Methods and applications of colorimetric analysis of antibiotics using paper-based detection chips

Abstract

This invention discloses a method and application for colorimetric analysis of antibiotics using a paper-based detection chip. The method includes: preparing a paper-based detection unit; immersing the paper-based detection unit in a NiCo@C HNs solution modified with nucleic acid aptamers; drying to obtain a paper-based detection chip; adding samples containing different concentrations of antibiotics for reaction; then adding a solution containing TMB and H2O2 for colorimetric reaction; using a mobile phone to identify color intensity to obtain RGB or grayscale values; selecting the linear curve with the highest fitting degree between antibiotic concentration and RGB or grayscale values ​​as the antibiotic detection standard curve; and then quantitatively detecting the antibiotic content based on the RGB or grayscale values ​​of the measured sample. Antibiotics can also be qualitatively detected based on colorimetric analysis. This method has advantages such as low cost, simple operation, fast response, strong anti-interference ability, and on-site detection capability, and can be applied to the detection of antibiotic residues in the environment and food.

Description

Technical Field

[0001] This invention belongs to the field of colorimetric sensing technology and relates to a colorimetric analysis method for antibiotics, specifically a method and application of colorimetric analysis of antibiotics using a paper-based detection chip. Background Technology

[0002] Antibiotics are a class of drugs with antibacterial activity, widely used in animal husbandry and aquaculture due to their low cost and strong antibacterial properties. However, the increasing antibiotic resistance caused by overuse has drawn attention, and antibiotic residues in the environment and food pose toxicity and serious side effects to humans. Current methods for determining antibiotics include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA), etc. These methods suffer from problems such as complex equipment, cumbersome operation, high detection costs, or long detection cycles. Therefore, there is an urgent need to develop a method that does not rely on complex equipment, is simple to operate, and has high sensitivity and selectivity for the quantitative detection of antibiotics in the environment and food. Paper-based colorimetric analysis has attracted widespread attention due to its advantages such as simple operation, cost-effectiveness, visualization, and on-site application. However, current paper-based detection methods generally suffer from low selectivity, low sensitivity, and inaccurate colorimetric interpretation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method and application for colorimetric analysis of antibiotics using a paper-based detection chip that is low in cost, easy to operate, visible to the naked eye, can be detected on-site, has high selectivity and high sensitivity.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0005] A method for colorimetric analysis of antibiotics using a paper-based detection chip includes the following steps:

[0006] S1. Preparation of paper-based detection chip: Cut filter paper into multiple detection units, and then soak them in a solution of NiCo@CHNs modified with nucleic acid aptamers. NiCo@CHNs is a NiCo bimetallic oxide porous carbon composite nanomaterial with a hollow nanobox microstructure. After that, it is taken out and dried.

[0007] S2. Sample detection: Add samples containing different concentrations of antibiotics to different detection units of the paper-based detection chip and react for 10 min to 30 min. Then add a mixed solution containing TMB and H2O2 and perform a colorimetric reaction for 10 min to 30 min.

[0008] S3. Color Recognition: Take a picture of the color-developing paper-based detection chip with a mobile phone, read the color intensity and convert it into RGB or grayscale values, establish a linear response relationship between antibiotic concentration and RGB or grayscale values, and select the curve with the highest fitting degree as the antibiotic detection standard curve.

[0009] S4. Based on the antibiotic detection standard curve obtained in step S3 and the measured RGB or grayscale values ​​of the sample containing antibiotics, the concentration of antibiotics in the sample is obtained.

[0010] In the above-described method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, the antibiotic is enrofloxacin, the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No. 1, and the linear regression equation for the detection of antibiotic concentration and the grayscale value of the paper-based detection chip is as follows:

[0011] Gray = 130.6 - 1.55lgC (1)

[0012] In formula (1), Gray represents the gray value, and C is the concentration of antibiotic in the sample to be tested, in ng·mL. -1 The correlation coefficient R in equation (1) 2 =0.986, the linear range for antibiotic detection is 0.1 ng / mL. -1 ~50μg·mL -1 The detection limit is 0.13 ng / mL. -1 .

