A ratio fluorescence sensor, a preparation method thereof, and an application thereof

Abstract

The present invention discloses a ratiometric fluorescence sensor, a preparation method thereof and an application, belonging to the technical field of chemical detection. The ratiometric fluorescence sensor of the present invention is prepared by the following method: adding a g-C3N4 nanosheet solution, an Eu(NO3)3·6H2O solution and a sodium citrate solution into a Tris-HCl buffer solution in sequence, mixing evenly, and incubating; after the incubation is completed, freeze-drying the incubation solution to obtain a g-C3N4 / CitNa / Eu nanoprobe, that is, the ratiometric fluorescence sensor. The ratiometric fluorescence sensor of the present invention can realize the rapid, intuitive and quantitative determination of tetracycline antibiotics remaining in a sample, has high sensitivity, good visualization effect, and can be used in combination with a smart phone for portable detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and particularly relates to a ratio fluorescence sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Tetracycline antibiotics are widely used as feed additives in the livestock industry. However, the excessive use of antibiotics will produce high levels of drug residues in animal products (especially milk, eggs, and meat) and the external environment, posing a serious threat to food safety, the ecological environment, and human health. The increase in antibiotic residues in food has attracted global attention. The European Union (EU) and the US Food and Drug Administration (FDA) have stipulated that the maximum residue limits of tetracycline (TC) in milk are 100 ng / mL (225 nM) and 300 ng / mL (676 nM). Therefore, to ensure food safety and protect consumer health, there is an urgent need to develop an efficient, ultrasensitive, visual, and portable detection scheme for tetracycline antibiotics residues.

[0003] Currently, there are various methods for detecting tetracycline antibiotics, including high-performance liquid chromatography (HPLC), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), electrochemical analysis, resonance scattering, etc. In recent years, some emerging sensor devices have shown great potential in the field of analytical chemistry, including electrochemical sensors, nanozyme sensors, fluorescence sensors, etc. Among them, fluorescence sensors are considered an ideal means for detecting tetracycline antibiotics due to their simple operation, low cost, high sensitivity, fast response speed, and easy visual analysis. However, the response of traditional fluorescence sensors to analytes is usually based on a single fluorescence signal emission, which is easily interfered by background, instruments, and the environment. Therefore, it is of great significance to provide a scheme that can rapidly, intuitively, and quantitatively determine tetracycline antibiotics on-site. Summary of the Invention

[0004] The technical solution of the present invention is as follows:

[0005] The present invention provides a preparation method of a ratio fluorescence sensor, and the steps are as follows:

[0006] Add the g-C 3 N 4 nanosheet solution, Eu(NO 3 ) 3 ·6H 2 O solution and sodium citrate solution into the Tris-HCl buffer solution in sequence, mix evenly, and incubate; after the incubation ends, lyophilize the incubation solution, and the obtained powder is the g-C 3 N 4 / CitNa / Eu nanoprobe, that is, the ratio fluorescence sensor.

[0007] In the above preparation method, the g-C 3 N 4 nanosheet solution, Eu(NO 3 ) 3 ·6H 2 O solution and the sodium citrate solution have a volume ratio selected from 10:1:1; the concentration of the g-C 3 N 4 nanosheet solution is selected from 0.3 mg / mL, the concentration of Eu(NO 3 ) 3 ·6H 2 O solution is selected from 100 μM; the concentration of the sodium citrate solution is selected from 500 μM; the concentration of the Tris-HCl buffer solution is selected from 50 mM, and the pH is 8.0; the incubation time is selected from 10 min.

[0008] In the above preparation method, the g-C 3 N 4 nanosheets are prepared by the following method:

[0009] Melamine is calcined at 550-600 °C for 2-3 h. After the calcination is completed, it is cooled to room temperature to obtain a yellow solid product, which is thoroughly ground; then, the yellow solid product is dispersed in water, ultrasonically pulverized, centrifuged to remove the unpeeled aggregates; the supernatant is collected and dried to obtain g-C 3 N 4 nanosheets.

[0010] The above calcination temperature is preferably 550 °C; the calcination time is preferably 2 h.

[0011] The mass-volume ratio of the above yellow solid product to water is selected from 1:180-1:250, g:mL; preferably 1:200, g:mL.

[0012] The present invention provides a ratio fluorescence sensor prepared by the above method.

[0013] The present invention provides the application of the above ratio fluorescence sensor in the detection of tetracycline antibiotics residues in food. The tetracycline antibiotics are selected from doxycycline, tetracycline, chlortetracycline and oxytetracycline; preferably tetracycline.

[0014] The present invention provides a method for detecting tetracycline antibiotics residues in food using the above ratio fluorescence sensor, and the steps are as follows:

[0015] Add trichloroacetic acid solution to the sample to be tested and react under ultrasonic waves; after the reaction is completed, centrifuge, collect the supernatant, filter the supernatant, then add the ratiometric fluorescence sensor, mix well, and place it at room temperature for complete reaction; after the reaction is completed, place the reaction solution at an excitation wavelength of 275 nm and observe the change in fluorescence color; if tetracycline antibiotics are present, the fluorescence peak at 620 nm will be excited and the fluorescence color will turn red.

[0016] In the above detection method, the volume ratio of the ratiometric fluorescence sensor to the filtrate is selected from 8:25.

[0017] In the above detection method, the sample to be tested is selected from common foods such as milk, eggs, chicken, beef, etc.; preferably milk.

[0018] The present invention provides a preparation method of a g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, and the steps are as follows:

[0019] Dissolve polyacrylonitrile in DMF to obtain a polyacrylonitrile solution; then add g-C 3 N 4 / CitNa / Eu powder into the polyacrylonitrile solution, stir well to obtain a g-C 3 N 4 / CitNa / Eu / PAN electrospinning solution; then perform electrospinning to obtain a g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane.

[0020] The mass-volume ratio of the above g-C 3 N 4 / CitNa / Eu powder to the polyacrylonitrile solution is selected from 10:9 - 10:11, mg:mL; preferably 10:9, mg:mL.

[0021] The present invention provides a g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane prepared by the above method.

[0022] The present invention provides the application of the above g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane in the detection of tetracycline antibiotic residues in foods. The tetracycline antibiotics are selected from doxycycline, tetracycline, chlortetracycline and oxytetracycline; preferably tetracycline.

[0023] The present invention provides a method for using the above g-C 3 N 4Method for detecting tetracycline antibiotics residues in food using / CitNa / Eu / PAN electrospun membrane, the steps are as follows:

[0024] Drop the test sample solution onto the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, or immerse the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane into the test sample solution, react for 5 min, then irradiate the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane with a 365 nm ultraviolet lamp, and observe the change in fluorescence color; if there are tetracycline antibiotics, the electrospun membrane will turn red under the irradiation of the ultraviolet lamp.

