A nanoprobe, a preparation method and application thereof, and a nanoprobe specifically recognizing quinolones and a preparation method thereof

Abstract

The present application relates to the field of chemical fluorescence technology, and more particularly to a kind of nano probe and its preparation method and application and a kind of nano probe and its preparation method for specifically recognizing quinolone.The nano probe of the present application comprises the following components: hydroxylated polystyrene sodium acid doped with niobium nanosphere solution, N carbon dot solution, copper carbon dot solution;The initial density of the hydroxylated polystyrene sodium acid doped with niobium nanosphere solution is 1-3×10 14 ML;The initial molar concentration of the N carbon dot solution is 0.05-0.15M;The initial molar concentration of the copper carbon dot solution is 0.05-0.15M.The present application realizes the convenience and efficiency of drug detection by wrapping nano materials and combining with quinolone drug antibodies.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence technology, and more particularly to a nanoprobe, its preparation method, and its application, as well as a nanoprobe that specifically recognizes quinolones and its preparation method. Background Technology

[0002] Carbon nanodots (CDs) are a novel fluorescent material that has attracted much attention in recent years. They possess excellent luminescent properties, resistance to photobleaching, low toxicity, and high biocompatibility, thus demonstrating superiority in the field of optical sensing. The surface of carbon nanodots is rich in functional groups and easily modified, allowing for the construction of various sensing modes. They have been successfully applied to the fluorescence detection of metal ions, anions, and small biomolecules. Similarly, drugs such as tetracycline, sulfamethoxazole, and metronidazole can also be identified by the fluorescence of carbon nanodots.

[0003] Currently, the detection of quinolone drugs is achieved through quantitative detection using dispersive solid phase extraction (dSPE) ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS), which is usually cumbersome and cannot provide rapid and intuitive detection.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a nanoprobe with a simple detection procedure and high sensitivity for detecting quinolone drugs, and particularly relates to a nanoprobe, its preparation method, and its application, as well as a nanoprobe that specifically recognizes quinolones and its preparation method.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a nanoprobe comprising the following components:

[0008] Hydroxylated polystyrene-doped sodium niobate nanosphere solution, N carbon dot solution, copper carbon dot solution;

[0009] The initial density of the hydroxylated polystyrene sodium niobate nanosphere solution is 1–3 × 10⁻⁶. 14 cells / mL;

[0010] The initial molar concentration of the N carbon point solution is 0.05–0.15 M;

[0011] The initial molar concentration of the copper carbon dot solution is 0.05–0.15 M;

[0012] The volume ratio of the hydroxylated polystyrene sodium niobate nanosphere solution to the N carbon dot solution is 18-22:2-4;

[0013] The volume ratio of the hydroxylated polystyrene sodium niobate nanosphere solution to the copper carbon dot solution is 18–22:2–4.

[0014] Preferably, the hydroxylated polystyrene sodium niobate nanosphere solution is prepared by mixing hydroxylated polystyrene sodium niobate nanospheres with an aqueous acetone solution.

[0015] The volume ratio of acetone to water in the acetone aqueous solution is 1-2:1-2;

[0016] The preparation method of the hydroxylated polystyrene sodium niobate nanospheres includes the following steps:

[0017] Styrene, acrylic acid, sodium dodecyl sulfonate aqueous solution and sodium niobate nanosphere aqueous solution are mixed to obtain a mixture; the mixture is heated to 70-80°C, potassium persulfate solution is added, and the reaction is carried out under anaerobic conditions for 7.5-8.5 hours to obtain a reaction solution; the reaction solution is filtered, the filtrate is taken, dialyzed for 4-6 days, and the liquid in the dialysis bag is collected to obtain hydroxylated polystyrene doped sodium niobate nanospheres;

[0018] The final molar concentration of styrene is 9–11 mM;

[0019] The final molar concentration of the acrylic acid is 0.9–1.0 mM;

[0020] The initial molar concentration of the sodium dodecyl sulfonate aqueous solution is 0.4–0.5 mM;

[0021] The initial molar concentration of the sodium niobate nanosphere aqueous solution is 0.12–0.2 mM;

[0022] The volume ratio of the sodium dodecyl sulfonate aqueous solution to the sodium niobate nanosphere aqueous solution is 1:0.67-0.71;

[0023] The initial molar concentration of the potassium persulfate solution is 0.1–0.2 mM;

[0024] The potassium persulfate solution is an aqueous solution of potassium persulfate.

[0025] The volume ratio of the potassium persulfate solution to the sodium dodecyl sulfonate aqueous solution is 1:18-22.

[0026] Preferably, the N carbon dot solution is prepared by mixing N carbon dots with water;

[0027] The method for preparing the N carbon dots includes the following steps:

[0028] Citric acid, diethylenetriamine and water were mixed and left to stand for 10-14 hours to obtain an intermediate.

[0029] The intermediate was dialyzed for 70-74 hours to obtain dialysate; the dialysate was freeze-dried to obtain N carbon dots.

[0030] The mass-to-volume ratio of the citric acid to water is 1.0–1.4 g: 18–22 mL;

[0031] The volume ratio of the diethylenetriamine to water is 0.14–0.16:18–22;

[0032] The placement temperature is 220–240°C;

[0033] The dialysis method is dialysis using a dialysis bag; the dialysis bag is a 1000MWCO.

[0034] Preferably, the copper carbon dot solution is prepared by mixing copper carbon dots with water;

[0035] The method for preparing the copper carbon dots includes the following steps:

[0036] Citric acid, copper acetate, and diethylenetriamine were mixed with water and dissolved to obtain an intermediate solution. The intermediate solution was heated to 220–240°C and held for 10–12 hours, then dialyzed and freeze-dried to obtain copper carbon dots.

