Fluorescent graphite phase carbon nitride quantum dots, preparation method and application thereof

Abstract

The present invention discloses a fluorescent graphite-phase carbon nitride quantum dot, a preparation method and an application thereof, and belongs to the field of fluorescence detection technology. The fluorescent graphite-phase carbon nitride quantum dots of the present invention are prepared by the following method: mixing urea and sodium citrate, grinding them into powder; heating the mixture for reaction; after the reaction is completed, washing the reaction product, centrifuging, collecting the centrifugal precipitate, and drying to obtain fluorescent graphite-phase carbon nitride quantum dots. The g-CNQDs prepared by the present invention not only have the advantages of adjustable fluorescence emission, good water dispersibility, significant stability and low toxicity, but also show particularly bright green fluorescence. The present invention uses electrospinning technology to prepare an electrospun membrane from g-CNQDs. The electrospun membrane can provide a more convenient and easily accessible platform for detecting objects. At the same time, the present invention uses a smartphone color recognizer to capture and convert fluorescent color information, thereby enabling on-site and quantitative detection of tetracycline antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and in particular relates to fluorescent graphite phase carbon nitride quantum dots, a preparation method and application thereof. Background Art

[0002] Tetracycline antibiotics are widely used as feed additives in the livestock industry. However, excessive use of antibiotics can lead to high levels of drug residues in animal products (especially milk, eggs, and meat) and the external environment. These residues, as food and environmental pollutants, threaten the ecological environment and accumulate in food, increasing potential risks to human health, such as skeletal dysplasia, allergic symptoms, yellowing of teeth, and liver damage.

[0003] Currently, a wide range of analytical methods are available for detecting antibiotic residues in food, including high-performance liquid chromatography (HPLC), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), and electrochemical analysis. Although these methods offer high sensitivity, they typically require expensive instrumentation, cumbersome sample pretreatment, and highly trained operators. Furthermore, the lack of reproducible results negatively impacts large-scale sample analysis, posing a significant challenge to achieving rapid and convenient detection. Therefore, there is an urgent need to develop a simple, reliable, and efficient method for the detection of doxycycline.

[0004] With the advent of nanotechnology, several emerging sensor devices have shown great potential in analytical chemistry, including electrochemical sensors, fluorescence sensors, and nanozyme sensors. Fluorescence sensors, owing to their high sensitivity, good selectivity, ease of operation, and low cost, have been widely used for antibiotic detection. Various fluorescent nanoprobes, including quantum dots (QDs), metal nanoparticles, fluorescent polymers, organic fluorescent dyes, and metal-organic frameworks (MOFs), have been applied in food analysis with excellent detection performance. For example, Wang et al. developed a hydrophobic fluorescence sensing platform based on CsPbBr3 QDs, which enabled visual and selective detection of tetracyclines (TETs) in a honey matrix. Yang et al. successfully synthesized an innovative dual-emission ratiometric fluorescence sensor based on boron nitride quantum dots and europium particles, demonstrating the feasibility of efficient monitoring of tetracyclines in milk and beef. Furthermore, Liu et al. developed a novel MOF-derived composite luminescent material for rapid and sensitive detection of tetracyclines in water and egg samples. Although some progress has been made in these fluorescent nanoprobes, there are also some inevitable disadvantages in the complex synthesis process, including hazardous chemical precursors, the introduction of heteroatoms, harsh conditions, etc., which limit their further application. Therefore, the development of simple operation, high sensitivity, low toxicity, low cost, and environmentally friendly fluorescent probe synthesis strategies still needs further exploration.

[0005] Carbon nitride, an organic semiconductor composed of carbon and nitrogen, has been widely used in fields such as photocatalytic degradation and bioimaging. Over the past few decades, the unique optical properties of semiconductor quantum dots (QDs) have attracted significant attention. However, due to the involvement of heavy metals, these QDs may cause serious health and environmental concerns, which limits their widespread application. In recent years, graphitic carbon nitride quantum dots (g-CNQDs) have emerged as promising candidates to replace traditional QDs due to their bright fluorescence, excellent stability, good water solubility and biocompatibility, low cost, and low cytotoxicity. They have also become novel fluorescent probes for biological and biochemical applications. For example, Achadu et al. prepared g-CNQDs and their 2,2,6,6-tetramethyloxy derivatives as fluorescent on / off probes for ascorbic acid detection. Susmit et al. studied functionalized two-dimensional carbon nitride nanodots to construct a fluorescent sensor for the detection of lead in the environment. However, g-CNQDs still face some challenges, such as a limited number of synthesis methods and low quantum yield. Until now, the preparation of graphitic carbon nitride has typically relied on the thermal decomposition of nitrogen-rich precursors such as melamine, cyanamide, and dicyandiamide. While this strategy is simple, the synthesis temperature is too high, between 450 and 600°C, resulting in large carbon nitride particles, poor luminescence properties, and unsuitable for optical applications. Therefore, the development of a simple and efficient method to prepare fluorescent g-CNQDs with high quantum yield is highly desirable. Summary of the Invention

[0006] To achieve the above objectives, the present invention uses a low-temperature solid-phase synthesis strategy to synthesize a graphene-phase carbon nitride quantum dot fluorescent probe with strong green fluorescence at extremely low temperatures using urea as a precursor. Simultaneously, a novel, simple sensing platform based on fluorescence quenching was constructed for the detection of tetracycline antibiotics in animal products. Based on this, the present invention proposes the following technical solutions:

[0007] The present invention provides a method for preparing fluorescent graphite phase carbon nitride quantum dots, comprising the following steps:

[0008] The urea and sodium citrate are mixed and ground into powder; the mixture is heated to react; after the reaction is completed, the reaction product is washed and centrifuged, the centrifugal precipitate is collected, and the centrifugal precipitate is dried to obtain g-CNQDs powder, i.e., the fluorescent graphite phase carbon nitride quantum dots.