[0025] The present invention provides a method for quantitatively detecting tetracycline antibiotics residues in food using a smart phone, the steps are as follows:

[0026] Drop the test sample solution onto the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, or immerse the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane into the test sample solution, react for 5 min, then irradiate the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane with a 365 nm hand-held ultraviolet lamp, use the smart phone to take a picture of the fluorescence of the excitation system, then use the color recognizer loaded in the smart phone to convert the photo color signal into color information (RGB value), calculate the ratio of the green and blue channels (G / B value), and substitute the G / B value into the standard curve to obtain the concentration of tetracycline antibiotics.

[0027] The above-mentioned color recognizer is selected from the ColorDesk application program.

[0028] The above-mentioned standard curve can be constructed by the following method:

[0029] Drop solutions of tetracycline antibiotics with different concentrations onto the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, or immerse the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane into solutions of tetracycline antibiotics with different concentrations, react for 5 min, then irradiate the g-C 3 N 4The / CitNa / Eu / PAN electrospun membrane is irradiated with a 365-nm handheld ultraviolet lamp, and the fluorescence of the excitation system is photographed using a smartphone. Then, the color recognition device loaded in the smartphone is used to convert the photo color signal into color information (RGB value), and the ratio of the green and blue channels (G / B value) is calculated. With the concentration of tetracycline antibiotics as the abscissa and the G / B value as the ordinate, a concentration-G / B value standard curve is constructed.

[0030] The concentration gradients of the above tetracycline compound solutions can be selected from 0 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM.

[0031] The beneficial effects of the present invention are as follows:

[0032] The dual-signal ratio fluorescence sensor (g-C 3 N 4 / CitNa / Eu) designed in the present invention can be used for on-site visual detection of tetracycline antibiotics. g-C 3 N 4 with blue luminescence ability can not only serve as the framework for Eu 3+ coordination, but also as the recognition unit for tetracycline antibiotics. The coordinated unsaturated red fluorescence Eu 3+ (λ em = 620 nm) is combined on the surface of g-C 3 N 4 and becomes a specific tetracycline antibiotic recognition element due to the antenna effect (AE). In the presence of tetracycline antibiotics, the ratio fluorescence sensor exhibits dual response and reverse response signals, accompanied by obvious multi-color width color changes (blue - purple - pink - red), achieving ultra-high sensitivity detection with a detection limit of 1.961 nM. In addition, by loading the ratio fluorescence sensor on the nanofiber film through electrospinning technology, a portable flexible sensor can be prepared. The successful combination of the flexible sensor and the smartphone greatly reduces the detection cost and time, providing a promising method for on-site qualitative identification and quantitative detection of tetracycline antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Emission spectra of bulk g-C 3 N 4 and g-C 3 N 4 nanosheets;

[0034] Figure 2 For g-C 3 N 4Characterization of nanosheets; among them, Figure A is the fluorescence spectrum, and the inset is g-C 3 N 4 Photographs of the nanosheet solution under ultraviolet and sunlight; Figure B is the TEM image; Figure C is the AFM image; Figure D is g-C 3 N 4 AFM image height profile of the nanosheets; Figure E is the bulk g-C 3 N 4 and g-C 3 N 4 XRD pattern of the g-C 3 N 4 nanosheet bulk; Figure F is the bulk g-C 3 N 4 FT-IR spectrum of the g-C 3 N 4 nanosheet bulk; Figure G is the fitted g-C 3 N 4 High-resolution XPS spectrum of C1s of the nanosheets; Figure H is the fitted g-C 3 N 4 High-resolution XPS spectrum of N1s of the nanosheets; Figure I is g-C 3+ N 3 , Eu 4 g-C 3 N 4 / CitNa / Eu, TC, CitNa and g-C

[0035] Figure 3 is the fluorescence stability of g-C 3 N 4 nanosheets under different conditions; among them, Figure A is the storage time stress; Figure B is the UV irradiation time; Figure C is the solution pH; Figure D is the NaCl concentration;

[0036] Figure 4 is the XPS spectrum of g-C 3 N 4 nanosheets; among them, Figure A is the full XPS spectrum, and the inset is g-C 3 N 4 Relative contents of C, N, and O atoms in the nanosheets; Figure B is the fitted g-C 3 N 4 High-resolution O1s spectrum of the nanosheets;

[0037] Figure 5 is the feasibility study of TC detection; among them, Figure A is the fluorescence excitation spectrum (a), emission spectrum (b) of g-C 3 N 4 nanosheets and the UV-visible absorption spectrum (c) of TC; Figure B is g-C3 N 4 CIE chromaticity coordinates of N (a) and CIE chromaticity coordinates of Eu / CitNa / TC (b); Figure C shows g-C 3 N 4 and the emission spectra of Eu / CitNa / TC (λex = 275 nm). The inset shows g-C 3 N 4 nanosheet solution (left) and Eu / Cit / TC solution (right) under irradiation of a 365 nm UV lamp; Figure D shows the fluorescence spectra of the various systems shown.

[0038] Figure 6 is for the addition of different concentrations of Eu 3+ and then the fluorescence spectra (Figure A) and changes in fluorescence intensity (Figure B) of g-C 3 N 4 ;

[0039] Figure 7 is the mechanism for TC detection; among them, Figure A is the absorption spectra of the various systems shown; Figure B is the fluorescence lifetimes of the various materials shown; Figure C is the observed (Eobsd, a) and corrected (Ecor, b) fluorescence quenching efficiencies after adding different concentrations of TC; Figure D is the schematic diagram of the emission level of g-C 3 N 4 / CitNa / Eu and the energy transfer from TC to Eu 3+ ; Figure E is the principle of the ratiometric fluorescence method for detecting TC based on g-C 3 N 4 / CitNa / Eu;

[0040] Figure 8 is the optimization of the amount of CitNa used; among them, Figure A is the fluorescence intensity; Figure B is the fluorescence intensity ratio, and the inset shows the photos of the corresponding solutions under irradiation of a 365 nm UV lamp;

[0041] Figure 9 is the optimization of the pH value of the ratiometric fluorescence detection TC sensing system;

[0042] Figure 10 is the incubation time optimization; among them, Figure A is the fluorescence intensity; Figure B is the fluorescence intensity ratio;