[0013] In the preferred embodiment of the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, in step S2, when a sample without antibiotics is added to the paper-based detection chip and incubated, and then H2O2 and TMB are added to react, the paper-based chip turns light blue; when a test sample containing antibiotics is incubated on the paper-based detection chip, and then H2O2 and TMB are added to react, the blue color of the paper-based detection chip deepens, thereby qualitatively detecting whether the test sample contains antibiotics.

[0014] In the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S2, the concentration of H2O2 is 5mM to 50mM, and the concentration of TMB is 0.1mM to 2mM.

[0015] In the aforementioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S1, the concentration of the nucleic acid aptamer-modified NiCo@C HNs solution is 10 μg·mL. -1 ~50μg·mL -1 .

[0016] In the aforementioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, the preparation method of the nucleic acid aptamer-modified NiCo@C HNs solution in step S1 includes the following steps:

[0017] S1.1. Disperse nickel nitrate and sodium citrate in water, then add potassium cobalt cyanide aqueous solution, incubate at room temperature, centrifuge and dry to obtain NiCo precursor;

[0018] S1.2 Disperse the NiCo precursor in a buffer solution, then add dopamine and react at room temperature to obtain a NiCo precursor with a polydopamine coating.

[0019] S1.3. The NiCo precursor with polydopamine coating obtained in step S1.2 is pyrolyzed to obtain NiCo@C HNs, a NiCo bimetallic oxide porous carbon composite nanomaterial with a hollow nanobox structure.

[0020] S1.4. Disperse the NiCo@C HNs obtained in step S1.3 in sodium acetate-acetic acid buffer, and then add the nucleic acid aptamer to the sodium acetate-acetic acid buffer containing NiCo@C HNs for mixing and incubation to obtain a nucleic acid aptamer-modified NiCo@C HNs solution.

[0021] In the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S1.1, the molar ratio of nickel nitrate to sodium citrate is 1:1 to 3, and the molar ratio of nickel nitrate to potassium cobalt cyanide is 1:0.5 to 1.

[0022] In the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S1.2, the buffer solution is Tris-HCl, and the mass ratio of dopamine to NiCo precursor is 1:1 to 3.

[0023] In the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S1.3, the pyrolysis temperature is 300℃~500℃ and the pyrolysis time is 2h~4h.

[0024] In the above-mentioned method for colorimetric analysis of antibiotics using a paper-based detection chip, preferably, in step S1.4, the concentration of the sodium acetate-acetic acid buffer is 0.1M to 0.2M, the concentration of the nucleic acid aptamer in the sodium acetate-acetic acid buffer is 0.1μM to 1μM, and the incubation is performed at room temperature for 10min to 30min.

[0025] As a general technical concept, the present invention also provides an application of the above-mentioned paper-based detection chip colorimetric analysis method for antibiotics in the detection of antibiotics in the environment or food.

[0026] In this invention, in step S1, the soaking time is 10 min to 30 min.

[0027] In this invention, in step S1.1, the room temperature is 25℃~30℃, and the incubation time is 18h~24h.

[0028] In this invention, HNs in NiCo@C HNs is an abbreviation for hollow nanobox.

[0029] In this invention, the mobile phone is preferably a smartphone, and the color intensity can be read using an existing smartphone APP.

[0030] In this invention, the concentrations of nickel nitrate, sodium citrate, and potassium cobalt cyanide in water are not limited, as long as they are dissolved in water.