[0009] In the above preparation method, the mass ratio of urea to sodium citrate is selected from 5:3 to 5:6, preferably 5:4.

[0010] In the above preparation method, the heating reaction conditions are selected from: heating to 180-200° C. and continuing the reaction for 1-2 hours; preferably heating to 180° C. and continuing the reaction for 1 hour.

[0011] The present invention provides fluorescent graphite phase carbon nitride quantum dots prepared by the method.

[0012] The present invention provides the use of the fluorescent graphite-phase carbon nitride quantum dots in the detection of tetracycline antibiotic residues in food. The tetracycline antibiotic is selected from doxycycline, tetracycline, aureotetracycline, and oxytetracycline, preferably doxycycline.

[0013] The present invention provides a method for detecting tetracycline antibiotic residues in food using the fluorescent graphite phase carbon nitride quantum dots, the steps of which are as follows:

[0014] Trichloroacetic acid solution was added to the sample to be tested and ultrasonic reaction was carried out. After the reaction was completed, centrifugation was performed and the supernatant was collected. The pH of the supernatant was adjusted to neutral and then mixed with the g-CNQDs solution and allowed to react completely at room temperature. After the reaction was completed, the reaction solution was placed under an excitation wavelength of 360nm and the fluorescence color change was observed. If tetracycline antibiotics were present, quenching occurred and the fluorescence of the mixture became lighter.

[0015] In the above detection method, the sample to be tested is selected from common foods such as milk, eggs, chicken, beef, etc.; preferably, it is milk.

[0016] The present invention provides a method for preparing a g-CNQDs / PAN nanofiber membrane, comprising the following steps:

[0017] Polyacrylonitrile was dissolved in DMF to obtain a polyacrylonitrile solution; g-CNQDs powder was then added to the polyacrylonitrile solution and stirred thoroughly to obtain a g-CNQDs / PAN electrospinning solution; and electrostatic spinning was then performed to obtain a g-CNQDs / PAN nanofiber membrane.

[0018] The mass volume ratio of the g-CNQDs to the polyacrylonitrile solution is selected from 10:9 to 11, mg:mL; preferably 10:9, mg:mL.

[0019] The present invention provides a g-CNQDs / PAN nanofiber membrane prepared by the above method.

[0020] The present invention provides the use of the g-CNQDs / PAN nanofiber membrane in the detection of tetracycline antibiotic residues in food. The tetracycline antibiotic is selected from doxycycline, tetracycline, aureotetracycline, and oxytetracycline, preferably doxycycline.

[0021] The present invention provides a method for detecting tetracycline antibiotic residues in food using the g-CNQDs / PAN nanofiber membrane, the steps of which are as follows:

[0022] The sample solution to be tested was added dropwise to the g-CNQDs / PAN nanofiber membrane, or the g-CNQDs / PAN nanofiber membrane was immersed in the sample solution to be tested and reacted for 5 minutes. The g-CNQDs / PAN nanofiber membrane was then placed under 365nm ultraviolet light to observe the change in fluorescence color. If tetracycline antibiotics were present, quenching occurred and the fluorescence of the g-CNQDs / PAN nanofiber membrane became lighter.

[0023] The present invention provides a method for quantitatively detecting tetracycline antibiotic residues in food using a smartphone, comprising the following steps:

[0024] The sample solution to be tested was added dropwise to the g-CNQDs / PAN nanofiber membrane, or the g-CNQDs / PAN nanofiber membrane was immersed in the sample solution to be tested and reacted for 5 minutes. The g-CNQDs / PAN nanofiber membrane was then placed under 365nm ultraviolet light, and the fluorescence of the system was photographed using a smartphone. The color signal of the photo was then converted into color information (RGB value) using a color recognizer loaded in the smartphone. The ratio of the green and blue channels (G / B value) was calculated, and the G / B value was substituted into the standard curve to obtain the concentration of tetracycline antibiotics.

[0025] The color identifier above is selected from the ColorDesk application

[0026] The above standard curve can be constructed by the following methods:

[0027] Tetracycline antibiotic solutions of different concentrations were added dropwise to the g-CNQDs / PAN nanofiber membrane, or the g-CNQDs / PAN nanofiber membrane was immersed in tetracycline antibiotic solutions of different concentrations and reacted for 5 minutes. The g-CNQDs / PAN nanofiber membrane was then placed under 365nm ultraviolet light, and the fluorescence of the system was photographed using a smartphone. The color signal of the photo was then converted into color information (RGB value) using a color recognizer loaded in the smartphone, and the ratio of the green and blue channels (G / B value) was calculated. The concentration-G / B value standard curve was constructed with the tetracycline antibiotic concentration as the horizontal axis and the G / B value as the vertical axis.

[0028] The concentration gradient of the tetracycline antibiotic solution can be selected from 0 μM, 5 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, and 150 μM.

