CN116574503A - Ratio fluorescence sensor, preparation method and application thereof - Google Patents

Ratio fluorescence sensor, preparation method and application thereof Download PDF

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Publication number
CN116574503A
CN116574503A CN202310550007.XA CN202310550007A CN116574503A CN 116574503 A CN116574503 A CN 116574503A CN 202310550007 A CN202310550007 A CN 202310550007A CN 116574503 A CN116574503 A CN 116574503A
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solution
citna
fluorescence
ratio
pan
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马永超
孙瑞清
董英佳
杨庆利
侯秀丹
刘萍
葛家成
张凤艳
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Qingdao Agricultural University
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/0883—Arsenides; Nitrides; Phosphides
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a ratio fluorescence sensor, a preparation method and application thereof, and belongs to the technical field of chemical detection. The ratio fluorescent sensor is prepared by the following method: will g-C 3 N 4 Nanosheet solution, eu (NO) 3 ) 3 ·6H 2 Sequentially adding the O solution and the sodium citrate solution into Tris-HCl buffer solution, uniformly mixing, and incubating; after the incubation is finished, freeze-drying the incubation solution to obtain g-C 3 N 4 CitNa/Eu nanoprobe, i.e., ratio fluorescence sensor. The ratio fluorescence sensor can realize the rapid, visual and quantitative determination of the residual tetracycline antibiotics in the sample, and has high sensitivity and visual effectThe portable detection device is good, can be used together with a smart phone, and is portable in detection.

Description

Ratio fluorescence sensor, preparation method and application thereof
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to a ratio fluorescence sensor, a preparation method and application thereof.
Background
Tetracyclines are widely used as feed additives in animal husbandry. However, excessive use of antibiotics can create high levels of drug residues in animal products (especially milk, eggs, meat) and in the external environment, posing a serious threat to food safety, ecological environment and human health. The increase in antibiotic residues in foods has attracted global attention. The European Union (EU) and the United states Food and Drug Administration (FDA) prescribe maximum residual limits for Tetracycline (TC) in milk as 100ng/mL (225 nM) and 300ng/mL (676 nM). Therefore, to ensure food safety and to protect consumer health, there is an urgent need to develop an efficient, ultrasensitive, visual and portable tetracycline antibiotic residue detection scheme.
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 immunoassay (ELISA), capillary Electrophoresis (CE), electrochemical analysis, resonance scattering, and the like. In recent years, some emerging sensor devices show great potential in the field of analytical chemistry, including electrochemical sensors, nanoenzyme sensors, fluorescence sensors, and the like. The fluorescence sensor is considered to be an ideal tetracycline antibiotic detection means due to the advantages of simple operation, low cost, high sensitivity, high response speed and easy visual analysis. However, the response of conventional fluorescence sensors to analytes is typically based on a single fluorescent signal emission, subject to background, instrumentation, and environmental interference. Therefore, the scheme for rapidly, intuitively and quantitatively measuring the tetracycline antibiotics on site is of great significance.
Disclosure of Invention
The technical scheme of the invention is as follows:
the invention provides a preparation method of a ratio fluorescence sensor, which comprises the following steps:
will g-C 3 N 4 Nanosheet solution, eu (NO) 3 ) 3 ·6H 2 Sequentially adding the O solution and the sodium citrate solution into Tris-HCl buffer solution, uniformly mixing, and incubating; after the incubation is finished, freeze-drying the incubation solution to obtain g-C powder 3 N 4 CitNa/Eu nanoprobe, i.e., ratio fluorescence sensor.
In the preparation method, g-C 3 N 4 Nanosheet solution, eu (NO) 3 ) 3 ·6H 2 The volume ratio of the O solution to the sodium citrate solution is selected from 10:1:1; g-C 3 N 4 The concentration of the nanosheet solution is selected from 0.3mg/mL, eu (NO 3 ) 3 ·6H 2 The concentration of the O solution is selected from 100. Mu.M; the concentration of the sodium citrate solution is selected from 500 μm; the concentration of Tris-HCl buffer is selected from 50mM, pH 8.0; the incubation time was selected from 10min.
In the above preparation method, the g-C 3 N 4 The nano-sheet is prepared by the following method:
calcining melamine at 550-600 ℃ for 2-3 h, cooling to room temperature after calcining, obtaining a yellow solid product, and fully grinding; then dispersing the yellow solid product in water, carrying out ultrasonic crushing, centrifuging and removing non-stripped aggregates; collecting supernatant, drying to obtain g-C 3 N 4 A nano-sheet.
The calcination temperature is preferably 550 ℃; the calcination time is preferably 2 hours.
The mass volume ratio of the yellow solid product to water is selected from 1:180-1:250, g: mL; preferably 1:200, g: mL.
The present invention provides a ratiometric fluorescence sensor prepared by the above method.
The invention provides application of the ratio fluorescence sensor in detection of tetracycline antibiotics residues in foods. The tetracycline antibiotic is selected from doxycycline, tetracycline, aureotetracycline and oxytetracycline; tetracycline is preferred.
The invention provides a method for detecting tetracycline antibiotics residues in food by using the ratio fluorescence sensor, which comprises the following steps:
adding trichloroacetic acid solution into a sample to be detected, and performing ultrasonic reaction; after the reaction is finished, centrifuging, collecting supernatant, filtering the supernatant, adding a ratio fluorescence sensor, fully mixing, and completely reacting at room temperature; after the reaction is finished, placing the reaction solution under the excitation wavelength of 275nm, and observing the change of fluorescent color; if tetracycline antibiotics exist, a fluorescent peak at 620nm is excited, and the fluorescent color becomes red.
In the above detection method, the volume ratio of the ratio fluorescence sensor to the filtrate is selected from 8:25.
In the detection method, the sample to be detected is selected from common foods such as milk, eggs, chicken, beef and the like; preferably milk.
The invention provides a g-C 3 N 4 The preparation method of the/CitNa/Eu/PAN electrospun membrane comprises the following steps:
dissolving polyacrylonitrile in DMF to obtain a polyacrylonitrile solution; then g-C 3 N 4 adding/CitNa/Eu powder into polyacrylonitrile solution, stirring thoroughly to obtain g-C 3 N 4 A CitNa/Eu/PAN electrospun solution; then carrying out electrostatic spinning to obtain g-C 3 N 4 CitNa/Eu/PAN electrospun membrane.
g-C as described above 3 N 4 The mass-volume ratio of the CitNa/Eu powder to the polyacrylonitrile solution is selected from 10:9-10:11, mg:mL; preferably 10:9, mg/mL.
The present invention provides g-C prepared by the above method 3 N 4 CitNa/Eu/PAN electrospun membrane.