[0043] Figure 11 is the ratiometric fluorescence method for detecting TC; among them, Figure A is the fluorescence spectra of g-C 3 N 4 / CitNa / Eu solution at different TC dosages; Figure B is the corresponding intensity changes of g-C 3 N 4 / CitNa / Eu solution at different TC dosages; Figure C is the fluorescence intensity ratio (F 620 / F450 ) Linear relationship with TC concentration; Figure D shows g-C 3 N 4 / CitNa / Eu solution under ultraviolet light (λ = 365 nm); Figure E shows the CIE chromaticity diagram at different TC concentrations; Figure F shows the chromaticity of CIE in the presence of different concentrations of TC;

[0044] Figure 12 For the single-signal determination of TC; among them, Figure A shows the fluorescence spectra of the g-C 3 N 4 -based fluorescence sensing system after adding different concentrations of TC; Figure B shows the linear relationship between TC concentration and (F 0 -F) / F 0 , where F 0 and F represent the fluorescence intensities of the sensing system before and after adding TC, respectively; Figure C shows the fluorescence color change photos of g-C 3 N 4 solution under ultraviolet light (λ = 365 nm);

[0045] Figure 13 For the TC specificity test; among them, Figure A shows the fluorescence responses of the g-C 3 N 4 / CitNa / Eu sensor to TC and interferents; Figure B shows the fluorescence responses of the g-C 3 N 4 / CitNa / Eu sensor to TC and interferents; Figure C shows the relevant photos under ultraviolet light, with the concentrations of TC and interferents both being 50 μM; Figure D shows the relevant photos under ultraviolet light, with the concentrations of TC and interferents both being 50 μM; Figure E shows the anti-interference experiment of the g-C 3 N 4 / CitNa / Eu sensor on TC, with the TC concentration being 50 μM and the interferent concentration being 150 μM; Figure F shows the anti-interference experiment of the g-C 3 N 4 / CitNa / Eu sensor on TC, with the TC concentration being 50 μM and the interferent concentration being 150 μM;

[0046] Figure 14 Ultraviolet-visible absorption spectra of TC, DOX, OXY, and CTE at a concentration of 45 μM; among them, at 325 nm, the curves from top to bottom represent TC, OXY, DOX, and CTE in turn;

[0047] Figure 15 Chemical structures of tetracycline drugs; among them, tetracycline (TC) is at the upper left, doxycycline (DOX) is at the upper right, oxytetracycline (OXY) is at the lower left, and chlortetracycline (CTE) is at the lower right. The four compounds have similar chemical structures;

[0048] Figure 16 For g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane and its intelligent applications; among them, Figure A is a schematic diagram of preparing nanofibers by electrospinning method; Figure B is a scanning electron microscope image of g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane; Figure C is a photograph of g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane; Figure D is a photograph of g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane under sunlight; Figure E is a photograph of g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane under ultraviolet light; Figure F is a photograph of the electrospun membrane after adding 15 μL of TC solutions with different concentrations (from left to right are 0 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM); Figure G is a photograph of the electrospun membrane after adding 15 μL of 200 μM different antibiotic solutions, from left to right are AmL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM2, DOX, CTE, OXY and TC; Figure H is a simplified flowchart of TC detection based on g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane and smartphone sensor; Figure I is the fluorescence color image of different concentrations of TC solutions of the designed visual sensor based on g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane under handheld ultraviolet lamp irradiation. Detailed implementation manners

[0049] The reagents, chemicals and instruments used in the present invention are as follows:

[0050] (Hydroxymethyl)aminomethane (Tris), melamine, Eu(NO 3 ) 3 ·6H 2O, sodium citrate (CitNa, 98%), polyacrylonitrile (PAN, Mw = 150,000), tetracycline (TC), chlortetracycline (CTE), oxytetracycline (OXY), doxycycline (DOX), roxithromycin (ROX), kanamycin sulfate (KM), thiamphenicol (THI), amoxicillin (AmL), luteolin (LUT), metronidazole (MNZ), lactose (Lac), lysine (Lys), ascorbic acid (Vc), aspartic acid (Asp), glutamic acid (Glu), tryptophan (Trp), D-tartaric acid (D-Ta), 10% trichloroacetic acid solution, and other ions were purchased from Macklin Biochemical Technology Co., Ltd. Ampicillin (AMP), streptomycin sulfate (STR), and ceftriaxone sodium (CTR) were purchased from Solarbio Science & Technology Co., Ltd. N,N-dimethylformamide (DMF), cysteine (Cys), gallic acid (GAE), sulfamethazine (SM2), sucrose (Suc), and anhydrous glucose (Gl) were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used as received without further purification. TD-3700 (China) X-ray diffractometer (XRD), transmission electron microscope (TEM, JOEL JEM 2001), atomic force microscope (AFM) SPM-9700HT instrument (China), X-ray photoelectron spectroscopy (XPS) ESCA-3Mark II spectrometer (VG Scientific Ltd., England), Fourier transform infrared (FTIR) spectroscopy Nicolet islo FTIR spectrometer (USA), SpectraMax i3x multifunctional microplate reader (USA), F-2700 fluorescence spectrophotometer (Hitachi, Japan), F-4600 spectrometer (Hitachi, Japan), XO-1000D ultrasonic cell disruptor, Zeiss scanning electron microscope (SEM, Japan), YFSP-T (Tianjin Yunfan, China) electrospinning equipment.

[0051] Other materials used in the present invention, unless otherwise specified, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise stated, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be described in further detail below with reference to specific examples and data. The following examples are only for illustrative purposes and do not limit the scope of the present invention in any way.

[0052] Example 1

[0053] g-C 3 N 4 Preparation of g-C

[0054] Put 3 g of melamine into an alumina crucible, then place it in a muffle furnace and heat it at a rate of 10 °C / min to 550 °C. Calcinate for 2 h. After cooling to room temperature, grind the obtained yellow solid product thoroughly. Then, disperse 2.25 g of the yellow solid product in 450 mL of ultrapure water, and then use an ultrasonic cell disruptor to crush it at a power of 1000 w for 48 h. Then centrifuge at 3000 rpm for 10 min to remove the unpeeled aggregates. Collect the supernatant containing g-C 3 N 4 nanosheets, dry it, and store it at 4 °C for later use.