[0029] The beneficial effects of the present invention are:

[0030] Most of the carbon dots reported in the prior art show blue fluorescence, while the g-CNQDs prepared by the present invention not only have the advantages of adjustable fluorescence emission, good water dispersibility, significant stability and low toxicity, but also show particularly bright green fluorescence. Through the inner filter effect (IFE), the quenching performance of tetracycline antibiotics can be observed. Based on this, the present invention loads the prepared strong luminescent g-CNQDs into electrospun nanofibers to prepare g-CNQDs / PAN nanofiber membranes. The electrospun membrane can provide a more convenient and easily accessible platform for detecting objects. At the same time, the present invention combines and applies a smartphone color recognizer to capture and convert fluorescent color information, thereby enabling on-site and quantitative detection of tetracycline antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Characterization of g-CNQDs; Figure a is a TEM image, and the inset is a representative HRTEM image of a single carbon nitride dot; Figure b is an AFM image of g-CNQDs deposited on a mica substrate; Figure c is a height profile; Figure d is an FT-IR spectrum; Figure e is an XPS spectrum; Figure f is a C1s high-resolution spectrum; Figure g is a N1s high-resolution spectrum; Figure h is an O1s high-resolution spectrum;

[0032] Figure 2 XRD patterns of g-CNQDs and sodium citrate; the upper part is g-CNQDs and the lower part is sodium citrate;

[0033] Figure 3 Optical properties of g-CNQDs; wherein, Figure a is the UV-visible absorption (red line), fluorescence excitation (purple line) and emission (green line) spectra of g-CNQDs, and the insets are photos of g-CNQDs under visible light and UV light (365 nm), respectively; Figure b is the excitation-emission matrix spectrum of g-CNQDs; Figure c is the excitation-dependent fluorescence spectrum of g-CNQDs; Figure d is the fluorescence spectrum of g-CNQDs in the absence (bottom) or presence (top) of DOX, and the insets show photos of g-CNQDs without (left) or with (right) DOX under 365 nm UV light irradiation;

[0034] Figure 4 Fluorescence stability of g-CNQDs under different solution pH (a), UV irradiation time (b), NaCl concentration (c), and storage time stress (d);

[0035] Figure 5 Effects of reaction time (a), temperature (b), and solution pH (c) on F0 / F in the presence of DOX;

[0036] Figure 6is the sensing mechanism of g-CNQDs; wherein, Figure a is the UV-visible absorption spectrum of DOX, the fluorescence excitation spectrum and emission spectrum of g-CNQDs, wherein, at 400nm, the curves are Ex, Em, and UV from top to bottom; Figure b is the absorption spectrum of DOX, g-CNQDs and g-CNQDs+DOX, wherein, at 450nm, the curves are g-CNQDs, g-CNQDs+DOX and DOX from top to bottom; Figure c is the photoluminescence lifetime of g-CNQDs and g-CNQDs+DOX mixture at room temperature; Figure d is the observation and correction of the quenching efficiency of DOX on g-CNQDs;

[0037] Figure 7 The results of the DOX detection test are shown in Figure 1. Figure a shows the fluorescence spectra of g-CNQDs under 360 nm excitation in the presence of different concentrations of DOX, and the inset is the corresponding fluorescence photograph taken under 365 nm light. Figure b shows the relationship between F0 / F and the relevant calibration curve of DOX detection at 475 nm.

[0038] Figure 8 Specific analysis of g-CNQDs; Figure a shows the fluorescence response of g-CNQDs to different types of antibiotics, molecules, and metal cations; Figure b shows the relative emission intensity F0 / F of g-CNQDs in the presence of various interfering substances;

[0039] Figure 9 UV-visible absorption spectra of CTE, DOX, TET, and OXY at a concentration of 37 μM. Within 250 nm, the curves from top to bottom represent CTE, OXY, DOX, and TET, respectively.

[0040] Figure 10 The chemical structures of tetracycline drugs; among them, doxycycline (DOX) on the upper left, tetracycline (TC) on the upper right, aureotetracycline (CTE) on the lower left, and oxytetracycline (OXY) on the lower right. The four compounds have similar chemical structures.

[0041] Figure 11 Preparation and application of g-CNQDs / PAN nanofiber membrane; Figure a is a schematic diagram of the preparation principle of g-CNQDs / PAN nanofiber membrane; Figure b is a scanning electron microscope image of g-CNQDs / PAN nanofiber membrane; Figure c is a g-CNQDs / PAN nanofiber membrane; Figure d is a schematic diagram of the smartphone color recognition for detecting DOX; Figure e is the change of fluorescence color with DOX concentration (photographed under 365nm ultraviolet light); Figure f is a linear relationship diagram of the color change (G / B) of the fluorescent probe solution with DOX concentration in the range of 0 to 150μM. DETAILED DESCRIPTION

[0042] The reagents, chemicals and instruments used in the present invention are as follows:

[0043] Urea was purchased from Shanghai Bioengineering Co., Ltd. Trisodium citrate, polyacrylonitrile (PAN, Mw = 150,000), tetracycline (TET), chlortetracycline (CTE), oxytetracycline (OXY), doxycycline (DOX), roxithromycin (ROX), kanamycin sulfate (KM), thiamphenicol (THI), amoxicillin (AML), norfloxacin (NOC), luteolin (LUT), metronidazole (MNZ), lactose (Lac), lysine (Lys), ascorbic acid (Vc), aspartic acid (Asp), and trichloroacetic acid solution (10%) were purchased from MacLean Biochemical Technology Co., Ltd. Ampicillin (AMP), streptomycin sulfate (STR), and ceftriaxone sodium (CTR) were purchased from Solebao Technology Co., Ltd. N,N-dimethylformamide (DMF), cysteine ​​(Cys), sulfamethazine (SM2), sucrose (Suc), and anhydrous glucose (Glu) were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used as received without further purification. X-ray diffraction (XRD) was performed on a TD-3700 instrument (China), transmission electron microscopy (TEM, JOEL JEM2001), atomic force microscopy (AFM) was performed on an SPM-9700HT instrument (China), X-ray photoelectron spectroscopy (XPS) was performed on an ESCA-3Mark II spectrometer (VG Scientific Ltd., England), Fourier transform infrared (FTIR) spectroscopy was performed on a Nicolet iso FTIR spectrometer (USA), a SpectraMax i3x multi-function microplate reader (USA), an F2700 fluorescence spectrophotometer (Hitachi, Japan), a Zeiss scanning electron microscope (SEM, Japan), and an electrospinning device, YFSP-T (Yunfan, Tianjin, China).

[0044] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0045] Example 1

[0046] Preparation of g-CNQDs:

[0047] 0.101 g of urea and 0.081 g of sodium citrate were mixed in an agate mortar and ground into a uniform powder. The mixture was placed in a reactor and heated to 180°C in an oven for 1 hour. The resulting yellow mixture was washed with ethanol and centrifuged (12,000 rpm, 10 minutes), repeated three times. The centrifugal precipitate was collected and dried in a 60°C oven overnight to obtain g-CNQDs powder.

[0048] The present invention provides a new solid-phase reaction strategy, using urea and sodium citrate as nitrogen and carbon sources, and synthesizing highly fluorescent g-CNQDs by a low-temperature solid-phase method. In the reaction system, urea was selected as a precursor due to its low cost, abundance and nitrogen-rich properties. In addition, urea is an active molecule under thermal treatment, and a large amount of graphitic carbon nitride is usually achieved by heating urea or thiourea at high temperatures (>400°C). Few reports show that highly fluorescent g-CNQDs can be synthesized from urea at low temperatures. Compared with the traditional pyrolysis method, the present invention achieves the preparation of g-CNQDs from urea at lower temperatures. In addition, g-CNQDs synthesized from urea have unique properties of non-toxicity, good water solubility and suitability for biological applications.

[0049] Characterization of the prepared g-CNQDs:

[0050] like Figure 1 As shown in a, the TEM image shows that the prepared g-CNQDs have uniform size and good monodispersity. Figure 1 The inset in a shows a representative image of a single nanoparticle, indicating high crystallinity with a lattice parameter of 0.34 nm, which is consistent with the 002 plane of graphitic carbon nitride, and the well-ordered lattice fringes reflect the excellent crystallinity of g-CNQDs.

[0051] The planar structure of the as-prepared g-CNQDs was further characterized by AFM analysis ( Figure 1 b), revealing a typical topographic height of 2.5–4 nm ( Figure 1 c), indicating that g-CNQDs are composed of several layers of CN sheets. XRD characterization further demonstrated three characteristic peaks at 22.3, 24.84 and 29.36 ( Figure 2 ), which is largely related to amorphous carbon and citrate. The strong peak at 22.3 represents the characteristic interplanar stacking of aromatic systems, and graphitic carbon nitride is indexed as the (002) peak.

[0052] like Figure 1As shown in Figure e, the chemical structure and composition of g-CNQDs were characterized using XPS. The survey spectrum showed three binding energy peaks at 284.8, 399.7, and 531.25 eV, corresponding to the elements C1s, N1s, and O1s, respectively. In contrast to previously reported g-CNQDs, a large amount of oxygen was measured in the obtained g-CNQDs.

[0053] Specifically, Figure 1 The high-resolution XPS spectrum of C1s in f can be deconvoluted into three peaks at 284.80, 286.36 and 288.38 eV; among them, the binding energy peak at 284.8 eV is attributed to the sp2 cc bond, the binding energy peak at 288.38 eV is attributed to the sp2 NC=N bond; and the binding energy peak at 286.36 eV is attributed to the CO bond, indicating the presence of oxygen-rich groups.

[0054] Figure 1 g shows the XPS spectrum of N1s, which can be divided into three peaks, indicating the presence of three different types of N in the prepared g-CNQDs; the binding energy of 398.3eV is associated with the C=NC group, the binding energy of 399.65eV can be assigned to pyridine CNC, and the peak at 400.5eV corresponds to N-(C)3. The results of C and N show that the basic structure of the prepared g-CNQDs is (tris-s-)triazine units.

[0055] Figure 1 The O1s spectrum in h shows four peaks at 531.15, 531.95, 532.92 and 535.52 eV. The peaks at 531.15, 531.95 and 532.92 eV belong to –OH, C-OH and C=O, respectively, which originate from H2O or CO2 adsorbed on the g-CNQD surface. The peak position at 535.52 eV corresponds to COO – .

