Preparation of a ratiometric fluorescent probe based on L / J-CDs and its application in the detection of cetirizine hydrochloride

Abstract

The invention discloses the preparation of a ratiometric fluorescent probe based on L / J-CDs and its application to the detection of cetirizine hydrochloride, belonging to the field of chemical synthesis and analysis technology. The ratiometric fluorescent probe is composed of a composite system of a carbon dot L-CDs solution emitting blue fluorescence and a carbon dot J-CDs solution emitting orange fluorescence. The addition of cetirizine hydrochloride molecules quenches the blue fluorescence of the L-CDs in the composite system, while the J-CDs serve as a reference signal and the fluorescence color does not change. Cross-reference correction is performed through the intensity ratio of two emission peaks at the same excitation wavelength, thereby achieving the effect of reducing errors and improving accuracy. In addition, the ratiometric fluorescent probe can also realize visual qualitative detection based on the naked eye under ultraviolet light and with the assistance of a smartphone.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis and analysis, and particularly relates to a ratiometric fluorescent probe based on L / J-CDs and a preparation method thereof. The fluorescent probe is used as a detection reagent for visual detection and quantitative detection of cetirizine hydrochloride. Background Art

[0002] Cetirizine hydrochloride (CTZ) is a nonsteroidal anti-inflammatory drug that relieves allergic reactions by interacting with H1 receptors and inhibiting the release of histamine molecules. It is also used to treat chronic urticaria. However, excessive use of cetirizine hydrochloride can cause adverse symptoms such as drowsiness, headaches, and cognitive impairment. Furthermore, cetirizine hydrochloride, which repeatedly enters the environment through various water circulation pathways, is a persistent organic pollutant that induces reactions in aquatic organisms and leads to aquatic ecological imbalances, including damage at the cellular level, altered enzyme activity during oxidative stress, and enhanced immunotoxicity.

[0003] Currently, instrumental techniques such as liquid chromatography-tandem mass spectrometry, high-performance liquid chromatography, capillary electrophoresis, and electrochemical sensors have been used for the detection of cetirizine hydrochloride. However, these methods suffer from long pretreatment times, high technical skills requirements, the need for dedicated equipment operation and maintenance, and high testing costs, limiting their application for rapid trace detection. Therefore, fluorescent probes, with their advantages of cost-effectiveness, rapid response, portability, and ability to perform in situ monitoring in various environments, have become candidates for real-time sample analysis.

[0004] Carbon dots (CDs) are unique zero-dimensional luminescent nanomaterials with an average diameter of less than 10 nm. They possess outstanding biocompatibility, optoelectronic properties, radiation stability, widespread availability, and low toxicity, and have been widely used in various fields. Heteroatom doping is an effective method for regulating the intrinsic properties of CDs. Nitrogen doping can improve the photoluminescence properties of CDs, such as increasing quantum yield and improving self-quenching by surface passivation.

[0005] Although a few papers have reported the application of single-emission CDs fluorescent nanomaterials as probes for the analytical detection of cetirizine hydrochloride, these few papers are based on measuring changes in a single fluorescence signal, which is easily affected by environmental and instrumental factors. Therefore, it is necessary to develop rapid and sensitive analytical methods for the detection of trace amounts of cetirizine hydrochloride in environmental water. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a preparation of a ratiometric fluorescent probe based on L / J-CDs and its application in the detection of cetirizine hydrochloride.

[0007] The rapid visual ratiometric fluorescent probe, which serves as the core of the probe system, achieves selective quantitative detection of cetirizine hydrochloride through the integrated design of L-CDs and J-CDs. It has good reproducibility and a wide linear range, and can achieve visual qualitative detection based on the naked eye under ultraviolet light and with the assistance of a smartphone. The addition of cetirizine hydrochloride causes the blue emission fluorescence of L-CDs at 445nm to be quenched, while the orange fluorescence emission of J-CDs at 578nm, which serves as a reference signal, remains almost unchanged, resulting in a clear ratiometric color change from lavender to orange in the probe detection system. Cross-reference correction is performed by the intensity ratio of the two emission peaks at the same excitation wavelength to achieve the effect of reducing errors and improving accuracy. This ratiometric fluorescent probe detection system provides a new method for constructing a visualization and quantitative system for the detection of trace drug residues.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a ratiometric fluorescent probe based on L / J-CDs, the method comprising the following steps:

