Electrochemical aptamer sensor for detecting kanamycin in food and detection method thereof

Abstract

The invention provides an electrochemical aptamer sensor for detecting kanamycin in food. According to the invention, Fe3O4 magnetic beads (DNAzyme (MBS)) of a composite material AuNPs-Tb / COF / KB loaded DNAzyme are used as a DNA walking leg, and the DNA walking leg can move along the surface of MBs modified by a substrate DNA by virtue of DNAzyme cutting driven by Mg < 2 + >, so that a target object kanamycin is converted into a large amount of Output DNA chains, and sensitive amplification is realized; then MXene-PEI-MoS2 is used as a sensing interface, the immobilization amount of the capture probe is increased, signal amplification is further achieved, and kanamycin in food is detected.

Description

Technical Field

[0001] The invention belongs to the technical field of nanocomposite materials and relates to kanamycin detection, and in particular to an electrochemical aptamer sensor for detecting kanamycin in food and a detection method thereof. Background Art

[0002] Kanamycin (KNM) is a broad-spectrum aminoglycoside antibiotic that inhibits protein synthesis and is used to treat serious infections caused by Gram-positive and Gram-negative bacteria, and is therefore widely used in agriculture and veterinary medicine. However, overuse or misuse can lead to KNM residues in foods such as milk and meat, which eventually bioaccumulate in the human body through the biological circulation system, leading to significant negative health effects, including nephrotoxicity, allergic reactions, ototoxicity, and even the emergence of antibiotic resistance. Therefore, many countries and organizations have set maximum residue limits (MRLs) for different foods. For example, China has established an MRL of 200 μg / kg for KNM in milk and 100 μg / kg for meat, while the European Union (EU) has established an MRL of 150 μg / kg for KNM in milk and 200 μg / kg for KNM in meat.

[0003] KNM is currently detected using a variety of methods, including high performance liquid chromatography (HPLC), colorimetry, capillary electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA), etc. However, these analytical techniques still have the disadvantages of cumbersome operation, high cost and long operation time. Therefore, it is of great significance to develop a more accessible and cost-effective method for the quantitative analysis of trace KNM in food. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides an electrochemical aptamer sensor for detecting kanamycin in food.

[0005] A DNA walker is a molecular machine that can run autonomously on a designated track. Due to its higher DNA loading capacity, 3D DNA walkers exhibit higher walking efficiency compared to one-dimensional (1D) and two-dimensional (2D) ones. However, the application of 3D DNA walkers in electrochemical aptamer sensors has some limitations because the walking legs move in a "step-by-step" manner on the same nanoparticle as the substrate strand and are easily derailed during the translocation process. When the walking legs are separated from the DNA track, the walking stop signal amplification also stops, which results in lower sensitivity than expected for sensors based on 3D DNA walkers.

[0006] The present invention uses the composite AuNPs-Tb / COF / KB loaded DNAzyme Fe3O4 magnetic beads (DNAzyme@MBs) as DNA walking legs, and uses Mg 2+ Driven by DNAzyme cutting, the DNA walking chain can move along the surface of the MBs modified by the substrate DNA, converting the target kanamycin into a large number of output DNA chains, thus achieving sensitive amplification. 2+ The system relies on DNA enzyme-mediated cutting as the forward driving force, acting as a thermally stable and programmable driving motor, and combines it with efficient magnetic separation and concentration processes to further achieve high DNA enrichment and signal amplification. Then, the sensing interface MXene-PEI-MoS2 is combined to increase the solid loading of the capture probe, further achieve signal amplification, and detect kanamycin in food.

[0007] The present invention adopts the following technical solution:

[0008] An electrochemical aptamer sensor for detecting kanamycin in food is characterized in that it is constructed by the following method: a MoS2-PEI-MXene solution is dripped onto an electrode surface electrochemically activated with H2SO4, and after drying at room temperature, the electrode is immersed in a HAuCl4 solution for deposition for 20-40 seconds; a CP solution is then dripped onto the electrode surface and incubated at 3-5°C for 10-15 hours; a bovine serum albumin (BSA) solution is then dripped onto the electrode surface and incubated at 3-5°C for 35-55 minutes; an output chain solution is then dripped onto the electrode surface and incubated at room temperature for 0.5-1.5 hours; and finally, an SP / AuNPs-Tb / COF / KB signal probe solution is dripped onto the electrode surface and incubated at room temperature for 2 hours, thereby obtaining an electrochemical sensor for detecting kanamycin.

[0009] Preparation of Output DNA Solution:

[0010] Adding target KNM and Mg to DNAzyme@MBs and S@MBs nanosolutions based on ferroferric oxide nanospheres 2+ , incubate on a shaker at room temperature for 60-100 minutes to activate the DNA Rolling cycle; then separate with a magnet, discard the magnetic beads at the bottom, collect the supernatant rich in the sheared DNA fragments, and obtain the Output DNA solution.

[0011] Preparation of DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres: ferroferric oxide magnetic nano-microspheres with amino groups on their surfaces were diluted with PBS buffer to a concentration of 1-3 mg / mL; the carboxyl-modified DNAzyme chain and the Aptamer chain were mixed and placed at 90-100°C for 3-7 minutes, and then cooled to room temperature to hybridize and form a D / A double-stranded structure; the substrate chain (S chain) also modified with carboxyl groups and the above-mentioned D / A double-stranded chain were added to an MES buffer medium containing EDC and NHS, and treated for 0.5-1.5 hours to activate the carboxyl group; ferroferric oxide magnetic nano-magnetic beads (MBs) solution was added, stirred at room temperature for 15-18 hours, and then washed three times with PBS buffer solution, and the supernatant was discarded by magnetic separation; finally, the magnetic beads were dispersed in Tris-HCl buffer to obtain DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres.