[0055] 1. Characterization of g-C 3 N 4 nanosheets

[0056] The present invention prepares g-C 3 N 4 nanosheets by calcination polymerization and ultrasonic exfoliation method. As Figure 1 can be seen, ultrasonic exfoliation enhances the emission intensity of g-C 3 N 4 nanosheets. In addition, due to the quantum confinement effect, the position of its emission peak is slightly blue-shifted compared with that of bulk g-C 3 N 4 , with a blue shift of about 12 nm. The PL excitation and emission spectra of g-C 3 N 4 nanosheets, as shown in Figure 2 A, show that at different excitation wavelengths, the PL emission position does not change significantly; the optimal emission and excitation wavelengths of g-C 3 N 4 nanosheets are 450 nm and 275 nm, respectively; in addition, the obtained g-C 3 N 4 nanosheets show good dispersibility in solution and emit bright blue fluorescence under UV. The fluorescence stability of g-C 3 N 4 nanosheets, as shown in Figure 3 , shows that the fluorescence intensity remains relatively consistent, indicating that the g-C 3 N 4 nanosheet solution has excellent fluorescence stability; in addition, the quantum yield of the prepared g-C 3 N 4 nanosheet solution is 5.23%. The TEM image of g-C 3 N 4 nanosheets, as shown in Figure 2 B, shows a typical regular flake structure. The AFM image of g-C 3 N 4 nanosheets, as shown in Figure 2 C andFigure 2 As shown in D, the AFM image further verified g-C 3 N 4 The nanosheets are sheet-like structures with a uniform thickness of about 3 nm. Figure 2 E shows an obvious characteristic XRD peak corresponding to g-C 3 N 4 The typical graphite interlayer deposition (002) peak. Compared with bulk g-C 3 N 4 After ultrasonic treatment, the XRD peak of g-C 3 N 4 The nanosheets changed from 27.3° to 27.5°, indicating that the bulk g-C 3 N 4 The separation was successful. The present invention further characterized the functional groups of g-C 3 N 4 The nanosheets, such as Figure 2 As shown in F, bulk g-C 3 N 4 And g-C 3 N 4 The nanosheets showed similar absorption peaks, confirming that ultrasonic exfoliation did not change the g-C 3 N 4 The structure of the nanosheets. XPS analysis further confirmed the elemental composition of g-C 3 N 4 The nanosheets, such as Figure 4 As shown in A, the synthesized g-C 3 N 4 The nanosheets are composed of three main chemical elements (C, N, and O). Figure 2 G and Figure 2 H are the fitted high-resolution C1s and N1s, respectively. The C1s spectrum showed three peaks at 284.6 eV (graphite carbon), 286.2 eV (C-OH), and 288.0 eV (N-C=N), respectively. The N1s band was composed of quaternary nitrogen (401.1 eV), tertiary nitrogen (399.8 eV), and C=N-C (398.44 eV). The deconvoluted O1s band indicated the presence of C-OH (532.2 eV), as Figure 4 Shown in B. In summary, ultrasonic exfoliation reduced the size of the g-C 3 N 4 Bulk and increased its emission intensity.

[0057] 2. Feasibility study of TC detection

[0058] First, the present invention measured the absorption spectrum of TC and the excitation spectrum of g-C 3 N 4 The nanosheets, such as Figure 5As shown in A, the two have a large spectral overlap, which enables the fluorescence of g-C 3 N 4 to be greatly quenched by TC through IFE, because the efficiency of IFE depends on the spectral overlap between the absorption of the quencher and the excitation or emission of the fluorophore.

[0059] Secondly, the present invention explores the effect of sodium citrate on the fluorescence of Eu 3+ The specific experimental process is as follows: (1) Concentration of Eu: 200 μL of 0.3 mg / mL g-C 3 N 4 solution is mixed with 20 μL of Eu at different concentrations 3+ and 780 μL of 50 mM pH = 8 Tric-HCl solution, and the fluorescence spectrum is recorded under excitation at 275 nm. (2) Concentration of CitNa: 200 μL of 0.3 mg / mL g-C 3 N 4 solution is mixed with 20 μL of 100 μM Eu 3+ and 20 μL of CitNa at different concentrations and 510 μL of 50 mM pH = 8 Tric-HCl and 250 μL of 50 μM TC solution, and the fluorescence spectrum is recorded under excitation at 275 nm. The experimental results are as Figure 6 shown. TC can effectively enhance the fluorescence of Eu 3+ with the assistance of CitNa, because TC and CitNa can chelate with Eu 3+ and transfer energy to Eu 3+ , thereby sensitizing the luminescence of Eu 3+ . The ligand (TC) chelated with lanthanide ions (Eu 3+ ) acts as an "antenna", absorbs photons and transfers energy to the lanthanide ions (Eu 3+ ), thereby sensitizing its luminescence. This process is called the antenna effect. Therefore, g-C 3 N 4 nanosheets and Eu 3+ can both be used as TC recognition units to achieve dual and reverse response signals (fluorescence of g-C 3 N 4 weakens, fluorescence of Eu 3+ enhances), which provides a prerequisite for ratiometric fluorescence sensing.

[0060] Then, it can be seen from the CIE chromaticity diagram that there is a large color difference between the fluorescence of g-C 3 N 4 nanosheets (0.15592, 0.12369) and Eu3+ (0.65324, 0.33518) ( Figure 5B). This significant color evolution from blue to red provides great advantages for the visual analysis of TC.

[0061] Finally, g-C 3 N 4 nanosheets and Eu 3+ 's other optical properties are also beneficial for the development of an effective ratiometric fluorescence sensor. For example, the optimal excitation wavelength of Eu / CitNa / TC (mixing 20 μL of 100 μM Eu, 20 μL of 500 μM CitNa, 710 μL of 50 mM pH = 8 Tric-HCl, and 250 μL of 50 μM TC solution, and recording the fluorescence spectrum under 275 nm excitation) is 275 nm. g-C 3 N 4 and Eu 3+ have similar excitation wavelengths at 275 nm. Therefore, the fluorescence of both g-C 3 N 4 and Eu 3+ can be excited at 275 nm and show well-resolved dual emission bands to achieve effective ratiometric fluorescence detection of TC.

[0062] In addition, as Figure 5 shown in C, g-C 3 N 4 shows a fluorescence maximum at 450 nm and bright blue fluorescence under UV light (left figure), while the maximum emission wavelength of Eu / CitNa / TC is 620 nm, showing red fluorescence (right figure). The emission peak of g-C 3 N 4 at 450 nm compared with the emission spectrum of Eu 3+ at 620 nm shows a large emission spectrum shift (about 170 nm) due to 5 D 0 → 7 F 2 transitions. The large peak shift not only easily enables a wide and sensitive color change but also effectively avoids spectral overlap of different emission peaks. In summary, the properties of g-C 3 N 4 nanosheets and Eu 3+ can meet the requirements for developing an effective ratiometric fluorescence method for the visual determination of TC.