The invention provides the g-C 3 N 4 Application of/CitNa/Eu/PAN electrospun membrane in detecting tetracycline antibiotics residue in food. The tetracycline antibiotic is selected from doxycycline, tetracycline, aureotetracycline and oxytetracycline; tetracycline is preferred.
The invention provides a method for preparing the high-purity zinc alloy by using the g-C 3 N 4 The method for detecting tetracycline antibiotics residues in food by using the CitNa/Eu/PAN electrospun membrane comprises the following steps:
dripping the sample solution to be tested into g-C 3 N 4 on/CitNa/Eu/PAN electrospun film, alternatively, g-C 3 N 4 Immersing the/CitNa/Eu/PAN electrospun membrane into the sample solution to be tested, reacting for 5min, and then immersing g-C 3 N 4 the/CitNa/Eu/PAN electrospun film was irradiated with 365nm ultraviolet lamp to observe the change of fluorescence color; if the tetracycline antibiotics are present, the electrospun film turns red under the irradiation of the ultraviolet lamp.
The invention provides a method for quantitatively detecting tetracycline antibiotics residues in food by using a smart phone, which comprises the following steps:
dripping the sample solution to be tested into g-C 3 N 4 on/CitNa/Eu/PAN electrospun film, alternatively, g-C 3 N 4 Immersing the/CitNa/Eu/PAN electrospun membrane into the sample solution to be tested, reacting for 5min, and then immersing g-C 3 N 4 The CitNa/Eu/PAN electrospun film is irradiated by a 365nm hand-held ultraviolet lamp, fluorescence of an excitation system is shot by a smart phone, then a color identifier loaded in the smart phone is used for converting a photo color signal into color information (RGB value), the ratio (G/B value) of green and blue channels is calculated, and the G/B value is substituted into a standard curve to obtain the concentration of the tetracycline antibiotics.
The color identifier is selected from the ColorDesk application.
The standard curve may be constructed by a method selected from the group consisting of:
dripping tetracycline antibiotics solution with different concentrations into g-C 3 N 4 on/CitNa/Eu/PAN electrospun film, alternatively, g-C 3 N 4 immersing/CitNa/Eu/PAN electrospun membrane in tetracycline antibiotics solution with different concentrations, reacting for 5min, and then adding g-C 3 N 4 the/CitNa/Eu/PAN electrospun film was illuminated with 365nm hand held in-vitro light, the fluorescence of the excitation system was photographed using a smart phone, and the photograph was then colored using a color identifier loaded into the smart phoneThe signal is converted into color information (RGB value), the ratio (G/B value) of the green and blue channels is calculated, the concentration of the tetracycline antibiotics is taken as the abscissa, the G/B value is taken as the ordinate, and a concentration-G/B value standard curve is constructed.
The concentration gradient of the tetracycline compound solution may be selected from 0. Mu.M, 0.5. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M.
The beneficial effects of the invention are as follows:
the invention designs a dual-signal ratio fluorescence sensor (g-C) 3 N 4 CitNa/Eu) can be used for in situ visual detection of tetracycline antibiotics. g-C with blue luminous ability 3 N 4 Not only can be used as Eu 3+ The coordinated skeleton can also be used as a recognition unit of tetracycline antibiotics. Coordinated unsaturated red fluorescent Eu 3+ (λ em =620 nm) is bound at g-C 3 N 4 On the surface, a specific tetracycline antibiotic recognition element is formed due to the Antenna Effect (AE). In the presence of tetracycline antibiotics, the ratiometric fluorescence sensor exhibited dual and inverted response signals with a distinct multicolour width colour change (blue-violet-pink-red), enabling ultra-high sensitivity detection with a detection limit of 1.961nM. In addition, the portable flexible sensor can be prepared and obtained by loading the ratio fluorescence sensor on the nanofiber membrane through an electrostatic spinning technology. The successful combination of the flexible sensor and the smart phone greatly reduces the detection cost and time, and provides a promising method for qualitative identification and quantitative detection of the tetracycline antibiotics on site.
Drawings
FIG. 1 is a block g-C 3 N 4 And g-C 3 N 4 The emission spectrum of the nanoplatelets;
FIG. 2 is g-C 3 N 4 Characterization of the nanosheets; wherein, the A picture is fluorescence spectrum, the insertion picture is g-C 3 N 4 Photographs of nanoplatelet solutions under uv and sunlight; b is a TEM image; panel C is an AFM image; d is g-C 3 N 4 AFM image height profile of the nanoplatelets; e is block g-C 3 N 4 And g-C 3 N 4 XRD spectrum of the nanoplatelets; f is block g-C 3 N 4 And g-C 3 N 4 FT-IR spectrum of the nanosheets; g graph is fitted with G-C 3 N 4 High-resolution XPS spectrum of C1s of the nano-sheet; h graph is fitted with g-C 3 N 4 N1s high-resolution XPS spectrum of the nano-sheet; i is g-C 3 N 4 、Eu 3+ 、g-C 3 N 4 CitNa/Eu, TC, citNa and g-C 3 N 4 Zeta potential of/CitNa/Eu/TC;
FIG. 3 is g-C 3 N 4 Fluorescence stability of the nanoplatelets under different conditions; wherein, A is the storage time stress; panel B shows the ultraviolet irradiation time; panel C shows the pH of the solution; panel D shows NaCl concentration;
FIG. 4 is g-C 3 N 4 XPS spectrogram of the nano-sheet; wherein, A is XPS full spectrum, and the illustration is g-C 3 N 4 The relative content of C, N, O atoms in the nanoplatelets; panel B is fitted g-C 3 N 4 High-resolution O1s spectrum of the nano-sheet;
FIG. 5 is a TC detection feasibility study; wherein, A is g-C 3 N 4 Fluorescence excitation spectrum (a), emission spectrum (b) and ultraviolet-visible absorption spectrum (c) of TC of the nanoplatelets; b is g-C 3 N 4 CIE chromaticity coordinates (a) and CIE chromaticity coordinates (b) of Eu/CitNa/TC; c is g-C 3 N 4 And Eu/CitNa/TC emission spectra (λex=275 nm), the inset shows g-C under 365nm UV lamp irradiation 3 N 4 Photographs of nanoplatelet solutions (left) and Eu/Cit/TC solutions (right); panel D shows the fluorescence spectra of the various systems shown.