[0012] According to one embodiment of the present invention, the preparation of the SP / AuNPs-Tb / COF / KB signal probe solution:

[0013] SP containing TCEP dissolved in TES buffer was added to the AuNPs-Tb / COF / KB dispersion, and the mixture was stirred in an ice bath for 10-15 hours. After centrifugation and washing, the precipitate was redispersed in ultrapure water to obtain a signal probe solution of SP / AuNPs-Tb / COF / KB.

[0014] According to one embodiment of the present invention, the preparation of the AuNPs-Tb / COF / KB dispersion:

[0015] Add COF / KB / Tb dispersion to 1-3% HAuCl4 solution, stir for 10-20 min, then add 3-5 mg / mL NaBH4 solution, stir in an ice bath for 40-50 min to obtain AuNPs-Tb / COF / KB dispersion.

[0016] According to one embodiment of the present invention, the preparation of the COF / KB / Tb dispersion is:

[0017] Tb is dissolved in ultrapure water, mixed with the COF / KB dispersion and stirred for 10-15 hours, centrifuged, washed with ultrapure water, and then the precipitate is dispersed in ultrapure water to obtain a COF / KB / Tb dispersion;

[0018] COF and KB were dissolved in ultrapure water, ultrasonicated for 10-15 min at room temperature, and then continuously stirred with magnetic stirring for 10-15 h to obtain a COF / KB dispersion;

[0019] Preparation of COF:

[0020] Tp and Pa were dissolved in a Shrek tube containing mesitylene, 1,4-dioxane and acetic acid, the mixture was ultrasonically treated for 10-15 minutes, and then the Shrek was frozen under 77K liquid nitrogen. After three freeze-thaw degassing cycles, the reaction was heated at 110-130°C for 70-75h; after cooling to room temperature, it was washed with N,N-dimethylformamide (DMF), acetone and tetrahydrofuran, respectively, and vacuum dried at 75-85°C to obtain a bright red COF powder.

[0021] According to one embodiment of the present invention, the MoS2-PEI-MXene solution is prepared by: dissolving MoS2 in ultrapure water, adding PEI and stirring for 1-3 hours; then adding MXene dissolved in ultrapure water, mixing and stirring for 10-15 hours, centrifuging, and washing with ultrapure water to obtain a MoS2-PEI-MXene composite material; and redispersing in ultrapure water to obtain a MoS2-PEI-MXene solution.

[0022] According to one embodiment of the present invention, the electrochemical aptamer sensor for detecting kanamycin in food is constructed by the following method:

[0023] (1) Preparation of signal probe SP / AuNPs-Tb / COF / KB solution;

[0024] 1) COF: 63 mg Tp and 48 mg Pa were dissolved in 1.5 mL mesitylene and 1.5 mL

[0025] The mixture was ultrasonically treated for 10 minutes in a schlenk tube containing 1,4-dioxane and 0.5 mL of acetic acid. The schlenk tube was then frozen in liquid nitrogen and subjected to three freeze-thaw degassing cycles. The mixture was heated at 120°C for 72 hours. After cooling to room temperature, the mixture was washed three times with N,N-dimethylformamide (DMF), acetone and tetrahydrofuran, respectively, and then dried under vacuum at 80°C to obtain a bright red COF powder.

[0026] 2) COF / KB: 1 mg of COF prepared in step 1) and 1 mg of KB were weighed and dissolved in 2 mL of ultrapure water. After ultrasonication at room temperature for 10 min, the mixture was continuously stirred with magnetic stirring for 12 h to obtain COF / KB;

[0027] 3) Tb / COF / KB dispersion: 1 mg Tb was weighed and dissolved in 1 mL ultrapure water, mixed with 1 mL COF / KB prepared in step 1) and stirred for 12 h, centrifuged, washed with ultrapure water, and then the precipitate was dispersed in 1 mL ultrapure water to obtain a Tb / COF / KB dispersion;

[0028] 4) AuNPs-Tb / COF / KB: 200 μL of 1% HAuCl4 was added to 1 mL of the COF / KB / Tb dispersion prepared in step 3) and stirred for 15 min, then 400 μL of 4 mg / mL NaBH4 was added and stirred in an ice bath for 45 min. After HAuCl4 was reduced by NaBH4, AuNPs were generated and adsorbed on the surface of Tb / COF / KB to obtain a composite solution of AuNPs-Tb / COF / KB;

[0029] 5) SP / AuNPs-Tb / COF / KB dispersion: Take 1 mL of the AuNPs-Tb / COF / KB dispersion prepared in step 3) and add 2 mM SP dissolved in TES buffer containing 10 mM TCEP, stir in an ice bath for 12 h, centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the signal probe SP / AuNPs-Tb / COF / KB solution, which is stored at 4°C for future use;

[0030] (2) Preparation of substrate material MoS2-PEI-MXene solution:

[0031] 1) Weigh 1 mg MoS2 and dissolve it in 1 mL ultrapure water, add 200 μL 1% PEI and stir for 2 h;

[0032] 2) Weigh 1 mg of MXene and dissolve it in 1 mL of ultrapure water, add 1 mL of step 1) and mix and stir for 12 h. After 3 centrifugations and washing with ultrapure water, a MoS2-PEI-MXene composite material is obtained, which is redispersed in 1 mL of ultrapure water to obtain a MoS2-PEI-MXene solution and maintained at 4 °C;

[0033] (3) Target KNM stimulates DNA rolling cycle to produce the preparation of output DNA solution:

[0034] 1) Preparation of DNARolling Nano-Roller: 200 μL of 5 mg / mL surface-amino-modified FeO magnetic nano-microspheres were washed three times with 0.1 M PBS buffer, and then diluted to 1 mL with 0.1 M PBS buffer to a concentration of 1 mg / mL; the carboxyl-modified DNAzyme chain and the Aptamer chain were mixed and placed at 95°C for 5 minutes, and then slowly cooled to room temperature to hybridize and form a D / A double-stranded structure with a concentration of 2 μM; next, 400 μL of the substrate S chain with the same carboxyl modification and 20 μL of the above D / A double-stranded were added to the MES buffer medium containing 0.4 M EDC and 0.1 M NHS, and treated for 1 hour to activate the carboxyl group; 1 mL of 1 mg / mL FeO magnetic nano-beads (MBs) solution was added, stirred at room temperature for 16 hours, and then treated with 0.1 M The beads were washed three times with PBS buffer solution, and the supernatant was discarded by magnetic separation. Finally, the magnetic beads were dispersed in 1 mL of 10 mM Tris-HCl buffer solution to obtain DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres.

[0035] 2) Triggering of DNA Rolling cycle: Take 80 μL of the DNAzyme@MBs nano solution obtained in step 1), add 10 μL of 1 μM target KNM and 1 μL of magnesium ions, and incubate on a shaker at room temperature for 80 min to activate the DNA Rolling cycle; then separate with a magnet, discard the magnetic beads at the bottom, and collect the supernatant rich in the sheared DNA fragments, which is named Output DNA solution;

[0036] (4) Construction of sensor:

[0037] 1) Dilute the capture probe CP chain with 20 mM Tris-HCl buffer at pH = 7.4, add 10 mM TES buffer containing TCEP, pre-treat for 1 hour to break the disulfide bonds in the CP chain, dilute the CP chain concentration to 2 μM, and store at 4°C for future use;

[0038] 2) Soak the bare electrode in piranha solution (98% H2SO4 / 30% H2O2 in a volume ratio of 3:1) for 30 minutes, then rinse with ultrapure water for later use;

[0039] 3) polishing the electrode obtained in step 2) to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powder respectively, and then ultrasonically treating the electrode in the order of ultrapure water, anhydrous ethanol, anhydrous ethanol, and ultrapure water respectively, and drying for later use;

[0040] 4) electrochemically activating the electrode obtained in step 3) in 0.5M H2SO4, then rinsing with ultrapure water and drying;

[0041] 5) 10 μL of the MXene-PEI-MoS2 solution prepared in step (2) was added dropwise onto the electrode surface prepared in step 4) and dried at room temperature;

[0042] 6) Immerse the electrode obtained in step 5) in 1 mL of 1% HAuCl4 solution for deposition for 30 seconds and dry at room temperature;

[0043] 7) 10 μL of CCP was added dropwise onto the electrode prepared in step 6) and incubated at 4°C for 12 h;

[0044] 8) Add 10 μL of 0.25% bovine serum albumin (BSA) solution onto the electrode obtained in step 7) at 4°C

[0045] Incubate for 45 min.

[0046] 9) Add 10 μL of the Output DNA solution prepared in step (3) onto the electrode obtained in step 8) and incubate at room temperature for 1 h;

[0047] 10) 10 μL of the SP / AuNPs-Tb / COF / KB signal probe solution prepared in step (1) was added dropwise to the electrode prepared in step 9) and incubated at room temperature for 2 h to obtain an electrochemical sensor for kanamycin detection.

[0048] In a second aspect, the present invention provides a method for detecting kanamycin in food using the electrochemical aptamer sensor.

[0049] The method for detecting kanamycin in food using the electrochemical aptamer sensor is characterized by comprising the following steps:

[0050] 1) dropping the output DNA solution and the signal probe generated by the DNA Rolling cycle stimulated by different concentrations of kanamycin onto the electrode of the above aptamer sensor;

[0051] 2) The electrode was placed in 0.1 M PBS buffer solution for characterization and its current value was measured;

[0052] 3) drawing a working curve according to the linear relationship between the current value obtained in step 2) and the logarithm of the kanamycin concentration;

[0053] 4) The sensor is used to detect the sample to be tested, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the concentration of kanamycin in the sample to be tested.

[0054] The present invention discloses a dual signal amplification strategy combining nanomaterials and DNA rolling, and develops a new aptamer sensor for detecting kanamycin (KNM). Firstly, AuNPs-Tb / COF / KB composite material was synthesized, which has a stable structure and can effectively amplify the detection signal. Secondly, Fe3O4 magnetic beads loaded with DNAzyme (DNAzyme@MBs) are used as DNA walking legs, and Mg is used to amplify the detection signal. 2+ Driven by DNAzyme cutting, the DNA walking legs can move along the surface of MBs modified with substrate DNA, converting the target kanamycin into a large number of output DNA chains, realizing sensitive amplification. Finally, MXene-PEI-MoS2 is used as the sensing interface to increase the solid loading amount of the capture probe, further realizing signal amplification. Through the above means, the prepared electrochemical aptamer sensor is successfully used for ultra-sensitive detection of kanamycin by electrochemical aptamer sensor. Compared with the traditional HPLC detection method, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, easy operation, low price of equipment and materials, and no pollution, thereby providing a new detection method for the detection of kanamycin in food.

[0055] The beneficial effects of the present invention are:

[0056] (1) Using COF to load KB can greatly improve the conductivity of COF and enhance the solubility of KB. Finally, the electrochemical signal molecule toluidine blue (Tb) with stable redox activity is introduced to synthesize the Tb / COF / KB composite material to achieve signal amplification and improve the detection sensitivity.

