Method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system

Abstract

The present invention discloses a method for detecting antibiotics based on an upconversion nanoparticle - silk fibroin nanoprobe system. The method includes preparing upconversion nanoparticles synthesized from lanthanide elements, coating water - soluble silica on the upconversion nanoparticles, followed by washing, collecting, and vacuum drying. Fibrous silk fibroin is dissolved, filtered, dialyzed, and concentrated to obtain an aqueous solution of silk fibroin. An antibiotic and a chromogenic agent are mixed to obtain an aqueous solution of a chromogenic agent - antibiotic complex. The upconversion nanoparticles, the aqueous solution of silk fibroin, and the aqueous solution of the chromogenic agent - antibiotic complex are mixed to obtain upconversion nanoparticles coated with silk fibroin and antibiotics. The obtained upconversion nanoparticles are deionized and recovered, and then the fluorescence intensity is measured. The concentration of the antibiotic to be detected is judged according to the fluorescence intensity to achieve the detection of antibiotics. The process of the present invention is simple, easy to operate, low in cost, and high in sensitivity, and has broad application prospects in the fields of quantitative detection of pollutants, antibiotic drugs, etc.

Description

Technical Field

[0001] The present invention discloses an antibiotic detection method belonging to the technical field of chemical analysis and detection, in particular to a method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system. Background Art

[0002] Antibiotics can be used to treat bacterial infections and are widely used in fields such as animal husbandry and fishery. In recent years, the problem of bacterial drug resistance caused by the abuse of antibiotics cannot be ignored. Accurately detecting the antibiotics present in soil and water resources is of great significance for the assessment of bacterial drug resistance. In the analysis and detection of antibiotics, currently commonly used methods include microbiological assay, mass spectrometry (MS), and chromatography (including thin-layer chromatography TLC, high-performance liquid chromatography HPLC, gas chromatography GC, capillary electrochromatography CEC), etc. The microbiological assay does not require the separation of multiple active components of antibiotics, but has disadvantages such as cumbersome steps, high cost, and large errors. Chromatography separates first and then detects, with advantages such as sensitivity, accuracy, and fast analysis speed. Combining with mass spectrometry can also improve selectivity and sensitivity. However, instruments such as HPLC, GC, and MS are expensive and have high equipment maintenance costs, which greatly limit their popularization. Traditional spectrophotometry is economical and practical, but has disadvantages such as high detection limits and many interfering factors.

[0003] Therefore, the prior art lacks a method that is simple in steps, low in cost, sensitive and accurate for effectively detecting antibiotics. Summary of the Invention

[0004] In order to overcome the above deficiencies existing in the existing antibiotic detection, the present invention proposes a method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system, which has the advantages of simple steps, low cost, low detection limit, sensitivity and accuracy, and provides a new method for antibiotic detection.

[0005] Based on the background art situation, the present invention uses the characteristics of silk fibroin being easy to adsorb and denature. After mixing upconversion nanoparticles (UCNP), silk fibroin (SF), and chromogenic agent-antibiotic (CA-ABX), ethanol is added to induce the denaturation of silk fibroin, forming upconversion nanoparticles coated with a silk fibroin / chromogenic agent-antibiotic blend film (UCNP@SF / CA-ABX). Fixing the input amounts of upconversion nanoparticles and silk fibroin, as the concentration of antibiotics in the chromogenic agent-antibiotic complex increases, the fluorescence intensity of the emission peak of the upconversion nanoparticles at the wavelength overlapping with the absorption peak of the chromogenic agent-antibiotic decreases.

[0006] The technical solution of the present invention is as follows:

[0007] (1) Synthesize upconversion nanoparticles (UCNP) doped with lanthanide elements;

[0008] (2) The upconversion nanoparticles obtained in step (1) are dissolved in cyclohexane, and then ammonia water is added and stirred for 30 min. Tetraethoxysilane (TEOS) is slowly added to react to form water-soluble silica, which coats the upconversion nanoparticles. Then, it is washed successively with a mixed solution of cyclohexane and absolute ethanol, absolute ethanol, and deionized water, collected with deionized water after washing, and dried in vacuum after collection.