[0063] To verify the feasibility of g-C 3 N 4 , CitNa, and Eu 3+ in the determination of TC, the present invention conducted relevant experiments as follows: a: 200 μL of 0.3 mg / mL g-C 3 N 4The solution is mixed with 510 μL of Tric-HCl with pH = 8 and 290 μL of ultrapure water. b: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 20 μL of 100 μM Eu 3+ + 510 μL of Tric-HCl with pH = 8 and 270 μL of ultrapure water. c: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 20 μL of 500 μM CitNa + 510 μL of Tric-HCl with pH = 8 and 270 μL of ultrapure water. d: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 20 μL of 500 μM CitNa + 20 μL of 100 μM Eu 3+ + 510 μL of Tric-HCl with pH = 8 and 250 μL of ultrapure water. e: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 250 μL of 50 μM TC + 510 μL of Tric-HCl with pH = 8 and 40 μL of ultrapure water. f: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 20 μL of 100 μM Eu 3+ + 250 μL of 50 μM TC + 510 μL of Tric-HCl with pH = 8 and 20 μL of ultrapure water. g: 200 μL of 0.3 mg / mL g-C 3 N 4 The solution + 20 μL of 500 μM CitNa + 20 μL of 100 μM Eu 3+ + 250 μL of 50 μM TC + 510 μL of Tric-HCl with pH = 8. Record the fluorescence spectrum under excitation at 275 nm.

[0064] The test results are as Figure 5 shown in

[0065] Eu 3+ and CitNa have little effect on the fluorescence of g-C 3 N 4 (curves a - d). After adding TC to the g-C 3 N 4 / Eu 3+ solution, the fluorescence of g-C 3 N 4 at 450 nm decreases significantly, while Eu 3+The fluorescence at 620 nm increased slightly (curve f). This is because the chelation of TC with Eu 3+ sensitized the luminescence of Eu 3+ and TC induced fluorescence quenching of g-C 3 N 4 by IFE. However, due to the asynchronous change in fluorescence intensity, the solution showed an inconspicuous color change ( Figure 5 D inset). To achieve effective ratio fluorescence sensing and more obvious visual analysis, CitNa was introduced as an auxiliary ligand into the g-C 3 N 4 / Eu 3+ solution. The fluorescence of Eu 3+ at 620 nm increased significantly (curve g) and the solution showed an obvious color change from blue to red ( Figure 5 D inset), which may be due to CitNa replacing the chelated water molecules and chelating with Eu 3+ / TC to inhibit the quenching effect caused by the chelated water molecules. These results indicate that the above design is feasible, and introducing an auxiliary ligand into the g-C 3 N 4 / Eu 3+ detection system can improve the detection limit of the system and make the color change more obvious.

[0066] For the above reasons, the present invention selects g-C 3 N 4 nanosheets, CitNa and Eu 3+ to develop a ratio fluorescence sensor for TC detection.

[0067] 3. Revelation of the mechanism for TC detection

[0068] The introduction of TC enhanced the red fluorescence of Eu 3+ and simultaneously quenched the blue fluorescence of g-C 3 N 4 at the same time.

[0069] First, the present invention confirmed the mechanism for the enhanced fluorescence of Eu 3+ . As shown in Figure 7 A, after adding Eu 3+ , the absorption peak of TC showed an obvious red shift and increased significantly compared with the absorbance of TC, indicating that Eu 3+ had a specific chelation with TC containing the β-diketone configuration. After adding the auxiliary ligand CitNa to the Eu 3+ / TC solution, the absorbance further increased, indicating that CitNa formed a complex with Eu 3+ / TC. The results showed that TC and CitNa chelated with Eu 3+The complexation and energy transfer sensitize the luminescence of Eu 3+ and thus enhance the red fluorescence of Eu 3+ .

[0070] Secondly, the present invention elucidates the fluorescence quenching mechanism of g-C 3 N 4 and measures the relevant fluorescence lifetimes. Generally, in the fluorescence resonance energy transfer (FRET) process, the fluorescence lifetime of the fluorophore decreases due to the transfer of energy from the fluorophore to the quencher, while for IFE, the fluorescence lifetime remains unchanged. As Figure 7 shown in Figure B and Table 1, the fluorescence lifetime of g-C 3 N 4 remains relatively stable after adding different concentrations of TC, indicating that the TC-induced fluorescence quenching of g-C 3 N 4 is mainly caused by IFE rather than FRET.

[0071] Table 1 Fluorescence lifetimes of g-C 3 N 4 under different conditions (λem = 450 nm)

[0072] Sample τ (ns) <![CDATA[g-C 3 N 4 > 6.25 <![CDATA[g-C 3 N 4 +Eu 3+ > 6.21 <![CDATA[g-C 3 N 4 +Eu 3+ +CitNa]]> 6.43 <![CDATA[g-C 3 N 4 +Eu 3+ +CitNa+10μM TC]]> 6.01 <![CDATA[g-C 3 N 4 +Eu 3+ +CitNa+50μM TC]]> 5.75

[0073] To further evaluate the role of IFE in fluorescence quenching, relevant corrections were made using the formula. Figure 7 Figure C shows that the fluorescence quenching efficiency of TC on g-C 3 N 4 is 42.8% before and after correction under the action of IFE, which means that there are other quenching mechanisms.

[0074] Figure 7 Figure D depicts the energy transfer schematic diagram. Energy is excited from the valence band to the reduction band, and PET can occur from g-C 3 N 4 to TC, which leads to fluorescence quenching. The triplet energy level of the ligand is higher than the vibrational energy level of the rare earth ion. This energy difference is conducive to the transfer of energy from the ligand to Eu 3+ . Therefore, the bright red emission at 620 nm can be explained as the transfer of energy from TC to the Eu 3+ center, resulting in the D-F transition between Eu 3+ -TC. The shift of the ultraviolet absorption peak indicates the existence of a coordination relationship between TC and the probe molecule. The above results show that the fluorescence enhancement of Eu 3+ is due to the complexation of TC and CitNa with Eu 3+ and the energy transfer that sensitizes its luminescence. Therefore, in the present invention, PET is another quenching mechanism. It can be seen that after adding TC to the sensing system, g-C 3N 4 The fluorescence quenching of is related to both IFE and PET. For Eu 3+ the fluorescence enhancement is related to AE. From Figure 7 The schematic diagram of the E ratio fluorescence method for detecting TC can more intuitively understand the mechanism underlying this experiment.