[0010] (1) Synthesis of L-CDs: 1.50 g of sodium citrate and 0.78 g of acrylamide nitrogen dopant were dispersed in 30 mL of deionized water and ultrasonicated for 10 min until completely dissolved. The resulting mixture was transferred to a 50 mL Teflon reactor and then reacted at 200 °C for 3 h. After cooling to room temperature, the product was filtered to remove larger particles. Finally, the obtained solution was diluted 10 to 1000 times with deionized water and stored at 4 °C. The resulting solution is the nitrogen-doped L-CDs solution.

[0011] (2) Synthesis of J-CDs: 0.75 g of rhodamine B was ultrasonically dispersed in 30 mL of deionized water. The resulting solution was transferred to a 50 mL Teflon reactor and treated at 160°C for 2 h. After cooling to room temperature, the resulting product was centrifuged at 10,000 rpm for 10 min. After purification by dialysis and freeze-drying, the resulting powder was dispersed in 20 L of deionized water and stored at 4°C. The resulting solution was the J-CDs solution.

[0012] (3) Preparation of L / J-CDs ratio fluorescent probe: The L-CDs solution obtained in step (1) and the J-CDs solution obtained in step (2) were fully mixed at a volume ratio of 2:3 to 5:3 to obtain the L / J-CDs ratio fluorescent probe.

[0013] In step (1), the filter membrane is a 0.22 μm water-based microporous filter membrane.

[0014] Preferably, the final solution is diluted 100 times with deionized water.

[0015] In step (2), the dialysis purification is performed by filtering through a 0.22 μm water-based microporous filter membrane and dialysis purification using a dialysis bag with a molecular weight cut-off of 1000 Da.

[0016] Preferably, in step (3), the volume ratio of the L-CDs solution to the J-CDs solution is 1:1.

[0017] In the second aspect, the present invention provides a ratio fluorescent probe based on L / J-CDs, wherein the ratio fluorescent probe is prepared by the above method, and the ratio fluorescent probe is composed of a composite system of L-CDs emitting blue fluorescence and J-CDs emitting orange fluorescence.

[0018] In a third aspect, the present invention provides an application of a ratiometric fluorescent probe based on L / J-CDs in the preparation of a detection reagent, wherein the detection reagent is used for visual detection or quantitative detection of cetirizine hydrochloride.

[0019] The core of the present invention's visual and quantitative detection of cetirizine hydrochloride residues lies in the designed L / J-CDs ratiometric fluorescence probe detection system. The introduction of cetirizine hydrochloride into the L / J-CDs ratiometric fluorescence detection system quenches the blue fluorescence emission of the L-CDs at 445 nm, while the orange fluorescence emission of the J-CDs, serving as a reference signal, at 578 nm remains virtually unchanged. This results in a distinct ratiometric color change from lavender to orange in the probe detection system, enabling visual detection of cetirizine hydrochloride using the ratiometric fluorescence probe detection system. The cetirizine hydrochloride content in the sample is then determined by the degree of fluorescence change in the ratiometric fluorescence system.

[0020] A visual detection method for cetirizine hydrochloride residues based on an L / J-CDs ratiometric fluorescent probe comprises: taking 0.6 mL of the ratiometric fluorescent probe as a detection reagent and dropping it into a 5 mL plastic centrifuge tube (EP tube); adding 1 mL of PBS buffer solution; and then adding 2.4 mL of a cetirizine hydrochloride test solution; mixing and incubating in a water bath at a temperature of 20 to 65° C. for 0 to 15 minutes; and observing a significant fluorescence color change under ultraviolet light, thereby achieving visual detection of the cetirizine hydrochloride test solution;

[0021] The concentration of the buffer solution is 10 mM;

[0022] The pH range of the buffer solution is 3 to 9, preferably 3;

[0023] Preferably, the water bath temperature parameter is 25°C;

[0024] Preferably, the incubation time parameter is 1 min;

[0025] Fluorescence images and RGB values ​​were obtained using software under ultraviolet light source, and a relationship graph between the ratio of R to G and the concentration of cetirizine hydrochloride was established to assist in visual detection.