[0009] (3) The fibrous silk fibroin formed after degumming the silkworm cocoons is dissolved, and after filtration, dialysis, and concentration in sequence, an aqueous solution of silk fibroin (SF) is obtained, and the concentration of silk fibroin in the aqueous solution of silk fibroin is adjusted to 10 mg / mL.

[0010] (4) The antibiotic to be detected and the chromogenic agent are mixed to prepare an aqueous solution of the chromogenic agent-antibiotic complex (CA-ABX).

[0011] (5) The upconversion nanoparticles obtained in step (2) are dissolved in deionized water and ultrasonically dispersed, the aqueous solution of silk fibroin in step (3) and the aqueous solution in step (4) are added, and after stirring, an equal volume of the inducing reagent is added to promote the self-assembly of silk fibroin SF, obtaining upconversion nanoparticles coated with silk fibroin and antibiotics (UCNP@SF / CA-ABX).

[0012] (6) The upconversion nanoparticles coated with silk fibroin and antibiotics are centrifuged and recovered with deionized water. After recovery, the fluorescence intensity is measured under the excitation light, and the concentration of the antibiotic to be detected is judged according to the fluorescence intensity to achieve antibiotic detection.

[0013] In step (1) described above, the upconversion nanoparticles are synthesized by the high-temperature thermal precipitation method or the thermal decomposition method, but are not limited thereto.

[0014] In step (1) described above, the size of the synthesized upconversion nanoparticles is 10 - 100 nm.

[0015] In step (2) described above, the reaction time for tetraethoxysilane (TEOS) to react to form water-soluble silica is 6 - 24 h.

[0016] In step (2) described above, the temperature of vacuum drying is 40 - 100 °C, and the time of vacuum drying is 4 - 48 h.

[0017] In step (4) described above, the antibiotics used include cephalosporin antibiotics such as ceftriaxone, cefadroxil, cefazolin; quinolone antibiotics such as pefloxacin mesylate, gatifloxacin, levofloxacin hydrochloride, ofloxacin, norfloxacin, ciprofloxacin; macrolide antibiotics such as roxithromycin, erythromycin, oleandomycin, spiramycin, tylosin, erythromycin, ethyl succinate, erythromycin ethylsuccinate, clarithromycin, azithromycin; other antibiotics such as clotrimazole, ampicillin sodium, etc.

[0018] In step (4), the color reagent is a color reagent such that the absorption peak of the color reagent - antibiotic complex after the combination of the antibiotic and the color reagent overlaps with the emission peak of the upconversion nanoparticles.

[0019] When the antibiotic is a cephalosporin antibiotic, the color reagent can be p - benzoquinone, acceptor 7,7,8,8 - tetracyano - p - benzoquinone (TCNQ), 2,4 - dinitrophenol, phenylfluorone;

[0020] When the antibiotic is a quinolone antibiotic, the color reagent can be 7,7,8,8 - tetracyano - p - benzoquinone (TCNQ) and tetrafluorobenzoquinone (TFQ), p - nitrophenol, chloranilic acid, alizarin red, 2,3 - dicyano - 5,6 - dichloro - p - benzoquinone (DDQ);

[0021] When using a macrolide antibiotic, the color reagent can be alizarin red, p - dimethylaminobenzaldehyde, xanthydrol, sodium alizarin sulfonate, quinizarin, pyrene, cresol red, chloranilic acid;

[0022] When using other types of antibiotics, the color reagent used is included within the above range.

[0023] After the combination of the antibiotic and the color reagent, the absorption peak shifts to the visible light region. The upconversion nanoparticles can be adjusted in size and type. By selecting different types of upconversion nanoparticles, the absorption peak of the selected antibiotic can be made to overlap with the emission peak of the UCNP.

[0024] In step (4), the color reagent can be changed, and the corresponding antibiotic can also be adjusted, as long as it meets the condition that the absorption peak of the color reagent - antibiotic complex overlaps with the emission peak of the upconversion nanoparticles.

[0025] In step (5), the rotation speed of stirring is 100 - 1000 rpm, and the stirring time is 2 - 24 h.

[0026] In step (5), the inducing reagent is ethanol, methanol or glycerol, but not limited to these, and is used to induce the self - assembly of silk fibroin.