[0057] (2) By utilizing a fast and efficient enzyme-free nucleic acid signal amplification strategy, only the target kanamycin can trigger the cycle, converting the detection of the target into the detection of small molecule target DNA. The sandwich method is used to construct the sensor, so that the detection is not limited by the aptamer binding site and the number of bound aptamers, thus providing a new research method and analysis method for the detection of trace antibiotics.

[0058] (3) The materials involved can be synthesized under laboratory conditions, with simple operation. The raw materials are inexpensive, low in toxicity, environmentally friendly, and the amount used each time is extremely small, which reduces the experimental cost.

[0059] (4) The entire detection and analysis method has clear and simple steps, high sensitivity, and rapid signal response.

[0060] (5) The electrochemical aptamer sensor prepared by this method can provide a new direction for the detection of kanamycin in food: The electrochemical sensor prepared by this invention can also be used in other aspects such as food, medicine and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Cyclic voltammetry (A) and impedance characterization (B) of different modified electrodes in 0.1 M PBS (pH 7.0) at a voltage range of -0.2 to 0.6 V at a scan rate of 100 mV / s.

[0062] Figure 2 There is Mg 2+ No target (a), with target but no Mg 2+ (b) and Mg 2+ In the presence of target (1pM) (c), after DNA Rolling cycle, the modified electrode was placed in 0.1M PBS solution for DPV characterization, and the resulting electrical signal diagram.

[0063] Figure 3 This is the result diagram of the sensor after the DNA Rolling cycle is triggered by different concentrations of kanamycin (A), and the calibration curve diagram of the concentration logarithm and sensor current value (B).

[0064] Figure 4 The long-term stability of the sensor was obtained by detecting target signals at three concentrations (10 nM, 100 pM and 100 fM) using the prepared sensor under optimal conditions and performing signal detection every five days until the 20th day. Figure 5 The intra-batch and inter-batch differences are obtained by scanning sensors obtained by incubating 1 nM kanamycin with different glassy carbon electrodes simultaneously and for five consecutive days under the same conditions.

[0065] Figure 6 This is the specific detection diagram of the electrochemical sensor. Under the same conditions, the bar graph of the current value is detected after DNA rolling is triggered by 1nM kanamycin and other aminoglycoside antibiotic interferors: gentamicin sulfate (GEN, 100nM), streptomycin (STR, 100nM) and other interferors: chloramphenicol (CAP, 100nM), tetracycline (TET, 100nM) and glucose (GLU, 100nM). DETAILED DESCRIPTION

[0066] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further illustrated by specific embodiments below, and it should be understood that these embodiments only help to understand the present invention and should not be regarded as specific limitations of the present invention. The raw materials and reagents used in the present invention are all commercially available products. Unless otherwise specified, the parts described in the present invention are all weight parts, and the percentages are all mass percentages.

[0067] Example 1

[0068] The main chemical reagents used in the embodiments of the present invention are as follows:

[0069] Chloroauric acid (HAuCl4), (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC), N-hydroxysuccinimide (NHS), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), 1,3,5-triformylphloroglucinol (Tp), and p-phenylenediamine (Pa) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Layered Ti3C2Tx MXene, molybdenum disulfide (MoS2), amino-modified Fe3O4 magnetic nanospheres (MBs), and Ketjen black (KB) were purchased from Xianfeng Nanotechnology Co., Ltd. (Nanjing, China). Bovine serum albumin (BSA) and toluidine blue (Tb) were purchased from Beijing Bailingwei Technology Co., Ltd. (Beijing, China). Ethanol was purchased from Chuandong Chemical Group Co., Ltd. N,N-dimethylformamide (DMF) was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China); ultrapure water with a resistivity of 18.2 MΩ·cm was used throughout the experiments.

[0070] The DNA sequences involved have been purified by HPLC and synthesized by Shanghai Bioengineering Co., Ltd. The specific sequences are as follows:

[0071]

[0072] Equipment and technical parameters used:

[0073] Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were performed using a MetrohmAutolab BV electrochemical workstation (Modular Instruments, Switzerland). A three-electrode system was used for electrochemical detection: a modified glassy carbon electrode (4 mm in diameter) was used as the working electrode, a platinum wire was used as the counter electrode, and a saturated calomel electrode (SCE) was used as the reference electrode. A pH meter was used to monitor pH (S210 Seven Compact, Mettler-Toledo, Shanghai, China). The electrochemical three-electrode system was scanned at 100 mV / s in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0074] Example 1 Preparation of signal probes, substrate materials and output DNA solution generated by DNA Rolling cycles.

[0075] Follow these steps:

[0076] (1) Preparation of signal probes;

[0077] 1) Covalent organic structure COF: 63 mg Tp and 48 mg Pa were dissolved in a Shrek tube containing 1.5 mL mesitylene, 1.5 mL 1,4-dioxane and 0.5 mL acetic acid. To ensure complete homogeneity, the mixture was sonicated for 10 minutes. Next, the Shrek was frozen under 77K liquid nitrogen, and after three freeze-thaw degassing cycles, it was heated at 120 ° C for 72 h. After cooling to room temperature, it was washed three times with N, N-dimethylformamide (DMF), acetone and tetrahydrofuran, respectively, and then vacuum dried at 80 ° C to obtain a bright red COF powder.

[0078] 2) COF / KB: 1 mg of COF prepared in step 1) and 1 mg of KB were weighed and dissolved in 2 mL of ultrapure water. After ultrasonication at room temperature for 10 min, continuous magnetic stirring was performed for 12 h to obtain COF / KB.

