Iridium metal complex fluorescent probe for detecting norfloxacin, molecular imprinting modified iridium probe fluorescent sensor and preparation method and application thereof

Abstract

The application discloses an iridium metal complex fluorescent probe for detecting norfloxacin, a molecular imprinting modified iridium probe fluorescent sensor and a preparation method and application thereof, and has the characteristics that the chemical structural formula of the iridium metal complex fluorescent probe is shown in the following formula (I), the preparation method comprises the steps that hydrated iridium trichloride and 2-phenylquinoline are dissolved in 2-ethoxyethanol to obtain orange crude compounds, the crude compounds are reacted with anhydrous sodium carbonate dissolved in 2-ethoxyethanol to obtain the probe (I); further, the probe (I), a crosslinking agent and an initiator are sequentially injected into a homogeneous dispersion liquid of cyclohexane and Triton X-100 to obtain a microemulsion system after reaction; a template molecule norfloxacin and a functional monomer are added into the cyclohexane medium to be mixed to form a stable complex; the complex is added into the microemulsion system to be reacted, and quantum dots are further added to be reacted, so that the fluorescent sensor is obtained, and the advantages are that the synthesis difficulty can be reduced, and the fluorescent sensor has good selectivity, high sensitivity and high accuracy.

Description

Technical Field

[0001] This invention relates to the technology for detecting norfloxacin, and in particular to an iridium metal complex fluorescent probe, an iridium probe fluorescent sensor, and their preparation methods and applications for detecting norfloxacin. Background Technology

[0002] Iridium metal complexes possess typical triplet emission and unique photophysical properties, including three excited states: 3MLCT, 3LLCT, and 3IL. They exhibit excellent photophysical and photochemical properties as well as a long lifetime. As fluorescent probes, iridium complexes can detect the presence of target substances by detecting changes in fluorescence signals. Molecular imprinting (MIP) is an effective method to improve the selectivity of sensors for target analytes. MIPs, also known as artificial antibodies, involve completely polymerizing a target analyte template through the action of cross-linking agents and functional monomers, followed by template elution to form an imprinted cavity. Combining molecularly imprinted polymers with fluorescence analysis techniques effectively improves the sensitivity and selectivity of detection methods.

[0003] Iridium metal complex probes have demonstrated excellent performance in practical applications for detecting antibacterial drugs. They are easy to operate, do not require expensive instruments, and are well-suited for real-time or in-situ detection of antibacterial drugs. However, iridium metal complex probes typically require complex synthesis steps, increasing the difficulty of probe preparation. Furthermore, iridium metal complex probes exhibit poor stability and selectivity, and may be affected by interference in certain complex samples or extreme pH conditions. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an iridium metal complex fluorescent probe, an iridium probe fluorescent sensor, and their preparation method and application for detecting norfloxacin, which can reduce the difficulty of synthesis and have good selectivity, sensitivity and accuracy.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: an iridium metal complex fluorescent probe for detecting norfloxacin, wherein the chemical structural formula of the iridium metal complex fluorescent probe is shown in formula (I).

[0006] (I).

[0007] The present invention also provides a method for preparing the above-mentioned iridium metal complex fluorescent probe for detecting norfloxacin, comprising the following steps:

[0008] Step 1: Iridium trichloride hydrate and 2-phenylquinoline were dissolved in 2-ethoxyethanol and ultrapure water and reacted to obtain an orange crude compound with the chemical structure shown in formula (II).

[0009] (II);

[0010] Step 2: The orange crude compound 4,4'-(1,10-phenanthroline-2,9-diyl)diphenylamine and anhydrous sodium carbonate were dissolved in 2-ethoxyethanol and reacted under heating. Then, NH4PF6 was added and stirred. Silica gel column chromatography was performed using cyclohexane-ethyl acetate as the eluent to obtain an iridium metal complex fluorescent probe for the detection of norfloxacin. Its chemical structure is shown in formula (I).

[0011] (I).

[0012] Furthermore, the specific steps are as follows:

[0013] Step 1: Dissolve 0.05–0.2 mg of iridium trichloride hydrate and 0.2–0.4 g of 2-phenylquinoline in 10–20 mL of 2-ethoxyethanol and 2–10 mL of ultrapure water and add the solution to a pressure-resistant tube. Heat the solution to 95–100 °C and stir for 10–15 hours. Cool the resulting mixture to room temperature, dilute it with 40–60 mL of ultrapure water, centrifuge it 2–4 times to remove the supernatant, collect the orange precipitate, and vacuum dry it to obtain the orange crude compound, the chemical structure of which is shown in Formula (II).

