CN113150772A

CN113150772A - Zn/H2aip fluorescent probe, preparation and application thereof in detection of tetracycline antibiotics

Info

Publication number: CN113150772A
Application number: CN202110389888.2A
Authority: CN
Inventors: 王素华; 陈宏霞; 彭俊翔; 余龙; 孙明泰
Original assignee: Guangdong University of Petrochemical Technology
Current assignee: Guangdong University of Petrochemical Technology
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2021-07-23
Anticipated expiration: 2041-04-12
Also published as: CN113150772B

Abstract

The invention relates to a Metal Organic Frameworks (MOFs) fluorescent probe for detecting tetracycline antibiotics (TCs) and a preparation method thereof, and simultaneously comprises the steps of providing a fluorescence integration method and designing, analyzing and identifying an application program of the whole pollution degree of the tetracycline antibiotics. The fluorescent probe is prepared from 5-amino isophthalic acid (H)₂aip) is a stable three-dimensional framework organic ligand, and the transition metal Zn (II) is used as a metal source to synthesize fluorescent probes with unique response to TCs. Meanwhile, based on a fluorescence integration method, the method is not limited to identifying single antibiotics, the pollution degree of TCs can be quantified through a TCs Analysis application program, and a new method is provided for measuring the whole pollution degree of TCs through precise data processing and intelligent Analysis, so that the method is suitable for portable Analysis and detection.

Description

Zn/H2aip fluorescent probe, preparation and application thereof in detection of tetracycline antibiotics

Technical Field

The invention belongs to the technical field of antibiotic detection and analysis, and particularly relates to synthesis of a fluorescent probe for identifying Tetracycline (TCs), a fluorescence integration method and an application program for identifying tetracycline antibiotics by using the fluorescence integration method.

Background

Metal-organic frameworks (MOFs) are ordered combinations of organic ligands and metal ions, also known as porous coordination polymers. The MOFs have large internal specific surface area, a multi-topology network structure, low density, uniform cavity, permanent porosity, acid resistance, alkali resistance and high thermal stability, so that the MOFs become a widely explored novel supramolecular research material and are applied to multiple fields such as gas separation, catalysis, drug delivery, optics, electronic sensing and the like, 5-amino isophthalic acid is an excellent organic synthesis intermediate in the MOFs, two carboxylic acid groups at

positions

1 and 3 have abundant coordination and conjugation, and an infinite structure compound can be formed with metal ions through covalent connection, for example, a porous metal-organic framework consisting of 5-amino isophthalic acid is used for detecting nitro explosives and antibiotics. However, the use of fluorescence detection based on the induction of the aggregation of MOFs is still in its infancy. Several examples have been reported, such as metal organic frameworks enhancing aggregation-induced fluorescence of aureomycin and detection applications; multi-responsive aggregation-inducing active tetraphenylethylene-functional salicylaldehyde-p-Zn²⁺And CO₃ ^2-And (6) detecting. Therefore, the analysis method based on the fluorescent MOFs has outstanding advantages in the aspects of sensitivity and selectivity, has great research value in the application of environment and biological imaging, and has attractive prospect in designing a multifunctional chemical sensor capable of detecting TCs with high selectivity and sensitivity. All of the above reported MOFs sensors have no specific distinction between multiple cognate analytes, which is the most challenging part of designing sensors. TCs are often used as drugs and feed additives for treating human infectious diseases, are prone to produce large amounts of residues and are found in food. Reported detection methods include electrochemistry, ultraviolet-visible spectroscopy, and high performance liquid chromatography. TCs have similar chemical structures, so it is difficult to clearly distinguish tetracycline antibiotics such as TC, OTC, DOX, and CTC using probes in the same environment.

