Sensor for detecting tetracycline by electrochemiluminescence method based on inner filter effect, its preparation method and application

Abstract

The present invention relates to a sensor for detecting antibiotics by electrochemiluminescence method based on inner filter effect, its preparation method and application, including NH₂-AgBr-N-Ti₃C₂ as the luminescent body and COOH-dWO₃·H₂O as the absorber. The luminescent body and the absorber are connected by two complementary DNA strands, one of which is the aptamer of tetracycline, connected to the luminescent body and modified on the glassy carbon electrode, and the other complementary strand is connected to the absorber. The two DNA strands are connected together by base complementary pairing. Based on the inner filter effect existing between NH₂-AgBr-N-Ti₃C₂ and COOH-dWO₃·H₂O, the quantitative detection of antibiotics is achieved. The sensor has the advantages of high sensitivity, low detection limit, wide linear range, etc., and is of great significance for the detection of antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemiluminescence analysis and detection, and relates to a sensor for detecting tetracycline by electrochemiluminescence method based on inner filter effect, its preparation method and application. Specifically, it relates to a method of using NH2-AgBr-N-Ti3C2 as a luminescent body, an aptamer (apt) with specific recognition as a recognition element, and COOH-dWO3·H2O as a complementary absorber, which are co-modified on a glassy carbon electrode (GCE), and then using COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE as a working electrode to quantitatively detect tetracycline in water by electrochemiluminescence analysis method. Background Art

[0002] Tetracycline (TCN) is a representative antibiotic in the broad-spectrum tetracycline antibiotics, which has broad antibacterial activity against Gram-positive and Gram-negative microorganisms. Due to its overuse in animal husbandry, aquaculture and human disease prevention, it accumulates in the human body, which can cause allergic reactions, gastrointestinal diseases, inhibition of bone growth, etc., bringing serious problems to human health. At present, the traditional methods for detecting TCN mainly include: high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA) and capillary electrophoresis (CE), etc. However, these traditional detection methods require expensive large-scale instruments, with cumbersome operation methods and high costs. Therefore, developing a rapid, simple, highly sensitive and selective tetracycline detection method is of great significance for ensuring ecological environment safety and maintaining human health.

[0003] Electrochemiluminescence (ECL), also known as electrochemically induced chemiluminescence, not only combines the advantages of chemiluminescence and electrochemical technologies, but also does not require any external light source, and has low background signal and high selectivity, showing great potential in food safety testing and environmental monitoring. Inner filter effect (IFE) is considered a wise strategy for designing new ECL sensors. When the absorption spectrum of the absorber overlaps with the ECL emission spectrum, the absorber absorbs the ECL emission light in the detection system and converts the absorption information into an ECL response. Compared with another quenching mechanism (resonance energy transfer (RET)), IFE has been proven to significantly improve sensitivity and selectivity. More importantly, the IFE quenching mechanism is independent of distance, and there is no restricted chemical connection between the ECL emitter and the absorber, which provides considerable flexibility and simplicity for constructing sensors. How to prepare a biosensor with high accuracy and strong selectivity for tetracycline detection is the technical problem to be solved by the present invention. Summary of the Invention

[0004] The object of the present invention is to provide an electrochemiluminescence sensor based on the inner filter effect, its preparation method and application, aiming at the deficiencies of the existing technologies for antibiotic detection. The electrochemiluminescence sensor based on the inner filter effect of the present invention includes a luminescent body NH2-AgBr-N-Ti3C2 nanocomposite and an absorber COOH-dWO3·H2O nanosheet, which are successively modified on the electrode surface from the inside out; the luminescent body and the absorber are connected by two complementary DNA strands, one of which is an aptamer connecting the luminescent body, and the other complementary DNA strand connects the absorber.

[0005] The preparation method of the above-mentioned electrochemiluminescence sensor based on the inner filter effect includes the following steps:

[0006] A PBS buffer solution containing NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride) is drop-coated on a clean bare glassy carbon electrode to obtain an activated glassy carbon electrode. Subsequently, a DMF dispersion solution of the NH2-AgBr-N-Ti3C2 composite material is dropped on the activated glassy carbon electrode to obtain an NH2-AgBr-N-Ti3C2 / GCE modified electrode; then, a Tris-HCl solution containing an aptamer (apt) and a COOH-dWO3·H2O / cDNA colloidal solution are successively drop-coated to obtain the electrochemiluminescence sensor based on the inner filter effect COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE; the aptamer is a DNA strand for connecting the luminescent body.

[0007] Furthermore, the concentration of the aptamer is 3.0 - 8.0 μmol / L, and the concentration of the COOH-dWO3·H2O / cDNA colloidal solution is 3.0 - 8.0 μmol / L.