[0026] A method for quantitative detection of cetirizine hydrochloride residues based on an L / J-CDs ratio fluorescent probe comprises:

[0027] 0.6 mL of the L / J-CDs ratiometric fluorescent probe was dripped into a 5 mL plastic centrifuge tube (EP tube), 1 mL of PBS buffer solution was added, and then 2.4 mL of the cetirizine hydrochloride test solution was added. The mixture was mixed and incubated in a water bath at a temperature of 20 to 65°C for 0 to 15 minutes. The fluorescence emission spectrum of the solution was recorded in the range of 380 to 680 nm using an excitation wavelength of 355 nm. By establishing a linear relationship between the change in the fluorescence emission peak intensity ratio and the cetirizine hydrochloride concentration, quantitative detection of the cetirizine hydrochloride test solution was achieved.

[0028] The concentration of the buffer solution is 10 mM;

[0029] The pH range of the buffer solution is 3 to 9, preferably 3;

[0030] Preferably, the water bath temperature parameter is 25°C;

[0031] Preferably, the incubation time parameter is 1 min.

[0032] The fluorescence intensity ratio is (I 445 / I 578 )0 / (I 445 / I 578 ), specifically, (I 445 / I 578 ) is the intensity ratio of the double emission peaks of the fluorescence spectrum after adding different concentrations of cetirizine hydrochloride, (I 445 / I 578 )0 is the intensity ratio of the double emission peaks of the blank group.

[0033] Compared with existing detection technologies, the beneficial effects of the present invention are as follows:

[0034] (1) The ratiometric fluorescence probe detection system of the present invention detects cetirizine hydrochloride. Compared with other single-emission fluorescence detection methods, it effectively avoids the instability of monochromatic fluorescence intensity due to matrix effects, environmental factors and instrument factors, and has better fluorescence detection reproducibility.

[0035] (2) The present invention demonstrates the effect of visual qualitative detection by directly irradiating with ultraviolet light and analyzing the RGB values ​​of the fluorescence image using a smartphone.

[0036] (3) The present invention establishes a linear relationship between the fluorescence intensity ratio and the concentration of cetirizine hydrochloride through methodological research, thereby broadening the linear range of cetirizine hydrochloride content detection.

[0037] (4) The L / J-CDs ratio fluorescent probe prepared by the present invention has a simple preparation method, low material cost, is portable and responds quickly to cetirizine hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Transmission electron microscopy images of L-CDs (A) and J-CDs (B), and the insets are the particle size distribution under dynamic light scattering technology.

[0039] Figure 2 FT-IR infrared spectra of L-CDs (blue line) and J-CDs (pink line).

[0040] Figure 3 X-ray photoelectron spectroscopy (XPS) spectra of L-CDs ((A) full-range XPS spectrum, (B) high-resolution XPS spectrum of C1s, (C) high-resolution XPS spectrum of N1s, (D) high-resolution XPS spectrum of O1s).

[0041] Figure 4 X-ray photoelectron spectroscopy (XPS) patterns of J-CDs ((A) full-range XPS spectrum, (B) high-resolution XPS spectrum of C1s, (C) high-resolution XPS spectrum of N1s, (D) high-resolution XPS spectrum of O1s).

[0042] Figure 5 (A) Ex, Em fluorescence spectra and UV-visible absorption spectra of L-CDs, with the insets showing images under natural light (left) and UV light (right), respectively; (B) fluorescence emission spectra at an excitation wavelength of 295-385 nm; (C) Ex, Em fluorescence spectra and UV-visible absorption spectra of J-CDs, with the insets showing images under natural light (left) and UV light (right), respectively; (D) fluorescence emission spectra at an excitation wavelength of 520-570 nm.