[0079] 3) Tb / COF / KB dispersion: Weigh 1 mg Tb and dissolve it in 1 mL ultrapure water, mix it with 1 mL COF / KB prepared in step 1) and stir for 12 hours. After centrifugation and washing with ultrapure water, the precipitate is dispersed in 1 mL ultrapure water to obtain COF / KB / Tb dispersion.

[0080] 4) AuNPs-Tb / COF / KB: 200 μL HAuCl4 (1%) was added to 1 mL of COF / KB / Tb dispersion prepared in step 3) and stirred for 15 min. Then 400 μL NaBH4 (4 mg mL -1 ) was stirred in an ice bath for 45 min. After HAuCl4 was reduced by NaBH4, AuNPs were generated and adsorbed on the surface of Tb / COF / KB to obtain a composite solution of AuNPs-Tb / COF / KB

[0081] 5) SP / AuNPs-Tb / COF / KB solution: Take 1 mL of the AuNPs-Tb / COF / KB dispersion prepared in step 3), add 2 mM SP pretreated with TES buffer (containing 10 mM TCEP), stir in an ice bath for 12 h, centrifuge and wash with water, then redisperse the precipitate in 1 mL of ultrapure water to obtain the signal probe solution of SP / AuNPs-Tb / COF / KB, and store at 4°C for future use.

[0082] (2) Preparation of substrate materials;

[0083] 1) Weigh 1 mg MoS2 and dissolve it in 1 mL ultrapure water, add 200 μL PEI (1%) and stir for 2 h;

[0084] 2) Weigh 1 mg of MXene and dissolve it in 1 mL of ultrapure water, add 1 mL of step 1) and mix and stir for 12 h. After 3 centrifugations and ultrapure water washing, the MoS2-PEI-MXene composite material is redispersed in 1 mL of ultrapure water and maintained at 4 °C.

[0085] 3) Drop 10 μL of the MoS2-PEI-MXene composite material prepared in step 2) on the electrode surface and immerse it in 1 mL of 1% HAuCl4 solution for deposition for 30 seconds.

[0086] (3) Target KNM stimulates DNA rolling cycle to produce the preparation of output DNA solution:

[0087] 1) Preparation of DNAzyme@MBs and S@MBs: 200 μL (5 mg / mL) of ferroferric oxide magnetic nanospheres with amino groups on the surface were washed three times with 0.1M PBS buffer, and then diluted to 1 mL with 0.1M PBS buffer to a concentration of 1 mg / mL. The DNAzyme chain and the Aptamer chain with carboxyl groups were mixed and placed at 95°C for 5 minutes, and then slowly cooled to room temperature to hybridize and form a D / A double-stranded structure with a concentration of 2 μM. Next, the substrate chain (S chain) (400 μL, 2 μM) with the same carboxyl group modification and 20 μL of the above D / A double-stranded structure were added to the MES buffer medium containing EDC (0.4M) and NHS (0.1M) and treated for 1 hour to activate the carboxyl group. 1 mL, 1 mg / mL ferroferric oxide magnetic nanoparticles (MBs) solution was added, stirred at room temperature for 16 hours, and then washed three times with 0.1M PBS buffer solution, magnetic separation was performed, and the supernatant was discarded. Finally, the magnetic beads were dispersed in 1 mL, 10 mM Tris-HCl buffer to obtain DNAzyme@MBs and S@MBs nanosolutions.

[0088] 2) Triggering of DNA Rolling cycle: Take 80 μL of the DNAzyme@MBs nano solution obtained in step 1) and add target KNM (10 μL, 1 μM), Mg 2+ (1 μL), incubate on a shaker at room temperature for 80 min to activate the DNA Rolling cycle. Then separate with a magnet, discard the magnetic beads at the bottom, and collect the supernatant rich in sheared DNA fragments, which is named Output DNA solution.

[0089] Example 2 Preparation of an electrochemical aptamer sensor for detecting kanamycin in food.

[0090] Follow these steps:

[0091] 1) Dilute the capture probe (CP chain) with 20 mM Tris-HCl (pH = 7.4) buffer, add 10 mM TES buffer (containing TCEP), pre-treat for 1 hour to break the disulfide bonds in the CP chain, and finally dilute the CP chain concentration to 2 μM and store at 4°C for future use;

[0092] 2) Soak the bare electrode in piranha solution (98% H2SO4 / 30% H2O2=3:1, v / v) for 30 min and then rinse with ultrapure water for later use;

[0093] 3) polishing the electrode obtained in step 2) to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powder respectively, and then ultrasonically treating the electrode in the order of ultrapure water, anhydrous ethanol, anhydrous ethanol, and ultrapure water respectively, and drying for later use;

[0094] 4) electrochemically activating the electrode obtained in step 3) in 0.5M H2SO4, then rinsing with ultrapure water and drying;

[0095] 5) 10 μL of MXene-PEI-MoS2 solution was added dropwise onto the electrode surface prepared in step 4) and dried at room temperature;

[0096] 6) Immerse the electrode obtained in step 5) in 1 mL of 1% HAuCl4 solution for deposition for 30 seconds and dry at room temperature;

[0097] 7) 10 μL of CCP was added dropwise onto the electrode prepared in step 6) and incubated at 4°C for 12 h;

[0098] 8) Add 10 μL of 0.25% bovine serum albumin (BSA) solution dropwise onto the electrode obtained in step 7) and incubate at 4° C. for 45 min;

[0099] 9) 10 μL of the final Output DNA solution obtained in Example 1 was added dropwise to the electrode obtained in step 8) and incubated at room temperature for 1 hour;

[0100] 10) 10 μL of the SP / AuNPs-Tb / COF / KB signal probe solution described in Example 1 was dropped onto the electrode prepared in step 9) and incubated at room temperature for 2 h to obtain an electrochemical sensor for kanamycin detection.