[0031] The detection principle of this invention mainly lies in the following: This invention utilizes NiCo@CHNs composite nanomaterials with excellent peroxidase-like activity. These composite nanomaterials can catalyze the oxidation of TMB to a blue color in the presence of H2O2. By functionalizing NiCo@CHNs with nucleic acid aptamers, the peroxidase-like activity of NiCo@CHNs is reduced because the nucleic acid aptamers mask some of the active sites of NiCo@CHNs. On the other hand, because the nucleic acid aptamers can specifically recognize the target analyte, their higher affinity causes the aptamers to desorb from the NiCo@CHNs, gradually restoring their enzyme-like activity. Different concentrations of the target analyte result in different enzyme activities, corresponding to different depths of color. By combining nucleic acid aptamers to improve the selectivity of NiCo@CHNs and regulate its enzyme activity, the nucleic acid aptamer-functionalized NiCo@CHNs are integrated with paper-based analytical equipment. The color changes generated by the paper-based detection chip are read and recognized by a smartphone and converted into readable RGB or grayscale values, thereby achieving the purpose of on-site visualization, qualitative, and quantitative detection of the target analyte.

[0032] Compared with the prior art, the advantages of the present invention are as follows:

[0033] (1) This invention provides a method for colorimetric analysis of antibiotics using a paper-based detection chip. The method utilizes a NiCo@CHNs paper-based detection chip modified with nucleic acid aptamers to specifically identify antibiotics and perform a colorimetric reaction to achieve antibiotic detection. The NiCo@CHNs used in this invention is a nanozyme with peroxidase-like activity, capable of catalyzing the oxidation of some chromogenic substrates, such as 3,3',5,5'-tetramethylbenzidine (TMB), in the presence of H2O2. Due to the special hollow nanobox structure of NiCo@CHNs and the numerous pores distributed on its surface, it provides a larger specific surface area and catalytic active sites. Meanwhile, Ni and Co are inexpensive transition metal elements. Their synergistic effect and reversible multivalent state endow them with high peroxidase-like activity. That is, under low content NiCo@C HNs conditions (μg / mL level), they can catalytically oxidize TMB colorimetric reagent to generate a blue product in the presence of H2O2, which has high detection sensitivity. Moreover, the analytical method is fast-responding, and the colorimetric analysis method can observe a visible color change within 5 minutes, which greatly shortens the detection time.

[0034] The NiCo@C HNs preparation process used in this invention is simple, low-cost, highly stable, and environmentally friendly, making it suitable for large-scale preparation. The paper-based detection chip based on NiCo@C HNs is simple to prepare and inexpensive, which can greatly reduce the detection cost. Furthermore, by using widely available smartphones to read the paper color, it can achieve rapid, accurate, and visual on-site detection. The device is small and portable, meeting the needs of the general public for operation, and is suitable for widespread application. It overcomes the limitations of large indoor instruments for detection and analysis, such as high cost, inability to perform on-site detection, and the need for professional technicians to operate.

[0035] The method of this invention is based on the peroxidase activity of nucleic acid aptamers-functionalized NiCo@C HNs to catalyze the oxidation and color development of TMB, and is integrated into a paper-based analytical device. It has the advantages of simple operation, low cost, visualization, and on-site application. A blank control solution and the sample solution to be tested are added to the paper-based detection chip. The presence of antibiotics in the sample can be determined by comparing the color development of the sample and the control solution. By adding different concentrations of antibiotics to the paper-based detection chip, a linear relationship between antibiotic concentration and RGB or grayscale value can be established, enabling rapid on-site quantitative analysis of antibiotics. Therefore, the colorimetric analysis method of antibiotics using the paper-based detection chip of this invention can be used for qualitative and quantitative detection of antibiotics, and has good application value and market prospects.

[0036] (2) This invention utilizes MOFs, i.e., NiCo precursors, coated with a polydopamine (PDA) layer, and pyrolyzes them at high temperature to obtain NiCo@CHNs nanozymes with high catalytic activity. During pyrolysis, PDA transforms into a porous carbon shell, and the NiCo precursor transforms into NiCo bimetallic oxide. The PDA-derived carbon shell effectively protects the framework structure of the NiCo precursor, ultimately forming a NiCo bimetallic oxide porous carbon composite nanomaterial with a hollow nanobox structure. Combining nucleic acid aptamers enhances the selectivity of NiCo@CHNs and regulates its enzyme activity. Nucleic acid aptamer-functionalized NiCo@CHNs are integrated with paper-based analytical equipment. The color changes generated by the paper-based detection chip are read and identified by a smartphone and converted into readable RGB or grayscale values, thereby achieving on-site visualization, qualitative, and quantitative detection of the target analyte.