[0043] Figure 6 Fluorescence spectrum (A) and corresponding chromaticity diagram (B) of the L / J-CDs ratio fluorescent probe for the detection of cetirizine hydrochloride under optimized conditions.

[0044] Figure 7Figure 3. Effect of the volume ratio of L-CDs to J-CDs in the ratiometric fluorescence probe on the ability of the ratiometric fluorescence detection system to recognize cetirizine hydrochloride. (A) When the volume ratio of L-CDs to J-CDs is 5:6, the fluorescence spectrum of the ratiometric fluorescence probe detection system changes before and after the addition of cetirizine hydrochloride. (B) Curve diagram showing the effect of different volume ratios of L-CDs to J-CDs on the fluorescence intensity ratio of cetirizine hydrochloride detected by the ratiometric fluorescence probe.

[0045] Figure 8 The effect of the pH value of the system on the ability of the ratio fluorescence probe detection system to recognize cetirizine hydrochloride; (A) When the pH is 5, the fluorescence spectrum of the ratio fluorescence probe detection system changes before and after the addition of cetirizine hydrochloride; (B) The effect of pH value on the fluorescence intensity ratio of the ratio fluorescence probe detecting cetirizine hydrochloride.

[0046] Figure 9 The effect of incubation temperature on the ability of the ratiometric fluorescent probe detection system to recognize cetirizine hydrochloride; (A) Changes in the fluorescence spectrum of the ratiometric fluorescent probe detection system before and after the addition of cetirizine hydrochloride at an incubation temperature of 65°C; (B) Curve diagram of the effect of incubation temperature on the fluorescence intensity ratio of cetirizine hydrochloride detected by the ratiometric fluorescent probe.

[0047] Figure 10 The fluorescence spectra of L / J-CD complex system with different concentrations of cetirizine hydrochloride (A) (the inset is the color change diagram under ultraviolet light) and the corresponding chromaticity diagram (B); (C) and (D) are the fluorescence intensity ratios of the ratiometric fluorescent probe L / J-CDs complex system (I 445 / I 578 )0 / (I 445 / I 578 ) and the concentration of cetirizine hydrochloride.

[0048] Figure 11 (A) Schematic diagram of smartphone color recognition when different concentrations of cetirizine hydrochloride are added to the L / J-CDs ratiometric fluorescent probe composite system; (B) is a graph showing the relationship between the color change (R / G) value of the ratiometric fluorescent probe solution and the concentration of cetirizine hydrochloride.

[0049] Figure 12 Selectivity and anti-interference diagram of the ratiometric fluorescent probe for the detection of cetirizine hydrochloride.

[0050] Figure 13 When L-CDs is used as a fluorescent probe alone, the fluorescence spectrum changes before and after the addition of cetirizine hydrochloride (A) and the corresponding chromaticity diagram (B).

[0051] Figure 14 When J-CDs is used alone as a fluorescent probe, the fluorescence spectrum changes before and after the addition of cetirizine hydrochloride. DETAILED DESCRIPTION

[0052] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention. The following description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0053] Example 1

[0054] The preparation steps of L / J-CDs ratio fluorescent probe are as follows:

[0055] (1) Preparation of L-CDs: 1.50 g of sodium citrate and 0.78 g of acrylamide nitrogen dopant were dispersed in 30 mL of deionized water and ultrasonicated for 10 min until completely dissolved. The resulting mixture was transferred to a 50 mL Teflon reactor and then reacted at 200 °C for 3 h. After naturally cooling to room temperature, the product was filtered through a 0.22 μm aqueous microporous filter membrane to remove larger particles. Finally, the obtained solution was diluted 100 times with deionized water and stored at 4 °C. The resulting solution was the nitrogen-doped L-CDs solution.

[0056] (2) Preparation of J-CDs: 0.75 g of rhodamine B was ultrasonically dispersed in 30 mL of deionized water. The resulting solution was transferred to a 50 mL Teflon reactor and treated at 160 °C for 2 h. After naturally cooling to room temperature, the obtained deep red product was centrifuged at 10,000 rpm for 10 min. After filtration through a 0.22 μm aqueous microporous filter membrane and purification by dialyzation with a 1000 Da molecular weight cutoff dialysis bag and freeze-dried, the obtained powder was dispersed in 20 L of deionized water and stored at 4 °C. The resulting solution was the J-CDs solution.