[0027] In the present invention, in step (5), the upconversion nanoparticles, silk fibroin, and color reagent - antibiotic are blended and then ethanol is added to induce the self - assembly of silk fibroin, and a silk fibroin / color reagent - antibiotic blend film layer with a thickness of 1 nm - 5 nm can be formed outside the nanoparticles.

[0028] In step (6), the wavelength of the excitation light is 980 nm, and the measurement band range of the fluorescence intensity is 500 nm - 600 nm.

[0029] The present invention discovers and establishes a linear relationship among the fluorescence intensity, fluorescence quenching rate, and antibiotic of upconversion nanoparticles coated with silk fibroin and antibiotics (the fluorescence quenching rate increases with the increase in antibiotic). This linear relationship is obtained through pre-calibration. The fluorescence intensity obtained corresponding to the antibiotic to be measured is converted into a fluorescence quenching rate, and the fluorescence quenching rate is substituted into the linear relationship to obtain an accurate result of the concentration of the antibiotic to be measured.

[0030] The specific expression of the linear relationship is as follows:

[0031] Y = ax + b

[0032] Wherein, x represents the concentration of the antibiotic, Y represents the conversion of the fluorescence intensity affected by the chromogenic agent-antibiotic complex generated by the antibiotic to be measured into a fluorescence quenching rate, and a and b respectively represent the first and second fitting parameters.

[0033] The principle of the present invention is that functional functional groups (such as carboxyl groups, hydroxyl groups, amino groups, etc.) in the silk fibroin peptide chain adsorb the chromogenic agent-antibiotic and form hydrogen bonds with the active groups on the surface of the upconversion nanoparticles, thereby coating on the particle surface to form upconversion nanoparticles coated with a silk fibroin / chromogenic agent-antibiotic blend film (UCNP@SF / CA-ABX), wherein the thickness of the silk / chromogenic agent-antibiotic film layer is about 1-5 nm. The characteristic absorption peak of the chromogenic agent-antibiotic overlaps with the emission peak of the upconversion nanoparticles. When the concentration of the chromogenic agent is fixed and the concentration of the antibiotic is increased, the peak value of the characteristic absorption peak of the chromogenic agent-antibiotic complex increases. As the concentration of the antibiotic increases, the fluorescence quenching of the upconversion nanoparticles enhances and the fluorescence intensity weakens.

[0034] The antibiotic detection performance of the present invention is stronger than that of ultraviolet spectrophotometry. The functional functional groups in the silk fibroin peptide chain are beneficial to the adsorption of drugs. By combining the antibiotic with the chromogenic agent, the characteristic absorption peak of the antibiotic can be changed to overlap with the emission peak of the selected upconversion nanoparticles. The nano-probe system of the present invention has a sensitive fluorescence response to antibiotics at different concentrations, significantly improving the detection limit of the colorimetric method for detecting antibiotics. This method has a simple process, is easy to operate, has low cost and high sensitivity, and has broad application prospects in the fields of quantitative detection of pollutants and antibiotic drugs.

[0035] Due to the application of the above technical solutions, the present invention has the following outstanding features and beneficial effects compared with the prior art:

[0036] (1) Low cost. The extraction method of silk fibroin is mature; the extraction process is simple and efficient, without a complex and cumbersome preparation process;

[0037] (2) Do not use toxic reagents, and the preparation process does not cause harm to the environment and experimental personnel;

[0038] (3) Low detection limit and high sensitivity. The detection in actual samples is remarkable, which helps to design it into a sensor for on-site detection of antibiotics in actual samples.

[0039] (4) The upconversion nanoparticles used have strong luminescence, good stability, are not easily photobleached, and the emitted light in the near-infrared region can effectively reduce the interference of background fluorescence, improve the signal-to-noise ratio, and enhance the sensitivity. Thus, by using the inner filter effect, that is, the coincidence and matching of the absorption spectrum of antibiotics and the emission spectrum of fluorescent nanomaterials, the selectivity of the fluorescent nanoprobe can be improved, and it has strong tissue penetration and no light damage. Description of the Drawings

[0040] Figure 1 It is the TEM morphology diagram of UCNP@SF / Ail-RXM in Example 1.