Disclosure of Invention

The invention relates to Zn/H₂aip fluorescent probe, preparation and application thereof in detecting tetracycline antibiotics. The method is applied to a fluorescence integration method by synthesizing a fluorescent probe for identifying Tetracycline (TCs), and is also applied to the identification of tetracycline antibiotics by using the fluorescence integration method. The specific technical scheme is as follows:

Zn/H as defined in the invention₂aip the preparation method of the fluorescent probe comprises the following steps:

taking 0.05g of ZnO, 0.08-0.12g of 5-amino isophthalic acid and 6-10mL of DMF, carrying out ultrasonic treatment on the mixture, placing the mixture into a reaction kettle for reaction, sequentially washing the mixture with DMF (N, N-dimethylformamide) and absolute ethyl alcohol, then carrying out vacuum drying in an oven at 50-70 ℃, and grinding the dried mixture for later use.

In the above preparation method, preferably, the reaction is carried out in a reaction kettle for 48 hours at a reaction temperature of 120 ℃.

The preparation method can obtain Zn/H₂aip fluorescent probe, the Zn/H₂aip application of fluorescent probe in detecting tetracycline antibiotics.

The application can adopt the following method steps: preparing a fluorescent probe into a 1mg/mL stock solution, adding the stock solution into a Tris-HCl buffer solution with the pH value of 8 and the concentration of 10mM, sequentially adding tetracycline antibiotics with different concentrations, recording a fluorescence spectrum, and establishing a linear relation between the fluorescence intensity and the concentration of TCs. And performing fluorescence detection on the sample to be detected, and calculating to obtain the concentration of TCs in the corresponding sample according to the linear relation between the fluorescence intensity and the concentration of TCs.

The application can also adopt the following method steps: preparing a fluorescent probe into a 1mg/mL stock solution, quantitatively adding the stock solution into 10mM Tris-HCl buffer solution with the pH value of 8, sequentially adding tetracycline antibiotics with different concentrations, recording a fluorescence spectrum, and performing integral evaluation on the area under the curve from a specified position to a position where a peak is formed in the fluorescence spectrum (the integral evaluation can be realized by common software such as Matlab and the like) to obtain an integral area and TCs concentration, thereby forming an integral area-concentration relation curve. And (3) performing fluorescence detection analysis on the sample to be detected, and calculating to obtain the concentration of TCs in the corresponding sample according to the integral area-concentration relation curve. Meanwhile, an application program 'TCs Analysis' can be designed to display the pollution degree of TCs in the sample in real time on a terminal device interface such as a mobile phone app application interface and a computer interface. The application program 'TCs Analysis' records the relevant data of the integrated area-concentration relation curve (txt note file and the like), when the fluorescence data obtained by the sample fluorescence detection is recorded in the application program, the Analysis instruction is started, and after the internal calculation of the program, a new interface is popped up to display the pollution degree of TCs in real time: XX-XX. mu.M "data.

The tetracycline antibiotics of the invention include but are not limited to the following drugs and mixtures thereof- - - -TC: a tetracycline; OTC: oxytetracycline; DOX: doxycycline; CTC: chlortetracycline, and the like.

The invention selects 5-amino isophthalic acid (H)₂aip) as organic ligands for stable three-dimensional framework MOFs, transition metal Zn (II) as metal source, synthesizing probes with unique response to TCs and named [ Zn/H ]₂aip]. In the present invention, the probe [ Zn/H ]₂aip]The three-dimensional structure of (a) was analyzed by SEM, XRD, FITR, TG, while fluorescence spectroscopy, uv-vis spectroscopy, were used as qualitative and quantitative core analysis data for TCs. Meanwhile, the pollution quantitative Analysis of the TCs is established, the fluorescence integration method is applied, and the concentration range of the total pollution contained in the TCs of the sample can be quickly obtained through a 'TCs Analysis' application program, so that the excellent combination of intelligent processing and fluorescence detection is effectively realized. The fluorescence integration method provides a new method for quantifying the contamination level of TCs by designing a "TCs Analysis" application program, and quantifies the contamination level through sophisticated data processing and intelligent Analysis. The present invention synthesizes fluorescent probes that have unique responses to TCs. Meanwhile, based on a fluorescence integration method, the method is not limited to identifying single antibiotics, the pollution degree of TCs can be quantified through a TCs Analysis application program, and a new method is provided for measuring the whole pollution degree of TCs through data processing and intelligent Analysis, so that the method is suitable for portable Analysis and detection.