[0037] (3) The paper-based detection chip colorimetric analysis method of the present invention has good detection recovery rate when applied to the detection of antibiotics in the environment and food. Compared with traditional colorimetric detection technology, the colorimetric detection method of the present invention has the advantages of simple operation, low reagent volume required, fast response, high accuracy, good selectivity, strong anti-interference ability and instant detection. Attached Figure Description

[0038] Figure 1 This is a physical image of the paper-based detection chip prepared in Embodiment 1 of the present invention.

[0039] Figure 2 The image shows the color development of the paper-based detection chip after adding different concentrations of enrofloxacin in Embodiment 1 of the present invention.

[0040] Figure 3 This is a schematic diagram of a paper-based detection chip detection scenario in Embodiment 1 of the present invention.

[0041] Figure 4 This is an interface display diagram of the smartphone reading the RGB values ​​of the paper-based detection chip after color development, different fitting methods, and drawing of linear curves in Embodiment 1 of the present invention.

[0042] Figure 5 This is a transmission electron microscope image of the NiCo@C HNs enzyme-like composite nanomaterial prepared in Example 1 of this invention.

[0043] Figure 6 The image shows the X-ray photoelectron spectrum of the NiCo@C HNs enzyme-like composite nanomaterial prepared in Example 1 of this invention.

[0044] Figure 7This is the colorimetric image of different antibiotics detected by colorimetric analysis using a paper-based detection chip in Embodiment 2 of the present invention. In this image, OFL is ofloxacin, CIP is ciprofloxacin, CAP is chloramphenicol, KAN is kanamycin, GEN is gentamicin, TOB is tobramycin, and ENR is enrofloxacin.

[0045] Legend:

[0046] 1. Paper-based detection chip; 2. Tray; 3. Detection chamber; 4. Auxiliary support; 5. Smartphone. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the raw materials and instruments used in the following embodiments are commercially available.

[0048] In the following examples, the room temperature is between 25°C and 30°C.

[0049] In the following examples, the sodium acetate-acetic acid buffer solution is mainly prepared by sodium acetate and acetic acid in a certain ratio. Preferably, the molar ratio of sodium acetate to acetic acid is 1:5 to 6, but it is not limited to this. The sodium acetate-acetic acid buffer solution is also commercially available.

[0050] Example 1

[0051] A method for colorimetric analysis of antibiotics using a paper-based detection chip according to the present invention includes the following steps:

[0052] S1. Preparation of paper-based detection chip: Whatman No. 1 filter paper is cut into circular pieces, then soaked in a solution containing 50 μg / mL of nucleic acid aptamer-modified NiCo@C HNs for 10 min, and then removed and dried.

[0053] Paper-based detection chips that have been soaked in a NiCo@C HNs solution modified with nucleic acid aptamers and then dried are shown below. Figure 1 As shown.

[0054] S2. Sample Detection: Samples containing different concentrations of enrofloxacin (0.1 ng / mL) were tested. -1 1 ng·mL -1 10 ng·mL -1 100 ng·mL -1 1 μg·mL -1 10 μg·mL -1 50 μg·mL -1 0.1 mg·mL -1An acetate-sodium acetate buffer solution was added dropwise to a paper-based detection chip and reacted for 30 minutes. Then, a mixed solution containing 0.5 mM TMB and 10 mM H₂O₂ was added dropwise, and the colorimetric reaction was allowed to proceed for another 20 minutes. Figure 2 As shown, after 20 minutes of color development reaction, the paper-based detection chips with added enrofloxacin all showed a relatively uniform blue color. However, the higher the concentration of enrofloxacin added, the deeper the blue color became. This is because higher concentrations of enrofloxacin specifically recognize the nucleic acid aptamers on the surface of NiCo@CHNs, causing the nucleic acid aptamers to be detached from the surface of NiCo@CHNs. This increases the affinity of NiCo@CHNs for the color development substrate TMB, resulting in a stronger catalytic ability to TMB and producing a deeper blue color.