[0037] The mass-to-volume ratio of the citric acid to water is 1.0–1.4 g: 18–22 mL;

[0038] The mass-to-volume ratio of the copper acetate to water is 0.13–0.17 g: 18–22 mL;

[0039] The volume ratio of the diethylenetriamine to water is 0.14–0.16:18–22;

[0040] The copper acetate is a copper acetate with a dimer unit structure;

[0041] The dissolution is ultrasonic dissolution; the ultrasonic dissolution time is 10-12 min; the dialysis method is dialysis bag dialysis; the dialysis bag is 1000MWCO.

[0042] The present invention also provides a method for preparing the aforementioned nanoprobe, comprising the following steps:

[0043] (1) Mix the hydroxylated polystyrene sodium niobate nanosphere solution, N carbon dot solution and copper carbon dot solution, heat to 55-65℃, react in the dark for 9-11 h, and then react at 20-25℃ for 1.8-2.2 h to obtain the reaction solution;

[0044] (2) After distilling and dialysis of the reaction solution, the liquid in the dialysis bag is collected to obtain the nanoprobe.

[0045] Preferably, the distillation is vacuum distillation; the dialysis bag is 1000MWCO; and the dialysis time is 4–6 days.

[0046] The present invention also provides the application of the nanoprobe described herein or the nanoprobe prepared by the method described herein in the preparation of detection products that specifically recognize quinolones.

[0047] The present invention also provides a nanoprobe that specifically recognizes quinolones, wherein the nanoprobe that specifically recognizes quinolones includes the nanoprobe described above or the nanoprobe prepared by the preparation method described above, as well as a quinolone antibody;

[0048] The quinolone antibody is a norfloxacin monoclonal antibody.

[0049] The present invention also provides a method for preparing the nanoprobe that specifically recognizes quinolones, comprising the following steps:

[0050] (1) Activate the nanoprobe or the nanoprobe prepared by the preparation method to obtain an activated nanoprobe;

[0051] (2) Dissolve the activated nanoprobe in borate buffer to obtain a solution; mix the solution with a quinolone antibody and react for 11-13 h to obtain a reaction solution;

[0052] (3) Centrifuge the reaction solution, collect the precipitate, and dissolve the precipitate in phosphate buffer to obtain a nanoprobe that specifically recognizes quinolone.

[0053] The initial molar concentration of the borate buffer solution in step (2) is 0.01–0.02 M;

[0054] The initial pH of the borate buffer solution is 7.8–8.2;

[0055] The mass-to-volume ratio of the quinolone antibody to the solution is 0.2–0.3 mg: 5 mL;

[0056] The centrifuge speed is 11000-13000 rpm;

[0057] The centrifugation time is 9–11 minutes;

[0058] The phosphate buffer also contains trehalose and bovine serum albumin;

[0059] The mass ratio of trehalose added to the volume ratio of phosphate buffer is 1-2 g: 100 mL.

[0060] The ratio of the added bovine serum albumin to the volume of phosphate buffer is 1.5–2.5 g: 100 mL.

[0061] The initial molar concentration of the phosphate buffer is 0.01–0.02 M;

[0062] The initial pH of the phosphate buffer is 7.2 to 7.6.

[0063] Preferably, the activation step in step (1) is as follows:

[0064] The nanoprobes described above or prepared by the aforementioned method are diluted to a density of 1.0–3.0 × 10⁻⁶. 12 The nanoprobes were diluted to a concentration of 1 / mL. The diluted nanoprobes were then sonicated and mixed with an aqueous solution of carbodiimide. The mixture was reacted for 13–17 min, centrifuged, and the precipitate was collected to obtain the activated nanoprobes.

[0065] The solution used to dilute the nanoprobes is a 0.01–0.02 M borate buffer solution;

[0066] The power of the ultrasound is 300-500W;

[0067] The duration of the ultrasound is 25–35 seconds;

[0068] The initial concentration of the carbodiimide aqueous solution is 14–16 mg / mL;

[0069] The volume ratio of the diluted nanoprobe to the carbodiimide aqueous solution is 48–52:1.

[0070] The reaction temperature is 20–25°C;

[0071] The centrifuge speed is 14,000 to 16,000 rpm;

[0072] The centrifugation time is 8 to 12 minutes.

[0073] A nanoprobe, its preparation method, and its application are disclosed. This invention relates to a nanoprobe specifically recognizing quinolones and its preparation method. Based on the fluorescence recognition of quinolones using carbon dots, it utilizes the principle that the complex formed by the binding of quinolones and copper ions absorbs the fluorescence of carbon dots. A fluorescence quenching recognition mode for quinolone determination is established based on the internal filtration effect. This invention achieves convenient and efficient drug detection by encapsulating nanomaterials with quinolone drug antibodies.

[0074] In this invention, sodium niobate is added during the preparation of nanomaterials. Due to its chemical and thermal stability, sodium niobate can significantly improve the material properties when applied to the preparation of nanomaterials. It also has excellent luminescent properties, so when combined with carbon dots, it forms a fluorescent nanoprobe, which enhances the fluorescence effect.

[0075] The specific nanoprobe of this invention is a supramolecular complex composed of a dimeric unit structure of copper acetate and citric acid. This carbon dot exhibits a tendency to bind to copper ions, which effectively quench the carbon dot fluorescence through photoinduced electron transfer. Furthermore, the introduction of a quinolone, due to its strong binding to copper ions, causes the copper ions to leave the carbon dot surface, thus restoring the fluorescence. Attached Figure Description

[0076] Figure 1 This is a transmission electron microscope image of a nanoprobe.