The fluorescent probe provided by the invention can be used for qualitatively and quantitatively detecting tetracycline family antibiotics, and the fluorescence can be obviously enhanced under ultraviolet light, so that the qualitative detection of TCs is realized; by establishing a linear relation between the fluorescence intensity and the concentration of TCs, the concentration of TCs can be quantitatively detected;

under the condition of synthesizing a fluorescent probe, the invention establishes a linear relation by using a fluorescence spectrum and uses an integral method for fitting, thereby realizing the effectiveness of data processing;

the invention quantifies the pollution degree of TCs by designing a 'TCs Analysis' application program, provides a new method, and measures the pollution degree through precise data processing and intelligent Analysis. The existing pollution quantification mainly depends on fluorescence intensity, the peak of a fluorescence spectrum cannot be very clear in fluorescence detection of mixed pollutants, the peak of the fluorescence intensity is difficult to select, corresponding errors can be brought, and the precise integration algorithm does not need to pay attention to the peak, so that the mentioned defects can be compensated. That is, the single tetracycline antibiotic pollutant can be quantitatively detected by the ratio fluorescence intensity-concentration method, but the peak of the fluorescence spectrum cannot be very clear when the fluorescence of the mixed pollutants (containing one or more tetracycline antibiotics) is detected, and it is difficult to determine the total pollution degree of the tetracycline antibiotics in the pollutants by the ratio fluorescence intensity-concentration method. In addition, a standard curve can be made through a wider concentration interval and antibiotic types and uploaded to the background of 'TCs Analysis' as a reference standard.

Drawings

FIG. 1 is a fluorescence spectrum and a linear relationship chart of the probe of the present invention and TC and OTC with different concentrations, wherein the figure is a visual effect chart before and after adding TC and OTC to the probe respectively;

FIG. 2 is a graph of fluorescence spectra and linear relationship between the probe of the present invention and DOX and CTC of different concentrations, wherein the graph is a graph of visualization effect before and after adding DOX and CTC to the probe;

FIG. 3 is a curve of an integral fit of probes of the invention to TCs;

FIG. 4 is an illustration of an Analysis interface for a "TCs Analysis" application to quantify the contamination level of TCs.

FIG. 5 is Zn/H of the present invention₂aip structural characterization of the probe material.

FIG. 6 is a spectrum of a selectivity study experiment.

Detailed Description

The invention relates to synthesis of a fluorescent probe for identifying tetracycline antibiotics, application of the fluorescent probe to a fluorescence integration method, and an application program for identifying the tetracycline antibiotics by using the fluorescence integration method. The invention is further described below with reference to the accompanying drawings and examples.

FIG. 1 is a fluorescence spectrum and a linear relationship diagram of the probe of the present invention and TC and OTC with different concentrations, wherein the excitation wavelength is 365nm, the emission wavelengths are 517nm and 520nm, respectively, and the figure is a visual effect diagram before and after adding TC and OTC to the probe (under a 365nm ultraviolet lamp); in the graphs A and C, the TCs concentration corresponding to each fluorescence curve is gradually increased from bottom to top; B. in the diagram D, the three columns at the lower right corner represent the change of the fluorescence color of the solution before and after the probe of the invention is added respectively- -in the diagram B, before the probe is added: the probe solution is blue, the TCs solution is earthy yellow, and after the probe solution is added: the mixed solution is light green; in panel D, before addition: the probe solution is blue, the TCs solution is earthy yellow, and after the probe solution is added: the mixed solution is light green;