[0075] 4. Optimization of TC detection test conditions

[0076] To achieve the best sensing performance of g-C 3 N 4 / CitNa / Eu for TC, the relevant variables were optimized, including the concentrations of Eu 3+ and CitNa, the pH value of the system, and the reaction time.

[0077] First, since the concentration of Eu 3+ is related to the linear range of the method, the concentration of Eu 3+ was optimized. As Figure 6 shown, the concentration of Eu 3+ has little effect on the g-C 3 N 4 nanosheets. When the concentration of Eu 3+ increases from 0 μM to 500 μM, the fluorescence change of g-C 3 N 4 is negligible. Considering the linear range and cost, 100 μM Eu 3+ was used in the subsequent sensing system.

[0078] Secondly, as mentioned above, the auxiliary ligand CitNa plays a key role in sensitizing the luminescence of Eu 3+ . To achieve effective ratio fluorescence sensing, the amount of CitNa was optimized in this invention. As Figure 8 shown in A, as the concentration of CitNa increases, the fluorescence of the sensing system at 620 nm gradually increases. When the concentration of CitNa reaches 500 μM, the fluorescence intensity ratio (F 620 / F 450 ) tends to be constant ( Figure 8 B). Therefore, 500 μM CitNa was used for the subsequent experiments. Subsequently, the pH value of the sensing system was optimized using a Tris-HCl buffer with a pH of 5.0 to 9.0. The fluorescence intensity ratio (F 620 / F 450 ) has the strongest and most stable fluorescence at pH = 8.0. Therefore, pH 8.0 was selected as the optimal pH value for the subsequent experiments ( Figure 9 ).

[0079] Finally, the response time and stability of the ratio fluorescence probe were studied. Figure 10 ​A shows the change of fluorescence of the sensing system with time after adding TC, revealing that the fluorescence response can reach equilibrium rapidly after 10 min, and after reaching equilibrium, the fluorescence intensity ratio (F 620 / F 450 ) remains basically unchanged, indicating good stability( Figure 10 B). These results show that the fluorescence sensing system can detect TC rapidly and stably, and its optimal test conditions are as follows: the concentration of Eu 3+ is 100 μM; the concentration of CitNa is 500 μM; the pH is 8.0; the incubation time is 10 min.

[0080] Example 2

[0081] Preparation of the ratio fluorescence sensor:

[0082] 200 μL of 0.3 mg / mL g-C 3 N 4 solution, 20 μL of 100 μM Eu(NO 3 ) 3 ·6H 2 O solution and 20 μL of 500 μM sodium citrate solution were successively added to 260 μL of Tris-HCl buffer (50 mM, pH = 8.0), mixed evenly, and incubated for 10 min; after the incubation ended, the incubation solution was freeze-dried to obtain g-C 3 N 4 / CitNa / Eu nanoprobe, that is, the ratio fluorescence sensor.

[0083] I. Detection of TC by ratio fluorescence method

[0084] 200 μL of g-C 3 N 4 solution, 20 μL of 100 μM Eu(NO 3 ) 3 ·6H 2 O solution and 20 μL of 500 μM sodium citrate solution were successively added to 260 μL of Tris-HCl buffer (50 mM, pH 8.0), mixed evenly, and incubated for 10 min. Then, different concentrations of TC solutions (0 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM) were added to the above incubation solution. The mixture was diluted to 1.0 mL with ultrapure water. Then the mixture was mixed thoroughly, incubated at room temperature for 10 min, and added to a black 96-well microplate. The fluorescence spectrum with an excitation wavelength of 275 nm was obtained.

[0085] As shown Figure 11 in Figures A Figure 11 and B, as the TC concentration increased from 0 μM to 100 μM, the fluorescence of the sensing system at 450 nm gradually decreased, while the fluorescence at 620 nm gradually increased. Figure 11 Figure C shows the relationship between the TC concentration and the fluorescence intensity ratio (), with a good linear relationship in the range of 0 - 100 μM (R 2 = 0.9937). The linear equation can be: F 620 / F 450 = 0.08632x + 0.01106. According to the 3σ / slope (σ is the standard deviation of blank samples (n = 9)) rule, the detection limit (LOD) was calculated to be as low as 1.961 nM, lower than the maximum residue limit of TC in milk (225 nM) stipulated by the European Union.

[0086] In addition, the detection performance of this method (such as detection time, linear range, and LOD) is also comparable to most of the methods reported in recent years. More importantly, based on g-C 3 N 4 / CitNa / Eu ratio fluorescence method showed a significant color change from blue to red with the increase of TC concentration, which could be clearly identified and distinguished by the naked eye under 365 nm UV light irradiation, as shown Figure 11 in Figure D. CIE chromaticity coordinates were used to further verify the related color change. As shown Figure 11 in Figure E, when the TC concentration increased from 0 μM to 100 μM, the CIE coordinates continuously moved from the blue coordinate (0.1553, 0.1244) to the red coordinate (0.5262, 0.2811). The color rendering property of the TC sensing system provided a prerequisite for the development of a portable TC visual sensor.

[0087] As a comparison, the present invention also used g-C 3 N 4 as a fluorescence probe for single-signal determination of TC. As shown Figure 12 in Figure A, the fluorescence intensity of g-C 3 N 4 at 450 nm had a good linear relationship with the TC concentration in the range of 0 - 100 μM (R 2 = 0.9951), and the detection limit was 24 nM. Its linear equation was: (F 0 -F) / F = 0.03925x + 0.01631 ( Figure 12 Figure B). Compared with the single fluorescence signal response, based on g-C 3 N 4The ratio fluorescence method of / CitNa / Eu has a wider linear range and higher sensitivity, which benefits from the self-calibration and background-free characteristics of the ratio fluorescence sensor. More importantly, based on g-C 3 N 4 / CitNa / Eu ratio fluorescence method shows significant color evolution for different concentrations of TC, while the single-signal sensing based on g-C 3 N 4 can only show the brightness change towards TC ( Figure 12 C). It is difficult to distinguish the TC concentration through the fluorescence color change ( Figure 11 F). The above results indicate that the ratio fluorescence method based on g-C 3 N 4 / CitNa / Eu has great practical application potential in the visual detection of TC.

[0088] II. TC Specificity Test

[0089] To evaluate the selectivity of the above g-C 3 N 4 / CitNa / Eu sensor for TC, under the same experimental conditions, different potential interfering substances were added to the sensing system, including other common antibiotics (AmL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM2, L-PA, CTE, DOX and OXY), molecules (Gl, Suc, Lac, Cys, Asp, Vc, Lys, Glu, Trp, D-Ta, GAE) and some common cations and anions (K + , Na + , Mn 2+ , Mg 2+ , Zn 2+ , Co 2+ , Al 3+ , Ca 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Cl - , SO 4 2- , NO 3 2- , PO 4 3- ).