[0077] Figure 2 This is a dynamic light scattering nanoparticle size distribution diagram of the nanoprobe.

[0078] Figure 3 Solutions with different carbon dots were prepared.

[0079] Figure 4 The images show the UV-Vis absorption spectra of solutions with different carbon dots.

[0080] Figure 5 The fluorescence emission spectra of the nanoprobe that specifically recognizes quinolones in Example 1 are shown under excitation light of different wavelengths.

[0081] Figure 6 Stability testing of the nanoprobe that specifically recognizes quinolones in Example 1.

[0082] Figure 7 The fluorescence emission spectrum of the quinolone drug detected by the nanoprobe that specifically recognizes quinolones in Example 1 is shown. Detailed Implementation

[0083] This invention provides a nanoprobe comprising the following components: a solution of hydroxylated polystyrene doped with sodium niobate nanospheres, an N-carbon dot solution, and a copper carbon dot solution; wherein the initial density of the hydroxylated polystyrene doped with sodium niobate nanosphere solution is 1–3 × 10⁻⁶. 14 Cells / mL, preferably 2×10 14 The initial molar concentration of the N carbon dot solution is 0.05–0.15 M, preferably 0.1 M; the initial molar concentration of the copper carbon dot solution is 0.05–0.15 M, preferably 0.1 M; the volume ratio of the hydroxylated polystyrene-doped sodium niobate nanosphere solution to the N carbon dot solution is 18–22:2–4, preferably 20:3; the volume ratio of the hydroxylated polystyrene-doped sodium niobate nanosphere solution to the copper carbon dot solution is 18–22:2–4, preferably 20:3.

[0084] In this invention, the preparation method of the hydroxylated polystyrene sodium niobate nanosphere solution is as follows: hydroxylated polystyrene sodium niobate nanospheres are prepared by mixing with an acetone aqueous solution; the volume ratio of acetone to water in the acetone aqueous solution is 1-2:1-2, preferably 1:1; the preparation method of the hydroxylated polystyrene sodium niobate nanospheres includes the following steps: mixing styrene, acrylic acid, sodium dodecyl sulfonate aqueous solution and sodium niobate nanosphere aqueous solution to obtain a mixed solution; heating the mixed solution to 70-80°C, adding potassium persulfate solution, and reacting under anaerobic conditions for 7.5-8.5 hours to obtain a reaction solution; filtering the reaction solution, taking the filtrate, dialyzing for 4-6 days, collecting the liquid in the dialysis bag, and obtaining the hydroxylated polystyrene sodium niobate nanospheres; the final molar concentration of styrene is 9... The final molar concentration of the acrylic acid is 0.9-1.0 mM, preferably 0.95 mM; the initial molar concentration of the sodium dodecyl sulfonate aqueous solution is 0.4-0.5 mM, preferably 0.45 mM; the initial molar concentration of the sodium niobate nanosphere aqueous solution is 0.12-0.2 mM, preferably 0.16 mM; the volume ratio of the sodium dodecyl sulfonate aqueous solution to the sodium niobate nanosphere aqueous solution is 1:0.67-0.71, preferably 1:0.69; the initial molar concentration of the potassium persulfate solution is 0.1-0.2 mM, preferably 0.15 mM; the potassium persulfate solution is an aqueous solution of potassium persulfate; the volume ratio of the potassium persulfate solution to the sodium dodecyl sulfonate aqueous solution is 1:18-22, preferably 1:20.

[0085] In this invention, the sodium niobate nanospheres are prepared as follows: 0.35g of niobium pentoxide is mixed with 50mL of 12mol / L NaOH solution, and sonicated for 0.5h. A rotor is added, and the mixture is magnetically stirred for 2h to ensure thorough mixing. The mixture is then placed in a 50mL high-pressure reactor, which is then placed in an oven preheated to 150℃ and cooled to room temperature. The resulting white precipitate is centrifuged or filtered, and washed with double-distilled water and ethanol until the pH reaches 7. The product is then placed in a vacuum drying oven and dried at 60℃ for 8h to obtain sodium niobate nanospheres.

[0086] In this invention, the N carbon dot solution is prepared by mixing N carbon dots with water;

[0087] The method for preparing the N-carbon dots includes the following steps: mixing citric acid, diethylenetriamine, and water, and allowing the mixture to stand for 10–14 hours to obtain an intermediate; dialyzing the intermediate for 70–74 hours to obtain a dialysate; and freeze-drying the dialysate to obtain the N-carbon dots. The mass-to-volume ratio of the citric acid to water mixture is 1.0–1.4 g: 18–22 mL, preferably 1.2 g: 20 mL; the volume ratio of the diethylenetriamine to water mixture is 0.14–0.16: 18–22, preferably 0.15:20; the standing temperature is 220–240 °C, preferably 230 °C; the standing time is preferably 12 hours; the dialysis method is dialysis using a dialysis bag; the dialysis bag is a 1000 MWCO; and the dialysis time is preferably 72 hours.

[0088] In this invention, the copper carbon dot solution is prepared by mixing copper carbon dots with water;

[0089] The method for preparing the copper carbon dots includes the following steps:

[0090] Citric acid, copper acetate, and diethylenetriamine are mixed with water and dissolved to obtain an intermediate solution. The intermediate solution is heated to 220–240°C and held for 10–12 hours, then dialyzed and freeze-dried to obtain copper carbon dots. The mass-to-volume ratio of citric acid to water is 1.0–1.4 g:18–22 mL, preferably 1.2 g:20 mL; the mass-to-volume ratio of copper acetate to water is 0.13–0.17 g:18–22 mL, preferably 0.15 g:20 mL; the volume ratio of diethylenetriamine to water is 0.14–0.16:18–22, preferably 0.15:20; the copper acetate is a dimerized unit copper acetate; the dissolution is ultrasonic dissolution; the ultrasonic dissolution time is 10–12 min, preferably 11 min; the dialysis method is dialysis using a dialysis bag; the dialysis bag is a 1000 MWCO. The heating temperature is preferably 230°C, and the holding time is preferably 11 hours.