FIG. 2 is a fluorescence spectrogram and a linear relation graph of the probe of the invention and DOX and CTC with different concentrations, wherein the excitation wavelength is 365nm, the emission wavelengths are 513nm and 525nm respectively, and the figure is a visual effect graph before and after the DOX and CTC are added to the probe (under a 365nm ultraviolet lamp); in the graphs A and C, the TCs concentration corresponding to each fluorescence curve is gradually increased from bottom to top; B. in the diagram D, the three columns at the lower right corner represent the change of the fluorescence color of the solution before and after the probe of the invention is added respectively- -in the diagram B, before the probe is added: the probe solution is blue, the TCs solution is earthy yellow, and after the probe solution is added: the mixed solution is light green; in panel D, before addition: the probe solution was blue, the TCs solution was dark green, and after addition: the mixed solution is light green;

FIG. 3 is an integral fit curve of probes of the present invention to TCs, the area under the curve from 436nm (designated position) to 700nm (end peak position) being evaluated integrally to obtain a mathematical equivalent and TCs concentration, forming an integral area-concentration relationship curve;

FIG. 4 is an illustration of an Analysis interface for a "TCs Analysis" application to quantify the contamination level of TCs, as shown, the main menu of the application "TCs Analysis" includes three sections (FIG. 4). The first part is for reading txt files. The second part is "analysis". Once the instruction is initiated, "TCs contamination level" is displayed in the new interface popped up after internal calculation of the program: XX-XX. mu.M ". The third part shows 'application information', operation process of the reminding program and the statement of the version.

FIG. 5 is Zn/H of the present invention₂aip structural characterization of the probe material. Wherein (A) Zn/H₂aip. (B) ZnO, Zn/H₂aip and H₂aip XRD analysis. (C) Zn/H₂aip TGA profile. (D) Zn/H₂aip and H₂aip infrared spectrum. Zn/H of the invention₂aip, the SEM shows a unique cubic structure and is not a common Zn coordinating polyhedron structure. And, 2 θ peaks at 31.74 °, 34.42 °, and 36.29 ° correspond to typical wurtzite structure Bragg peaks (100), (002), and (101) of ZnO. At the same time, the material shows new high intensity diffraction at 20.05 ° and 22.83 °. In the TG spectrum, the decomposition starts at 429 ℃ and the stabilization is reached at 486 ℃. FTIR strong carboxylic acid coordination stretching vibration at 1553cm^-1And 1363cm^-1。

FIG. 6 is a spectrum of a selectivity study experiment. (A) [ Zn/H ] in the Presence of DOX, CTC, OTC and TC (16. mu.M total)₂aip](0.05mg/ml) fluorescence spectrum. (B) Probe [ Zn/H ]₂aip]Selective studies (0.05mg/mL) on other antibiotics and metal ions in Tris (pH 8). TC, OTC, CTC, DOX, AMP (ampicillin), LPA (L-penicillamine), SMS (streptomycin sulfate), Ca²⁺、K⁺、Na⁺The concentrations were the same and 10. mu.M each. As seen, AMP (ampicillin), LPA (L-penicillamine), SMS (streptomycin sulfate), Ca²⁺、K⁺、Na⁺The presence of (c) does not affect the selected integral area of the invention (from 436nm to 700 nm),reflecting the effectiveness and feasibility of the invention for detecting tetracycline antibiotics.

Example 1

(1) Synthesis of fluorescent probes

A mixture of ZnO (0.05g) and 5-aminoisophthalic acid (0.1g) and 8mL of DMF was sonicated and placed in a 120 ℃ reaction vessel for 48 hours. Taking out, washing with DMF and anhydrous ethanol in sequence, vacuum drying in a 60 deg.C oven, and grinding to obtain Zn/H₂aip fluorescent probes. The probe material is prepared into a stock solution of 1mg/mL, and can be stored in a refrigerator for later use.