[0101] Example 3 Detection of kanamycin using electrochemical biosensor

[0102] The electrochemical biosensor constructed in Example 2 was used to detect kanamycin in the following steps:

[0103] 1. Draw the working curve

[0104] 1) The modified electrodes from step 4) to step 10) of Example 2 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV and EIS characterization, and their current response signals were measured. The results are as follows: Figure 1 (A) shows: (a) bare electrode; (b) adding capture probe; (c) adding BSA to block; (d) adding Output DNA. The impedance response signal is measured, and the results are as follows: Figure 1 (B) shows: (a) bare electrode; (b) adding capture probe; (c) adding BSA for blocking; (d) adding Output DNA. The modified electrode in step 8) was placed in 0.1M PBS solution for DPV characterization. The results are shown in Figure 2 As shown: Mg 2+ No target (a) With target but no Mg 2+ (b) and Mg 2+ And there is an electrical signal generated by the target (1pM) (c).

[0105] 2) 10 μL of target kanamycin of different concentrations was added dropwise to the DNA Rolling circulation system constructed in Example 2, and then the electrochemical response signals were measured respectively. Figure 3 As shown in (A): the concentrations of a→h are: 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 100 nM.

[0106] 3) According to the linear relationship between the obtained current value and the logarithmic value of kanamycin concentration, a working curve was drawn. The measurement results showed that the electrical signal response value and the logarithmic value of kanamycin concentration had a good linear relationship in the concentration range of 10fM-100nM, with a linear correlation coefficient of 0.9986 and a detection limit of 0.66fM. For details, see Figure 3 (B).

[0107] 2. Sensor long-term stability test

[0108] The sensor prepared under the optimal conditions in Example 2 was used to detect three concentrations of target signals (10nM, 100pM and 100fM), and the signal detection was performed every five days until the 20th day. The current intensity measured on the 20th day still accounted for more than 80% of the initial value (e.g. Figure 4 This indicates that the sensor has good stability.

[0109] 3. Sensor reproducibility test

[0110] After electrochemical measurement (such as Figure 5The relative standard deviations of intra-batch and inter-batch differences were 0.41% and 1.10%, respectively, indicating that the sensor had good reproducibility.

[0111] 4. Sensor Specificity Test

[0112] In order to study the specificity of the proposed sensor, the interfering substances that may exist in serum were used: under the same conditions, 1nM kanamycin and other aminoglycoside antibiotic interfering substances: gentamicin sulfate (GEN, 100nM), streptomycin (STR, 100nM) and other interfering substances: chloramphenicol (CAP, 100nM), tetracycline (TET, 100nM) and glucose (GLU, 100nM) were used to trigger DNA rolling and then the current response value was detected in 0.1M PBS (pH=7.0). The results showed that ( Figure 6 As shown), the proposed electrochemical aptasensor based on the high specific reaction of kanamycin has good specificity.

[0113] 5. Actual sample analysis application

[0114] In order to evaluate the practical applicability and accuracy of the proposed electrochemical biosensor, various concentrations of kanamycin KNM (as shown in Table 1) were added to milk diluted 10 times, and the prepared electrochemical aptamer sensor was used for detection. The test results are shown in Table 1, with a relative standard deviation ranging from 1.6% to 4.6%, and a recovery rate of 93.0% to 106.1%. The results show that the aptamer sensor prepared by the present invention is feasible for detecting kanamycin and can meet the needs of actual food analysis.

[0115] Table 1 Determination of Kanamycin KNM in milk samples by using the electrochemical aptasensor prepared by the present invention (n=3)

[0116]

[0117] In summary, the present invention provides a dual-amplification electrochemical aptamer sensor based on DNA enzyme-driven rolling nanomachine and nanomaterial technology for KNM detection. First, the target KNM reactivates the pre-locked DNAzyme, 2+In the presence of DNA, the DNA enzyme-driven rolling process is initiated, and then a large amount of output DNA is generated, which is released and enriched by magnetic separation technology to achieve target recognition and initial signal amplification in the solution. A composite material composed of polyethyleneimine functionalized molybdenum disulfide and titanium carbide (MoS2-PEI-MXene) is introduced as a sensing interface to further anchor additional capture probes (CP), while also promoting electron transfer. Then, the Tb / COF / KB complex is modified with AuNPs and labeled with a signal probe (SP) to form a tracer label (SP-AuNPs-Tb / COF / KB), which is incubated with the output DNA and connected to CPs to form a sandwich structure, generating a larger electrochemical signal, and the KNM sensitive detection aptamer sensor. Therefore, the present invention uses DNA Rolling to achieve the amplification of the target kanamycin, and SP-AuNPs-Tb / COF / KB is used as a signal probe to synergistically amplify the electrical signal of the electrochemical sensor to achieve ultra-sensitive detection of kanamycin, providing a new method for the detection of kanamycin in food.