[0091] The present invention also provides a method for preparing the aforementioned nanoprobe, comprising the following steps:

[0092] (1) A solution of hydroxylated polystyrene doped sodium niobate nanospheres, an N carbon dot solution, and a copper carbon dot solution are mixed and heated to 55–65°C. After reacting in the dark for 9–11 hours, the mixture is further reacted at 20–25°C for 1.8–2.2 hours to obtain a reaction solution. (2) The reaction solution is distilled and dialyzed, and the liquid in the dialysis bag is collected to obtain a nanoprobe. In this invention, the heating temperature is preferably 60°C; the reaction time is preferably 10 hours; the re-reaction temperature is preferably 22.5°C, and the re-reaction time is preferably 2.0 hours.

[0093] In this invention, the distillation is vacuum distillation; the dialysis bag is 1000MWCO; and the dialysis time is 4 to 6 days, preferably 5 days.

[0094] The present invention also provides the application of the nanoprobe described herein or the nanoprobe prepared by the method described herein in the preparation of detection products that specifically recognize quinolones.

[0095] The present invention also provides a nanoprobe that specifically recognizes quinolones, wherein the nanoprobe that specifically recognizes quinolones includes the nanoprobe described above or the nanoprobe prepared by the preparation method described above, as well as a quinolone antibody;

[0096] The quinolone antibody is a norfloxacin monoclonal antibody.

[0097] The present invention also provides a method for preparing the nanoprobe that specifically recognizes quinolones, comprising the following steps:

[0098] (1) Activate the nanoprobe or the nanoprobe prepared by the preparation method to obtain activated nanoprobe; (2) Dissolve the activated nanoprobe in borate buffer to obtain a solution; mix the solution with a quinolone antibody and react for 11-13 h to obtain a reaction solution; (3) Centrifuge the reaction solution, collect the precipitate, and dissolve the precipitate in phosphate buffer to obtain a nanoprobe that specifically recognizes quinolones; In step (2), the initial molar concentration of the borate buffer is 0.01-0.02 M, preferably 0.015 M; the initial pH of the borate buffer is 7.8-8.2, preferably 8.0; the mass-to-volume ratio of the quinolone antibody to the solution is 0.2-0.3 mg:5 mL. The preferred concentration is 0.25 mg: 5 mL; the centrifugation speed is 11000–13000 rpm, preferably 12000 rpm; the centrifugation time is 9–11 min, preferably 10 min; the phosphate buffer also contains trehalose and bovine serum albumin; the mass ratio of trehalose to phosphate buffer is 1–2 g: 100 mL, preferably 1.5 g: 100 mL; the mass ratio of bovine serum albumin to phosphate buffer is 1.5–2.5 g: 100 mL, preferably 2.0 g: 100 mL; the initial molar concentration of the phosphate buffer is 0.01–0.02 M, preferably 0.015 M; the initial pH of the phosphate buffer is 7.2–7.6, preferably 7.4.

[0099] In this invention, the activation step in step (1) is as follows:

[0100] The nanoprobes described above or prepared by the aforementioned method are diluted to a density of 1.0–3.0 × 10⁻⁶.12 The nanoprobes were diluted to a concentration of 1 / mL; the diluted nanoprobes were sonicated, mixed with an aqueous solution of carbodiimide, reacted for 13–17 min, centrifuged, and the precipitate was collected to obtain the activated nanoprobes; the solution used for diluting the nanoprobes was a 0.01–0.02 M borate buffer; the preferred dilution density was 2.0 × 10⁻⁶. 12 The concentration of the nanoprobe / mL is: 300–500 W, preferably 400 W; the ultrasound duration is: 25–35 s, preferably 30 s; the initial concentration of the carbodiimide aqueous solution is: 14–16 mg / mL, preferably 15 mg / mL; the volume ratio of the diluted nanoprobe to the carbodiimide aqueous solution is: 48–52:1, preferably 50:1; the reaction temperature is: 20–25 °C, preferably 22.5 °C; the centrifugation speed is: 14000–16000 rpm, preferably 15000 rpm; the centrifugation time is: 8–12 min, preferably 10 min.

[0101] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0102] The copper acetate described in this embodiment of the invention has a dimer unit structure, and the copper acetate was purchased from Shanghai Chuangsai Technology Co., Ltd.

[0103] Example 1

[0104] Preparation of nanoprobes that specifically recognize quinolones

[0105] Under room temperature and light-protected conditions, 1.2 g of citric acid, 20 mL of deionized water and 0.15 mL of diethylenetriamine were mixed and placed in a 100 mL reactor. The mixture was placed at 230 °C for 12 h, then dialyzed through a 1000 MWCO dialysis bag for 72 h, and finally freeze-dried to obtain N carbon dot powder.

[0106] 1.2 g of citric acid and 0.17 g of copper acetate were mixed and dissolved in 20 mL of deionized water. Then, 0.15 mL of diethylenetriamine was added, and the mixture was sonicated for 10 min to obtain a clear and transparent solution. This solution was transferred to a 100 mL autoclave and heated at 230 °C for 11 h, followed by natural cooling. The solution was dialyzed through a 1000 MWCO dialysis bag for 72 h and then freeze-dried to obtain solid copper carbon dots.