(2) Qualitative and quantitative determination of TCs

Probe of the invention Zn/H₂aip fluorescence response with TCs was performed in Tris-HCl buffer (10mM, pH 8). 100 μ L of Probe Zn/H₂aip (1mg/mL) was added to a cuvette containing 2mL of buffer, and incubation was carried out for 30 minutes to wait for a final concentration of 0.05mg/mL of probe, and then TCs were added to the solution. The final concentrations of TCs were 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 μ M, respectively. Recording the fluorescence spectrum from 385nm to 700nm with 365nm as the excitation wavelength, and taking I₅₁₇/I₄₀₃、I₅₂₀/I₄₀₃、I₅₁₃/I₄₀₃And I₅₂₅/I₄₀₃The ratio fluorescence intensities of TC tetracycline, OTC oxytetracycline, DOX doxycycline and CTC aureomycin are used as linear data of final quantitative analysis to obtain a ratio fluorescence intensity-concentration relation curve and a linear equation, such as fig. 1 and fig. 2.

(3) Application program "TCs Analysis" design

And intelligent analysis on the pollution degree is introduced to realize the identification of the pollution degree of the TCs. Different TCs with unknown concentrations are added into the prepared probe solution to obtain corresponding fluorescence spectra, and the fluorescence spectra are compared with a standard curve after integration treatment. Finally, the estimated contamination concentration is displayed on the newly created window.

The first step is the standard curve. Selecting TCs fluorescence spectrum of 0-28 μ M between 436nm and 700nm according to the fluorescence spectrograms shown in the figure 1 and the figure 2 in the step (2), and obtaining an integral area-concentration relation curve and a linear equation of the four tetracycline antibiotics fluorescence detection by an area integration method, wherein the integral area-concentration relation curve and the linear equation are shown in figure 3.

The second step is theoretical design. The method comprises the steps of carrying out fluorescence detection on a sample with unknown concentration to obtain a fluorescence spectrum, carrying out area integration on a fluorescence spectrum curve between 436nm and 700nm to obtain a mathematical equivalent value, namely an integrated area value, comparing the integrated area value with an integrated area-concentration relation standard curve of four tetracycline antibiotics to obtain concentration values corresponding to the integrated area value on the four standard curves, and taking out the highest concentration and the lowest concentration as a pollution range, namely obtaining an index of pollution degree.

The third step is the smart application. Meanwhile, according to data processing and the intelligent client, the obtained information is processed in the background and the result of intelligent operation is presented. Software installed on the desktop reads the txt file of the fluorescence spectrum and the concentration of contamination is displayed on the newly created window.

By means of an application named "TCs Analysis", the concentration range of the total contamination of TCs contained in the sample can be obtained quickly. The detection is based on automatic matching after an exact comparison with the standard curve.

Example 2

Doxycycline sample solutions were prepared and placed in 2mL of Tris-HCl buffer solution (10mM, pH 8) to a final concentration of 4 μ M. The integral area method is adopted for detection. First 100. mu.L of probe Zn/H₂aip (1mg/mL) was added to the above cuvette of 2mL Tris-HCl buffer, and incubation was performed for 30 minutes with a final concentration of 0.05mg/mL for the probe, fluorescence detection was performed at 365nm as the excitation wavelength, the fluorescence spectrum was recorded from 385nm to 700nm, and the area under the curve between 436nm and 700nm was integrated to obtain an area integral value, and then the concentration range of the TCs sample solution was calculated to be 4.01-12.12. mu.M based on the four standard curve readings as shown in FIG. 3. The application program "TCs Analysis" can directly display the "TCs pollution degree" on the interface: 4.01-12.12. mu.M ".

Example 3

A doxycycline sample solution was prepared and placed in 2mL of Tris-HCl buffer solution to a final concentration of 8. mu.M. The concentration of the TCs sample solution obtained by detection using the method described in example 2 was 8.05-21.4. mu.M. The application program "TCs Analysis" can directly display the "TCs pollution degree" on the interface: 8.05-21.4. mu.M ".