FIG. 6 shows the addition of Eu at various concentrations 3+ Post g-C 3 N 4 Fluorescence spectrum (a plot) and fluorescence intensity variation (B plot);
FIG. 7 is a mechanism of TC detection; wherein, A is the absorption spectrum of various systems shown in the figure; panel B shows fluorescence lifetime of various materials shown; panel C shows fluorescence quenching efficiency after addition of different concentrations of TC for observation (Eobsd, a) and correction (Ecor, b); d is g-C 3 N 4 Emission level diagram of/CitNa/Eu and TC-Eu 3+ Energy transfer of (a); e is based on g-C 3 N 4 Principle of detecting TC by using/CitNa/Eu ratio fluorescence method;
FIG. 8 is an optimization of CitNa usage; wherein, graph A is fluorescence intensity; panel B is the fluorescence intensity ratio, and the inset shows a photograph of the corresponding solution under 365nm ultraviolet lamp irradiation;
FIG. 9 is an optimization of the pH of a ratiometric fluorescence detection TC sensing system;
FIG. 10 is an optimization of incubation time; wherein, graph A is fluorescence intensity; panel B shows the fluorescence intensity ratio;
FIG. 11 shows detection of TC by ratiometric fluorescence; wherein, A is g-C under different TC dosage 3 N 4 Fluorescence spectrum of the/CitNa/Eu solution; panel B shows g-C at different TC dosages 3 N 4 Corresponding intensity variation of the/CitNa/Eu solution; panel C shows the fluorescence intensity ratio (F 620 /F 450 ) Linear relation to TC concentration; d is g-C at different TC dosages 3 N 4 Fluorescence color change photograph of the CitNa/Eu solution under uv lamp (λ=365 nm); e-chart is CIE chromaticity diagram at different TC concentrations; diagram F shows the chromaticity of CIE in the presence of different concentrations of TC;
FIG. 12 is a single signal measurement of TC; wherein, A is g-C after adding TC with different concentrations 3 N 4 Fluorescence spectrum of the base fluorescence sensing system; panel B shows TC concentration and (F) 0 -F)/F 0 In a linear relationship, F 0 And F represents the fluorescence intensity of the sensing system before and after adding TC, respectively; panel C shows the g-C of different concentrations of TC under UV lamp irradiation (λ=365 nm) 3 N 4 Fluorescence color-changing photograph of the solution;
FIG. 13 is a TC specificity assay; wherein, A is g-C 3 N 4 Fluorescence response of the CitNa/Eu sensor to TC and interferents; b is g-C 3 N 4 Fluorescence response of the CitNa/Eu sensor to TC and interferents; panel C is a photograph of the correlation under ultraviolet, the concentrations of TC and interferents are 50. Mu.M; panel D shows a UV correlation photograph, with TC and interferent concentrations of 50. Mu.M; e is based on g-C 3 N 4 Anti-interference experiment of the CitNa/Eu sensor on TC, wherein the TC concentration is 50 mu M, and the interference concentration is 150 mu M; f graph is based on g-C 3 N 4 /CitNan anti-interference experiment of the a/Eu sensor on TC, wherein the TC concentration is 50 mu M, and the interference concentration is 150 mu M;
FIG. 14 is an ultraviolet-visible absorption spectrum of TC, DOX, OXY and CTE at 45 μm concentration; wherein, at 325nm, each curve represents TC, OXY, DOX, CTE from top to bottom in turn;
FIG. 15 shows the chemical structure of tetracyclines; wherein, the upper left is Tetracycline (TC), the upper right is Doxycycline (DOX), the lower left is Oxytetracycline (OXY) and the lower right is aureotetracycline (CTE), and the four compounds have similar chemical structures;
FIG. 16 is g-C 3 N 4 CitNa/Eu/PAN electrospun membrane and intelligent application; wherein, the A diagram is a schematic diagram of the electrostatic spinning method for preparing the nanofiber; b is g-C 3 N 4 Scanning electron microscope image of/CitNa/Eu/PAN electrospun film; c is g-C 3 N 4 Photographs of/CitNa/Eu/PAN electrospun films; panel D shows g-C under sunlight 3 N 4 Photographs of the CitNa/Eu/PAN electrospun films; e graph is g-C under ultraviolet light 3 N 4 Photographs of the CitNa/Eu/PAN electrospun films; FIG. F is a photograph of an electrospun film after adding 15. Mu.L of TC solutions of different concentrations (0. Mu.M, 0.5. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, respectively from left to right); FIG. G is a photograph of an electrospun membrane after adding 15. Mu.L of 200. Mu.M of different antibiotic solutions, amL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM, DOX, CTE, OXY and TC in that order from left to right; h diagram is based on g-C 3 N 4 TC detection simplified flow diagrams of the CitNa/Eu/PAN electrospun membrane and the smart phone sensor; i is based on g-C 3 N 4 Visual sensor of CitNa/Eu/PAN electrospun film fluorescent color image of TC solutions of different concentrations under hand-held uv lamp irradiation.
Detailed Description
The reagents, chemicals and instruments employed in the present invention are as follows:
(hydroxymethyl) aminomethane (Tris), melamine, eu (NO) 3 ) 3 ·6H 2 O, sodium citrate (CitNa, 98%), polyacrylonitrile (PAN, mw=150000), tetracycline (TC), aureomycin (CTE), geotrichumOther ions such as biotin (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), trichloroacetic acid solution (10%) were purchased from microphone biochemical technologies limited. Ampicillin (AMP), streptomycin Sulfate (STR), ceftriaxone sodium (CTR) were purchased from sorafenib technologies. N, N-Dimethylformamide (DMF), cysteine (Cys), gallic Acid (GAE), sulfadimidine (SM 2), sucrose (Suc), and anhydrous glucose (Gl) were purchased from Country chemical reagent Co. All reagents were used as received without further purification. TD-3700 (China) X-ray diffraction (XRD), transmission electron microscopy (TEM, JOEL JEM 2001), atomic Force Microscopy (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 spectroscope (U.S.), spectraMax i3X multifunctional enzyme-labeled instrument (U.S.), F2700 fluorescence spectrophotometer (Hitachi, japan), F4600 spectrometer (Japan, hitachi), XO-1000D ultrasonic cell pulverizer, zeiss scanning electron microscope (SEM, japan), YFSP-T (Tianjin cloud of China) electrostatic spinning device.
Other materials used in the present invention, such as those not specifically stated, are available through commercial sources. Other terms used herein, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
g-C 3 N 4 Preparation of nanosheets:
3g of melamine is placed in an alumina crucible, then placed in a muffle furnace, heated to 550 ℃ at a speed of 10 ℃/min, calcined for 2 hours, cooled to room temperature, and the obtained yellow solid product is fully ground. Then, 2.25g of a yellow solid product was addedDispersed in 450mL of ultrapure water, and then pulverized with an ultrasonic cell pulverizer at a power of 1000w for 48 hours. The non-exfoliated aggregates were then removed by centrifugation at 3000rpm for 10min. Collecting the mixture containing g-C 3 N 4 The supernatant of the nanoplatelets was dried and stored at 4 ℃ for later use.