[0057] (3) Preparation of L / J-CDs ratio fluorescent probe: The L-CDs solution obtained in step (1) and the J-CDs solution obtained in step (2) were fully mixed at a volume ratio of 1:1 to obtain the L / J-CDs ratio fluorescent probe.

[0058] Considering that the detection sensitivity of the probe to the analyte is related to its own properties, FT-IR, TEM, DLS, and XPS were used to determine the morphology and elemental composition of the components of the ratio fluorescence probe system. Figure 1 It can be seen that the prepared L-CDs and J-CDs are uniformly dispersed, with average particle sizes of 1.07nm and 2.45nm respectively. Figure 2 It can be seen that there are active functional groups such as amino and hydroxyl groups on the surfaces of L-CDs and J-CDs. Figure 3 It can be seen that L-CDs are composed of C, N, and O, and there are amino and pyrrolic nitrogen on the surface, which proves that nitrogen element is successfully doped in the preparation of L-CDs. Figure 4 It also confirms the structural characteristics of J-CDs in the infrared spectrum.

[0059] In addition, the spectral characteristics of the ratiometric fluorescent probe system components were studied by UV-vis and fluorescence spectroscopy. From the fluorescence excitation and emission spectra of L-CDs, it can be seen that at the optimal excitation wavelength of 355nm, the strongest emission peak is at 445nm ( Figure 5 A), and also exhibited strong blue fluorescence under 365nm ultraviolet light source ( Figure 5 A illustration), Figure 5 B shows that the position of the fluorescence emission peak of L-CDs does not change with the change of excitation wavelength, indicating that it has no excitation dependence, which indicates that the size distribution and surface state of L-CDs are uniform. From the fluorescence excitation and emission spectra of J-CDs, it can be seen that under the optimal excitation wavelength of 555nm, the strongest emission peak is at 578nm ( Figure 5 C), and also exhibited strong orange fluorescence under 365nm ultraviolet light source ( Figure 5 C illustration), Figure 5 D shows that the J-CDs have no excitation dependence, which indicates that the size distribution and surface state distribution of J-CDs are also uniform.

[0060] The above optical property research and morphology characterization results can prove the successful preparation of the ratiometric fluorescent probe system components L-CDs and J-CDs, and they have excellent optical properties.

[0061] Example 2

[0062] 0.6 mL of the L / J-CDs ratiometric fluorescent probe prepared in step (3) of Example 1 was added dropwise to a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 3) was added, and then 2.4 mL of cetirizine hydrochloride solution (concentration in the EP tube was 200 μM) was added. The mixture was mixed and incubated in a water bath at 25°C for 1 min. The fluorescence emission spectrum of the solution in the range of 380 to 680 nm was recorded using an excitation wavelength of 355 nm. Figure 6 It can be seen that the fluorescence emission peak at 445 nm is significantly reduced after the addition of cetirizine hydrochloride.

[0063] Example 3

[0064] The L-CDs synthesized in step (1) of Example 1 and the J-CDs synthesized in step (2) were fully mixed at a volume ratio of 5:6, and 0.6 mL was added dropwise to a 5 mL EP tube. 1 mL of PBS buffer solution (10 mM, pH = 3) was added, followed by 2.4 mL of cetirizine hydrochloride solution (concentration of 200 μM). The mixture was mixed and incubated in a water bath at 25°C for 1 minute. The fluorescence emission spectrum of the solution in the range of 380 to 680 nm was recorded at an excitation wavelength of 355 nm. The effect of the volume ratio of L-CDs to J-CDs on the ability of the ratiometric fluorescent probe detection system to recognize cetirizine hydrochloride was also investigated. Figure 7 It can be seen that the recognition effect of cetirizine hydrochloride is best when L-CDs and J-CDs are mixed in a volume ratio of 1:1 in the ratiometric fluorescent probe composite system.