[0014] Step 2: Dissolve 0.4–0.6 g of the orange crude compound 1,4,4'-(1,10-phenanthroline-2,9-diyl)diphenylamine and 1–2 g of anhydrous sodium carbonate in 15–25 ml of 2-ethoxyethanol and add the solution to a pressure-resistant tube. Heat the mixture to 95–100 °C and stir vigorously for 15–25 h. Then add NH4PF6 and stir for 1–3 h. After the pressure-resistant tube has cooled to room temperature, use cyclohexane-ethyl acetate (a mixture of cyclohexane and ethyl acetate in a volume ratio of 5:1) as the eluent for silica gel column chromatography. Take the orange-yellow portion of the silica gel column solution and evaporate it to dryness at 20–30 °C to obtain a dry solid, which is the iridium metal complex fluorescent probe for detecting norfloxacin. Its chemical structure is shown in formula (I).

[0015] This invention also provides a method for detecting norfloxacin using the above-mentioned iridium metal complex fluorescent probe. This method is not intended for disease diagnosis or treatment and includes the following steps:

[0016] Step 1: Dissolve the iridium metal complex fluorescent probe in dimethyl sulfoxide to prepare a 5-30 mg / L iridium metal complex fluorescent probe solution;

[0017] Step 2: Using a Varioskan Flash microplate reader, set the excitation wavelength to 360 nm, the scanning range to 400-700 nm, and the slit width to 5 nm. Measure the fluorescence intensity of the iridium metal complex fluorescent probe solution at 495 nm and record it as F0. Add the sample solution to be tested to the iridium metal complex fluorescent probe solution at a volume ratio of 1:99 to obtain a mixture. After reacting for 4-6 min, measure the fluorescence intensity of the mixture at 495 nm and record it as F. Calculate the fluorescence enhancement intensity F / F0.

[0018] Step 3: Calculate the concentration of norfloxacin in the sample solution based on the linear relationship between norfloxacin concentration and fluorescence sensitization intensity F / F0.

[0019] This invention also provides a method for preparing a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin, comprising the following steps:

[0020] Step 1: After mixing cyclohexane and Triton X-100 to form a homogeneous dispersion, the probe (I), crosslinking agent tetraethoxysilane, and ammonia initiator are injected sequentially for pre-crosslinking reaction to obtain a microemulsion system;

[0021] Step 2: Add the template molecule norfloxacin and the functional monomer 3-aminopropyltriethoxysilane to a cyclohexane medium and mix to form a stable complex;

[0022] Step 3: After adding the complex to the microemulsion system, in-situ polymerization was carried out under light-protected conditions. Then, CdSe@ZnS quantum dots were added for reaction. After the reaction was completed, acetone was added to demulsify and terminate the reaction. The precipitate was collected by centrifugation. The precipitate was washed with ultrapure water to remove residual monomers. Then, multiple rounds of ultrasonic elution were performed with an ethanol-acetonitrile mixture until LC-UV detection confirmed that the template molecules were completely removed, resulting in a molecularly imprinted modified iridium probe fluorescent sensor for detecting norfloxacin.

[0023] Furthermore, the specific steps are as follows:

[0024] Step 1: Mix 6-10 mL of cyclohexane and 1-3 mL of Triton X-100, and stir magnetically at 300-500 r / min for 8-12 min to form a homogeneous dispersion. Then, sequentially inject 40-60 μL of probe (I) solution with a concentration of 0.5-2 mg / mL, 50-70 μL of crosslinking agent tetraethoxysilane, and 150-250 μL of ammonia initiator with a concentration of 20-30 wt%. After the pre-crosslinking reaction is carried out by stirring magnetically at 300-500 r / min for 1-3 h, a microemulsion system is obtained.

[0025] Step 2: Add 200-400 L of the template molecule norfloxacin with a concentration of 5-20 mg / mL and 50-60 μL of the functional monomer 3-aminopropyltriethoxysilane to 100-300 L of cyclohexane medium, and stir magnetically at 300-500 r / min for 1-3 h to form a stable complex.

[0026] Step 3: After adding the complex to the microemulsion system, in-situ polymerization was carried out under light-protected conditions with magnetic stirring at 300-500 r / min for 10-12 h. Then, CdSe@ZnS quantum dots were added and magnetic stirring was carried out at 300-500 r / min for 2-300-500 h. After the reaction was completed, 8-12 mL of acetone was added to demulsify and terminate the reaction. The precipitate was collected by centrifugation. The precipitate was washed with ultrapure water to remove residual monomers. Then, multiple rounds of ultrasonic elution were performed using an ethanol-acetonitrile mixture (ethanol and acetonitrile in a volume ratio of 8:2) until LC-UV detection confirmed that the template molecules were completely removed, resulting in a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin.

