, add 10 -6 M cDNA to the centrifuge tube and place it in a shaker at 37 °C overnight again. Then, wash Z-800 with 10 mM phosphate buffer solution with a pH of 6.0 to remove the excess unbound cDNA to Z-800, and obtain cDNA-functionalized Z-800. Transfer the cDNA-functionalized Z-800 to 2.0 mL of 1 mM phosphate buffer solution with a pH of 7.4, add 160 μL of 10 mM luminol to the solution, and add 110 μL of 10 -6 M aptamer solution and incubate at 37 °C for 6 hours to obtain a porous carbon material encapsulating luminol with aptamers.

[0044] Application Example 1 Add 200 μL of enrofloxacin sample to 100 μL of the porous carbon material encapsulating luminol with aptamers. After stirring at 25 °C for 30 min, determine the CL signal of the supernatant by magnetic separation. Adjust the pH of the supernatant to 10 with 0.1 M sodium hydroxide and add it to 100 μL of 20 mM hydrogen peroxide solution containing immiscible solution drops of Bnbzo (4 μL) to generate an amplified CL signal, which reflects the content of enrofloxacin in the sample.

[0045] Result Analysis CL Detection Principle of Enrofloxacin The detection platform principle of the binding of enrofloxacin to CL is as Figure 1As shown. The whole process is divided into three parts. The first part is to prepare luminol-encapsulated cage Z-800; in the second part, carboxylated Z-800 is combined with amino-modified cDNA through amidation reaction, then luminol is added and shaken for a period of time. After luminol enters the carrier pores, enrofloxacin aptamer is added to make it complementary to the cDNA bases, completing the encapsulation of luminol. In the absence of enrofloxacin, luminol is locked in a cage and cannot be released. The supernatant reacts with hydrogen peroxide, producing only a weak CL signal. Once enrofloxacin appears, the aptamer accurately recognizes it, and enrofloxacin specifically binds to the aptamer, opening the switch for encapsulating luminol, resulting in the release of luminol into the supernatant, enabling it to react with hydrogen peroxide to produce a strong CL signal. The third part is about droplet-enhanced CL. As long as a certain volume of immiscible organic droplets is added to the solution, the CL signal can be greatly enhanced. The phenomenon of droplet-enhanced CL was confirmed in this step. The immiscible organic droplets have a high refractive index. Adding high-refractive-index droplets to the solution not only increases the refractive index of the entire liquid environment but also uses multiple reflections of light to improve the collection efficiency of the light signal. After adding Bnbzo droplets to the solution environment, the light radiation generated by CL directly leads to a multiple increase in the CL signal, thereby improving the sensitivity of CL detection. To achieve the CL signal enhancement effect, in the last step, immiscible organic droplets are pre-added to the reaction solution, which has great potential for improving the sensitivity of CL detection of small samples. The above three parts constitute Figure 1 the complete CL detection enrofloxacin system shown in

[0046] Characterization of ZIF-8 and Z-800 The structures and morphologies of ZIF-8 and Z-800 in different states were observed by transmission electron microscopy (TEM). Figure 2 As shown in A of Figure 2 , ZIF-8 as a precursor presents a regular dodecahedron shape with a size of about 100 nm. As shown in Figure 3 B of Figure 3 , Z-800 produced after carbonization is the same as ZIF-8, presenting a uniform regular dodecahedron shape. PXRD and N2 adsorption-desorption isotherms are shown in Figure 3 A and Figure 3As can be seen in C, the Brunauer-Emmett-Teller (BET) surface area of Z-800 is 755.347 m 2 g -1 , and there are a large number of micropores with a size of about 1.3 nm on the surface, which is confirmed by N2 adsorption-desorption technology. Therefore, Z-800 is a good encapsulation material for luminol. Fourier transform infrared spectroscopy (FTIR) was used to verify the success of the next step of the carboxylation reaction of Z-800, as shown in Figure 3 D. For carboxylated Z-800, due to the generation of carboxyl groups, a new infrared characteristic peak appears at 1602 cm -1 , which is mainly due to the stretching vibration of C=O. The results show that Z-800 has been successfully modified by carboxyl groups.