1、g-C 3 N 4 Characterization of nanoplatelets
The invention adopts the calcination polymerization and ultrasonic stripping method to prepare the g-C 3 N 4 As can be seen from FIG. 1, the ultrasonic exfoliation enhances g-C 3 N 4 Emission intensity of nanoplatelets. In addition, due to quantum confinement effect, the position of the emission peak is higher than that of the block g-C 3 N 4 Slightly blue shifted by about 12nm. g-C 3 N 4 PL excitation and emission spectra of the nanoplatelets, as shown in fig. 2A, show no significant change in PL emission position at different excitation wavelengths; g-C 3 N 4 The optimal emission and excitation wavelengths of the nanoplatelets are 450nm and 275nm, respectively; in addition, the g-C obtained 3 N 4 The nanoplatelets exhibit good dispersibility in solution and fluoresce bright blue under UV. g-C 3 N 4 Fluorescence stability of nanoplatelets, as shown in FIG. 3, fluorescence intensity remained relatively uniform, indicating g-C 3 N 4 The nanosheet solution has excellent fluorescence stability; in addition, g-C prepared 3 N 4 The quantum yield of the nanoplatelet solution was 5.23%. g-C 3 N 4 TEM images of the nanoplatelets, as shown in FIG. 2B, show a typical regular lamellar structure. g-C 3 N 4 AFM image of nanoplatelets, as shown in FIGS. 2C and 2D, AFM images further verify g-C 3 N 4 The nano-sheet has a sheet-like structure with a thickness of about 3 nm. FIG. 2E shows a distinct characteristic XRD peak corresponding to g-C 3 N 4 Is a typical graphite interlayer deposition (002) peak. With block g-C 3 N 4 In contrast, g-C after sonication 3 N 4 XRD peak of the nanoplatelets changed from 27.3 to 27.5, indicating bulk g-C 3 N 4 The separation was successful. The invention further characterizes g-C by utilizing infrared spectrum 3 N 4 Functional group of nanosheetsAs shown in FIG. 2F, the block shape g-C 3 N 4 And g-C 3 N 4 The nanoplatelets show similar absorption peaks, confirming that ultrasonic stripping does not alter g-C 3 N 4 The structure of the nanoplatelets. XPS analysis further confirmed g-C 3 N 4 The elemental composition of the nanoplatelets, as shown in FIG. 4A, synthetic g-C 3 N 4 Nanoplatelets consist of three main chemical elements (C, N and O). Fig. 2G and 2H are fitted high resolution C1s and N1s, respectively, with three peaks in the C1s spectra at 284.6eV (graphitic carbon), 286.2eV (C-OH) and 288.0eV (N-c=n), respectively. The N1s band is quaternary nitrogen (401.1 eV), tertiary nitrogen (399.8 eV) and c=n-C (398.44 eV), respectively. Deconvolution of the O1s band indicates the presence of C-OH (532.2 eV), as shown in FIG. 4B. In conclusion, the ultrasonic stripping reduces g-C 3 N 4 The size of the block increases its emission intensity.
2. TC detection feasibility study
First, the present invention measures the absorption spectrum of TC and g-C 3 N 4 The excitation spectra of the nanoplatelets, as shown in FIG. 5A, have a large spectral overlap, which results in g-C 3 N 4 The fluorescence energy of (c) is greatly quenched by the IFE by TC, since the efficiency of IFE depends on the extent of spectral overlap of the quencher's absorption and excitation or emission of the fluorophore.
Secondly, the invention explores the sodium citrate to Eu 3+ Influence of fluorescence. The specific test procedure is as follows: (1) Eu concentration: 200 mu L g-C of 0.3mg/mL 3 N 4 Solution and 20. Mu.L of Eu of different concentrations 3+ And 780 μl of 50mM pH=8 Tric-HCl solution, and fluorescence spectra were recorded under 275nm excitation. (2) CitNa concentration: 200 mu L g-C of 0.3mg/mL 3 N 4 Solution and 20. Mu.L of Eu at 100. Mu.M 3+ And 20. Mu.L of different concentrations of CitNa and 510. Mu.L of 50mM Tris-HCl, pH=8, and 250. Mu.L of 50. Mu.M TC solution, fluorescence spectra were recorded under excitation at 275 nm. As shown in FIG. 6, TC can effectively enhance Eu with the aid of CitNa 3+ Since TC and CitNa can be combined with Eu 3+ Chelating and energy transfer to Eu 3+ Thereby making Eu 3+ And (5) luminescence sensitization. With lanthanide ions (Eu) 3+ ) Chelating agentsThe combined ligand (TC) acts as an "antenna", absorbing photons and transferring energy to the lanthanide ion (Eu) 3+ ) Thereby sensitizing its luminescence, a process known as antenna effect. Thus, g-C 3 N 4 Nanoplatelets and Eu 3+ Can be used as a TC identification unit to realize double and reverse response signals (g-C 3 N 4 Fluorescence is weakened, eu 3+ Fluorescence enhancement), which provides a precondition for ratiometric fluorescence sensing.
Then, as can be seen from the International Commission on illumination (CIE) chromaticity diagram, g-C 3 N 4 There is a large color difference in fluorescence between nanoplatelets (0.15592,0.12369) and eu3+ (0.65324,0.33518) (fig. 5B). This phenomenon of significant color evolution from blue to red provides great advantages for visual analysis of TC.
Finally, g-C 3 N 4 Nanoplatelets and Eu 3+ Is also advantageous for developing an efficient ratiometric fluorescence sensor. For example, eu/CitNa/TC (mixing 20. Mu.L of 100. Mu.M Eu with 20. Mu.L of 500. Mu.M CitNa with 710. Mu.L of 50mM pH=8 Tric-HCl with 250. Mu.L of 50. Mu.M TC solution, recording fluorescence spectrum under 275nm excitation) has an optimal excitation wavelength of 275nm, g-C 3 N 4 And Eu 3+ Has a similar excitation wavelength at 275nm, thus g-C 3 N 4 And Eu 3+ Can be excited at 275nm and exhibit well resolved dual emission bands to achieve effective rate fluorescence detection of TC.
In addition, g-C as shown in FIG. 5C 3 N 4 Shows a fluorescence maximum at 450nm and a bright blue fluorescence under a UV lamp (left panel), whereas Eu/CitNa/TC has a maximum emission wavelength of 620nm, red fluorescence (right panel). g-C 3 N 4 Emission peak at 450nm with Eu 3+ Compared with the emission spectrum at 620nm, due to 5 D 0 → 7 F 2 The transition shows a large emission spectrum shift (about 170 nm), and a large peak shift not only facilitates wide and sensitive color changes, but also can effectively avoid spectral overlap of different emission peaks. In conclusion, g-C 3 N 4 Nanoplatelets and Eu 3+ Properties of (3)Can meet the requirements of developing an effective ratio fluorescence method for visual determination of TC.