[0041] Figure 2 It is the ultraviolet absorption diagram of the complex of alizarin red - different concentrations of roxithromycin (Ail-RXM) in Example 1.

[0042] Figure 3 It is the fluorescence intensity diagram of UCNP@SF / Ail-RXM in Example 1.

[0043] Figure 4 It is the ultraviolet absorption diagram of the complex of pyronin - different concentrations of roxithromycin (Pup-RXM) in Example 2.

[0044] Figure 5 It is the fluorescence intensity diagram of UCNP@SF / Pup-RXM in Example 2.

[0045] Figure 6 It is the ultraviolet absorption diagram of the complex of alizarin red - different concentrations of azithromycin (Ail-AZM) in Example 3.

[0046] Figure 7 It is the fluorescence intensity diagram of UCNP@SF / Ail-AZM in Example 3. Detailed Embodiments

[0047] The present invention will be further described in detail below through examples. The following examples are explanations of the present invention, and the present invention is not limited to the following examples.

[0048] The embodiments of the present invention are as follows:

[0049] Example 1

[0050] The preparation method of the probe of silk fibroin, roxithromycin (alizarin red - roxithromycin) co-blended upconversion nanoparticles for detecting antibiotics includes the following steps in sequence:

[0051] (1) Synthesize upconversion nanoparticles (GUCNP) of sodium yttrium tetrafluoride doped with ytterbium, erbium, and gadolinium, NaYF4:Yb / Er / Gd@NaYF4, with a size of 28 nm by high-temperature thermal precipitation method. Under 980 nm near-infrared light irradiation, it emits green light, and the strongest emission peak is between 542 nm and 548 nm.

[0052] (2) Dissolve the upconversion nanoparticles obtained in step (1) in cyclohexane, add ammonia water and stir for 30 min, slowly add tetraethoxysilane (TEOS) and react for 12 h to generate water-soluble silica and coat it on the upconversion nanoparticles. Slowly add Aptes to the system and react for 3 h to graft amino groups on the upconversion nanoparticles. Then wash successively with a mixture of cyclohexane and absolute ethanol, absolute ethanol, and deionized water. After washing, collect with deionized water, and after collection, dry in vacuum at 60 °C for 12 h.

[0053] (3) Dissolve the fibrous silk fibroin formed after degumming the silkworm cocoons, and obtain an aqueous solution of silk fibroin after filtration, dialysis, and concentration in sequence. Adjust the concentration of silk fibroin in the aqueous solution of silk fibroin to 10 mg / mL.

[0054] (4) Fix the concentration of alizarin red (Ail) at 10 μg / mL, add 0 - 400 μg / mL of roxithromycin (RXM) to alizarin red, and prepare an alizarin red-roxithromycin (Ail-RXM) complex. The highest absorption peak of the complex is between 520 and 550 nm, and the absorption peak of the complex overlaps with the emission peak (535 nm - 550 nm) of the NaYF4:Yb / Er / Gd@NaYF4 upconversion nanoparticles under 980 nm laser irradiation.

[0055] (5) Dissolve the upconversion nanoparticles obtained in step (2) with deionized water and disperse them by ultrasonic treatment. Add the aqueous solution of silk fibroin obtained in step (3) and the aqueous solution of alizarin red-roxithromycin with different ratios in step (4). After mixing and stirring at 300 rpm for 2 h, add an equal volume of ethanol to promote the self-assembly of silk fibroin.

[0056] (6) Centrifuge the upconversion nanoparticles (UCNP@SF / Ail-RXM) coated with a silk fibroin / alizarin red-roxithromycin film layer obtained in step (5) at 20000 g for 30 min, and then recover with deionized water.

[0057] The TEM morphological results of the upconversion nanoparticles (UCNP@SF / Ail-RXM) coated with a silk fibroin / alizarin red-roxithromycin film layer are as Figure 1 shown. The right figure is a detailed display of the left figure. It can be seen in the direction of the arrow in the right figure that the outer layer of the UCNP particles is coated with a silk fibroin / alizarin red-roxithromycin film layer with a thickness of 1.5 - 2.5 nm.