[0090] The test procedure is as follows:

[0091] Add 200 μL of g-C 3 N 4 solution, 20 μL of 100 μM Eu(NO 3 ) 3·6H 2 The 6H O solution and 20 μL of 500 μM sodium citrate solution were successively added to 260 μL of Tris-HCl buffer (50 mM, pH = 8.0), mixed evenly, and incubated for 10 min. Then, various interfering substances at 150 μM were added to the above incubation solution. The mixture was diluted to 1.0 mL with ultrapure water. Then the mixture was thoroughly mixed, incubated at room temperature for 10 min, and added to a black 96-well microplate. A fluorescence spectrum with an excitation wavelength of 275 nm was obtained.

[0092] The test results are as Figure 13 shown:

[0093] As Figure 13 shown in Figure 13 A and 620 / F 450 ), except for CTE, DOX, and OXY, the fluorescence responses (F Figure 14 and Figure 15 ) of other potential interfering substances did not change significantly. This is because TC, OXY, DOX, and CTE are all tetracycline antibiotics. They have similar absorption spectra and chemical structures ( Figure 13 and Figure 13 ). As

[0094] shown in 620 / F 450 ), only TC can trigger a significant fluorescence change in the sensing system from blue to red compared to other potential interfering substances. This obvious fluorescence color change can be observed by the naked eye with the help of a portable ultraviolet lamp (365 nm), which provides a promising method for on-site qualitative identification of TC. These results indicate that the sensor prepared by the present invention has good selectivity for the detection of tetracycline antibiotics. Figure 13 In addition, the anti-interference test was carried out using other interfering substances coexisting with TC in the present invention, and it was observed that the response ratio F Figure 13 / F

[0095] III. Practicality test

[0096] To verify the feasibility and practicality of this method in actual samples, g-C 3 N 4The / CitNa / Eu ratio fluorescence sensor is used for the detection of TC in animal-derived food (milk). The milk was purchased from a local supermarket. The standard addition method was used to determine TC in milk. 5 mL of 1% (v / v) trichloroacetic acid solution was added to 5 mL of pure milk, and the sample was ultrasonically treated for 30 min and then placed in a refrigerator at 4 °C for 1 h to fully react, so as to separate organic substances such as proteins and lipids. Then, it was centrifuged at 12,000 rpm for 10 min to remove the precipitate. The supernatant was collected and further filtered through a 0.22 μm filter membrane. Subsequently, the obtained solution was diluted 5-fold for the detection of actual samples, which could effectively avoid the interference of the endogenous fluorescence of pure milk on the probe. Finally, the milk samples spiked with TC were detected. The detection method was as follows: 240 μL of g-C 3 N 4 / CitNa / Eu ratio fluorescence sensor was added to the above 750 μL milk sample, mixed well, incubated at room temperature for 10 min, and then added to a black 96-well microplate. 200 μL of the liquid to be tested was added to each well. Under the excitation of 275 nm, the fluorescence spectrum was measured with a microplate reader.

[0097] The test results are shown in Table 2:

[0098] The recovery rate range of the milk samples was 95.75 - 102.95%, and the relative standard deviation (RSD) was 0.33 - 3.67%, showing good accuracy and reliability. The results indicate that the ratio fluorescence sensor of the present invention is suitable for the actual detection of TC in animal-derived food samples.

[0099] Table 2 Detection of TC in milk samples (n = 3)

[0100]

[0101] IV. g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane

[0102] In recent years, flexible intelligent sensors have attracted extensive attention due to their simple manufacturing process, outstanding plasticity, and excellent sensing performance. Electrospun thin films have a large specific surface area, good flexibility, high porosity, and excellent mechanical properties, and are considered to be one of the simplest and superior methods for preparing flexible intelligent sensors.

[0103] The present invention uses an electrospinning device (such as Figure 16 A) to fix the g-C 3 N 4 / CitNa / Eu nanoprobe on the surface of polyacrylonitrile by in-situ growth to prepare a fluorescent electrospun membrane ( Figure 16C), it can be seen that the electrospun membrane has a large surface area and good flexibility, and can adjust the shape of the flexible sensor as needed. When using polyacrylonitrile (PAN) as the matrix, the obtained electrospun membrane becomes very flexible in water and has super hydrophilicity. Water droplets can pass through in 1.5 s, which is beneficial to the formation of close contact between the electrospun membrane and TC in the aqueous solution, enabling TC to be evenly distributed on the surface of the electrospun membrane and penetrate into the interior, thereby improving the sensing performance. Figure 16 B depicts the SEM image of the prepared electrospun membrane, which is composed of randomly arranged nanofibers with numerous pores of different sizes, providing a large specific surface area and abundant sensing sites. This indicates that PAN at 10 wt% (the PAN ratio refers to the ratio of the amounts of PAN and DMF solution, taking 1 g of PAN and 9 mL of DMF, which is the most suitable ratio) is appropriate and has good electrospinnability. The above results show that the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane can be used as a solid-state flexible sensor to achieve efficient detection of TC.

[0104] The above g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane is prepared as follows:

[0105] Dissolve polyacrylonitrile (PAN) (Mw = 150000) in DMF to a concentration of 10 wt%, and stir vigorously at 90 °C for 2 h to obtain a polyacrylonitrile solution. Add 10 mg of g-C 3 N 4 / CitNa / Eu powder into 9 mL of the polyacrylonitrile solution, stir well to obtain a uniform pale yellow g-C 3 N 4 / CitNa / Eu / PAN electrospinning solution. Using a YFSP-T electrospinning device, use a syringe pump to inject the electrospinning solution into the needle at a rate of 0.002 mm / s for electrospinning to obtain a g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane.

[0106] To evaluate the effectiveness of the electrospun membrane in detection, cut the electrospun membrane into circular pieces with a diameter of 1 cm, and then drop 15 μL of solutions with different TC concentrations (concentrations are 0 μM, 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM). As Figure 16 D and Figure 16 E show, g-C 3 N 4The / CitNa / Eu ratio fluorescence probe was successfully immobilized on the electrospun membrane, and the electrospun membrane showed bright blue fluorescence under 365 nm ultraviolet light irradiation. As the concentration of TC increased, an obvious change in fluorescence color could be clearly observed by the naked eye, from blue to purplish red and then to red, as shown in Figure 16 Figure F. Even at a TC concentration of 50 nM, the emission of blue-violet fluorescence could be clearly observed. Then, the selectivity of the electrospun membrane was evaluated by adding 15 μL of 200 μM TC and other antibiotics to the electrospun membrane. As shown in Figure 16 Figure G, only tetracycline antibiotics (such as DOX, CTE, OXY, and TC) could trigger a significant color change from blue to red, while the effects of other antibiotics (AmL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM2) were negligible. These results indicate that the g-C 3 N 4 / CitNa / Eu electrospun membrane of the present invention can be used for the visual and rapid quantitative detection of TC. The realization of the fluorescence electrospun membrane for TC detection expands the applications in the fields such as flexible intelligent wearable devices for human health monitoring.