To verify g-C 3 N 4 CitNa and Eu 3+ Feasibility in TC determination, the present invention has carried out related experiments, specifically as follows: a:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +510. Mu.L of Tric-HCl with pH=8 and +290. Mu.L of ultra pure water were mixed. b:200 mu L0.3mg/mL g-C 3 N 4 Solution +20. Mu.L 100. Mu.M Eu 3+ +510. Mu.L of Tric-HCl with pH=8 and 270. Mu.L of ultra pure water were mixed. c:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +20. Mu.L 500. Mu.M CitNa +510. Mu.L Tric-HCl of pH=8 +270. Mu.L ultra pure water. d:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +20. Mu.L 500. Mu.M CitNa +20. Mu.L 100. Mu.M Eu 3+ +510. Mu.L of Tric-HCl with pH=8 and 250. Mu.L of ultra pure water were mixed. e:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +250. Mu.L 50. Mu.M TC +510. Mu.L Tric-HCl pH=8 +40. Mu.L ultra pure water was mixed. f:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +20. Mu.L 100. Mu.M Eu 3+ +250. Mu.L of 50. Mu.M TC+510. Mu.L of Tric-HCl pH=8+20. Mu.L of ultrapure water. g:200 mu L of 0.3mg/mL g-C 3 N 4 Solution +20. Mu.L 500. Mu.M CitNa +20. Mu.L 100. Mu.M Eu 3+ +250. Mu.L of 50. Mu.M TC+510. Mu.L of Tric-HCl pH=8. Fluorescence spectra were recorded at 275nm excitation.
The test results are shown in fig. 5D:
Eu 3+ and CitNa vs g-C 3 N 4 Has little effect on fluorescence (a-d curves). In g-C 3 N 4 /Eu 3+ After TC is added into the solution, g-C 3 N 4 Fluorescence at 450nm is significantly reduced, whereas Eu 3+ Fluorescence at 620nm increases slightly (curve f). This is because TC and Eu 3+ Sensitization of Eu by chelation of 3+ And TC induces g-C by IFE 3 N 4 Fluorescence quenching of (2). However, the solution showed no apparent color change due to unsynchronized fluorescence intensity changes (fig. 5D inset). To achieve efficient ratiometric fluorescence sensing and more pronounced visual analysis, citNa was introduced as an ancillary ligand into g-C 3 N 4 /Eu 3+ In solution. Eu (Eu) 3+ Fluorescence at 620nm increases significantly (g curve) and the solution exhibits a clear color change from blue to red (inset of fig. 5D), probably due to CitNa substituting chelate water molecules and Eu 3+ TC chelate to inhibit quenching effects by the chelate water molecules. These results indicate that the above design is feasible, at g-C 3 N 4 /Eu 3+ The auxiliary ligand is introduced into the detection system, so that the detection limit of the system can be improved, and the color change is more obvious.
For the above reasons, the present invention selects g-C 3 N 4 Nanoplatelets, citNa and Eu 3+ To develop a ratiometric fluorescence sensor for TC detection.
3. Mechanism of TC detection reveals
The introduction of TC enhances Eu 3+ Red fluorescence of (C) while quenching g-C 3 N 4 Blue fluorescence of (c).
First, the present invention is directed to Eu 3+ The fluorescence enhancement mechanism of (2) was confirmed, as shown in FIG. 7A, eu was added 3+ The absorption peak of the rear TC is obviously red-shifted and obviously increased compared with the absorbance of the TC, which indicates Eu 3+ Specific chelation occurs with TC containing the β -diketone configuration. At Eu 3+ After adding the auxiliary ligand CitNa to the TC solution, the absorbance further increased, indicating that the CitNa and Eu 3+ the/TC forms a complex. The results show that TC and CitNa and Eu 3+ Eu is caused by complexation and energy transfer of Eu 3+ Is sensitized by luminescence of (2) to enhance Eu 3+ Red fluorescence of (2).
Next, the present invention illustrates g-C 3 N 4 And the associated fluorescence lifetime was measured. Generally, during Fluorescence Resonance Energy Transfer (FRET), the fluorescence lifetime of a phosphor decreases due to energy transfer from the phosphor to the quencher, whereas for IFE the fluorescence lifetime is unchanged. As shown in FIG. 7B and Table 1, g-C 3 N 4 The fluorescence lifetime of (C) remained relatively stable after addition of TC at various concentrations, indicating g-C 3 N 4 The TC-induced fluorescence quenching of (2) is mainly caused by IFE rather than FRET.
TABLE 1 g-C under different conditions 3 N 4 Fluorescent lifetime of (λem=450 nm)
Sample of τ(ns)
g-C 3 N 4 6.25
g-C 3 N 4 +Eu 3+ 6.21
g-C 3 N 4 +Eu 3+ +CitNa 6.43
g-C 3 N 4 +Eu 3+ +CitNa+10μM TC 6.01
g-C 3 N 4 +Eu 3+ +CitNa+50μM TC 5.75
To further evaluate the role of IFE in fluorescence quenching, correlation corrections were made using formulas. FIG. 7C shows TC versus g-C before and after correction by IFE 3 N 4 The fluorescence quenching efficiency of (2) was 42.8%, which means that other quenching mechanisms were present.
FIG. 7D depicts a schematic of energy transfer from the valence band to the reduction band, PET can be from g-C 3 N 4 By TC, this occurs, which results in fluorescence quenching. The triplet energy level of the ligand being higher than that of the rare earth ionA kinetic energy stage. This energy difference facilitates energy transfer from the ligand to Eu 3+ . Thus, bright red emission at 620nm can be interpreted as energy transfer from TC to Eu 3+ Center, leading to Eu 3+ -D-F transitions between TCs. The shift of the ultraviolet absorption peak indicates that there is a coordination relationship between TC and the probe molecule. The above results indicate that Eu 3+ The fluorescence enhancement of (C) is due to TC and CitNa and Eu 3+ Is caused by the complexation and energy transfer of the fluorescent dye. Thus, PET is another quenching mechanism in the present invention. It can be seen that g-C after TC is added to the sensor system 3 N 4 Fluorescence quenching related to both IFE and PET, eu 3+ The fluorescence enhancement of (2) is related to AE. The mechanism underlying this experiment can be more intuitively understood from the schematic diagram of the detection of TC by the ratio fluorescence method of FIG. 7E.
4. TC detection test condition optimization
To achieve g-C 3 N 4 Optimal sensing performance of TC by CitNa/Eu, optimization of related variables including Eu 3+ And the concentration of CitNa, pH of the system, reaction time.