[0055] S3, Color Recognition: The paper-based detection chip detection process is as follows... Figure 3 As shown, the colored paper-based detection chip 1 is placed on the tray 2, and the tray 2 is pushed into the detection chamber 3. The top of the detection chamber 3 is equipped with an auxiliary support 4, which supports a smartphone 5. The smartphone 5 takes a picture, and the color of the paper-based chip is read through a mobile app. The color is then converted into RGB or grayscale values. Based on different fitting results, the logarithm of the enrofloxacin concentration shows the highest good fit with the grayscale value, such as... Figure 4 As shown, the corresponding fitting equation is:

[0056] Gray = 130.6 - 1.55lgC (1)

[0057] In formula (1), Gray represents the gray value, and C is the concentration of the antibiotic in the test solution, with the unit being ng·mL. -1 The correlation coefficient R in equation (1) 2 =0.986, the linear range for antibiotic detection is 0.1 ng / mL. -1 ~50μg·mL -1 The detection limit is 0.13 ng / mL. -1 .

[0058] S4. Based on the antibiotic detection standard curve obtained in step S3 and the measured RGB or grayscale values ​​of the sample containing antibiotics, the concentration of antibiotics in the sample can be calculated, thereby achieving quantitative detection of antibiotics.

[0059] In this embodiment, the nucleic acid aptamer-modified NiCo@C HNs are mainly prepared by the following steps:

[0060] S1.1. Disperse nickel nitrate and sodium citrate in water, then add 0.4 mM potassium cobalt cyanide aqueous solution. The molar ratio of nickel nitrate to sodium citrate is 1:2, and the molar ratio of nickel nitrate to potassium cobalt cyanide is 1:1. Incubate at room temperature for 20 h, centrifuge and dry to obtain NiCo precursor.

[0061] S1.2. The NiCo precursor was redispersed in a buffer solution, and then dopamine was added. The mass ratio of dopamine to NiCo precursor was 1:1. The reaction was carried out at room temperature for 6 hours to obtain a NiCo precursor with a polydopamine coating.

[0062] S1.3 The NiCo precursor with polydopamine coating obtained above is pyrolyzed at 350°C for 3 hours to obtain NiCo bimetallic oxide porous carbon composite nanomaterial with hollow nanobox microstructure, namely NiCo@C HNs enzyme-like composite nanomaterial.

[0063] S1.4. NiCo@C HNs were dispersed in 0.2M sodium acetate-acetic acid buffer. Then, the nucleic acid aptamer was added to the sodium acetate-acetic acid buffer containing NiCo@C HNs and mixed. The mixture was incubated at room temperature for 30 min to obtain nucleic acid aptamer-modified NiCo@C HNs. The concentration of the nucleic acid aptamer in the sodium acetate-acetic acid buffer was 0.5 μM, and the concentration of NiCo@C HNs in the sodium acetate-acetic acid buffer was 50 μg / mL. -1 .

[0064] The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No. 1, specifically:

[0065] CCCAT CAGGG GGCTA GGCTA ACACG GTTCG GCTCT CTGAG CCCGG GTTAT TTCAGGGGGA

[0066] like Figure 5 As shown, electron microscopy imaging analysis of NiCo@CHNs revealed that they exhibit a hollow nanobox microstructure with a diameter of approximately 250 nm, and numerous pores on the surface. These pores increase the specific surface area and provide more active sites. This demonstrates the successful fabrication of NiCo@CHNs with a unique hollow structure.