[0058] The results of the ultraviolet absorption tests of Ail-RXM with the above different concentration gradients are as Figure 2 shown. When the concentration of the fixed chromogenic agent Ail is visible in the figure, as the concentration of roxithromycin RXM increases, the characteristic peak intensity of alizarin red-roxithromycin Ail-RXM at 450-575 nm increases.

[0059] (7) Measure the fluorescence intensity in the 500-600 nm band under 980 nm excitation light. The fluorescence intensity results are as Figure 3 shown, Figure 3 as shown in, the fluorescence quenching rate of the upconversion nanoparticles coated with silk fibroin / alizarin red-roxithromycin film layer increases with the increase of the antibiotic concentration, and the fluorescence intensity decreases with the increase of the roxithromycin concentration. It can be seen from this that there is a linear relationship between the fluorescence intensity of the upconversion nanoparticles coated with silk fibroin / alizarin red-roxithromycin film layer and the roxithromycin concentration.

[0060] The linear relationship in this example is specifically expressed as:

[0061] Y = 0.008346x + 0.02805

[0062] where x represents the concentration of roxithromycin, and Y represents the conversion of the fluorescence intensity affected by the alizarin red-antibiotic complex formed by roxithromycin into the fluorescence quenching rate.

[0063] Example 2

[0064] A method for preparing a probe of silk fibroin and roxithromycin (pyrrole-roxithromycin) co-doped upconversion nanoparticles for detecting antibiotics successively includes the following steps:

[0065] (1) Synthesize sodium yttrium tetrafluoride NaYF4:Yb / Er / Gd@NaYF4 upconversion nanoparticles (GUCNP) doped with ytterbium, erbium, and gadolinium with a size of 28 nm according to the high-temperature thermal precipitation method. Under 980 nm near-infrared light irradiation, it emits green light, and the strongest emission peak is between 542 nm and 548 nm.

[0066] (2) Dissolve the upconversion nanoparticles obtained in step (1) in cyclohexane, then add ammonia water and stir for 30 min. Slowly add tetraethyl orthosilicate TEOS and react for 12 h to form water-soluble silica and coat it on the upconversion nanoparticles. Slowly add Aptes to the system and react for 3 h to graft amino groups on the upconversion nanoparticles. Then wash with a mixed solution of cyclohexane and absolute ethanol, absolute ethanol, and deionized water in sequence. After washing, collect with deionized water, and after collection, vacuum dry at 60 °C for 12 h.

[0067] (3) Dissolve the fibrous silk fibroin formed after degumming the silkworm cocoons, and obtain an aqueous solution of silk fibroin through filtration, dialysis, and concentration in sequence. Adjust the concentration of silk fibroin in the aqueous solution of silk fibroin to 10 mg / mL.

[0068] (4) Fix the concentration of pyrroline (Pup) at 20 μg / mL, add 0 - 500 μg / mL of roxithromycin (RXM) to pyrroline, and prepare a pyrroline-roxithromycin (Pup-RXM) complex. The highest absorption peak of the complex is located between 510 - 540 nm, and the absorption peak of the complex overlaps with the emission peak (535 nm - 550 nm) of NaYF4:Yb / Er / Gd@NaYF4 upconversion nanoparticles under 980 nm laser irradiation.

[0069] (5) Dissolve the upconversion nanoparticles obtained in step (2) with deionized water and disperse them by ultrasonic wave. Add the aqueous solution of silk fibroin obtained in step (3) and the aqueous solution of pyrroline-roxithromycin with different ratios in step (4). After mixing and stirring at 300 rpm for 2 h, add an equal volume of ethanol to promote the self-assembly of silk fibroin.

[0070] (6) Centrifuge the upconversion nanoparticles (UCNP@SF / Pup-RXM) coated with a silk fibroin / pyrroline-roxithromycin film layer obtained in step (5) at 20000 g for 30 min, and then recover with deionized water.

[0071] The results of ultraviolet absorption tests for the above Pup-RXM with different concentration gradients are as Figure 4 shown. It can be seen from the figure that when the concentration of the chromogenic agent pyrroline Pup is fixed, with the increase in the concentration of roxithromycin RXM, the intensity of the characteristic peak of pyrroline-roxithromycin Pup-RXM at 400 - 600 nm increases.