Claims

1. An electrochemical aptamer sensor for detecting kanamycin in food, characterized in that: The electrochemical sensor was constructed by the following method: a MoS2-PEI-MXene solution was added to the surface of an electrode electrochemically activated with H2SO4, and after drying at room temperature, the electrode was immersed in a HAuCl4 solution for deposition for 20-40 seconds; a CP solution was then added to the surface of the electrode and incubated at 3-5°C for 10-15 hours; a bovine serum albumin (BSA) solution was then added to the surface of the electrode and incubated at 3-5°C for 35-55 minutes; an Output DNA solution was then added to the surface of the electrode and incubated at room temperature for 0.5-1.5 hours; and a SP / AuNPs-Tb / COF / KB signal probe solution was added to the surface of the electrode and incubated at room temperature for 2 hours to obtain an electrochemical sensor for kanamycin detection. Preparation of Output DNA Solution: Adding target KNM and Mg into DNAzyme@MBs and S@MBs nanosolutions based on ferroferric oxide nanospheres 2+ , incubate on a shaker at room temperature for 60-100 min to activate the DNA Rolling cycle; then separate with a magnet, discard the magnetic beads at the bottom, collect the supernatant rich in sheared DNA fragments, and obtain the Output DNA solution; Preparation of DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres: ferroferric oxide magnetic nano-microspheres with amino groups on their surfaces were diluted with PBS buffer to a concentration of 1-3 mg / mL; the carboxyl-modified DNAzyme chain and the Aptamer chain were mixed and placed at 90-100°C for 3-7 min, and then cooled to room temperature to hybridize and form a D / A double-stranded structure; the substrate chain (S chain) also modified with carboxyl groups and the above-mentioned D / A double-stranded chain were added to the MES buffer medium containing EDC and NHS, and treated for 0.5-1.5 h to activate the carboxyl group; ferroferric oxide magnetic nano-magnetic beads (MBs) solution was added, stirred at room temperature for 15-18 h, and then washed three times with PBS buffer solution, magnetically separated and the supernatant was discarded; finally, the magnetic beads were dispersed in Tris-HCl buffer to obtain DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres.

2. The electrochemical aptasensor according to claim 1, characterized in that: The preparation method of the SP / AuNPs-Tb / COF / KB signal probe solution is as follows: SP containing TCEP dissolved in TES buffer is added to the AuNPs-Tb / COF / KB dispersion, stirred in an ice bath for 10-15 h, centrifuged, washed with water, and then the precipitate was redispersed in ultrapure water to obtain the SP / AuNPs-Tb / COF / KB signal probe solution.

3. The electrochemical aptamer sensor according to claim 2, characterized in that: The preparation method of the AuNPs-Tb / COF / KB dispersion is as follows: add COF / KB / Tb dispersion to HAuCl4 solution with a concentration of 1-3%, stir for 10-20 min, then add NaBH4 solution with a concentration of 3-5 mg / mL, stir in an ice bath for 40-50 min, and obtain AuNPs-Tb / COF / KB dispersion.

4. The electrochemical aptamer sensor according to claim 3, characterized in that: The preparation method of the COF / KB / Tb dispersion is: (1) Preparation of COF: Tp and Pa were dissolved in a Shrek tube containing mesitylene, 1,4-dioxane and acetic acid, and the mixture was sonicated for 10-15 min. The Shrek was then frozen under liquid nitrogen at 77 K. After three freeze-thaw degassing cycles, the mixture was heated at 110-130 °C for 70-75 h. After cooling to room temperature, the mixture was washed with N,N-dimethylformamide (DMF), acetone and tetrahydrofuran, respectively, and dried in vacuum at 75-85 °C to obtain a bright red COF powder. Preparation of COF / KB dispersion: COF and KB were dissolved in ultrapure water, ultrasonicated for 10-15 min at room temperature, and then continuously stirred with magnetic stirring for 10-15 h to obtain a COF / KB dispersion; Preparation of COF / KB / Tb dispersion: Tb is dissolved in ultrapure water, mixed with the COF / KB dispersion and stirred for 10-15 h, centrifuged, washed with ultrapure water, and then the precipitate is dispersed in ultrapure water to obtain a COF / KB / Tb dispersion.

5. The electrochemical aptamer sensor according to any one of claims 1 to 4, characterized in that: The preparation method of the MoS2-PEI-MXene solution is as follows: dissolve MoS2 in ultrapure water, add PEI and stir for 1-3 hours; then add MXene dissolved in ultrapure water, mix and stir for 10-15 hours, centrifuge, and wash with ultrapure water to obtain a MoS2-PEI-MXene composite material; and redisperse in ultrapure water to obtain a MoS2-PEI-MXene solution.