[0107] 0.35 g of niobium pentoxide was mixed with 50 mL of 12 mol / L NaOH solution and sonicated for 0.5 h. A rotor was then added, and the mixture was magnetically stirred for 2 h to ensure thorough mixing. The mixture was then placed in a 50 mL high-pressure reactor, which was then placed in an oven preheated to 150 °C and cooled to room temperature. The resulting white precipitate was centrifuged or filtered and washed with double-distilled water and ethanol until the pH reached 7. The product was then dried in a vacuum drying oven at 60 °C for 8 h to obtain sodium niobate nanospheres.

[0108] Styrene monomer, acrylic acid monomer, 10 mL of sodium dodecyl sulfate aqueous solution, and 6.8 mL of sodium niobate nanosphere aqueous solution were mixed in a round-bottom flask to achieve a styrene concentration of 10 mM and an acrylic acid concentration of 0.95 mM. The mixture was stirred evenly with a magnetic stirrer. The air in the round-bottom flask was removed with high-purity nitrogen, and the mixture was sealed and heated to 80°C. 0.5 mL of 0.15 mM potassium persulfate aqueous solution was added, and the mixture was sealed and stirred for 8 hours to obtain the reaction solution. The reaction solution was cooled to 25°C, filtered through Whatman 2V filter paper, and dialyzed against the filtrate for 5 days using a dialysis bag with a molecular weight cutoff of 25,000 Da. The liquid in the dialysis bag was collected, and 0.05% sodium azide was added before storage at 4°C to obtain hydroxylated polystyrene-doped sodium niobate nanospheres. The initial molar concentration of the sodium dodecyl sulfate aqueous solution was 0.4 mM; the initial molar concentration of the sodium niobate nanosphere aqueous solution was 0.15 mM.

[0109] Hydroxylated polystyrene sodium niobate nanospheres were mixed with an acetone aqueous solution to achieve a density of 2 × 10⁻⁶ hydroxylated polystyrene sodium niobate nanospheres in the mixture. 14 A solution of hydroxylated polystyrene-doped sodium niobate nanospheres was obtained by applying nanospheres per mL. The volume ratio of acetone to water in the acetone-water solution was 1:1.

[0110] The N carbon dots were diluted with water to 0.1 M to obtain an N carbon dot solution.

[0111] The copper carbon dots were diluted with water to a concentration of 0.1 M to obtain a copper carbon dot solution.

[0112] Take a density of 2×10 14 One mL of a solution of hydroxylated polystyrene doped with sodium niobate nanospheres (1 / mL) was added, along with 150 μL of N carbon dot solution and 150 μL of copper carbon dot solution. The mixture was heated to 60 °C and stirred in the dark for 10 h, then cooled to 20 °C and reacted for 2 h to obtain the reaction solution. The organic solvent in the reaction solution was removed by vacuum distillation. The distillate was dialyzed for 5 days to remove remaining small molecules. The liquid in the dialysis bag was collected, and 0.05% sodium azide was added. The solution was stored at 4 °C to obtain the nanoprobe. The morphology and particle size of the nanoprobe were observed by transmission electron microscopy (TEM). The results are as follows: Figures 1-2 As shown.

[0113] Figures 1-2 The results show that the nanoprobe particles exhibit a spherical morphology with a size of less than 5 nm; the size distribution of the nanoprobe particles ranges from 1.76 to 5.13 nm, with an average particle size of 3.22 nm.

[0114] Dissolve the nanoprobe in 10 mL of 0.01 M pH 8.0 borate buffer to achieve a nanoprobe density of 1.0 × 10⁻⁶. 12 The nanoprobes were diluted to 10 mL / mL. 10 mL of the diluted nanoprobes were sonicated in a 400 W ultrasonic oscillator for 30 s, then 200 μL of 15 mg / mL carbodiimide aqueous solution (EDC) was slowly added. The mixture was incubated at 25 °C with uniform stirring for 15 min, centrifuged at 15000 rpm for 10 min, and the precipitate was collected. The precipitate was washed three times repeatedly with 0.01 M pH 8.0 borate buffer to obtain the activated nanoprobes.

[0115] The activated nanoprobe was reconstituted in 5 mL of 0.01 M pH 8.0 borate buffer to obtain a solution. 5 mL of the solution was then added to 250 μg of norfloxacin monoclonal antibody, and the mixture was stirred at 4 °C for 12 h to obtain the reaction solution.

[0116] The reaction solution was centrifuged at 12,000 rpm for 10 min, and the precipitate was collected. The precipitate was redissolved in 0.01 M pH 7.4 phosphate buffer containing 1.5% (m / v) trehalose and 2% (m / v) bovine serum albumin to obtain the nanoprobe that specifically recognizes quinolones.

[0117] Example 2

[0118] Under light-protected conditions at room temperature, 1.0 g of citric acid, 20 mL of deionized water, and 0.15 mL of diethylenetriamine were mixed and placed in a 100 mL reactor. The mixture was then placed at 240 °C for 10 h, dialyzed through a 1000 MWCO dialysis bag for 70 h, and finally freeze-dried to obtain N carbon dot powder.

[0119] 1.0 g citric acid and 0.15 g copper acetate were mixed and dissolved in 20 mL of deionized water. Then, 0.15 mL of diethylenetriamine was added, and the mixture was sonicated for 12 min to obtain a clear and transparent solution. This solution was transferred to a 100 mL autoclave and heated at 240 °C for 10 h, followed by natural cooling. The solution was dialyzed through a 1000 MWCO dialysis bag for 70 h and then freeze-dried to obtain solid copper carbon dots.