[0056] These results also obtained Figure 1 d FT-IR spectrum shown in support. 656.7 cm -1 The characteristic peak at 926 cm can be attributed to the breathing mode of the carbon nitride s-triazine ring. -1 The characteristic bands near 1017cm are attributed to the heptazine unit. -1 The peak at 1630 cm-1 can be attributed to the -CO- and C-OH groups on the surface of g-CNQDs. -1 Around 1719 cm, which can be assigned to aromatic C=N stretching. -1 The strong peak at 2930 cm comes from the asymmetric C=O stretching vibration. -1The small peak at 3354 cm -1 The broad peaks around can also be attributed to the stretching vibration of NH groups, confirming the availability of these groups on the surface of g-CNQDs. These results all indicate that carboxylic acid and hydroxyl groups are located on the surface of g-CNQDs.

[0057] The optical properties of g-CNQDs are as follows:

[0058] Visually, the g-CNQDs solution appears light yellow under sunlight and emits strong green fluorescence under 365 nm ultraviolet light ( Figure 3 This phenomenon motivated us to explore the optical properties of g-CNQDs using UV-Vis and fluorescence spectroscopy. Figure 3 As shown in a, characteristic absorption bands of g-CNQDs at 253 nm and 326 nm (red line) can be observed. The former is attributed to the π-π* transition of aromatic C=C, and the latter may be due to surface chemical effects. Under optimal excitation at 360 nm (purple line), the fluorescence spectrum shows a maximum emission peak at 461 nm (green line), and the optimal excitation overlaps with the absorption at 326 nm, which is consistent with the excitation emission matrix spectrum ( Figure 3 b) is consistent, corresponding to the transition from an indirect band gap to a direct band gap semiconductor. Moreover, g-CNQDs exhibit a distinct excitation-dependent fluorescence behavior, which is similar to most luminescent carbon dots and graphene quantum dots. This means that as the excitation wavelength increases, the emission wavelength will undergo a red shift ( Figure 3 c). This may be attributed to the optical selection (quantum effect) of nanoparticles of different sizes and the distribution of emission holes on the surface of g-CNQDs. According to the test, the quantum yield of the prepared g-CNQDs is about 8.65%.

[0059] In addition, the present invention also studies the fluorescence stability of g-CNQDs under the stress of solution pH, ionic strength, UV irradiation time and storage time. The specific experimental process is as follows: (1) pH: The g-CNQDs solution is mixed with a solution with a pH of 2 to 12, and its fluorescence intensity at 461 nm is observed under the excitation of 360 nm. (2) Ionic strength: The g-CNQDs solution is mixed with solutions with different NaCl concentrations, and its fluorescence intensity at 461 nm is observed under the excitation of 360 nm. (3) UV irradiation: The g-CNQDs solution is placed under an ultraviolet light source for irradiation for 0 min, 20 min, 40 min, 60 min, 80 min, and 100 min, respectively. Under the excitation of 360 nm, the fluorescence intensity at 461 nm is recorded. (4) Storage time: The g-CNQDs solution was stored at room temperature for 0 days, 1 day, 3 days, 5 days, 7 days, 9 days, 15 days, 20 days, 25 days, and 30 days. The fluorescence intensity at 461 nm was recorded under 360 nm excitation. Figure 4 As shown in the figure, there is no significant difference in the fluorescence intensity of g-CNQDs, indicating that g-CNQDs have excellent fluorescence stability. In summary, the g-CNQDs synthesized in the present invention exhibit excellent optical properties.

[0060] In view of the excellent optical properties of g-CNQDs, the feasibility of g-CNQDs as a fluorescent probe for DOX was explored in this paper. 800 μL of g-CNQDs solution and 400 μL of a mixed solution of g-CNQDs and 400 μL of DOX were taken and the fluorescence curves of the two were measured under 360 nm excitation. Figure 3 As shown in Figure d, after the addition of DOX, the fluorescence intensity of the emission peak centered at 461 nm decreased significantly (the inset is the corresponding photograph under 365 nm UV light), confirming the feasibility of g-CNQDs as a fluorescent probe for detecting DOX.

[0061] In order to obtain the best analytical performance of DOX, the present invention optimizes the experimental conditions in the detection process, including reaction time, temperature and solution pH. The specific experimental process is as follows: (1) Reaction time: 400 μL g-CNQDs solution and 400 μL DOX solution are mixed, and the fluorescence intensity is tested at 1 min, 3 min, 5 min, 7 min and 9 min after adding DOX solution. (2) Temperature: 400 μL g-CNQDs solution and 400 μL DOX solution are mixed and placed in a water bath, and the temperature is controlled at 20°C, 30°C, 40°C, 50°C and 60°C. (3) pH: The g-CNQDs solution is mixed with a solution with a pH of 2 to 12 and then the DOX solution is added. Under excitation at 360 nm, the fluorescence intensity at 461 nm is observed. The above experiments all record F0 / F, where F refers to the fluorescence intensity at 461 nm after adding other substances, and F0 refers to the fluorescence intensity of the g-CNQDs solution at 461 nm. As Figure 5 As shown in Figure a, it was found that F0 / F increased rapidly within 5 minutes and then gradually stabilized, indicating that the reaction between the probe and the substance was basically completed after 5 minutes. In addition, the incubation temperature and the pH value of the solution are also important parameters that affect the sensitivity and accuracy of the probe. Generally, the fluorescence intensity of the probe decreases with increasing temperature, which is mainly due to the vibrational relaxation effect that causes the internal energy loss of the molecule during the transition from the excited state to the ground state. Figure 5 As shown in Figure 2b, F0 / F has a clear trend of increasing with increasing temperature, which is attributed to the acceleration of energy transfer in the reaction system caused by the addition of DOX. When the temperature reaches 30°C, F0 / F remains almost constant. Considering the practical application potential of the fluorescent probe, room temperature was selected as the subsequent detection condition. In addition, the present invention also investigated the effect of solution pH between 2 and 12, and the results are shown in Figure 2b. Figure 5 As shown in Figure c, under strong acid or strong base conditions, the fluorescence intensity changes slightly, which may be related to the different ionization degrees of -OH, -COOH and -NH2 on the surface of g-CNQDs to different environmental pH. The present invention also found that F0 / F is slightly higher under acidic conditions than under alkaline conditions, and pH = 6 is the best choice for fluorescence detection. A reasonable explanation for this phenomenon is that the change in pH value affects the charge state of the emission hole on the surface of the fluorescent group. However, in order to facilitate the experimental operation, ultrapure water (pH = 7) was selected instead of the buffer solution of pH = 6, thereby effectively avoiding the complicated preparation process of PBS. Therefore, the optimal parameters for subsequent experiments are: reaction time 5min, detection temperature at room temperature, and pH = 7.