[0107] V. Quantitative Detection of TC by Smart Phone

[0108] Although the g-C 3 N 4 / CitNa / Eu ratio sensor can achieve the ratio fluorescence sensing and visual analysis of TC, ratio fluorescence analysis requires expensive and bulky instruments and is not suitable for on-site analysis. In addition, it is difficult to quantify the TC concentration by visually evaluating the observed color change. To solve these problems, we used a portable smart phone as a signal reader and analyzer, converted the color information into RGB values for semi-quantitative analysis, thereby improving the accuracy and reliability of the results and realizing the portability of the platform.

[0109] Figure 16 Figure H shows the sensing process of detecting TC based on the g-C 3 N 4 / CitNa / Eu ratio sensor and smart phone. First, on the cut g-C 3 N 415 μL of TC with different concentrations was dropped onto the / CitNa / Eu / PAN electrospun membrane. After 5 min, the electrospun membrane was photographed using a smartphone under the illumination of a 365-nm ultraviolet lamp. As the TC concentration increased, the color of the electrospun membrane showed a continuous evolution from blue to light purple to light magenta to pink to red. Secondly, after a series of fluorescence images were captured, the emitted color information (RGB values) was analyzed using the color recognition application (ColorDesk) loaded in the smartphone, and the TC concentration could be evaluated by calculating the ratio of the red channel (R) and the blue channel (B). Finally, as Figure 16 shown in i, there was a two-stage linear relationship between the R / B value and the TC concentration. The linear equation in the range of 0 - 2.5 μM was: R / B = 0.29003x + 0.14365, R 2 = 0.973, and the LOD value was calculated to be 7.42 nM (LOD = 3σ / S, where σ is the standard deviation of 5 blank samples and S is the slope of the calibration curve). The relevant linear equation in the range of 2.5 - 200 μM was: R / B = 0.01095x + 0.879, R 2 = 0.983. The above results indicate that this portable smartphone-assisted platform has great application potential in the visual and on-site quantitative detection of TC.

[0110] As described above, it is only the preferred embodiment of the present invention, and it is not a limitation of the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of a ratio fluorescence sensor, Characterized in that, The steps are as follows: Add the g-C 3 N 4 nanosheet solution, Eu(NO 3 ) 3 ·6H 2 O solution and sodium citrate solution into the Tris-HCl buffer solution in sequence, mix evenly and incubate; after the incubation ends, lyophilize the incubation solution, and the obtained powder is the g-C 3 N 4 / CitNa / Eu nanosensor, namely the ratio fluorescence sensor; The g-C 3 N 4 nanosheet solution, Eu(NO 3 ) 3 ·6H 2 O solution and the sodium citrate solution have a volume ratio selected from 10:1:1; the concentration of the g-C 3 N 4 nanosheet solution is selected from 0.3 mg / mL, the concentration of the Eu(NO 3 ) 3 ·6H 2 O solution is selected from 100 μM; the concentration of the sodium citrate solution is selected from 500 μM; the concentration of the Tris-HCl buffer solution is selected from 50 mM and the pH is 8.0; the incubation time is selected from 10 min; The g-C 3 N 4 nanosheets are prepared by the following method: Put melamine in a furnace and calcine it at 550 - 600 °C for 2 - 3 h. After the calcination is completed, cool it to room temperature to obtain a yellow solid product, and grind it thoroughly. Then, disperse the yellow solid product in water, ultrasonically crush it, and centrifuge to remove the unpeeled aggregates. Collect the supernatant and dry it to obtain g-C 3 N 4 nanosheets.

2. A ratio fluorescence sensor prepared by the method described in claim 1.

3. A method for detecting the residue of tetracycline antibiotics in food by using the ratio fluorescence sensor described in claim 2, Characterized in that, The steps are as follows: Add trichloroacetic acid solution to the sample to be tested and react under ultrasonic waves; after the reaction is completed, centrifuge, collect the supernatant, filter the supernatant, then add the ratio fluorescence sensor, mix well, and place it at room temperature for complete reaction; after the reaction is completed, place the reaction solution at an excitation wavelength of 275 nm and observe the change in fluorescence color; if there are tetracycline antibiotics, the fluorescence peak at 620 nm will be excited and the fluorescence color will turn red.

4. A preparation method of g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane Characterized in that, The steps are as follows: Dissolve polyacrylonitrile in DMF to obtain a polyacrylonitrile solution; then add the ratiometric fluorescence sensor described in claim 2 to the polyacrylonitrile solution and stir well to obtain a g-C 3 N 4 / CitNa / Eu / PAN electrospinning solution; then perform electrospinning to obtain a g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane.

5. g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane prepared by the method according to claim 4.

6. The ratio fluorescence sensor according to claim 2 or the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane for detecting the residue of tetracycline antibiotics in food; the tetracycline antibiotics are selected from doxycycline, tetracycline, chlortetracycline and oxytetracycline.

7. A method for detecting the residues of tetracycline antibiotics in food by using the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, comprising the following steps: Drop the test sample solution onto the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane. Alternatively, immerse the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane into the test sample solution and react for 5 min. Then irradiate the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane with a 365-nm ultraviolet lamp and observe the change in fluorescence color. If tetracycline antibiotics are present, the electrospun membrane will turn red under the irradiation of the ultraviolet lamp.

8. A method for quantitatively detecting the residue of tetracycline antibiotics in food by using a smart phone, the steps are as follows: Drop the test sample solution onto the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane, or immerse the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane into the test sample solution, react for 5 min, and then irradiate the g-C 3 N 4 / CitNa / Eu / PAN electrospun membrane with a 365-nm handheld ultraviolet lamp, take a photo of the fluorescence of the excitation system using a smartphone, then convert the photo color signal into color information using the color identifier loaded in the smartphone, calculate the ratio of the green and blue channels, i.e., the G / B value, and then substitute the G / B value into the standard curve to obtain the concentration of tetracycline antibiotics.

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