[0065] Example 4

[0066] 0.6 mL of the L / J-CDs ratiometric fluorescent probe prepared in step (3) of Example 1 was added dropwise to a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 5) was added, and then 2.4 mL of cetirizine hydrochloride solution (concentration of 200 μM) was added. The mixture was mixed and incubated in a water bath at 25°C for 1 minute. The fluorescence emission spectrum of the solution in the range of 380 to 680 nm was recorded using an excitation wavelength of 355 nm. The effect of pH value on the ability of the ratiometric fluorescent probe detection system to recognize cetirizine hydrochloride was also investigated. Figure 8 It can be seen that the ratiometric fluorescent probe L / J-CDs composite system has the best recognition effect on cetirizine hydrochloride at pH = 3.

[0067] Example 5

[0068] 0.6 mL of the L / J-CDs ratiometric fluorescent probe prepared in step (3) of Example 1 was added dropwise to a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 3) was added, and then 2.4 mL of cetirizine hydrochloride solution (concentration of 200 μM) was added. The mixture was mixed and incubated in a water bath at 65°C for 1 minute. The fluorescence emission spectrum of the solution in the range of 380 to 680 nm was recorded using an excitation wavelength of 355 nm. The effect of different incubation temperatures on the ability of the ratiometric fluorescent probe detection system to recognize cetirizine hydrochloride was also investigated. Figure 9 It can be seen that the ratiometric fluorescent probe L / J-CDs composite system has the best recognition effect on cetirizine hydrochloride at an incubation temperature of 25°C.

[0069] Example 6

[0070] The L / J-CDs ratiometric fluorescent probe solution was used to detect cetirizine hydrochloride in the following steps:

[0071] (1) 0.6 mL of the L / J-CDs ratio fluorescent probe prepared in step (3) of Example 1 was dripped into a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 3) was added, and then 2.4 mL of cetirizine hydrochloride solution of different concentrations (concentration range is 0.05-300 μM) was added. The mixture was mixed and incubated in a water bath at 25°C for 1 min. The fluorescence emission spectrum of the solution in the range of 380-680 nm was recorded with an excitation wavelength of 355 nm. The fluorescence emission peak intensity ratio (I 445 / I 578 )0 / (I 445 / I 578 ) changes and the concentration of cetirizine hydrochloride to achieve quantitative detection of cetirizine hydrochloride.

[0072] (2) Drawing of standard curve: After adding different concentrations of cetirizine hydrochloride solution to the composite system of L / J-CDs ratio fluorescence probe, the fluorescence intensity was tested. The results showed that the blue fluorescence at 445 nm was gradually quenched, and the orange fluorescence at 578 nm remained basically unchanged. By establishing the fluorescence emission peak intensity ratio (I 445 / I 578 )0 / (I 445 / I 578 ) and the concentration of cetirizine hydrochloride, the quantitative detection of cetirizine hydrochloride can be achieved. Figure 10 It can be seen from the fluorescence spectrum that with the increase of the concentration of cetirizine hydrochloride added, the fluorescence emission peak intensity ratio (I 445 / I 578 )0 / (I 445 / I 578 ) and cetirizine hydrochloride concentration, with a calculated detection limit of 10.2 nM. Compared with previously reported single-emission carbon dot N-CDs detection systems for cetirizine determination, the linear range was broadened from 0.08-48 μM to 0.05-60 μM and 60-290 μM.

[0073] Example 7

[0074] The L / J-CDs ratiometric fluorescent probe solution was used for the visual detection of cetirizine hydrochloride. The steps are as follows:

[0075] (1) Construction of a smartphone-assisted visualization detection platform: including enzyme label strips, UV dark box, UV lamp and smartphone.

[0076] (2) Visual Detection of Cetirizine Hydrochloride: Using the preferred detection conditions of step (1) of Example 6, the ratiometric fluorescent probe solution and different concentrations of cetirizine hydrochloride were mixed and titrated onto enzyme-labeled strips. Fluorescence color changes were visually observed in a dark environment. Fluorescence images were captured using a smartphone, and the RGB values ​​of the images could be further identified.