[0047] Feasibility of luminol The successful encapsulation of luminol in the cage is very important for this system. Therefore, methods such as ultraviolet-visible spectroscopy, fluorescence spectroscopy, and CL intensity were used to verify whether luminol was successfully encapsulated. The experimental results are shown in Figure 4 . By comparing the ultraviolet-visible spectra before and after the ligation of Z-800 cDNA ( Figure 4 A), the new ultraviolet absorption peak at 260 nm proves that cDNA and Z-800 are integrally ligated through amidation reaction. Figure 4 The gel electrophoresis in D proves that cDNA is successfully combined with APT and can form an encapsulation switch for luminol. By comparing the ultraviolet-visible spectra ( Figure 4 B) and fluorescence spectra ( Figure 4 C) before and after the encapsulation of luminol, the characteristic peak height of luminol decreases. The CL intensity of the supernatant after encapsulation is also lower than that before encapsulation. It is not difficult to distinguish whether the content of luminol in the supernatant decreases. These results indicate that luminol is successfully encapsulated in the cage by the compound DNA double strand.

[0048] Characterization of enhanced CL by immiscible organic droplets When a chemical reaction generates CL in immiscible organic droplets with different refractive indices (n1, n2), CL signals with different intensities are reflected multiple times in the immiscible organic droplets with a higher refractive index, thereby producing better and more effective CL signal collection, improving the collection efficiency of CL signals, and ultimately generating enhanced CL ( Figure 4 E and F).

[0049] The refractive indices of deionized water and Bnbzo were measured with an Abbe refractometer to be 1.330 and 1.565, respectively. Figure 5The state of immiscible organic droplets Bnbzo in a liquid environment observed with a high-power optical microscope. The added immiscible organic droplets are spherical and relatively uniform in size. The spherical Bnbzo enhances the light refraction ability, changes the refractive index of the entire liquid environment, thereby increasing the multiple of the CL signal. The addition of Bnbzo droplets results in multiple reflections of light, which greatly improves the light signal acquisition efficiency, thus increasing the intensity of the CL signal. In actual detection, the Bnbzo-enhanced CL system is used.

[0050] Analytical performance of the enrofloxacin CL assay To evaluate the analytical performance of the constructed sensor, under the optimal experimental conditions, the CL response signals of different concentrations of enrofloxacin were tested in the presence and absence of Bnbzo droplets respectively. From Figure 6 it can be seen that regardless of the presence of Bnbzo, the intensity of CL is directly related to the concentration of enrofloxacin. As the concentration of enrofloxacin increases, the CL intensity also increases. Its intensity shows a good linear relationship with the concentration of enrofloxacin. In the absence of Bnbzo, the regression equation is I = 191.16932logc + 205.30238, and the correlation coefficient is 0.9985 (in the range of enrofloxacin concentration from 5 nM to 500 nM ( Figure 6 A and B in)). The detection limit is 2.0 nM. When Bnbzo droplets are added, the CL signal is significantly enhanced, and the CL signal intensity still maintains a good linear relationship with the concentration of enrofloxacin (from 0.1 nM to 500 nM) ( Figure 6 C and D in). The regression equation at this time is I = 512.87008logc + 1318.5918, and the correlation coefficient is 0.9988. The detection limit is 0.03 nM. Compared with the detection limit when Bnbzo droplets are not added, the detection limit is significantly reduced, indicating that the added immiscible organic droplets successfully achieve the amplification of the CL signal and reduce the detection limit of the system. Table 1 shows the performance comparison of different analytical methods for detecting enrofloxacin. This proves that the experimental results of detecting enrofloxacin by this strategy are very effective, accurate and reliable.

[0051] Specificity and reproducibility of enrofloxacin CL detection To detect the constructed CL detection method, the specificity of ciprofloxacin (CIP), ofloxacin (OFX), and norfloxacin (NOR) was used. Therefore, three drugs similar to enrofloxacin were selected in this invention for comparative study. As Figure 7 shown in A, the presence of enrofloxacin directly affects the size of the CL signal generated in this experiment. Only when enrofloxacin is present, the sensor will generate a relatively strong CL signal, indicating that this detection method has excellent specificity for enrofloxacin.

[0052] The repeatability of the method is an important indicator of the method, reflecting the stability of the method. The detection method repeatedly detected enrofloxacin at 100 nM, with a total of 6 detections. The results are as Figure 7 shown in B. The relative standard deviation (RSD) of the generated CL signal peak was calculated to be 2.6%, indicating that the CL intensity is quite stable. This is sufficient to show that the method has good reproducibility.

[0053] Detection of real samples Since there is no residual enrofloxacin in the prepared samples, a recovery experiment was adopted, and the CL method was used for detection to evaluate the accuracy of the method. The results showed that at concentrations of 1.0 nM to 100.0 nM, the spiked recoveries of enrofloxacin were 92.00% - 95.63%. Compared with the standard method, this method has higher accuracy in the detection of enrofloxacin in fish samples.