First, due to Eu 3+ Is related to the linear range of the method, and therefore for Eu 3+ Is optimized. As shown in FIG. 6, eu 3+ Concentration vs g-C 3 N 4 The influence of the nano-sheets is not great, when Eu is 3+ The concentration was increased from 0. Mu.M to 500. Mu.M, g-C 3 N 4 The fluorescence change of (2) is negligible. Taking into account the linear range and the cost, 100 μM Eu is used in the subsequent sensing system 3+ 。
Next, as described above, the auxiliary ligand CitNa is sensitized with Eu 3+ Plays a key role in luminescence. In order to achieve efficient ratiometric fluorescence sensing, the present invention optimizes the amount of CitNa. As shown in FIG. 8A, the fluorescence of the sensing system increases gradually at 620nm as the concentration of CitNa increases. When the CitNa concentration reached 500. Mu.M, the fluorescence intensity ratio (F 620 /F 450 ) Tends to be constant (fig. 8B). Thus, 500. Mu.M CitNa was used for subsequent experiments. Subsequently, the pH of the sensing system was optimized using 5.0 to 9.0 Tris-HCl buffer. Ratio of fluorescence intensity (F) 620 /F 450 ) There was the strongest and stable fluorescence at ph=8.0, so pH 8.0 was chosen as the optimal pH for the subsequent experiments (fig. 9).
Finally, response time and stability of the ratiometric fluorescent probes were studied. FIG. 10A shows the change of fluorescence of the sensor system over time after TC addition, revealing that after 10min the fluorescence response is rapidly reached and equilibrated, and the ratio of fluorescence intensity of the sensor system after equilibration (F 620 /F 450 ) Remains substantially unchanged and has better stability (fig. 10B). These results show that the fluorescence sensing system can rapidly and stably detect TC, and the optimal test conditions are as follows: eu (Eu) 3+ The concentration is 100 mu M; the concentration of CitNa was 500. Mu.M; the pH was 8.0; incubation time was 10min.
Example 2
Preparation of ratio fluorescence sensor:
200 mu L of 0.3mg/mL g-C 3 N 4 Solution, 20. Mu.L of 100. Mu.M Eu (NO) 3 ) 3 ·6H 2 Sequentially adding the O solution and 20 mu L of 500 mu M sodium citrate solution into 260 mu L of Tris-HCl buffer solution (50 mM, pH=8.0), uniformly mixing, and incubating for 10min; after the incubation is finished, freeze-drying the incubation solution to obtain g-C 3 N 4 CitNa/Eu nanoprobe, i.e., ratio fluorescence sensor.
1. Detection of TC by ratiometric fluorescence
Will be 200 mu L g-C 3 N 4 Solution, 20. Mu.L of 100. Mu.M Eu (NO) 3 ) 3 ·6H 2 The O solution and 20. Mu.L of 500. Mu.M sodium citrate solution were added sequentially to 260. Mu.L of Tris-HCl buffer (50 mM, pH 8.0), mixed well and incubated for 10min. Then, TC solutions (0. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 1.5. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, 100. Mu.M) were added to the above incubation solutions at different concentrations. The mixture was diluted to 1.0mL with ultrapure water. The mixture was then thoroughly mixed and incubated at room temperature for 10min and added to a black 96-well microplate. A fluorescence spectrum with an excitation wavelength of 275nm was obtained.
As shown in fig. 11A and 11B, as TC concentration increases from 0 μm to 100 μm, sensingThe fluorescence of the system at 450nm gradually decreases, while the fluorescence at 620nm gradually increases. FIG. 11C shows the relationship between TC concentration and fluorescence intensity ratio (), with good linearity in the range of 0 to 100. Mu.M (R 2 = 0.9937), the linear equation can be: f (F) 620 /F 450 =0.08632x+0.01106. According to the rule of 3σ/slope (σ is standard deviation of blank sample (n=9)), the detection Limit (LOD) was calculated as low as 1.961nM, below the maximum residual limit of TC in milk (225 nM) specified by the european union.
In addition, the detection performance (such as detection time, linear range, and LOD) of this method is also comparable to most of the methods reported in recent years. More importantly, based on g-C 3 N 4 The fluorescence method of the ratio of/CitNa/Eu exhibited a significant color change from blue to red with increasing TC concentration, which was clearly identifiable and distinguishable by the naked eye under irradiation of a 365nm UV lamp, as shown in FIG. 11D. The CIE chromaticity coordinates were used to further verify the relevant color change, as shown in fig. 11E, with the TC concentration increasing from 0 μm to 100 μm, the CIE coordinates continuously shifted from blue (0.1553,0.1244) to red (0.5262,0.2811). The color rendering of the TC sensing system provides preconditions for developing a portable TC vision sensor.
As a comparison, the invention also uses g-C 3 N 4 As fluorescent probes for single signal measurement of TC. As shown in FIG. 12A, g-C 3 N 4 There was a good linear relationship between fluorescence intensity at 450nm and TC concentration in the range of 0 to 100. Mu.M (R 2 = 0.9951), the limit of detection is 24nM, and the linear equation is: (F) 0 -F)/f=0.03925x+0.01631 (fig. 12B). Based on g-C compared to single fluorescent signal response 3 N 4 the/CitNa/Eu ratio fluorescence method has a wider linear range and higher sensitivity, which benefits from the self-correcting and background-free properties of the ratio fluorescence sensor. More importantly, based on g-C 3 N 4 the/CitNa/Eu ratio fluorescence method showed a significant color evolution for different concentrations of TC, based on g-C 3 N 4 Only the single signal sensing of (a) can show a change in brightness to TC (fig. 12C). The TC concentration was difficult to distinguish by fluorescence color change (fig. 11F). The above results indicate that the radicalsIn g-C 3 N 4 The ratio fluorescence method of/CitNa/Eu has great practical application potential in visual detection of TC.
2. TC specificity assay
To evaluate the above g-C 3 N 4 Selectivity of the CitNa/Eu sensor for TC under the same experimental conditions, different potential interfering substances including other common antibiotics (AmL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM, 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- )。
The test procedure was as follows:
will be 200 mu L g-C 3 N 4 Solution, 20. Mu.L of 100. Mu.M Eu (NO) 3 ) 3 ·6H 2 The O solution and 20. Mu.L of 500. Mu.M sodium citrate solution were added sequentially to 260. Mu.L of Tris-HCl buffer (50 mM, pH=8.0), mixed well and incubated for 10min. Then, 150. Mu.M of each interfering substance was added to the above incubation solution. The mixture was diluted to 1.0mL with ultrapure water. The mixture was then thoroughly mixed and incubated at room temperature for 10min and added to a black 96-well microplate. A fluorescence spectrum with an excitation wavelength of 275nm was obtained.