[0120] 0.35 g of niobium pentoxide was mixed with 50 mL of 12 mol / L NaOH solution and sonicated for 0.5 h. A rotor was then added, and the mixture was magnetically stirred for 2 h to ensure thorough mixing. The mixture was then placed in a 50 mL high-pressure reactor, which was then placed in an oven preheated to 150 °C and cooled to room temperature. The resulting white precipitate was centrifuged or filtered and washed with double-distilled water and ethanol until the pH reached 7. The product was then dried in a vacuum drying oven at 60 °C for 8 h to obtain sodium niobate nanospheres.

[0121] Styrene monomer, acrylic acid monomer, 10 mL of sodium dodecyl sulfate aqueous solution, and 6.7 mL of sodium niobate nanosphere aqueous solution were mixed in a round-bottom flask to achieve a styrene concentration of 9 mM and an acrylic acid concentration of 1 mM. The mixture was stirred evenly with a magnetic stirrer. The air in the round-bottom flask was removed with high-purity nitrogen, and the mixture was sealed and heated to 70°C. 0.5 mL of 0.2 mM potassium persulfate aqueous solution was added, and the mixture was sealed and stirred for 8 hours to obtain the reaction solution. The reaction solution was cooled to 20°C, filtered through Whatman 2V filter paper, and dialyzed against the filtrate for 6 days using a dialysis bag with a molecular weight cutoff of 25,000 Da. The liquid in the dialysis bag was collected, and 0.05% sodium azide was added before storage at 4°C to obtain hydroxylated polystyrene doped with sodium niobate nanospheres. The initial molar concentration of the sodium dodecyl sulfate aqueous solution was 0.5 mM; the initial molar concentration of the sodium niobate nanosphere aqueous solution was 0.2 mM.

[0122] Hydroxylated polystyrene sodium niobate nanospheres were mixed with an acetone aqueous solution to achieve a density of 2 × 10⁻⁶ hydroxylated polystyrene sodium niobate nanospheres in the mixture. 14 A solution of hydroxylated polystyrene-doped sodium niobate nanospheres was obtained by applying nanospheres per mL. The volume ratio of acetone to water in the acetone-water solution was 1:1.

[0123] The N carbon dots were diluted with water to 0.15 M to obtain an N carbon dot solution.

[0124] The copper carbon dots were diluted with water to a concentration of 0.15 M to obtain a copper carbon dot solution.

[0125] Take a density of 2×10 14 1 mL of a solution of hydroxylated polystyrene-doped sodium niobate nanospheres (1 / mL) was added, along with 150 μL of N carbon dot solution and 150 μL of copper carbon dot solution. The mixture was heated to 55 °C and stirred in the dark for 11 h, then cooled to 25 °C and reacted for 1.8 h to obtain the reaction solution. The organic solvent in the reaction solution was removed by vacuum distillation. The distillate was dialyzed for 5 days to remove the remaining small molecules. The liquid in the dialysis bag was collected, and 0.05% sodium azide was added and stored at 4 °C to obtain the nanoprobe.

[0126] The prepared nanoprobe was dissolved in 10 mL of 0.01 M pH 8.0 borate buffer to achieve a nanoprobe density of 1.0 × 10⁻⁶. 12 The nanoprobes were diluted to a concentration of 14 mg / mL. 10 mL of the diluted nanoprobes were sonicated in a 400 W ultrasonic oscillator for 30 s, then 200 μL of a 14 mg / mL carbodiimide aqueous solution (EDC) was slowly added. The mixture was incubated at 22 °C with uniform stirring for 17 min, centrifuged at 16000 rpm for 8 min, and the precipitate was collected. The precipitate was washed twice with 0.01 M pH 8.0 borate buffer to obtain the activated nanoprobes.

[0127] The activated nanoprobe was reconstituted in 5 mL of 0.01 M pH 8.0 borate buffer to obtain a solution. 5 mL of the solution was then added to 250 μg of norfloxacin monoclonal antibody, and the mixture was stirred at 4 °C for 13 h to obtain the reaction solution.

[0128] The reaction solution was centrifuged at 13,000 rpm for 9 min, and the precipitate was collected. The precipitate was redissolved in 0.01 M phosphate buffer (pH 7.4) containing 1.5% (m / v) trehalose and 2% (m / v) bovine serum albumin to obtain the nanoprobe that specifically recognizes quinolones.

[0129] Comparative Example 1

[0130] The nanoprobe of Comparative Example 1 was prepared according to the preparation process of the nanoprobe of Example 1. The difference from Example 1 is that iron carbon dots were used to replace copper carbon dots in the nanoprobe of Comparative Example 1. When preparing iron carbon dots, FeCl3 was used to replace copper acetate in Example 1. The remaining steps were the same as those in Example 1.

[0131] Experimental Example 1

[0132] The N carbon dots (N-CDs), nanoprobes (N,Cu-CDs) obtained in Example 1 and the N,Fe nanoprobes (N,Fe-CDs) obtained in Comparative Example 1 were prepared as follows: Figure 3 As shown.

[0133] The spectra of N-carbon dots (N-CDs), N-Cu-CDs, and N-Fe nanoprobes (N-Fe-CDs) obtained in Example 1 and Comparative Example 1 were detected using UV-Vis light. The specific detection method is as follows: Under UV irradiation, the prepared N-carbon dots, N-Fe nanoprobes, and N-Cu nanoprobes were dissolved in ultrapure water for photochromic performance testing. The analysis results are shown below. Figure 4 As shown, after 3 minutes of ultraviolet light irradiation, the N,Cu nanoprobe rapidly changed from yellow to colorless, and its absorbance decreased rapidly.

[0134] Test results as follows Figure 4As shown.

[0135] Figure 4 The nanoprobes exhibit broad absorption (blue) in the 300–500 nm range.