Example 4

A doxycycline sample solution was prepared and placed in 2mL of Tris-HCl buffer solution to a final concentration of 10. mu.M. The concentration of the TCs sample solution was 9.92-25.7. mu.M, as determined by the method described in example 2. The application program "TCs Analysis" can directly display the "TCs pollution degree" on the interface: 9.92-25.7. mu.M ".

The following table shows the corresponding data for different tetracycline antibiotics at different concentrations of the output interface.

Example 5

A mixed solution of four types of tetracycline (containing DOX, CTC, OTC and TC) was prepared and placed in 2mL of Tris-HCl buffer solution so that the final total concentration of the four types of tetracycline was 16. mu.M (4. mu.M each of DOX, CTC, OTC and TC). The fluorescence detection was performed by the integrated area method of the present invention as shown in FIG. 6 (A). First 100. mu.L of probe [ Zn/H ]₂aip](1mg/mL) was added to a cuvette containing 2mL of Tris-HCl buffer (10mM, pH 8), incubation was performed for 30 minutes waiting for a final concentration of 0.05mg/mL, and then the prepared TCs sample solution was added to the above solution, fluorescence detection was performed with 365nm as an excitation wavelength, a fluorescence spectrum was recorded from 385nm to 700nm, and an integrated area value was obtained by integrating an area under the curve between 436nm and 700 nm. The area integral of the curve was then taken and the concentration of TCs sample solution was obtained from the standard curve described in figure 3. The application program "TCs Analysis" can directly display the "TCs pollution degree" on the interface: 15.42-38.35. mu.M ".

Claims

1. Zn/H₂aip fluorescent probeThe preparation method is characterized by comprising the following specific steps:

taking 0.05g of ZnO, 0.08-0.12g of 5-amino isophthalic acid and 6-10mL of DMF, carrying out ultrasonic treatment on the mixture, placing the mixture into a reaction kettle for reaction, sequentially washing the mixture with DMF and absolute ethyl alcohol, then carrying out vacuum drying in an oven at 50-70 ℃, and grinding the dried mixture for later use.

2. The method according to claim 1, wherein the reaction is carried out in a reaction vessel for 48 hours at a temperature of 120 ℃.

3. Zn/H obtained by the production method according to claim 1 or 2₂aip fluorescent probes.

4. Zn/H according to claim 3₂aip application of fluorescent probe in detecting tetracycline antibiotics.

5. Use according to claim 4, characterized in that the following method steps are used: preparing a fluorescent probe into a 1mg/mL stock solution, adding the stock solution into a Tris-HCl buffer solution with the pH value of 8 and the concentration of 10mM, sequentially adding tetracycline antibiotics with different concentrations, recording a fluorescence spectrum, and establishing a linear relation between the fluorescence intensity and the concentration of TCs.

6. Use according to claim 4, characterized in that the following method steps are used: preparing a fluorescent probe into a 1mg/mL stock solution, adding the stock solution into 10mM Tris-HCl buffer solution with the pH value of 8, sequentially adding tetracycline antibiotics with different concentrations, recording a fluorescence spectrum, and performing area integration on a curve from a specified position to a position at which a peak is formed, so that an integrated area-concentration relation curve is formed by the obtained integrated area and the TCs concentration.

7. The application of claim 6, wherein the application program "TCs Analysis" is designed to display the contamination degree of TCs in the sample to be detected in real time on the terminal device interface; the application program 'TCs Analysis' records the relevant data of the integral area-concentration relation curve, when the fluorescence detection of the sample is carried out and the obtained fluorescence data is recorded in the application program, the Analysis instruction is started, and the TCs pollution degree data is displayed in real time after the internal calculation of the program is popped up in a new interface.