[0008] Furthermore, the pH value of the Tris-HCl solution containing the aptamer (apt) is 7 - 7.8, and more preferably 7.4.

[0009] More preferably, the dropping amount of NH2-AgBr-N-Ti3C2 is 6.0 μL, and the concentration is 2.0 mg / mL; the dropping amount of the aptamer is 6.0 μL, and the concentration is 3.0 μmol / L; the dropping amount of the COOH-dWO3·H2O / cDNA colloidal solution is 6.0 μL, and the concentration is 3.0 μmol / L.

[0010] Furthermore, the preparation of the NH2-AgBr-N-Ti3C2 composite material includes the following steps:

[0011] In an aqueous ammonia solution of silver nitrate uniformly dispersed with N-Ti3C2 nanomaterials and ethylene glycol, with 1-hexadecyl-3-methylimidazolium bromide as the bromine source, an AgBr-N-Ti3C2 nanocomposite material is in-situ prepared at the reaction temperature, and then amino modification is carried out through APTMS (3-aminopropyltrimethoxysilane), introducing NH2 into the AgBr-N-Ti3C2 nanocomposite material to obtain an NH2-AgBr-N-Ti3C2 composite material.

[0012] Specifically, the AgBr-N-Ti3C2 nanocomposite material is added to ethanol, ultrasonically dispersed thoroughly, APTMS (3-aminopropyltrimethoxysilane) is added, after being dispersed evenly, the mixture is heated to the reaction temperature for full reaction, after the reaction ends, the product is centrifuged, washed repeatedly with deionized water and ethanol, and vacuum dried to obtain a grayish-white powder, namely the NH2-AgBr-N-Ti3C2 composite material.

[0013] More specifically, 5 mL of a Ti3C2 (preferably with a size of 1-15 μm) dispersion liquid (2.5 mg / mL) and 30 mg of glycine are mixed and ultrasonically dispersed evenly for 1 h. The above precursor solution is transferred into an alumina porcelain boat, and in a tube furnace, it is heated to 350 °C at a rate of 5 °C / min under a nitrogen atmosphere and kept for 2 h. After cooling to room temperature, the sample is taken out from the porcelain boat with a spatula to obtain the N-Ti3C2 nanomaterials for standby. 87.74 mg of silver nitrate is weighed in a centrifuge tube and dissolved in 2 mL of ammonia water to obtain a reaction solution for standby; 600 μL of N-Ti3C2 (5 mg / mL) and 8 mL of ethylene glycol are added to a 50 mL round-bottom flask, ultrasonically treated for 20 min, then 200.10 mg of 1-hexadecyl-3-methylimidazolium bromide is added and stirred at room temperature for 30 min, and then the reaction solution is slowly added dropwise to the above round-bottom flask, and oil-bathed at 90 °C for 6 h; after the reaction ends, the product is centrifuged, washed, and freeze-dried for 12 h to obtain the AgBr-N-Ti3C2 nanocomposite material; finally, the AgBr-N-Ti3C2 nanocomposite material is added to ethanol, ultrasonically dispersed for 30 min, APTMS (3-aminopropyltrimethoxysilane) with a mass concentration of 4.5% (w / v) is added, after ultrasonically treating for 30 min, the mixture is stirred at 65 °C for 12 h, after the reaction ends, the product is centrifuged, washed repeatedly with deionized water and ethanol, and vacuum dried to obtain a grayish-white powder, namely the NH2-AgBr-N-Ti3C2 composite material, and the NH2-AgBr-N-Ti3C2 composite material is dispersed in DMF for use;

[0014] Further, the preparation of the COOH-dWO3·H2O / cDNA colloidal solution includes the following steps:

[0015] Add the Tris-HCl solution of cDNA to COOH-dWO3·H2O to obtain a suspension with a concentration of 0.2 mg / ml. Vigorously shake the reaction, centrifuge and separate, and dissolve the precipitate in the Tris-HCl solution. Stir well until COOH-dWO3·H2O and cDNA are fully connected to obtain COOH-dWO3·H2O / cDNA. The cDNA is a DNA strand used to connect the absorber.

[0016] More specifically, add 100 μL of the Tris-HCl solution of cDNA (pH = 7.4) to a centrifuge tube containing 20 mg of COOH-dWO3·H2O to obtain a suspension. After shaking the reaction for 16 h, centrifuge at a speed of 10000 rpm for 10 min, and dissolve the precipitate in 100 μL of the Tris-HCl solution (pH = 7.4). Ultrasonically treat the obtained colloidal solution for 10 min and shake it at room temperature for 1 h to obtain COOH-dWO3·H2O / cDNA. Finally, store it at 4 °C. The cDNA is a DNA strand used to connect the absorber.