[0077] (3) Drawing of the standard curve: In a dark environment, the RGB value of the fluorescent color was further identified by irradiating the fluorescent image with a 365nm UV lamp and capturing it with a smartphone. It was observed that the R / G value showed a linear relationship with the concentration of cetirizine hydrochloride ( Figure 11 ).

[0078] Example 8

[0079] L / J-CDs ratiometric fluorescent probe solution for specific detection of cetirizine hydrochloride

[0080] Using the preferred detection conditions in step (1) of Example 6, 200 μM of CTZ, promethazine hydrochloride, tobramycin sulfate, chlorpheniramine, fluconazole, fluoxetine hydrochloride and some amino acids (such as phenylalanine Phe, glycine Gly, threonine Thr, histidine His, arginine Arg, leucine Leu, methionine Met) were added to the ratiometric fluorescent probe detection system. Only CTZ significantly increased (I 445 / I 578 )0 / (I 445 / I 578 ) values. When different substrates (200 μM) coexisted with CTZ (200 μM), other substrates had almost no effect on the detection of CTZ. 500 μM of metal cations (Cd 2+ 、Al 3+ , K + Mg 2+ 、Zn 2+ 、Na +) and common anions (SO4 2- 、NO3 - 、Cl - ), fluorescence intensity ratio (I 445 / I 578 )0 / (I 445 / I 578 ) remains basically unchanged, such as Figure 12 The results showed that the system had good selectivity and anti-interference ability for cetirizine hydrochloride.

[0081] Comparative Example 1

[0082] L-CDs as a fluorescent probe for the detection of cetirizine hydrochloride

[0083] (1) Preparation of L-CDs: The preparation process of this step is the same as that of Example 1.

[0084] (2) 0.3 mL of L-CDs was dropped into a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 3), 0.3 mL of deionized water, and 2.4 mL of cetirizine hydrochloride solution (concentration of 200 μM) were added, and the mixture was mixed and incubated in a water bath at 25°C for 1 min. The fluorescence emission spectrum of the solution was recorded in the range of 380 to 680 nm using an excitation wavelength of 355 nm. Figure 13 It can be seen that the fluorescence emission intensity is obviously quenched after the addition of cetirizine hydrochloride.

[0085] Comparative Example 2

[0086] J-CDs as a fluorescent probe for the detection of cetirizine hydrochloride

[0087] (1) Preparation of J-CDs: The preparation process of this step is the same as that of Example 1.

[0088] (2) 0.3 mL of J-CDs was dropped into a 5 mL EP tube, 1 mL of PBS buffer solution (10 mM, pH = 3), 0.3 mL of deionized water, and 2.4 mL of cetirizine hydrochloride solution (concentration of 200 μM) were added, mixed and incubated in a water bath at 25°C for 1 min, and the fluorescence emission spectrum of the solution in the range of 380 to 680 nm was recorded using an excitation wavelength of 355 nm. Figure 14 It can be seen that the fluorescence emission intensity does not change substantially after the addition of cetirizine hydrochloride.

[0089] The present invention investigates the results of using the fluorescent probes prepared in Example 2, Comparative Example 1 and Comparative Example 2 for the detection of cetirizine hydrochloride. Figure 6 and Figure 13 It can be seen that after adding cetirizine hydrochloride, the fluorescence emission intensity at 445nm will be significantly reduced with the addition of cetirizine hydrochloride when L-CDs is used as a single fluorescent probe or when a ratio fluorescent probe is used. Figure 13 The fluorescence color change in the ratio fluorescence probe detection system shown in the CIE chromaticity diagram is larger than that of the single-emission fluorescence probe, which is conducive to visual resolution. Figure 14 The fluorescence emission intensity did not change significantly, and J-CDs could not be used alone for the detection of cetirizine hydrochloride.