[0067] like Figure 6 The X-ray photoelectron spectrum shows the presence of C, O, Co, and Ni elements in the NiCo@C HNs enzyme-like composite nanomaterial, indicating that the generated NiCo@C HNs contains NiCo bimetallic oxide and carbon.

[0068] As can be seen from the embodiments, the paper-based detection chip colorimetric analysis method for antibiotics of the present invention can be used for qualitative and quantitative detection of enrofloxacin, and the concentration of the enrofloxacin to be tested can be calculated based on the detection linear regression equation.

[0069] Example 2

[0070] The present invention discloses a method for colorimetric analysis of antibiotics using a paper-based detection chip, applicable to the detection of antibiotics in the environment and food.

[0071] The paper-based detection chip prepared in Example 1 was used to analyze antibiotics in actual samples. The detection accuracy of the colorimetric analysis method for antibiotics using the paper-based detection chip of the present invention was evaluated. The standard addition method was used to detect the target analyte in the actual sample, and a recovery rate experiment was conducted.

[0072] The samples to be tested were wastewater samples and milk samples with different concentrations of enrofloxacin added. The specific steps were as follows: After pretreatment such as filtration, the supernatant of each sample was diluted with sodium acetate-acetic acid buffer solution. The colorimetric detection method described in Example 1 was used to determine the enrofloxacin in the test solution. The results are listed in Table 1. The concentration of enrofloxacin added to the samples is shown in Table 1. When the concentration of enrofloxacin added to the wastewater was 0, the detection concentration was 22.36 ± 0.42 ng·mL. -1 This indicates that the wastewater contains a certain amount of enrofloxacin.

[0073] Table 1. Validation results of enrofloxacin recovery rate in the test solution.

[0074]

[0075] As shown in Table 1, the paper-based detection chip colorimetric analysis method for antibiotics of this invention achieves a recovery rate of approximately 97.66% to 118.11% within the measurable concentration range, indicating ideal measurement results. Compared to traditional colorimetric detection techniques, the colorimetric detection method of this invention is simple, rapid, and highly accurate. Table 1 also shows that the paper-based detection chip colorimetric analysis method for antibiotics of this invention can be used to detect enrofloxacin in actual samples, achieving excellent detection accuracy.

[0076] Anti-interference capability assessment

[0077] The interference resistance of the paper-based detection chip colorimetric analysis method for antibiotics of the present invention was evaluated using the colorimetric detection method of Example 1 against common interfering agents, such as ofloxacin, ciprofloxacin, chloramphenicol, kanamycin, gentamicin, and tobramycin, at higher concentrations (1 μg·mL⁻¹). -1 A colorimetric determination was performed, and the test results are as follows: Figure 7 As shown. By Figure 7 It is evident that the colorimetric analysis method for antibiotics using the paper-based detection chip of the present invention exhibits strong specificity for enrofloxacin. The color intensity of the added interfering substances is almost identical to that of the blank control, indicating that the colorimetric detection method of the present invention has good anti-interference ability and good selectivity for the target antibiotic.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention. sequence list <110> Hunan University <120> Methods and applications of colorimetric analysis of antibiotics using paper-based detection chips <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(60) <223> The nucleotide sequence designed according to experimental requirements to serve as an aptamer probe. <400> 1 cccatcaggg ggctaggcta acacggttcg gctctctgag cccgggttat ttcaggggga 60

Claims

1. A method for colorimetric analysis of antibiotics using a paper-based detection chip, characterized in that, Includes the following steps: S1. Preparation of paper-based detection chip: Cut filter paper into multiple detection units, and then soak them in a solution of NiCo@CHNs modified with nucleic acid aptamers. NiCo@CHNs is a NiCo bimetallic oxide porous carbon composite nanomaterial with a hollow nanobox microstructure. After that, it is taken out and dried. S2. Sample detection: Add samples containing different concentrations of antibiotics to different detection units of the paper-based detection chip and react for 10 min to 30 min. Then add a mixed solution containing TMB and H2O2 and perform a colorimetric reaction for 10 min to 30 min. S3. Color Recognition: Take a picture of the color-developing paper-based detection chip with a mobile phone, read the color intensity and convert it into RGB or grayscale values, establish a linear response relationship between antibiotic concentration and RGB or grayscale values, and select the curve with the highest fitting degree as the antibiotic detection standard curve. S4. Based on the antibiotic detection standard curve obtained in step S3 and the measured RGB or grayscale values ​​of the sample containing antibiotics, the concentration of antibiotics in the sample is obtained.

2. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 1, characterized in that, The antibiotic is enrofloxacin, and the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No.

1. The linear regression equation for the detection of antibiotic concentration and the grayscale value of the paper-based detection chip is as follows: Gray = 130.6 - 1.55 lgC (1) In equation (1), Gray represents the grayscale value, and C is the concentration of the antibiotic in the sample to be tested, in ng·mL. -1 The correlation coefficient R in equation (1) 2 =0.986, the linear range for antibiotic detection is 0.1 ng / mL. -1 ~50μg·mL -1 The detection limit is 0.13 ng / mL. -1 .

3. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 1, characterized in that, In step S2, when a sample without antibiotics is added to the paper-based detection chip and incubated, and then H2O2 and TMB are added to react, the paper-based chip turns light blue; when a test sample containing antibiotics is incubated on the paper-based detection chip, and then H2O2 and TMB are added to react, the blue color of the paper-based detection chip deepens, thereby qualitatively detecting whether the test sample contains antibiotics.

4. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 1, characterized in that, In step S2, the concentration of H2O2 is 5mM to 50mM, and the concentration of TMB is 0.1mM to 2mM.

5. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 1, characterized in that, In step S1, the concentration of the nucleic acid aptamer-modified NiCo@C HNs solution is 10 μg·mL. -1 ~50μg·mL -1 .

6. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to any one of claims 1 to 5, characterized in that, In step S1, the preparation method of the nucleic acid aptamer-modified NiCo@C HNs solution includes the following steps: S1.

1. Disperse nickel nitrate and sodium citrate in water, then add potassium cobalt cyanide aqueous solution, incubate at room temperature, centrifuge and dry to obtain NiCo precursor; S1.2 Disperse the NiCo precursor in a buffer solution, then add dopamine and react at room temperature to obtain a NiCo precursor with a polydopamine coating. S1.

3. The NiCo precursor with polydopamine coating obtained in step S1.2 is pyrolyzed to obtain NiCo@C HNs, a NiCo bimetallic oxide porous carbon composite nanomaterial with a hollow nanobox structure. S1.

4. Disperse the NiCo@C HNs obtained in step S1.3 in sodium acetate-acetic acid buffer, and then add the nucleic acid aptamer to the sodium acetate-acetic acid buffer containing NiCo@C HNs for mixing and incubation to obtain a nucleic acid aptamer-modified NiCo@C HNs solution.

7. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 6, characterized in that, In step S1.1, the molar ratio of nickel nitrate to sodium citrate is 1:1 to 3, and the molar ratio of nickel nitrate to potassium cobalt cyanide is 1:0.5 to 1. And / or, in step S1.2, the buffer solution is Tris-HCl, and the mass ratio of dopamine to NiCo precursor is 1:1 to 3.

8. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 6, characterized in that, In step S1.3, the pyrolysis temperature is 300℃~500℃ and the pyrolysis time is 2h~4h.

9. The method for colorimetric analysis of antibiotics using a paper-based detection chip according to claim 6, characterized in that, In step S1.4, the concentration of the sodium acetate-acetic acid buffer is 0.1M to 0.2M, the concentration of the nucleic acid aptamer in the sodium acetate-acetic acid buffer is 0.1μM to 1μM, and the incubation is performed at room temperature for 10min to 30min.

10. The application of a paper-based detection chip colorimetric analysis method for antibiotics as described in any one of claims 1 to 9 in the detection of antibiotics in the environment or food.

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