[0017] Application of the above electrochemiluminescence sensor based on the inner filter effect in the detection of antibiotics by electrochemiluminescence method. Taking tetracycline as an example, the corresponding aptamer is the tetracycline aptamer, and the nucleotide sequence is as follows: Aptamer: 5'-COOH CGTACG GAA TTC GCT AGC CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TGGATC CGA GCT CCA CGT G-3' cDNA: 5'-TGC-CGG-GGG-GCT-AGC-GAA-TTC-CGT-ACG-NH2-3'.

[0018] The present invention also provides a specific detection method, including the following steps:

[0019] Step 1, prepare a series of antibiotic standard solutions with different concentrations, and the concentration range is 1.0×10 -14 mol / L~1.0×10 -7 mol / L;

[0020] Step 2: Use the electrochemiluminescence sensor based on the inner filter effect as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. Immerse the three-electrode system in the series of standard antibiotic solutions with different concentrations prepared in Step 1 for a certain period of incubation to allow the aptamer to fully bind to the antibiotic (for example, when the antibiotic is tetracycline, the incubation time is at least 20 min). Within the electrochemical window range of -1.8 to 0 V, with the high voltage of the photomultiplier tube at 800 V and the scanning rate of 0.075 to 0.125 V / s (preferably 0.1 V / s), perform cyclic voltammetry scanning, record the potential-luminescence intensity curve, establish the linear relationship between the difference in luminescence intensity before and after adding the antibiotic and the logarithm of the antibiotic concentration, and obtain the corresponding linear regression equation;

[0021] Step 3: Sample detection. Conduct the test according to Step 2 above to obtain the luminescence intensity, and calculate the obtained luminescence intensity using the linear regression equation obtained in Step 2 to obtain the concentration of the antibiotic in the sample.

[0022] Preferably, the soaking time of the electrochemiluminescence sensor based on the inner filter effect in Step 1 is 20 min.

[0023] In this invention, NH2-AgBr-N-Ti3C2 is used as the luminescent material and COOH-dWO3·H2O is used as the complementary absorber. The luminescent and the absorber are connected by two DNA strands (apt and cDNA) and co-modified on the glassy carbon electrode (GCE), and then

[0024] An electrochemiluminescence (ECL) analysis method for quantitatively detecting antibiotics in water using COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE as the working electrode. Since nitrogen doping in Ti3C2 MXene can effectively improve the ECL performance, the NH2-AgBr-N-Ti3C2 / GCE modified electrode has not only high but also stable luminescence intensity. Based on the ECL inner filter effect between NH2-AgBr-N-Ti3C2 and COOH-dWO3·H2O, the ECL intensity is quenched. When the aptamer specifically binds to tetracycline, the inner filter effect between the absorber COOH-dWO3·H2O and the emitter NH2-AgBr-N-Ti3C2 is inhibited, thus restoring the light intensity, and the light intensity has a linear relationship with the concentration of antibiotics. This invention not only has the advantages of high sensitivity, wide linear range, and simple operation of ECL analysis, but also has important practical significance for the specific detection of antibiotics in water. The remarkable advantage of this invention is the development of an ECL sensor capable of detecting antibiotics. For the first time, dWO3·H2O is used as an absorber in the ECL field. Conventional absorbers such as noble metal nanoparticles like gold and platinum are restricted in practical applications due to high costs and narrow light absorption areas. As a non-metallic plasmon, incident light can induce the generation of excess free carriers on the surface of dWO3·H2O, causing collective oscillations, thereby resulting in strong and extensive light absorption, and the molar extinction coefficient of tungsten trioxide is several orders of magnitude higher than that of traditional absorbers. Compared with ordinary ECL sensors, it has the following remarkable advantages: the introduction of N-Ti3C2 material accelerates the electron transfer on the electrode surface and significantly enhances the light intensity of AgBr; dWO3·H2O is a heavily doped semiconductor and also has the LSPR effect of noble metals, avoiding the use of noble metals; the ECL sensing strategy based on the inner filter effect has higher accuracy and selectivity than the traditional ECL method based on resonance energy transfer, and the lowest detectable limit that can be achieved is 3.8 fM. Brief Description of the Drawings

[0025] Figure 1 It is a brief flow chart of the preparation of the sensor and the detection of tetracycline in the embodiment of this invention.

[0026] Figure 2 It is an ECL-time graph of different concentrations of tetracycline.

[0027] Among them, the concentrations of tetracycline from top to bottom according to the peak heights of the curves are: 1.0×10 -7 mol / L (a), 1.0×10 -8 mol / L (b), 1.0×10 -9 mol / L (c), 1.0×10 -10mol / L (d), 1.0×10 -11 mol / L (e), 1.0×10 - 12 mol / L (f), 1.0×10 -13 mol / L (g), and 1.0×10 -14 mol / L (h).