[0136] Experimental Example 2

[0137] The fluorescence emission spectra of the quinolone-specific nanoprobe from Example 1 under excitation light of different wavelengths were obtained. The specific detection method is as follows: Under ultraviolet light irradiation at different wavelengths (335nm, 345nm, 355nm, 365nm, 375nm, 385nm, 395nm, 405nm), the quinolone-specific nanoprobe from Example 1 was dissolved in ultrapure water for photochromic performance testing. The analysis results are as follows: Figure 4 As shown, after 3 minutes of UV irradiation, the fluorescence intensity excited by the nanoprobes that specifically recognize quinolones varied under UV light of different wavelengths.

[0138] Test results as follows Figure 5 As shown.

[0139] Figure 5 The nanoprobes that specifically recognize quinolones exhibited excitation-dependent fluorescence emission. The most pronounced fluorescence emission was observed at 450 nm under 365 nm excitation. The prepared nanoprobes that specifically recognize quinolones demonstrated excellent fluorescence properties, laying the foundation for their use as fluorescent probes for molecular recognition.

[0140] Experimental Example 3

[0141] The stability of the quinolone-specific nanoprobes prepared in Example 1 was tested by irradiating them with ultraviolet light, and by placing them at 25°C for 4 months. The test results are as follows: Figure 6 As shown in the image. The top image shows the nanoprobe that specifically recognizes quinolones, which was just prepared, and the bottom image shows the nanoprobe that specifically recognizes quinolones, which has been stored for 4 months.

[0142] Figure 6 The results showed that the quinolone-specific nanoprobe, after being stored for 4 months, still exhibited fluorescence, with the fluorescence intensity almost identical to that of the freshly prepared quinolone-specific nanoprobe, indicating that the quinolone-specific nanoprobe prepared in this invention is stable.

[0143] Application Example 1

[0144] When the absorption spectrum of a quinolone drug acting as a receptor significantly overlaps with the excitation or emission spectrum of the nanoprobe, the receptor can quench the fluorescence of the fluorescent probe through the IFE effect.

[0145] The quinolone-specific nanoprobes prepared in Example 1 were mixed with different concentrations of enoxacin (0 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL) to detect the effect of the specific nanoprobes on the quinolone drugs. The detection method was as follows: when the nanoprobes were mixed with enoxacin solutions of different concentrations, the fluorescence of the fluorescent probe was quenched by the binding of specific antibodies on the surface of the nanoprobes. The degree and time of fluorescence quenching varied with different concentrations of enoxacin solution.

[0146] Test results as follows Figure 7 As shown.

[0147] Figure 7 The results show that the nanoprobes prepared by this invention that specifically recognize quinolones have good photochromic and antibacterial properties and can specifically recognize quinolone drugs. Figure 7 The fluorescence spectra shown in the image, from top to bottom, are the fluorescence curves for detecting enoxacin at concentrations of 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL.

[0148] As can be seen from the above embodiments, this invention provides a nanoprobe, its preparation method, and its application, as well as a nanoprobe specifically for recognizing quinolones and its preparation method. This invention is based on the fluorescence recognition of quinolones using carbon dots. It utilizes the principle that the complex formed by the binding of quinolones and copper ions absorbs the fluorescence of carbon dots, and establishes a fluorescence quenching recognition mode for quinolone determination based on the internal filtration effect. This invention achieves convenient and efficient drug detection by encapsulating nanomaterials and binding them to quinolone drug antibodies.

[0149] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanoprobe, characterized in that, It includes the following components: Hydroxylated polystyrene-doped sodium niobate nanosphere solutions, N-carbon dot solutions, and copper carbon dot solutions; The initial density of the hydroxylated polystyrene sodium niobate nanosphere solution is 1~3×10⁻⁶. 14 cells / mL; The initial molar concentration of the N carbon point solution is 0.05~0.15M; The initial molar concentration of the copper carbon dot solution is 0.05~0.15M; The volume ratio of the hydroxylated polystyrene sodium niobate nanosphere solution to the N carbon dot solution is 18~22:2~4; The volume ratio of the hydroxylated polystyrene sodium niobate nanosphere solution to the copper carbon dot solution is 18~22:2~4; The preparation method of the hydroxylated polystyrene sodium niobate nanosphere solution is as follows: the hydroxylated polystyrene sodium niobate nanospheres are mixed with an aqueous acetone solution. The volume ratio of acetone to water in the acetone aqueous solution is 1~2:1~2; The preparation method of the hydroxylated polystyrene sodium niobate nanospheres includes the following steps: Styrene, acrylic acid, sodium dodecyl sulfonate aqueous solution and sodium niobate nanosphere aqueous solution are mixed to obtain a mixture; the mixture is heated to 70~80℃, potassium persulfate solution is added, and the reaction is carried out under anaerobic conditions for 7.5~8.5h to obtain a reaction solution; the reaction solution is filtered, the filtrate is taken, dialyzed for 4~6 days, and the liquid in the dialysis bag is collected to obtain hydroxylated polystyrene doped sodium niobate nanospheres; The final molar concentration of styrene is 9-11 mM; The final molar concentration of the acrylic acid is 0.9~1.0 mM; The initial molar concentration of the sodium dodecyl sulfonate aqueous solution is 0.4~0.5 mM; The initial molar concentration of the sodium niobate nanosphere aqueous solution is 0.12~0.2mM; The volume ratio of the sodium dodecyl sulfonate aqueous solution to the sodium niobate nanosphere aqueous solution is 1:0.67~0.71; The initial molar concentration of the potassium persulfate solution is 0.1~0.2 mM; The potassium persulfate solution is an aqueous solution of potassium persulfate. The volume ratio of potassium persulfate solution to sodium dodecyl sulfate aqueous solution is 1:18~22; The nanoprobe is used to detect quinolones; The preparation method of the nanoprobe is as follows: (1) a hydroxylated polystyrene sodium niobate nanosphere solution, an N carbon dot solution and a copper carbon dot solution are mixed, heated to 55~65℃, reacted in the dark for 9~11h, and then reacted at 20~25℃ for 1.8~2.2h to obtain a reaction solution; (2) the reaction solution is distilled and dialyzed, and the liquid in the dialysis bag is collected to obtain the nanoprobe.