[0062] On this basis, the present invention discusses the possible sensing mechanism of g-CNQDs. First, the absorption spectrum of DOX and the excitation spectrum of g-CNQDs have a large spectral overlap ( Figure 6a), which indicates that the absorption of DOX can effectively inhibit the excitation energy absorption of g-CNQDs, which makes the fluorescence of g-CNQDs greatly quenched by DOX through the inner filter effect (IFE), because the efficiency of IFE depends on the degree of spectral overlap between the absorption of the quencher and the excitation or emission of the fluorophore. Secondly, it can be seen from the UV absorption spectrum that no new absorption peak is generated after the addition of DOX ( Figure 6 b), indicating that the fluorescence quenching of g-CNQDs induced by DOX cannot be attributed to fluorescence resonance energy transfer (FRET). Finally, the fluorescence lifetime of the fluorophore decreases during the FRET process due to the energy transfer from the fluorophore to the quencher, while the fluorescence lifetime of IFE remains constant. Figure 6 c It can be seen that after the addition of DOX, the fluorescence lifetime of g-CNQDs changes slightly from 6.59 ns to 6.55 ns, which further indicates that the DOX-induced fluorescence quenching of g-CNQDs mainly comes from IFE rather than FRET.

[0063] In order to further evaluate the role of IFE in fluorescence quenching, relevant correction data and results were obtained by calculation. The obtained correction coefficients (CF) did not exceed 3, thus ensuring that the correction was convincing. The fluorescence quenching efficiency of DOX on g-CNQDs before and after correction is shown in Figure 2. Figure 6 d. This result further indicates that the fluorescence quenching of g-CNQDs caused by DOX mainly comes from IFE. All the above results indicate that the fluorescence quenching of g-CNQDs after adding DOX to the sensing system is related to IFE.

[0064] 1. DOX detection test

[0065] First, 1 mg / mL g-CNQDs and 1.125×10 -3mol / L DOX stock solution. 0.4 mL of g-CNQDs stock solution was mixed with different volumes of DOX stock solution in a test tube. The mixed solution was then diluted to 0.8 mL with ultrapure water to achieve a final concentration range of 0 to 150 μmol / L (0 μmol / L, 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 6 μmol / L, 8 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, and 150 μmol / L). The prepared test solution was allowed to react completely at room temperature for 5 minutes, then extracted and added to a black 96-well microplate. Fluorescence spectra of the different solutions were obtained in a microplate reader with an excitation wavelength of 360 nm. A calibration curve was constructed with DOX concentration as the horizontal axis and F0 / F (F0 is the fluorescence intensity at 475 nm without DOX, and F is the fluorescence intensity at 475 nm after adding different concentrations of DOX) as the vertical axis. Selectivity and anti-interference tests were conducted in the above detection system. For other interfering substances, DOX was replaced with the corresponding solution and the fluorescence measurement procedure was then repeated. All experiments were repeated three times. In addition, the fluorescence color changes of the mixtures were observed under 365 nm ultraviolet light.

[0066] The test results are as follows Figure 7 As shown:

[0067] like Figure 7 As shown in Figure a, the fluorescence intensity of g-CNQDs at 475 nm decreased significantly with the increase of DOX concentration (0-150 μmol / L). The same results were obtained in the corresponding photos under 365 nm UV light ( Figure 7 (a, inset). This phenomenon can be easily identified visually with the naked eye, indicating the superior performance of g-CNQDs in detecting DOX as a fluorescent nanoprobe.

[0068] like Figure 7 As shown in b, the fluorescence intensity ratio F0 / F of the g-CNQDs fluorescent probe has a good linear relationship with the concentration of DOX in the range of 0 to 150 μmol / L. The regression equation is F0 / F=1.04399+0.01039x, and the determination coefficient (R 2 ) was 0.992 (where x is the concentration of DOX). The limit of detection (LOD) was 0.012 μmol / L, according to the following equation: LOD = 3S B / K, where S Bis the standard deviation of the blank signal (n = 3) and K is the slope of the calibration curve. Compared with previously reported worksheets for DOX detection (Table 1), this method has a low detection limit and a comparable detection range. This significant analytical performance is likely related to the oxygen-containing groups on the probe surface providing more active sites for DOX response.