[0072] (7) Measure the fluorescence intensity in the 500 nm - 600 nm band under 980 nm excitation light. The fluorescence intensity results are as Figure 5 shown, Figure 5 which shows that the fluorescence quenching rate of the upconversion nanoparticles coated with a silk fibroin / pyrroline-roxithromycin film layer increases with the increase in the antibiotic concentration, and the fluorescence intensity decreases with the increase in the concentration of roxithromycin. It can be seen that there is a linear relationship between the fluorescence intensity of the upconversion nanoparticles coated with a silk fibroin / pyrroline-roxithromycin film layer and the concentration of roxithromycin.

[0073] The linear relationship in this example is specifically expressed as:

[0074] Y = 0.01127x + 0.01210

[0075] Among them, x represents the concentration of roxithromycin, and Y represents the conversion of the fluorescence intensity affected by the pyrrole-roxithromycin complex formed by roxithromycin into the fluorescence quenching rate.

[0076] Example 3

[0077] A method for preparing a probe of silk fibroin and azithromycin (alizarin red-azithromycin) co-doped upconversion nanoparticles for detecting antibiotics successively includes the following steps:

[0078] (1) Synthesize sodium yttrium tetrafluoride NaYF4:Yb / Er / Gd@NaYF4 upconversion nanoparticles (GUCNP) doped with ytterbium, erbium, and gadolinium with a size of 28 nm according to the high-temperature thermal precipitation method, which emits green light under the irradiation of 980 nm near-infrared light, and the strongest emission peak is between 542 nm and 548 nm.

[0079] (2) Dissolve the upconversion nanoparticles obtained in step (1) in cyclohexane, add ammonia water and stir for 30 min, slowly add tetraethyl orthosilicate TEOS and react for 12 h to generate water-soluble silica and coat it on the upconversion nanoparticles. Slowly add Aptes to the system and react for 3 h to graft amino groups on the upconversion nanoparticles. Then wash them successively with a mixed solution of cyclohexane and absolute ethanol, absolute ethanol, and deionized water, collect them with deionized water after washing, and vacuum dry them at 60 °C for 12 h.

[0080] (3) Dissolve the fibrous silk fibroin formed after degumming the silkworm cocoons, and obtain an aqueous solution of silk fibroin after filtration, dialysis, and concentration in sequence, and adjust the concentration of silk fibroin in the aqueous solution of silk fibroin to 10 mg / mL.

[0081] (4) Fix the concentration of alizarin red (Ail) at 5 μg / mL, add 0-150 μg / mL of azithromycin (AZM) to alizarin red, and prepare an alizarin red-azithromycin (Ail-AZM) complex. The highest absorption peak of the complex is between 520 and 550 nm, and the absorption peak of the complex overlaps with the emission peak (535 nm - 550 nm) of the NaYF4:Yb / Er / Gd@NaYF4 upconversion nanoparticles under the irradiation of 980 nm laser.

[0082] (5) Dissolve the upconversion nanoparticles obtained in step (2) with deionized water and disperse them by ultrasonic wave, add the aqueous solution of silk fibroin obtained in step (3) and the aqueous solution of alizarin red-azithromycin with different ratios in step (4), mix and stir at 300 rpm for 2 h, and then add an equal volume of ethanol to promote the self-assembly of silk fibroin.

[0083] (6) The upconversion nanoparticles (UCNP@SF / Ail-AZM) coated with the silk fibroin / alizarin red-azithromycin film layer in step (5) were centrifuged at 20,000 g for 30 min, and then recovered with deionized water.

[0084] The results of the UV absorption test of Ail-AZM at different concentration gradients are as follows: Figure 6 As shown in the figure, it can be seen that when the concentration of the color developer Alizarin Red Ail is fixed, as the concentration of azithromycin AZM increases, the characteristic peak intensity of pyrimethamine-roxithromycin Ail-AZM at 460-650nm increases.