[0090] Obviously, the use of L / J-CDs ratiometric fluorescent probe for the detection of cetirizine hydrochloride is conducive to the development of a simple method for visually assisted detection.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Persons skilled in the art may, without departing from the principles of the present invention, make minor improvements based on the above-mentioned technical content, and such improvements and variations are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a ratiometric fluorescent probe based on L / J-CDs, characterized in that: The method comprises the following steps: (1) Synthesis of L-CDs: 1.50 g of sodium citrate and 0.78 g of acrylamide nitrogen dopant were dispersed in 30 mL of deionized water and ultrasonicated for 10 min until completely dissolved. The resulting mixture was transferred to a 50 mL Teflon reactor and then reacted at 200 °C for 3 h. After cooling to room temperature, the product was filtered to remove larger particles. Finally, the obtained solution was diluted 10 to 1000 times with deionized water and stored at 4 °C. The resulting solution is the nitrogen-doped L-CDs solution. (2) Synthesis of J-CDs: 0.75 g of rhodamine B was ultrasonically dispersed in 30 mL of deionized water. The resulting solution was transferred to a 50 mL Teflon reactor and treated at 160°C for 2 h. After cooling to room temperature, the resulting product was centrifuged at 10,000 rpm for 10 min. After purification by dialysis and freeze-drying, the resulting powder was dispersed in 20 L of deionized water and stored at 4°C. The resulting solution was the J-CDs solution. (3) Preparation of L / J-CDs ratio fluorescent probe: The L-CDs solution obtained in step (1) and the J-CDs solution obtained in step (2) were fully mixed at a volume ratio of 2:3 to 5:3 to obtain the L / J-CDs ratio fluorescent probe.

2. The method for preparing a ratiometric fluorescent probe based on L / J-CDs according to claim 1, characterized in that: The filter membrane in step (1) is a 0.22 μm water-based microporous filter membrane.

3. The method for preparing a ratiometric fluorescent probe based on L / J-CDs according to claim 1, characterized in that: The dilution factor in step (1) is 100 times.

4. The method for preparing a ratiometric fluorescent probe based on L / J-CDs according to claim 1, wherein: The dialysis purification in step (2) is performed by filtering through a 0.22 μm microporous filter membrane and dialysis purification using a dialysis bag with a molecular weight cut-off of 1000 Da.

5. The method for preparing a ratiometric fluorescent probe based on L / J-CDs according to claim 1, characterized in that: In step (3), the volume ratio of the L-CDs solution to the J-CDs solution is 1:

1.

6. A ratiometric fluorescent probe based on L / J-CDs, characterized in that: The ratio fluorescent probe is prepared by the method according to any one of claims 1 to 5, and is composed of a composite system of L-CDs emitting blue fluorescence and J-CDs emitting orange fluorescence.

7. Use of the L / J-CDs-based ratiometric fluorescent probe according to claim 6 in the preparation of a detection reagent, characterized in that: The detection reagent is used for visual detection or quantitative detection of cetirizine hydrochloride.

8. The use according to claim 7, characterized in that The application includes the following steps: taking 0.6 mL of the ratio fluorescent probe as a detection reagent and dropping it into a 5 mL plastic centrifuge tube, adding 1 mL of PBS buffer solution, and then adding 2.4 mL of the cetirizine hydrochloride test solution, mixing and incubating for 0 to 15 minutes in a water bath temperature of 20 to 65° C., and observing obvious fluorescent color changes under ultraviolet light to achieve visual detection of the cetirizine hydrochloride test solution.

9. The use according to claim 8, characterized in that Fluorescence images and RGB values ​​were obtained using software under ultraviolet light source, and a relationship graph between the ratio of R to G and the concentration of cetirizine hydrochloride was established to assist in visual detection.

10. The use according to claim 7, characterized in that 0.6 mL of the ratiometric fluorescent probe is taken as a detection reagent and dropped into a 5 mL plastic centrifuge tube, 1 mL of PBS buffer solution is added, and then 2.4 mL of a cetirizine hydrochloride test solution is added. The mixture is mixed and incubated for 0 to 15 minutes in a water bath at a temperature of 20 to 65° C., and the fluorescence emission spectrum of the solution in the range of 380 to 680 nm is recorded with an excitation wavelength of 355 nm. By establishing a relationship between the change in the fluorescence emission peak intensity ratio and the cetirizine hydrochloride concentration, quantitative detection of the cetirizine hydrochloride test solution is achieved.

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