[0054] In summary, the present invention proposes an analytical strategy for porous carbon materials for the CL detection of enrofloxacin residues in fish samples with ZIF-8 as the precursor, luminol aptamer encapsulation. The porous carbon material generated with ZIF-8 as the precursor has good chemiluminescent reagent loading performance due to its large specific surface area, uniform pore size distribution, and good pore volume ratio. Combining with high-quality aptamer technology, the encapsulation of luminol was successfully achieved, which has high specificity for enrofloxacin and realizes the detection of enrofloxacin. Droplet-enhanced CL is a simple and effective CL signal amplification method, which improves the collection efficiency of the CL signal and makes the CL signal increase exponentially. Compared with the original traditional method, droplet-enhanced CL successfully reduced the detection limit of enrofloxacin from 2.0 nM to 0.03 nM. The application potential of quantitative detection is huge. This strategy is of great significance for the application of antibiotics such as enrofloxacin in on-site detection, laying a foundation for the rapid and simple detection of enrofloxacin in the future. This strategy can be applied to other fields of antibiotic detection. In summary, this strategy is a simple and reliable method for the detection of enrofloxacin in fish samples, and has broad development prospects and potential in the future fields of small sample and trace antibiotic detection and CL analysis.

[0055] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step methods are the same as those in the embodiments, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0056] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing a porous carbon material with aptamer encapsulated luminol, characterized in that: The following steps are involved: Dissolving a soluble zinc salt and 2-methylimidazole in water respectively to obtain a soluble zinc salt solution and a 2-methylimidazole solution, adding the soluble zinc salt solution to the 2-methylimidazole solution, stirring at room temperature, standing, and post-treating to obtain ZIF-8, and heating the ZIF-8 in a tube furnace to obtain Z-800 with a porous carbon framework; Adding the Z-800 with a porous carbon framework into an acidic solution for carboxylation to obtain carboxylated Z-800; The carboxylated Z-800 is activated with a phosphate buffer, and then the amino-modified cDNA is added to the carboxylated Z-800. The carboxylated Z-800 combines with the amino-modified cDNA through an amide reaction to obtain cDNA-functionalized Z-800. The cDNA-functionalized Z-800 is then transferred to a phosphate buffer, and luminol and an aptamer are added. After incubation, the luminol is encapsulated to obtain a porous carbon material in which the aptamer encapsulates luminol.

2. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: The incubation time is 37°C for 4 to 6 hours.

3. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: In phosphate buffer, the dosage ratio of cDNA functionalized Z-800, luminol and aptamer was 200 mg: 140 μL~160 μL: 90 μL~110 μL, where the concentration of luminol was 10 mM and the concentration of aptamer was 10 -6 M.

4. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: The mass ratio of soluble zinc salt to 2-methylimidazole is 1.1~1.3:21.8~23.6, the dosage ratio of soluble zinc salt to water is 1.1~1.3g:7.8~8.2 mL, and the dosage ratio of 2-methylimidazole to water is 21.8~23.6:78~82 mL.

5. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: The heating treatment method is to first treat at 145°C~155°C for 3 hours~5 hours to remove bound water, and then heat at 750°C~850°C for 1.5 hours~2.5 hours.

6. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: The acidic solution is prepared by adding ammonium persulfate and sulfuric acid into water, and the usage ratio of ammonium persulfate, sulfuric acid and water is 27.0 g~27.8 g:13.8 mL~14.2 mL:118 mL~122 mL.

7. The method for preparing the porous carbon material of aptamer encapsulated luminol according to claim 1, characterized in that: The phosphate buffer solution is a mixed solution composed of EDC and NHS, wherein the molar ratio of EDC to NHS is 1.9-2.1:0.95-1.

05.

8. A porous carbon material of luminol encapsulated by an aptamer obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the porous carbon material of aptamer encapsulated luminol according to claim 8 in specific detection of enrofloxacin, characterized in that: Enrofloxacin was added to the porous carbon material containing luminol encapsulated by the aptamer, and after stirring, the supernatant was used to measure the CL signal by magnetic separation, wherein the volume ratio of enrofloxacin to the porous carbon material was 1.9-2.1:0.95-1.05; The supernatant was adjusted to pH 9.0-12.5 and added to a H2O2 solution with immiscible Bnbzo droplets at the bottom to generate an amplified CL signal, wherein the volume ratio of Bnbzo droplets to H2O2 solution was 1:

25.

10. The use according to claim 9, characterized in that: The detection limit of enrofloxacin was 0.03 nM.

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