[0027] This invention also provides a method for detecting norfloxacin using a molecularly imprinted modified iridium probe fluorescent sensor prepared by the above method. This method is not intended for disease diagnosis or treatment and includes the following steps:

[0028] Step 1: Dissolve the molecularly imprinted iridium probe fluorescent sensor in dimethyl sulfoxide to prepare a 5-30 mg / L molecularly imprinted iridium probe fluorescent sensor solution;

[0029] Step 2: Using a Varioskan Flash microplate reader, set the excitation wavelength to 360 nm, the scanning range to 400-700 nm, and the slit width to 5 nm. Measure the fluorescence intensity of the molecularly imprinted modified iridium probe fluorescent sensor solution at wavelengths of 442 nm and 630 nm. Take the I0442 / I0630 value as the fluorescence intensity of Ir-NOR@MIP-CdSe@ZnS without the addition of template molecules, and record it as f0.

[0030] Step 3: Add the sample solution to be tested to the molecularly imprinted iridium probe fluorescent sensor solution at a volume ratio of 1:99 to obtain a mixture. After reacting for 4-6 min, measure the fluorescence intensity of the mixture at wavelengths of 442 nm and 630 nm. Take the I442 / I630 value as the fluorescence intensity of the molecularly imprinted iridium probe fluorescent sensor after incubation with the template molecule, and record it as f. Calculate the fluorescence enhancement intensity f / f0.

[0031] Step 4: Calculate the concentration of norfloxacin in the sample solution based on the linear relationship between norfloxacin concentration and fluorescence sensitization intensity f / f0.

[0032] Compared with the prior art, the advantages of the present invention are as follows:

[0033] 1. Iridium metal complex fluorescent probes exhibit high specificity and sensitivity: Norfloxacin possesses an extended conjugated structure, and its higher highest occupied molecular orbital (HOMO) energy level promotes charge transfer (LMCT) from the ligand to the metal center in iridium complex probes, rather than a static quenching process. This process, driven by energy level matching, significantly enhances the radiative transition probability of the system, generating a fluorescence enhancement effect at 495 nm through effective electron transfer, thereby achieving fluorescence detection of the analyte and achieving a detection limit of 1.04 μg / L.

[0034] 2. Molecularly imprinted iridium probe fluorescent sensors further improve specificity and sensitivity:

[0035] (1) "Lock and key" mechanism: The molecular imprinted cavity provides a unique "molecular lock-key" recognition function, which can effectively eliminate the interference of structural analogs, realize the specific recognition of norfloxacin, and ensure the high accuracy and reliability of the detection results.

[0036] (2) Synergy of dual-signal probes: The emission spectra of iridium complexes and quantum dots can be well separated, and their response modes to target molecules are different (e.g., one enhances and the other quenches), which can be used to construct ratiometric sensors. Ratiometric sensors quantify by measuring the ratio of signals from two wavelengths, which can effectively eliminate the interference of environmental factors such as light source fluctuations and probe concentration changes, and significantly improve the accuracy and sensitivity of detection.

[0037] (3) Physical protection: The MIP polymer matrix encapsulates the iridium complex and quantum dots, forming a robust "nanoreactor" or "protective shell." This can: prevent the leakage and aggregation of quantum dots and iridium complexes; and shield the signal probe from the physical adsorption and damage of macromolecules such as proteins and enzymes in complex samples, thus improving the stability of the probe in the biological environment.

[0038] (4) Provides anti-interference capability: Traditional iridium complex or quantum dot probes are easily quenched or interfered with by other substances in complex samples (such as serum, urine, and environmental water samples), resulting in false positive or false negative signals. The MIP layer acts as a highly efficient "pre-enrichment and screening layer", allowing only target molecules to approach the signal probe, fundamentally improving the probe's anti-interference capability.

[0039] In summary, this invention combines molecularly imprinted polymers with iridium metal complex probes and CdSe@ZnS quantum dots to construct a sensor that integrates high selectivity, high sensitivity, and multiple signal outputs, achieving a detection limit as high as 0.32 μg / L. Attached Figure Description

[0040] Figure 1 This is a synthetic route diagram of the iridium metal complex fluorescent probe in Specific Embodiment 1;

[0041] Figure 2 This illustrates the linear relationship between probe (I) and NOR concentration in Specific Example 2;

[0042] Figure 3 The results of the selective analysis of NOR by probe (I) in specific embodiment three are shown.

[0043] Figure 4 The effect of TEOS dosage on the molecularly imprinted sensor in specific embodiment five;

[0044] Figure 5 This illustrates the effect of the NOR to APTES molar ratio on the molecularly imprinted sensor in Specific Embodiment 5.

[0045] Figure 6 The present invention relates to the selectivity and competition analysis of norfloxacin by the molecularly imprinted sensor in Specific Embodiment Six, wherein A represents the selectivity analysis result and B represents the competition analysis result.