2. The nanoprobe according to claim 1, characterized in that, The N carbon dot solution was prepared by mixing N carbon dots with water; The method for preparing the N carbon dots includes the following steps: Citric acid, diethylenetriamine and water were mixed and left to stand for 10-14 hours to obtain an intermediate. The intermediate was dialyzed for 70-74 hours to obtain dialysate; the dialysate was freeze-dried to obtain N carbon dots. The mass-to-volume ratio of citric acid to water is 1.0~1.4g:18~22mL; The volume ratio of the diethylenetriamine to water is 0.14~0.16:18~22; The placement temperature is 220~240℃; The dialysis method is dialysis using a dialysis bag; the dialysis bag is 1000 MWCO.

3. The nanoprobe according to claim 1, characterized in that, The copper carbon dot solution was prepared by mixing copper carbon dots with water. The method for preparing the copper carbon dots includes the following steps: Citric acid, copper acetate, and diethylenetriamine were mixed with water and dissolved to obtain an intermediate solution. The intermediate solution was heated to 220-240°C and held for 10-12 hours, then dialyzed and freeze-dried to obtain copper carbon dots. The mass-to-volume ratio of citric acid to water is 1.0~1.4g:18~22mL; The mass-to-volume ratio of the copper acetate to water is 0.13~0.17g:18~22mL; The volume ratio of the diethylenetriamine to water is 0.14~0.16:18~22; The copper acetate is a copper acetate with a dimer unit structure; The dissolution is ultrasonic dissolution; the ultrasonic dissolution time is 10~12 min; the dialysis method is dialysis bag dialysis; the dialysis bag is 1000 MWCO.

4. The method for preparing the nanoprobe according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Mix the hydroxylated polystyrene sodium niobate nanosphere solution, N carbon dot solution and copper carbon dot solution, heat to 55~65℃, react in the dark for 9~11h, and then react at 20~25℃ for 1.8~2.2h to obtain the reaction solution; (2) After distilling and dialysis of the reaction solution, the liquid in the dialysis bag is collected to obtain the nanoprobe.

5. The preparation method according to claim 4, characterized in that, The distillation is performed under reduced pressure; the dialysis bag is 1000 MWCO; and the dialysis time is 4-6 days.

6. The application of the nanoprobe according to any one of claims 1-3 or the nanoprobe prepared by the preparation method according to any one of claims 4-5 in the preparation of detection products that specifically recognize quinolones.

7. A nanoprobe that specifically recognizes quinolones, characterized in that, The nanoprobe that specifically recognizes quinolones includes the nanoprobe according to any one of claims 1-3 or the nanoprobe prepared by the preparation method according to any one of claims 4-5, as well as quinolone antibodies; The quinolone antibody is a norfloxacin monoclonal antibody.

8. The method for preparing the nanoprobe that specifically recognizes quinolones according to claim 7, characterized in that, Includes the following steps: (1) Activate the nanoprobe according to any one of claims 1-3 or the nanoprobe prepared by the preparation method according to any one of claims 4-5 to obtain an activated nanoprobe; (2) Dissolve the activated nanoprobe in borate buffer to obtain a solution; mix the solution with a quinolone antibody and react for 11-13 h to obtain a reaction solution; (3) Centrifuge the reaction solution, collect the precipitate, dissolve the precipitate in phosphate buffer to obtain a nanoprobe that specifically recognizes quinolone; The initial molar concentration of the borate buffer solution in step (2) is 0.01~0.02M; The initial pH of the borate buffer solution is 7.8~8.2; The mass-to-volume ratio of the quinolone antibody to the solution is 0.2-0.3 mg: 5 mL; The centrifuge speed is 11000~13000 rpm; The centrifugation time is 9-11 minutes; The phosphate buffer also contains trehalose and bovine serum albumin; The mass ratio of trehalose added to the volume ratio of phosphate buffer is 1~2g:100mL; The ratio of the added bovine serum albumin to the volume of phosphate buffer is 1.5~2.5g:100mL; The initial molar concentration of the phosphate buffer is 0.01~0.02M; The initial pH of the phosphate buffer is 7.2 to 7.

6.

9. The preparation method according to claim 8, characterized in that, The activation step described in step (1) is as follows: The nanoprobes prepared by the method according to any one of claims 1-3 or claims 4-5 are diluted to a density of 1.0~3.0×10⁻⁶. 12 The nanoprobes were diluted by 1 / mL; the diluted nanoprobes were sonicated and then mixed with an aqueous solution of carbodiimide. The mixture was reacted for 13-17 min, centrifuged, and the precipitate was collected to obtain the activated nanoprobes. The solution used to dilute the nanoprobes was a 0.01–0.02 M borate buffer solution. The power of the ultrasound is 300~500W; The duration of the ultrasound is 25-35 seconds. The initial concentration of the carbodiimide aqueous solution is 14-16 mg / mL; The volume ratio of the diluted nanoprobe to the carbodiimide aqueous solution is 48~52:1; The reaction temperature is 20~25℃; The centrifuge speed is 14000~16000 rpm; The centrifugation time is 8-12 minutes.

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