[0028] Figure 3 is the standard curve of the difference in luminescence intensity before and after adding tetracycline and the logarithm of tetracycline concentration.

[0029] Figure 4 MoO 3-x / GCE and the ECL-time curve of dWO3·H2O / GCE in 0.1 mol / L PBS (pH 7.4) containing 0.1 mol / L K2S2O8.

[0030] Figure 5 is AgBr-N-T i3 C2 / GCE and the ECL potential curve of AgBr-Ti3C2 / GCE in 0.1 mol / L PBS (pH 7.4) containing 0.1 mol / L K2S2O8.

[0031] Figure 6 is the ECL emission curve of AgBr-N-Ti3C2 (a) and dWO3·H2O (b), MoO 3-x (c) UV absorption curve

[0032] Figure 7 is the influence of scan rate (A); pH value of buffer solution (B); concentration of aptamer (C); binding time of aptamer and TCN (D) on the performance of the sensor.

[0033] Figure 8 is the difference in ECL stability and intensity between AgBr (a) and AgBr-N-Ti3C2 (b).

[0034] Figure 9 is the ECL signal stability of the constructed ECL-IFE biosensor under continuous scanning for 14 cycles with 100 nM target TCN. Detailed implementation method

[0035] The present invention is further described in detail below in conjunction with embodiments:

[0036] Example 1: Preparation of COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE:

[0037] (1) Preparation of NH2-AgBr-N-Ti3C2 composite material:

[0038] 5mL Ti3C2 solution (2.5mg / mL) and 30mg glycine were mixed and ultrasonically dispersed for 1h, and the precursor solution was transferred to an alumina porcelain boat. The temperature was raised to 350℃ at a rate of 5℃ / min in a tube furnace under a nitrogen atmosphere and maintained for 2h. After cooling to room temperature, the sample was taken out from the porcelain boat with a medicine spoon to obtain N-Ti3C2 nanomaterial for use. 87.74mg silver nitrate was weighed in a centrifuge tube and dissolved in 2mL ammonia water to obtain a reaction solution for use; 600μL N-Ti3C2 (5mg / mL) and 8mL ethylene glycol were added to a 50mL round-bottom flask, ultrasonicated for 20min, and then 200.10mg 1-hexadecyl-3-methylimidazolium bromide was stirred at room temperature for 30 minutes, and then the reaction solution was slowly added dropwise to the above-mentioned round-bottom flask and incubated in an oil bath at 90°C for 6 hours; after the reaction, the product was centrifuged, washed, and freeze-dried for 12 hours to obtain an AgBr-N-Ti3C2 nanocomposite material; finally, the product was added to ethanol and ultrasonically dispersed for 30 minutes, and APTMS (3-aminopropyltrimethoxysilane) with a mass concentration of 4.5% (w / v) was added. After ultrasonication for 30 minutes, the mixture was stirred at 65°C for 12 hours. After the reaction, the product was centrifuged, repeatedly washed with deionized water and ethanol, and vacuum dried to obtain an off-white powder, i.e., an NH2-AgBr-N-Ti3C2 composite material, and the NH2-AgBr-N-Ti3C2 composite material was dispersed in DMF for use;

[0039] (2) Preparation of COOH-dWO3·H2O:

[0040] 0.5 mM Na₂WO₄·2H₂O, 0.75 mM CA, and 0.5 mM glucose were mixed with a certain amount of ultrapure water and stirred at room temperature to obtain a transparent solution. After adding 6 M HCl and stirring for 30 minutes, the mixture was poured into a 25 mL Teflon-lined autoclave and heated at 120°C for 24 hours. After cooling to room temperature, the supernatant was filtered and the precipitate was collected. The precipitate was washed at least four times with ethanol and water, respectively, and dried in a 60°C oven to obtain the dWO₃·H₂O nanomaterial for use. Finally, the dWO₃·H₂O and PEG (polyethylene glycol)-COOH were dissolved in methanol, stirred for 12 hours, and the precipitate was collected by centrifugation, washed multiple times with methanol, and dried in a 60°C oven. The COOH-dWO₃·H₂O composite was then dispersed in DMF for use.