[0085] (7) Measure the fluorescence intensity in the 500nm to 600nm band under 980nm excitation light. The fluorescence intensity results are as follows: Figure 7 As shown, Figure 7 It is shown in the figure that the fluorescence quenching rate of the upconversion nanoparticles increases with the increase of the antibiotic concentration, and the fluorescence intensity decreases with the increase of the azithromycin concentration. It can be seen that there is a linear relationship between the fluorescence intensity of the upconversion nanoparticles coated with silk fibroin / Alizarin red-azithromycin film and the azithromycin concentration.

[0086] The linear relationship of this embodiment is specifically expressed as:

[0087] Y=0.01536x+0.03909

[0088] Wherein, x represents the concentration of azithromycin, and Y represents the conversion of fluorescence intensity into fluorescence quenching rate affected by the Alizarin Red-azithromycin complex generated by azithromycin.

[0089] Finally, it should be noted that the above examples are only specific implementation examples of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system, characterized in that: The method sequentially includes the following steps: (1) Synthesize upconversion nanoparticles doped with lanthanide elements; (2) Dissolve the upconversion nanoparticles obtained in step (1) in cyclohexane, then add ammonia water and stir, add tetraethyl orthosilicate (TEOS) to react to generate water-soluble silica and coat it on the upconversion nanoparticles; then wash successively with a mixed solution of cyclohexane and absolute ethanol, absolute ethanol, and deionized water, collect with deionized water after washing, and dry in vacuum after collection; (3) Dissolve the fibrous silk fibroin formed after degumming the silkworm cocoon, and obtain an aqueous solution of silk fibroin after filtration, dialysis, and concentration in sequence, and adjust the concentration of silk fibroin in the aqueous solution of silk fibroin to 10 mg / mL; (4) Mix the antibiotic to be measured and a chromogenic agent to prepare an aqueous solution of a chromogenic agent-antibiotic complex; In the above step (4), the chromogenic agent is a chromogenic agent that can make the absorption peak of the chromogenic agent-antibiotic complex overlap with the emission peak of the upconversion nanoparticles; (5) Dissolve the upconversion nanoparticles obtained in step (2) with deionized water and disperse them by ultrasonic wave, add the aqueous solution of silk fibroin in step (3) and the aqueous solution in step (4), and add an equal volume of an inducing reagent after stirring to obtain upconversion nanoparticles coated with silk fibroin and antibiotics; (6) Centrifuge the upconversion nanoparticles coated with silk fibroin and antibiotics, recover them with deionized water, measure the fluorescence intensity under the excitation light after recovery, and judge the concentration of the antibiotic to be measured according to the fluorescence intensity to realize the detection of antibiotics.

2. The method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, wherein: In the above step (1), the upconversion nanoparticles are synthesized by a high-temperature thermal precipitation method or a thermal decomposition method.

3. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (1), the size of the synthesized upconversion nanoparticles is 10 - 100 nm.

4. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (2), the reaction time for adding tetraethyl orthosilicate (TEOS) to react to generate water-soluble silica is 6 - 24 h.

5. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (2), the temperature for vacuum drying is 40 - 100 °C, and the time for vacuum drying is 4 - 48 h.

6. The method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, wherein: In the above step (4), the antibiotics used include cephalosporin antibiotics, quinolone antibiotics, macrolide antibiotics, clotrimazole, and sodium ampicillin.

7. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 6, characterized in that: The cephalosporin antibiotics include ceftriaxone, cefadroxil, and cefazolin; the quinolone antibiotics include pefloxacin mesylate, gatifloxacin, levofloxacin hydrochloride, ofloxacin, norfloxacin, and ciprofloxacin; the macrolide antibiotics include roxithromycin, erythromycin, oleandomycin, spiramycin, tylosin, erythromycin, ethyl succinate, erythromycin ethylsuccinate, clarithromycin, and azithromycin.

8. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (5), the rotation speed for stirring is 100 - 1000 rpm, and the time for stirring is 2 - 24 h.

9. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (5), the inducing reagent is ethanol, methanol, or glycerol.

10. A method for detecting antibiotics based on an upconversion nanoparticle-silk fibroin nanoprobe system according to claim 1, characterized in that: In the above step (6), the wavelength of the excitation light is 980 nm, and the measurement band range of the fluorescence intensity is 500 nm - 600 nm.

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