6. The electrochemical aptasensor according to claim 1, characterized in that: Constructed by: (1) Preparation of signal probe SP / AuNPs-Tb / COF / KB solution; 1) COF: 63 mg Tp and 48 mg Pa were dissolved in a schlenk tube containing 1.5 mL mesitylene, 1.5 mL 1,4-dioxane and 0.5 mL acetic acid, and the mixture was sonicated for 10 min; the schlenk tube was then frozen in liquid nitrogen, and after three freeze-thaw degassing cycles, it was heated at 120 °C for 72 h. After cooling to room temperature, it was washed three times with N,N-dimethylformamide (DMF), acetone and tetrahydrofuran, respectively, and then vacuum dried at 80 °C to obtain bright red COF powder; 2) COF / KB: 1 mg of COF powder prepared in step 1) and 1 mg of KB were dissolved in 2 mL of ultrapure water, and then ultrasonicated at room temperature for 10 min and continuously stirred with magnetic stirring for 12 h to obtain COF / KB; 3) Tb / COF / KB dispersion: Weigh 1 mg Tb and dissolve it in 1 mL ultrapure water, mix it with 1 mL COF / KB prepared in step 1) and stir it for 12 h, centrifuge it, wash it with ultrapure water, and then disperse the precipitate in 1 mL ultrapure water to obtain Tb / COF / KB dispersion; 4) AuNPs-Tb / COF / KB: Take 200 μL of 1% HAuCl4 and add it to 1 mL of COF / KB / Tb dispersion prepared in step 3) and stir for 15 min. Then add 400 μL of 4 mg / mL NaBH4 and stir in an ice bath for 45 min. After HAuCl4 is reduced by NaBH4, AuNPs are generated and adsorbed on the surface of Tb / COF / KB to obtain a composite solution of AuNPs-Tb / COF / KB. 5) SP / AuNPs-Tb / COF / KB dispersion: Take 1 mL of the AuNPs-Tb / COF / KB dispersion prepared in step 3) and add 2 mM SP dissolved in TES buffer containing 10 mM TCEP. Stir in an ice bath for 12 h. Centrifuge and wash with water, then redisperse the precipitate in 1 mL of ultrapure water to obtain the signal probe SP / AuNPs-Tb / COF / KB solution. Store at 4 ºC for future use. (2) Preparation of substrate material MoS2-PEI-MXene solution: 1) Weigh 1 mg MoS2 and dissolve it in 1 mL ultrapure water, add 200 μL 1% PEI and stir for 2 h to obtain MoS2-PEI dispersion; 2) Weigh 1 mg of MXene and dissolve it in 1 mL of ultrapure water, add 1 mL of the MoS2-PEI dispersion prepared in step 1), mix and stir for 12 h, centrifuge 3 times, wash with ultrapure water to obtain a MoS2-PEI-MXene composite material, and redisperse it in 1 mL of ultrapure water and keep it at 4 °C; (3) Target KNM stimulates DNA rolling cycle to produce the preparation of Output DNA solution: 1) Preparation of DNA Rolling Nano-roller: 200 μL of 5 mg / mL surface-amino-modified FeO magnetic nano-microspheres were washed three times with 0.1M PBS buffer, and then diluted to 1 mL with 0.1M PBS buffer to a concentration of 1 mg / mL; the carboxyl-modified DNAzyme chain and the Aptamer chain were mixed and placed at 95°C for 5 minutes, and then slowly cooled to room temperature to hybridize and form a D / A double-stranded structure with a concentration of 2 μM; next, 400 μL of the substrate S chain with the same carboxyl modification and 20 μL of the above D / A double-stranded were added to the MES buffer medium containing 0.4M EDC and 0.1M NHS, and treated for 1 h to activate the carboxyl group; 1 mL of 1 mg / mL FeO magnetic nano-magnetic beads (MBs) solution was added, stirred at room temperature for 16 h, and then washed three times with 0.1M PBS buffer solution, magnetic separation and supernatant were discarded; finally, the magnetic beads were dispersed in 1 mL of Tris-HCl buffer with a concentration of 10 mM, and the DNAzyme@MBs and S@MBs nano-solutions based on ferroferric oxide nano-microspheres were obtained; 2) Triggering of DNA Rolling cycle: Take 80 μL of the DNAzyme@MBs nano solution obtained in step 1), add 10 μL of 1 μM target KNM and 1 μL of magnesium ions, and incubate on a shaker at room temperature for 80 min to activate the DNA Rolling cycle; then separate with a magnet, discard the magnetic beads at the bottom, and collect the supernatant rich in sheared DNA fragments, which is named OutputDNA solution; (4) Construction of sensors: 1) Dilute the capture probe CP chain with 20 mM Tris-HCl buffer at pH 7.4, add 10 mM TES buffer containing TCEP, pre-treat for 1 h to break the disulfide bonds in the CP chain, dilute the CP chain concentration to 2 μM, and store at 4 ºC for later use; 2) Soak the bare electrode in piranha solution (98% H2SO4 / 30% H2O2 in a volume ratio of 3:1) for 30 min, then rinse with ultrapure water for later use; 3) The electrodes soaked in the piranha solution in step 2) were polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrodes were ultrasonically treated in the order of ultrapure water, anhydrous ethanol, anhydrous ethanol, and ultrapure water, and dried for later use; 4) The electrode obtained by ultrasound in step 3) is electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried; 5) 10 μL of the MXene-PEI-MoS2 solution prepared in step (2) was added dropwise onto the electrode surface activated in step 4) and dried at room temperature; 6) Immerse the electrode dried in step 5) in 1 mL 1% HAuCl4 solution for 30 seconds and dry at room temperature; 7) 10 μL of CP was added to the electrode deposited with HAuCl4 in step 6) and incubated at 4 °C for 12 h; 8) Add 10 μL of 0.25% bovine serum albumin (BSA) solution onto the electrode incubated in step 7) and incubate at 4°C for 45 min; 9) Add 10 μL of the Output DNA solution prepared in step (3) onto the electrode treated with bovine serum albumin in step 8) and incubate at room temperature for 1 h; 10) 10 μL of the SP / AuNPs-Tb / COF / KB signal probe solution prepared in step (1) was added dropwise to the electrode treated with the Output DNA solution in step 9) and incubated at room temperature for 2 h to obtain an electrochemical sensor for kanamycin detection.

7. A method for detecting kanamycin in food using the electrochemical aptamer sensor according to any one of claims 1 to 6, characterized in that: The steps include: 1) dropping the output DNA solution and the signal probe generated by the DNA Rolling cycle stimulated by different concentrations of kanamycin onto the electrode of the aptamer sensor; 2) Place the electrode in 0.1M PBS buffer solution for characterization and measure its current value; 3) drawing a working curve based on the linear relationship between the current value obtained in step 2) and the logarithm of the kanamycin concentration; 4) The sensor is used to detect the sample to be tested, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the concentration of kanamycin in the sample to be tested.

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