[0041] (3) Preparation of COOH-dWO3·H2O / cDNA:

[0042] Add 100 μL of the cDNA Tris-HCl solution (pH = 7.4) to a centrifuge tube containing 20 mg of COOH-dWO3·H2O to obtain a suspension with a concentration of 20 mg / mL. After shaking and reacting for 16 h, centrifuge at 10,000 rpm for 10 min, and dissolve the precipitate in 100 μL of Tris-HCl solution (pH = 7.4). Ultrasonically treat the resulting colloidal solution for 10 min and shake at room temperature for 1 h to obtain COOH-dWO3·H2O / cDNA, and finally store it at 4 °C. The cDNA is the DNA strand used to link the absorber;

[0043] (4) Preparation of the electrochemiluminescence sensor COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE based on the inner filter effect:

[0044] Polish the glassy carbon electrode, ultrasonically treat it successively with nitric acid, absolute ethanol, and deionized water, and let it dry naturally for use. Under room temperature conditions, apply 6.0 μL of 0.01 M PBS buffer solution (pH 7.4) containing 0.005 M NHS and 0.01 M EDC dropwise onto the clean bare glassy carbon electrode for 1 h to activate the electrode. Subsequently, drop 6 μL of 2.0 mg / mL NH2-AgBr-N-Ti3C2 DMF solution onto the GCE to obtain the NH2-AgBr-N-Ti3C2 / GCE modified electrode. Then, successively drop 6.0 μL of the apt Tris-HCl solution (3.0 μM) and 6.0 μL of 3.0 mg / mL COOH-dWO3·H2O / cDNA colloidal solution to obtain the COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE modified electrode, that is, the electrochemiluminescence sensor based on the inner filter effect is obtained.

[0045] In the above sensor, the aptamer and cDNA sequences are as follows: (ordered from Sangon Biotech (Shanghai) Co., Ltd.)

[0046] Aptamer: 5'-COOH CGT ACG GAA TTC GCT AGC CCC CCG GCA GGC CAC GGC TTGGGT TGG TCC CAC TGC GCG TGG ATC CGA GCT CCA CGT G-3' cDNA: 5'-TGC-CGG-GGG-GCT-AGC-GAA-TTC-CGT-ACG-NH2-3'.

[0047] (5) Plotting of the standard curve

[0048] Prepare a series of tetracycline standard solutions with different concentrations. Use the electrochemiluminescence sensor COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE based on the inner filter effect as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. Immerse the three-electrode system in the above series of tetracycline standard solutions with different concentrations for 20 min, and use a 0.1 mol / L PBS buffer solution with 0.1 mol / L K2S2O8 and pH = 7.4 as the blank solution to detect the luminescence intensity.

[0049] Place the three-electrode system in a series of solutions with tetracycline concentrations (1.0×10 -14 mol / L, 1.0×10 -13 mol / L, 1.0×10 -12 mol / L, 1.0×10 -11 mol / L, 1.0×10 -10 mol / L, 1.0×10 -9 mol / L, 1.0×10 -8 mol / L, 1.0×10 - 7 mol / L). Within the electrochemistry window range of -1.8 to 0 V, with the high voltage of the photomultiplier tube at 800 V and the scanning rate at 0.1 V / s, record the time-luminescence intensity curve (E-ECL). Establish the linear relationship between the difference in luminescence intensity before and after adding tetracycline and the logarithm of the tetracycline concentration. The corresponding linear regression equation is: △I ECL = 7593.43417 + 487.53124LogC(mol / L), and the correlation coefficient (R) is 0.9705. The detection range of the linear regression equation is 1.0×10 -14 to 1.0×10 -7 mol / L, and the lowest detection limit is 3.8×10 -15 mol / L.

[0050] (6) Detection of samples

[0051] Take a certain amount of river water after filtration and impurity removal and add it to a 0.1 mol / L PBS buffer solution with 0.1 mol / L K2S2O8 and pH 7.4 for electrochemiluminescence detection. Calculate the tetracycline concentration in the sample to be detected according to the linear regression equation corresponding to the above step (5), and the results are listed in Table 1.

[0052] Comparative example 1

[0053] (1) Preparation of NH2-AgBr-N-Ti3C2 / GCE modified electrode

[0054] The glassy carbon electrode was polished, and successively ultrasonicated with nitric acid, absolute ethanol, and deionized water, and then air-dried for later use. 6.0 μL of the DMF solution of 2.0 mg / mL NH2-AgBr-N-Ti3C2 composite material (the preparation method of the NH2-AgBr-N-Ti3C2 composite material was as in Example 1) was dropped onto the surface of the clean glassy carbon electrode. After drying at room temperature, the NH2-AgBr-N-Ti3C2 / GCE modified electrode was obtained and used as the working electrode for electrochemiluminescence testing.

[0055] (2) Preparation of the standard curve

[0056] Using NH2-AgBr-N-Ti3C2 / GCE as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, a three-electrode system was formed for the detection of a series of tetracycline standard solutions with different concentrations. The detection method was the same as in Example 1.

[0057] The results are listed in Table 1.