[0069] Table 1

[0070]

[0071]

[0072] The references in Table 1 are as follows: [1] Food Chem, 2022, 374: 131774; [2] J Colloid Interface Sci, 2019, 539: 332-41; [3] Sensors and Actuators B: Chemical, 2022, 358; [4] Inorg Chem, 2022, 61(20): 8015-21; [5] Anal Chim Acta, 2022, 1197: 339530; [6] ACSS Sustainable Chemistry & Engineering, 2020, 8(46): 17185-93; [7] ACS Appl Mater Interfaces, 2020, 12(9): 11036-44; [8] Talanta, 2020, 208: 120342; [9] Food Chem, 2020, 304: 125377;

[10] Bioelectrochemistry, 2019, 128: 66-73.

[0073] 2. DOX specificity test

[0074] To evaluate the selectivity of the above fluorescent probe (g-CNQDs) for DOX, different potential interfering substances were added to the probe solution under the same experimental conditions, including other common antibiotics (TET, OXY, CTE, SM2, CTR, STR, AMP, ROX, MNZ, LUT, NOC, KM, THI), molecules (Cys, Lys, Vc, Aps, Glu, Suc, Lac) and metal cations (K + 、Na + 、Mn 2+ Mg 2+ 、Zn 2+ 、Co 2+ 、Al 3+ 、Fe 2+ 、Fe 3+, Ca 2+ 、Cu 2+ g-CNQDs concentration: 1 mg / mL; antibiotic and other substance concentration: 150 μmol / L.

[0075] The test process is as follows:

[0076] Mix 400 μL of g-CNQDs and 400 μL of an interfering substance. After 5 minutes of reaction, measure the fluorescence intensity at 360 nm. Record F0 / F (F refers to the fluorescence intensity at 461 nm after the addition of the interfering substance, and F0 refers to the fluorescence intensity of g-CNQDs at 461 nm without the addition of other substances). Perform three parallel experiments for each group.

[0077] The test results are as follows Figure 8 As shown:

[0078] like Figure 8 As shown in a, except for TET, OXY and CTE, the fluorescence responses of other potential interfering substances did not change significantly, and the effect on F0 / F was negligible. DOX, TET, OXY and CTE are all common tetracycline antibiotics with similar absorption spectra ( Figure 9 ) and chemical structure ( Figure 10 ), similar absorption spectra and chemical structures mean that TET, OXY, and CTE also trigger IFE in g-CNQD fluorescence sensing systems. At the same time, their detection performance varies. This is because the substituents (position and number of hydroxyl groups) of targets with the same parent structure vary slightly, affecting their interactions with g-CNQDs, such as hydrogen bonding. These results demonstrate that the g-CNQDs synthesized in this invention have excellent selectivity for tetracycline antibiotics.

[0079] In addition, when other interfering substances were added and coexisted with DOX, the response ratio F0 / F did not change significantly, e.g. Figure 8 Therefore, this fluorescent probe can be used for ultrasensitive and highly selective detection of DOX and has potential applications in various fields.

[0080] 3. Practicality Test

[0081] In order to evaluate the practicality and accuracy of g-CNQDs in actual food samples, g-CNQDs were used to determine DOX in pure milk. Milk was bought from a local supermarket. First, 10 mL of pure milk was diluted to 50 mL with ultrapure water in a volumetric flask, and then 2 mL of trichloroacetic acid solution (10%) was added. The sample was sonicated for 30 minutes and fully reacted for 1 hour. Then, centrifuged at 12000 rpm for 10 minutes to remove organic matter such as proteins and lipids. The supernatant was collected and NaOH (1 mol / L) solution was added to neutrality. The resulting solution was then diluted 50 times for actual sample detection, which can effectively avoid the interference of the intrinsic fluorescence of pure milk on the g-CNQDs probe. Finally, the DOX in the sample was quantitatively detected according to the method above.

[0082] Preliminary experiments confirmed that the sample did not contain DOX. The standard addition method was then used to detect DOX in milk. As shown in Table 2, the recovery rate of the milk sample ranged from 97.37 to 106.36%, and the relative standard deviation (RSD) was 0.20 to 2.29%, showing good accuracy and reliability. In addition, the present invention also found that the fluorescence detection results of different concentrations of DOX (0.5, 1, 5, 10, 20, 40, 60 μmol / L) in the same sample were significantly different (P≤0.05), indicating that the probe provides a feasible method for accurate trace detection of DOX in actual samples. In summary, the g-CNQDs probe developed by the present invention is suitable for practical applications in animal-derived food samples.

[0083] Table 2 DOX detection in actual samples (n=3)

[0084]

[0085] 4. g-CNQDs / PAN Nanofiber Membrane

[0086] In recent years, flexible smart sensors have attracted widespread attention due to their simple fabrication process, outstanding plasticity, and excellent sensing performance. Electrospun films with large specific surface area, good flexibility, high porosity, and excellent mechanical properties are considered to be one of the simplest and most superior methods for preparing flexible smart sensors.