The test results are shown in fig. 13:
as shown in fig. 13A and 13B, the fluorescence response (F 620 /F 450 ) There was no significant change. This is because TC, OXY, DOX and CTE are both tetracycline antibiotics. They have similar absorption spectra and chemical structures (fig. 14 and 15). As shown in fig. 13C and 13D, only TC may trigger a significant fluorescence change of the sensing system color from blue to red completely, as compared to other potentially interfering substances. Such a distinct change in fluorescence color can be achievedThis provides a promising method of qualitatively identifying TC in situ, with the aid of a portable ultraviolet lamp (365 nm) that is visible to the naked eye. These results indicate that the sensor prepared by the invention has good selectivity for tetracycline antibiotic detection.
In addition, the invention uses other interferents to coexist with TC to perform anti-interference test, and observe the response ratio F 620 /F 450 No significant changes (fig. 13E and 13F) demonstrated that the sensing performance was not disturbed by other substances. Therefore, the sensor can be used for ultra-sensitively and selectively detecting TC, and has potential application in various fields.
3. Practicality test
To verify the feasibility and practicality of the method in practical samples, g-C was used 3 N 4 The CitNa/Eu ratio fluorescence sensor is used for detecting TC in animal-derived foods (milk). Milk is purchased from a local supermarket. The TC in the milk was measured using a standard addition method. 5mL of 1% (v/v) trichloroacetic acid solution is added into 5mL of pure milk, the sample is placed in a refrigerator at 4 ℃ for full reaction for 1h after being subjected to ultrasonic treatment for 30min, and separation of organic matters such as protein, lipid and the like is realized. Then, the mixture was centrifuged at 12000rpm for 10min to remove the precipitate. The supernatant was collected and further filtered through a 0.22 μm filter. And then diluting the obtained solution by 5 times for actual sample detection, so that the interference of endogenous fluorescence of pure milk on the probe can be effectively avoided. Finally, detecting the TC-labeled milk sample, wherein the detection method comprises the following steps: will 240 mu L g-C 3 N 4 A/CitNa/Eu ratio fluorescence sensor was added to the above 750. Mu.L milk sample, thoroughly mixed, incubated at room temperature for 10min, then added to a black 96-well microplate, 200. Mu.L of the liquid to be measured was added to each well, and the fluorescence spectrum was measured with an ELISA reader under excitation at 275 nm.
The test results are shown in table 2:
the recovery rate of the milk sample ranges from 95.75 to 102.95 percent, and the Relative Standard Deviation (RSD) ranges from 0.33 to 3.67 percent, thus showing good accuracy and reliability. The results show that the ratio fluorescence sensor of the invention is suitable for the actual detection of TC in animal-derived food samples.
Table 2 detection of TC in milk sample (n=3)
4. g-C 3 N 4 CitNa/Eu/PAN electrospun film
In recent years, flexible intelligent sensors have attracted attention because of their simple manufacturing process, outstanding plasticity and excellent sensing performance. Electrospun films having a large specific surface area, good flexibility, high porosity and excellent mechanical properties are considered to be one of the simplest and superior methods of manufacturing flexible smart sensors.
The present invention uses an electrospinning apparatus (as in FIG. 16A) to grow g-C in situ 3 N 4 The fluorescent electrospun membrane (FIG. 16C) was prepared by immobilizing the CitNa/Eu nanoprobe on the surface of polyacrylonitrile, and it can be seen that the electrospun membrane has a large surface area and good flexibility, and the shape of the flexible sensor can be adjusted as required. When Polyacrylonitrile (PAN) is used as a matrix, the obtained electrospun membrane becomes very flexible in water and has super-strong hydrophilicity, water drops can pass through within 1.5 seconds, so that the electrospun membrane and TC form close contact in an aqueous solution, and the TC is uniformly distributed on the surface of the electrospun membrane and permeates into the inside, thereby improving the sensing performance. Fig. 16B depicts SEM images of the prepared electrospun membranes, consisting of nanofibers in a disordered array with innumerable pores of different sizes, which provided large specific surface area and rich sensing sites, indicating that PAN to 10wt% (PAN to refers to the ratio of the amount of PAN and DMF solution, PAN to 1g, DMF to 9mL, the most suitable ratio) is suitable and has good electrospinning. The above results indicate that the g-C prepared according to the present invention can be used 3 N 4 The CitNa/Eu/PAN electrospun membrane is used as a solid-state flexible sensor to realize the efficient detection of TC.
g-C as described above 3 N 4 Preparation of the/CitNa/Eu/PAN electrospun film, the steps were as follows:
polyacrylonitrile (PAN) (mw=150000) was dissolved in DMF to give a concentration of10wt% and vigorously stirred at 90℃for 2 hours to give a polyacrylonitrile solution. 10mg g-C 3 N 4 adding/CitNa/Eu powder into 9mL polyacrylonitrile solution, stirring thoroughly to obtain uniform yellowish g-C 3 N 4 CitNa/Eu/PAN electrospun solution. Adopting YFSP-T electrostatic spinning equipment, using a syringe pump to make the electrostatic spinning solution be fed into needle at the speed of 0.002mm/s so as to make electrostatic spinning so as to obtain g-C 3 N 4 CitNa/Eu/PAN electrospun membrane.
To evaluate the effectiveness of the electrospun membrane in the assay, the electrospun membrane was cut into 1cm diameter discs, and then 15. Mu.L of solutions of different TC concentrations (0. Mu.M, 0.5. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, respectively) were added dropwise. As shown in FIGS. 16D and 16E, g-C 3 N 4 The CitNa/Eu ratio fluorescent probe was successfully immobilized on an electrospun membrane, which showed bright blue fluorescence under 365nm UV lamp irradiation. As the TC concentration increased, a clear change in fluorescence color was clearly observed with the naked eye, from blue to mauve to red, as shown in fig. 16F. Blue-violet-emitting fluorescence was clearly observed even at a TC concentration of 50 nM. The selectivity of the electrospun membrane was then assessed by adding 15 μl of 200 μΜ TC and other antibiotics to the electrospun membrane. As can be seen in fig. 16G, only tetracycline antibiotics (e.g., DOX, CTE, OXY and TC) trigger a significant color change from blue to red, while the effect of other antibiotics (AmL, ROX, MNZ, LUT, THI, STR, KM, CTR, AMP, SM 2) is negligible. These results indicate that g-C of the present invention 3 N 4 the/CitNa/Eu electrospun membrane can be used for visual rapid quantitative detection of TC. The realization of the fluorescent electrospun membrane for TC detection expands the application in the fields of flexible intelligent wearing equipment for human health monitoring and the like.