[0058] Comparative Example 2

[0059] (1) Preparation of the NH2-AgBr-N-Ti3C2 / apt / GCE modified electrode

[0060] The glassy carbon electrode was polished, and successively ultrasonicated with nitric acid, absolute ethanol, and deionized water, and then air-dried for later use. 6.0 μL of the DMF solution of 2.0 mg / mL NH2-AgBr-N-Ti3C2 composite material (the preparation method of the NH2-AgBr-N-Ti3C2 composite material was as in Example 1) was dropped onto the surface of the clean glassy carbon electrode. After drying at room temperature, 6.0 μL of the Tris-HCl solution (2.0 μM) of apt was then dropped to obtain the NH2-AgBr-N-Ti3C2 / apt / GCE modified electrode, which was used as the working electrode for electrochemiluminescence testing.

[0061] (2) Preparation of the standard curve

[0062] Using NH2-AgBr-N-Ti3C2 / apt / GCE as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, a three-electrode system was formed for the detection of a series of tetracycline standard solutions with different concentrations. The detection method was the same as in Example 1.

[0063] The results are listed in Table 1.

[0064] Comparative Example 3:

[0065] (1) Preparation of the COOH-dWO3·H2O / GCE modified electrode

[0066] The glassy carbon electrode was polished, successively ultrasonicated with nitric acid, absolute ethanol, and deionized water, and then air-dried for later use. 6.0 μL of 2.0 mg / mL COOH-dWO3·H2O aqueous solution (the preparation method of the COOH-dWO3·H2O aqueous solution is as described in Example 1) was pipetted onto the surface of the clean glassy carbon electrode. After drying at room temperature, the COOH-dWO3·H2O / GCE modified electrode was obtained and used as the working electrode for electrochemiluminescence testing.

[0067] (2) Plotting the standard curve

[0068] Using the COOH-dWO3·H2O / GCE modified electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, a three-electrode system was formed for the detection of a series of tetracycline standard solutions with different concentrations. The detection method was the same as that in Example 1.

[0069] The results are listed in Table 1.

[0070] Comparative Example 4:

[0071] (1) Preparation of the COOH-dWO3·H2O / apt / GCE modified electrode

[0072] The glassy carbon electrode was polished, successively ultrasonicated with nitric acid, absolute ethanol, and deionized water, and then air-dried for later use. 6.0 μL of 2.0 mg / mL COOH-dWO3·H2O aqueous solution (the preparation method of the COOH-dWO3·H2O aqueous solution is as described in Example 1) was pipetted onto the surface of the clean glassy carbon electrode. After drying at room temperature, 6.0 μL of the Tris-HCl solution of apt was then added dropwise to obtain the COOH-dWO3·H2O / apt / GCE modified electrode, which was used as the working electrode for electrochemiluminescence testing.

[0073] (2) Plotting the standard curve

[0074] Using the COOH-dWO3·H2O / apt / GCE modified electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, a three-electrode system was formed for the detection of a series of tetracycline standard solutions with different concentrations. The detection method was the same as that in Example 1, and the results are listed in Table 1.

[0075] Table 1 Determination results of tetracycline in fish pond water

[0076]

[0077]

[0078] Remark: a is the average value of three determinations.

[0079] As shown in Table 1, the samples were detected in parallel 3 times, with a relative standard deviation less than 5%, and the spiked recovery rate ranged from 96% to 102%. The above results indicate that neither the bare glassy carbon electrode modified with dWO3·H2O or AgBr-N-Ti3C2 alone without the modification of COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE can detect tetracycline, and it is feasible to use the present invention to detect tetracycline in river water.

[0080] Based on Example 1, other test experiments were also carried out. For the specific situation and results, please refer to Figures 4 to 9 .

[0081] Figure 4 For MoO 3-x / GCE and dWO3·H2O / GCE in 0.1 mol / L PBS (pH 7.4) containing 0.1 mol / L K2S2O8. The ECL-time curves are shown. It can be seen from Figure 4 that as alternative absorbers, the ECL intensities of MoO 3-x and dWO3·H2O are similar. However, in the carboxylation of connecting the complementary strand cDNA, MoO 3-x will dissolve in the methanol solution and cannot be centrifuged out, so the subsequent experiments cannot be carried out, and finally dWO3·H2O is selected as the absorber.

[0082] Figure 5 For AgBr-N-T i3 C2 / GCE and AgBr-Ti3C2 / GCE in 0.1 mol / L PBS (pH 7.4) containing 0.1 mol / L K2S2O8. The ECL potential curves are shown. The ECL intensity of AgBr-Ti3C2 is 8300, and after doping with N, the ECL intensity can be increased to 10500. In the detection of actual samples, the concentration of the detected substance shows a linear relationship with the light intensity, and a high light intensity is beneficial for the detection of more concentrations. Therefore, the lowest detection limit of N-Ti3C2 is better than that of Ti3C2. The inner filter effect refers to the overlap between the absorption spectrum of the absorber and the emission spectrum of the luminescent substance, which is an effective strategy to improve the detection sensitivity and selectivity of the sensor, so it can effectively reduce the detection limit.