[0087] The present invention uses an electrostatic spinning machine (principle as Figure 11 (a) g-CNQDs nanoprobes were fixed on the surface of polyacrylonitrile to fabricate fluorescent electrospun membranes ( Figure 11 c) for highly selective recognition of DOX. When polyacrylonitrile (PAN) was used as the matrix, the obtained nanofibers became very flexible in water, making them very suitable for further studies. Figure 11b depicts an SEM image of the prepared electrospun membrane, which is composed of randomly arranged nanofibers. There are numerous pores of varying sizes, which provide a large specific surface area and abundant sensing sites, indicating that a PAN ratio of 10 wt% (PAN ratio refers to the ratio of the amounts of PAN and DMF, with 1 g of PAN and 9 mL of DMF being the most suitable ratio) is suitable and has good electrospinnability. These results indicate that the g-CNQDs / PAN nanofiber membrane prepared in this invention can be used as a solid-state flexible sensor for efficient detection of DOX.

[0088] The preparation steps of the g-CNQDs / PAN nanofiber membrane are as follows:

[0089] Polyacrylonitrile (Mw = 150,000), the polymer host, was dissolved in DMF to a concentration of 10 wt% and vigorously stirred at 90°C for 2 h. 10 mg of g-CNQD powder was then added to 9 mL of a 10 wt% PAN solution and stirred thoroughly at room temperature to obtain a light brown, uniform g-CNQDs / PAN electrospinning solution. A 10 mL syringe was used to draw the viscous liquid from the beaker into a needle. The needle was inverted to expel air and remove bubbles from the solution, preventing spin damage caused by bubbles during the electrospinning process. A syringe pump was used to feed the solution into the needle at a rate of 0.002 mm / s. Electrospinning was performed using a YFSP-T electrospinning apparatus at a high voltage of 25 kV, maintaining a 15 cm distance between the needle and a grounded collector wrapped in aluminum foil. Electrospun nanofiber membranes loaded with g-CNQDs, i.e., g-CNQDs / PAN nanofiber membranes, were obtained.

[0090] In order to evaluate the effectiveness of the fluorescent electrospun membrane in detection, the electrospun membrane was cut into 0.5 cm × 1 cm rectangular pieces and then immersed in a series of solutions with different DOX concentrations (0 μM, 5 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 150 μM). After 5 minutes, it was irradiated with 365 nm UV light. It can be clearly observed that the green fluorescence is gradually quenched with the increase of DOX concentration, as shown in Figure 2. Figure 11 These results demonstrate that the g-CNQDs / PAN nanofiber membrane prepared in this invention can be used for visual quantitative detection of DOX with a rapid response rate. The realization of fluorescent electrospun membranes for DOX detection expands applications in areas such as flexible smart wearable devices for human health monitoring.

[0091] 5. Quantitative detection of DOX using smartphones

[0092] It is difficult to quantify the DOX concentration by visually evaluating the color changes observed. Therefore, in order to improve the accuracy and reliability of the results and achieve the portability of the platform, the present invention combines a smartphone application to convert color information into RGB values ​​to achieve quantitative detection of DOX. Figure 11 As shown in (d), the RGB of the fluorescence image can be analyzed using a color identifier (ColorDesk) on a smartphone.

[0093] The specific steps are as follows:

[0094] Under the illumination of 365nm UV lamp, in the presence of different concentrations of DOX, a smartphone was used to take pictures of the fluorescence sensing system. As the concentration of DOX increases, the color of the sensing system shows a continuous evolution of gradual quenching. Therefore, after capturing a series of fluorescence images (such as Figure 11 e) After that, the color signal of the photo was converted into color information (RGB value) using a color recognition application loaded in the smartphone, and the DOX concentration could be estimated by calculating the ratio of the green and blue channels. Then, a standard curve was drawn based on the change in the ratio of the green channel to the blue channel (G / B) relative to the DOX concentration, with the equation Y = 0.00221X + 0.91306, R 2 =0.997; and the LOD was determined to be 0.285 μM, with a good linear relationship. Figure 11 As shown in f.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of g-CNQDs / PAN nanofiber membrane in the detection of doxycycline residues in food, characterized in that: The g-CNQDs / PAN nanofiber membrane was prepared by the following method: Polyacrylonitrile was dissolved in DMF to obtain a polyacrylonitrile solution; g-CNQDs powder was then added to the polyacrylonitrile solution and stirred thoroughly to obtain a g-CNQDs / PAN electrospinning solution; and then electrospinning was performed to obtain a g-CNQDs / PAN nanofiber membrane. The g-CNQDs powder was prepared by the following method: Mix urea and sodium citrate and grind them into powder; heat the mixture to react; after the reaction is completed, wash the reaction product, centrifuge, collect the centrifugal precipitate, and dry it to obtain g-CNQDs powder; The mass ratio of urea to sodium citrate is 101:81; the heating reaction conditions are: heating to 180° C. and continuing the reaction for 1 hour; The method for detecting doxycycline residues in food using g-CNQDs / PAN nanofiber membrane is as follows: The sample solution to be tested is added dropwise to the g-CNQDs / PAN nanofiber membrane, or the g-CNQDs / PAN nanofiber membrane is immersed in the sample solution to be tested and reacted for 5 minutes. The g-CNQDs / PAN nanofiber membrane is then placed under 365nm ultraviolet light, and the fluorescence of the system is photographed using a smartphone. The color signal of the photo is then converted into color information RGB value using a color identifier loaded in the smartphone, and the ratio G / B value of the green and blue channels is calculated. The G / B value is substituted into the standard curve to obtain the doxycycline concentration; the standard curve is a concentration-G / B value standard curve constructed with the doxycycline concentration as the horizontal coordinate and the G / B value as the vertical coordinate.

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