5. Quantitative detection TC of smart phone
Although based on g-C 3 N 4 the/CitNa/Eu ratio sensor can achieve ratio fluorescence sensing and visual analysis of TC, but ratio fluorescence analysis requires expensive and cumbersome instrumentation, which is not suitable for on-site analysis. In addition, it is difficult to evaluate the observed color change with naked eyesTo quantify TC concentration. In order to solve the problems, a portable intelligent mobile phone is used as a signal reader and an analyzer to convert color information into RGB values for semi-quantitative analysis, so that accuracy and reliability of results are improved, and portability of a platform is realized.
FIG. 16H shows g-C based 3 N 4 The CitNa/Eu ratio sensor and the smart phone detect the TC sensing process. First, in the tailored g-C 3 N 4 15. Mu.L of TC with different concentrations is dripped on the CitNa/Eu/PAN electrospun membrane, and after 5min, a smart phone is used for shooting the photo of the electrospun membrane under the illumination of 365nm ultraviolet lamp. As the TC concentration increases, the color of the electrospun film shows a continuous evolution from blue to light violet to light rose to pink to red. Second, after capturing a series of fluorescent images, the emission color information (RGB values) is analyzed using a color identifier application (ColorDesk) loaded in the smartphone, and TC concentration can be estimated by calculating the ratio of red channel (R) and blue channel (B). Finally, as shown in FIG. 16i, there is a two-stage linear relationship between R/B value and TC concentration. The linear equation in the range of 0 to 2.5 μm is: r/b=0.29003x+0.14365, R 2 The LOD value was calculated as 7.42nM (lod=3σ/S, σ is the standard deviation of 5 blank samples, S is the slope of the calibration curve) =0.973. The correlation linear equation in the range of 2.5 to 200 μm is: r/b=0.01095x+0.879, R 2 =0.983. The results show that the portable intelligent mobile phone auxiliary platform has great application potential in visual and on-site quantitative detection of TC.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (10)

1. A preparation method of a ratio fluorescence sensor is characterized by comprising the following steps:
will g-C 3 N 4 Nanosheet solution, eu (NO) 3 ) 3 ·6H 2 Sequentially adding the O solution and the sodium citrate solution into Tris-HCl buffer solution, uniformly mixing, and incubating; after the incubation is finished, freeze-drying the incubation solution to obtain g-C powder 3 N 4 CitNa/Eu nanoprobe, i.e., ratio fluorescence sensor.
2. The method according to claim 1, wherein g-C 3 N 4 Nanosheet solution, eu (NO) 3 ) 3 ·6H 2 The volume ratio of the O solution to the sodium citrate solution is selected from 10:1:1; g-C 3 N 4 The concentration of the nanosheet solution is selected from 0.3mg/mL, eu (NO 3 ) 3 ·6H 2 The concentration of the O solution is selected from 100. Mu.M; the concentration of the sodium citrate solution is selected from 500 μm; the concentration of Tris-HCl buffer is selected from 50mM, pH 8.0; the incubation time was selected from 10min.
3. The method of claim 1, wherein the g-C 3 N 4 The nano-sheet is prepared by the following method:
calcining melamine at 550-600 ℃ for 2-3 h, cooling to room temperature after calcining, obtaining a yellow solid product, and fully grinding; then dispersing the yellow solid product in water, carrying out ultrasonic crushing, centrifuging and removing non-stripped aggregates; collecting supernatant, drying to obtain g-C 3 N 4 A nano-sheet.
4. A ratiometric fluorescence sensor prepared by the method of any one of claims 1-3.
5. A method for detecting tetracycline antibiotic residues in food products using the ratiometric fluorescence sensor of claim 4, comprising the steps of:
adding trichloroacetic acid solution into a sample to be detected, and performing ultrasonic reaction; after the reaction is finished, centrifuging, collecting supernatant, filtering the supernatant, adding a ratio fluorescence sensor, fully mixing, and completely reacting at room temperature; after the reaction is finished, placing the reaction solution under the excitation wavelength of 275nm, and observing the change of fluorescent color; if tetracycline antibiotics exist, a fluorescent peak at 620nm is excited, and the fluorescent color becomes red.
6. g-C 3 N 4 The preparation method of the CitNa/Eu/PAN electrospun membrane is characterized by comprising the following steps:
dissolving polyacrylonitrile in DMF to obtain a polyacrylonitrile solution; then adding the ratio fluorescent sensor of claim 4 into the polyacrylonitrile solution, and stirring thoroughly to obtain g-C 3 N 4 A CitNa/Eu/PAN electrospun solution; then carrying out electrostatic spinning to obtain g-C 3 N 4 CitNa/Eu/PAN electrospun membrane.
7. A g-C prepared by the method of claim 6 3 N 4 CitNa/Eu/PAN electrospun membrane.
8. The ratio fluorescence sensor of claim 4 or the g-C of claim 7 3 N 4 Application of/CitNa/Eu/PAN electrospun membrane in detecting tetracycline antibiotics residue in food; the tetracycline antibiotic is selected from doxycycline, tetracycline, aureotetracycline, and oxytetracycline.
9. Use of a g-C as claimed in claim 7 3 N 4 The method for detecting tetracycline antibiotics residues in food by using the CitNa/Eu/PAN electrospun membrane comprises the following steps:
dripping the sample solution to be tested into g-C 3 N 4 on/CitNa/Eu/PAN electrospun film, alternatively, g-C 3 N 4 Immersing the/CitNa/Eu/PAN electrospun membrane into the sample solution to be tested, reacting for 5min, and then immersing g-C 3 N 4 the/CitNa/Eu/PAN electrospun film was irradiated with 365nm ultraviolet lamp to observe the change of fluorescence color; if tetracycline antibiotics exist, the electrospun film changes under the irradiation of an ultraviolet lampIs red.
10. A method for quantitatively detecting tetracycline antibiotic residues in food by using a smart phone comprises the following steps:
dripping the sample solution to be tested into g-C 3 N 4 on/CitNa/Eu/PAN electrospun film, alternatively, g-C 3 N 4 Immersing the/CitNa/Eu/PAN electrospun membrane into the sample solution to be tested, reacting for 5min, and then immersing g-C 3 N 4 The CitNa/Eu/PAN electrospun film is irradiated by a 365nm hand-held ultraviolet lamp, fluorescence of an excitation system is shot by a smart phone, then a color identifier loaded in the smart phone is used for converting a photo color signal into color information, the ratio of green and blue channels, namely a G/B value, is calculated, and then the G/B value is substituted into a standard curve to obtain the concentration of the tetracycline antibiotics.
CN202310550007.XA 2023-05-16 2023-05-16 Ratio fluorescence sensor, preparation method and application thereof Pending CN116574503A (en)

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