[0083] Figure 6 For the ECL emission curve of AgBr-N-Ti3C2 (a) and dWO3·H2O (b), MoO 3-x(c)'s ultraviolet absorption curve. The inner filter effect quenching mechanism is independent of distance and has no restricted chemical linkages, which provides considerable simplicity and flexibility in sensor construction. The key to maximizing the sensitivity of an inner filter effect-based ECL sensor is to expand the overlap between the absorption of the absorber and the emission of the luminophore, because the inner filter effect can only occur effectively when the absorption spectrum of the absorber completely overlaps with the emission spectrum of the ECL luminophore. As shown in the figure, the overlapping area between the ultraviolet absorption curve of dWO3·H2O and the ECL emission curve of AgBr-N-Ti3C2 is much larger than that of MoO 3-x , so hydrated defective tungsten trioxide is selected as the absorber.

[0084] Figure 7 The effects of scan rate (A); pH value of the buffer solution (B); concentration of the aptamer (C); binding time of the aptamer to TCN (D) on the sensor performance. The experimental conditions have a significant impact on the luminescence intensity of the ECL system. First, the ECL efficiency depends on the formation rate of the excited-state material and the diffusion rate of the coreactant. As Figure 7 shown in A, as the scan rate increases from 50 mV / s to 100 mV / s, the ECL signal continues to increase. After reaching the maximum value of 100 mV / s, as the scan rate is further increased, the ECL signal gradually decreases. The results show that overscanning accelerates the consumption of K2S2O8, resulting in a decrease in the ECL intensity. Therefore, 100 mV / s is selected as the optimal scan rate. Second, the effect of the pH value of the PBS solution on the ECL signal was further investigated. As Figure 7 shown in B, when the pH increases from 6.6 to 7.4, the ECL signal gradually increases. As the pH value further increases, the ECL signal weakens, so a weakly alkaline environment with a pH value of 7.4 is selected as the optimal pH value. At the same time, the aptamer concentration also affects the ECL intensity. For example, a PBS buffer solution containing NHS and EDC is dropped on a clean bare glassy carbon electrode to obtain an activated glassy carbon electrode. Subsequently, 6 μL of 2.0 mg / mL NH2-AgBr-N-Ti3C2 DMF solution is dropped on the GCE to obtain an NH2-AgBr-N-Ti3C2 / GCE modified electrode. Then, 6.0 μL of apt Tris-HCl solutions (0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 8.0 μM) and 6.0 μL of COOH-dWO3·H2O / cDNA colloidal solutions (0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 8.0 μM) are successively dropped, respectively, to obtain modified electrodes of different concentrations of COOH-dWO3·H2O / cDNA / apt / NH-AgBr-N-Ti3C2 / GCE, that is, an electrochemical luminescence sensor based on the inner filter effect is obtained, as Figure 7As shown in C, within the range of 0.5 μM to 3 μM, as the concentration of the TC aptamer increases, the ΔIECL signal gradually increases, indicating that more aptamer molecules are adsorbed on the electrode surface. When the aptamer concentration exceeds 3 μM, since the aptamer molecules reach the saturation point on the GCE, the ΔIECL intensity slightly decreases. Therefore, 3 μM is selected as the optimal concentration of the aptamer. As Figure 7 shown in D, during the increase from 5 min to 20 min, the ECL intensity gradually increases and reaches the highest level at 20 min. As the aptamer concentration continues to increase, the ECL signal tends to be stable. Therefore, we select 20 min as the optimal incubation time.

[0085] Figure 8 Differences in the ECL stability and intensity of AgBr (a) and AgBr-N-Ti3C2 (b). From Figure 8 it can be seen that the ECL emission of AgBr is unstable and relatively low, but the stability and ECL intensity of the AgBr-N-Ti3C2 nanocomposite are significantly enhanced, indicating that the N-Ti3C2 nanosheets can effectively improve the stability and luminescence intensity of AgBr NPs.

[0086] Figure 9 ECL signal stability of the constructed ECL-IFE biosensor under continuous scanning for 14 cycles with 100 nM target TCN. As Figure 9 shown, when TCN is monitored for 14 cycles, the relative standard deviation (RSD) of T is 2.60%, indicating that the prepared ECL sensor has good stability.

[0087] As described above, only the preferred specific embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. An electrochemiluminescent sensor for detecting antibiotics based on the inner filter effect, characterized in that: The luminescent body NH2-AgBr-N-Ti3C2 nanocomposite material and the absorber COOH-dWO3·H2O nanosheets are modified on the electrode surface from the inside out. The luminescent body and the absorber are connected by two complementary DNA chains, one of which is an aptamer connected to the luminescent body, and the other complementary DNA chain is connected to the absorber. The aptamer is connected to the DNA chain connected to the luminescent body.

2. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on the inner filter effect according to claim 1, wherein: The method includes dropwise coating a PBS buffer solution containing NHS and EDC on a clean bare glassy carbon electrode to obtain an activated glassy carbon electrode, then dropwise coating a DMF dispersion of an NH2-AgBr-N-Ti3C2 composite material on the activated glassy carbon electrode to obtain an NH2-AgBr-N-Ti3C2 / GCE modified electrode; and then sequentially dropwise coating a Tris-HCl solution and a COOH-dWO3·H2O / cDNA colloidal solution containing an aptamer to obtain the electrochemiluminescence sensor COOH-dWO3·H2O / cDNA / apt / NH2-AgBr-N-Ti3C2 / GCE based on the inner filter effect.

3. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on the inner filter effect according to claim 2, characterized in that: The concentration of the aptamer is 3.0-8.0 μmol / L, and the concentration of the COOH-dWO3·H2O / cDNA colloid solution is 3.0-8.0 μmol / L.

4. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on the inner filter effect according to claim 2, characterized in that: The pH value of the Tris-HCl solution containing the aptamer is 7-7.

8.

5. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on the inner filter effect according to claim 2, characterized in that: The pH value of the Tris-HCl solution containing the aptamer was 7.4; And / or, the coating volume of NH2-AgBr-N-Ti3C2 is 6.0 μL, and the concentration is 2.0 mg / mL; the coating volume of the aptamer is 6.0 μL, and the concentration is 3.0 μmol / L; the coating volume of the COOH-dWO3·H2O / cDNA colloidal solution is 6.0 μL, and the concentration is 3.0 μmol / L.

6. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on inner filter effect according to claim 2, characterized in that: The method comprises the following steps for preparing an NH2-AgBr-N-Ti3C2 composite material: in an ammonia solution of silver nitrate in which N-Ti3C2 nanomaterial and ethylene glycol are uniformly dispersed, using 1-hexadecyl-3-methylimidazolium bromide as a bromine source, in situ preparing an AgBr-N-Ti3C2 nanocomposite material at a reaction temperature, then performing amino modification through APTMS, introducing NH2 into the AgBr-N-Ti3C2 nanocomposite material, and obtaining the NH2-AgBr-N-Ti3C2 composite material.

7. The method for preparing a sensor for detecting antibiotics by electrochemiluminescence based on inner filter effect according to claim 2, characterized in that: The method comprises the following steps of preparing a COOH-dWO3·H2O / cDNA colloidal solution: adding a Tris-HCl solution of cDNA to COOH-dWO3·H2O to obtain a suspension having a concentration of 0.2 mg / ml, fully shaking the suspension for reaction, centrifuging the suspension to obtain a precipitate, dissolving the precipitate in a Tris-HCl solution, and fully stirring the suspension until the COOH-dWO3·H2O and the cDNA are fully connected to obtain COOH-dWO3·H2O / cDNA, wherein the cDNA is a DNA chain for connecting to an absorber.

8. The use of the sensor for detecting antibiotics by electrochemiluminescence method based on inner filter effect according to claim 1, characterized in that: For the detection of antibiotics in water, the following steps are included: Step 1: Prepare a series of antibiotic standard solutions with different concentrations, ranging from 1.0×10 -14 mol / L~1.0×10 -7 mol / L; Step 2: Using the inner filter effect-based electrochemiluminescence sensor as a working electrode, a platinum electrode as an auxiliary electrode, and Ag / AgCl as a reference electrode to form a three-electrode system, the three-electrode system is immersed in a series of standard solutions containing different concentrations of antibiotics prepared in step 1 and incubated for a certain time to allow the aptamer to fully bind to the antibiotics. Cyclic voltammetry is performed within the electrochemical window range of -1.8 to 0 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.075 to 0.125 V / s. The potential-luminescence intensity curve is recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of the antibiotic and the logarithm of the antibiotic concentration is established to obtain a corresponding linear regression equation; Step 3, sample testing, test according to the above step 2 and obtain the luminescence intensity, the obtained luminescence intensity is calculated using the linear regression equation obtained in step 2 to obtain the concentration of antibiotics in the sample.

9. The use of the sensor for detecting antibiotics by electrochemiluminescence method based on inner filter effect according to claim 1, characterized in that: The antibiotic was tetracycline, and the aptamer nucleotide sequence was: 5′-COOH CGT ACG GAA TTC GCT AGCCCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TGG ATC CGA GCT CCA CGTG-3′; cDNA sequence: 5'-TGC-CGG-GGG-GCT-AGC-GAA-TTC-CGT-ACG-NH2-3'; The incubation time was at least 20 min, and the scan rate was 0.1 V / s.

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