[0046] Figure 7 This illustrates the linear relationship between the molecularly imprinted sensor and the NOR concentration in Specific Embodiment Seven. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0048] Thin-layer chromatography uses silica gel plates with a diameter of 0.20 mm to 0.25 mm. Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the support.

[0049] Specific Example 1: An iridium metal complex fluorescent probe (hereinafter referred to as probe (I)) for detecting norfloxacin, the chemical structure of which is shown in formula (I).

[0050] (I).

[0051] The preparation method of the above-mentioned iridium metal complex fluorescent probe is as follows: Figure 1 As shown, it includes the following steps:

[0052] Step 1: Dissolve 0.1 mg iridium trichloride hydrate and 0.285 g 2-phenylquinoline in 15 mL of 2-ethoxyethanol and 5 mL of ultrapure water, and add the solution to a pressure-resistant tube. Heat to 100 °C and stir for 12 hours. Cool the resulting mixture to room temperature, dilute with 50 mL of ultrapure water, and then... 3Centrifuge for 10 minutes under g conditions, repeat three times to remove the supernatant, collect the orange precipitate, and vacuum dry to obtain the orange crude compound, whose chemical structure is shown in formula (II).

[0053] (II);

[0054] Step 2: Dissolve 0.49 g of the orange crude compound 4,4'-(1,10-phenanthroline-2,9-diyl)diphenylamine and 1.23 g of anhydrous sodium carbonate in 20 ml of 2-ethoxyethanol and add the solution to a pressure-resistant tube. Heat the mixture to 95°C and stir vigorously for 20 h. Then add NH4PF6 and stir for 2 h. After the pressure-resistant tube cools to room temperature, use cyclohexane-ethyl acetate (mixed with cyclohexane and ethyl acetate in a volume ratio of 5:1) as the eluent for silica gel column chromatography. Take the orange-yellow part of the silica gel column solution and evaporate it to dryness at 25°C to obtain a dry solid, which is the iridium metal complex fluorescent probe for detecting norfloxacin, abbreviated as probe (I), with a yield of 70%. Its chemical structure is shown in formula (I).

[0055] Specific Example 2: Sensitivity determination of the probe (I) prepared by the method of Specific Example 1 to norfloxacin.

[0056] The fluorescence sensitization intensity (F / F0) of probe (I) added to different concentrations (0.01–15.0 mg / L) of norfloxacin was analyzed, and the linear relationship between the sensitization intensity and the norfloxacin concentration was investigated to establish a corresponding standard curve. The linear range and precision were verified by plotting the standard curve and calculating the regression equation.

[0057] Experimental methods: A Varioskan Flash microplate reader (Thermo Fisher Scientific, model 3001 / 3001-1193) was used with an excitation wavelength of 360 nm, a scanning range of 400-700 nm, and a slit width of 5 nm. All experiments were performed with three repeated fluorescence scans. The probe (I) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / L probe (I) solution. The fluorescence intensity of the 10 mg / L probe (I) solution was measured and recorded as F0. Norfloxacin standard solutions with concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 30 mg / L, 50 mg / L and 100 mg / L were prepared. 10 μL of the norfloxacin standard solution of different concentrations was added to 990 μL of probe (I) solution with pH=7 and a concentration of 10 mg / L. After reacting for 5 min, the fluorescence intensity of the probe (I) solution with different concentrations of norfloxacin was measured at 495 nm and recorded as F. The fluorescence enhancement intensity F / F0 was calculated.

[0058] The results are shown in Figure 2. For probe (I), the fluorescence intensity changed linearly with increasing norfloxacin concentration, with the linear formula Y = 0.50475X + 0.10218 and the correlation coefficient R0. 2 =0.992, detection limit is 1.04 μg / L.

[0059] Specific Example 3: Selectivity determination of norfloxacin by probe (I) prepared by the method of Specific Example 1.

[0060] The selectivity of probe (I) for norfloxacin was determined by selecting other antibiotic substances: lomefloxacin (LFX), pefloxacin (PFX), ofloxacin (OFX), enrofloxacin (ENR), gatifloxacin (GAT), enoxacin (ENO), moxifloxacin (MXF), sparfloxacin (SPA), and ciprofloxacin (CIP), and preparing antibiotic solutions with a concentration of 10 mg / L.

[0061] Experimental method: Add 10 mg / L antibiotic solution to 0.2 mL of 10 mg / L probe (I) solution. After reacting for 5 min, record the fluorescence intensity at 400-700 nm (Ex = 360 nm). Repeat the measurement 3 times and calculate the fluorescence enhancement efficiency F / F0-1, where F0 is the fluorescence intensity of probe (I) solution and F is the fluorescence intensity after adding antibiotic solution to probe (I) solution.

[0062] The results are as follows Figure 3 As shown, the change in fluorescence intensity of probe (I) after adding other antibiotics to the probe (I) solution was weaker than the increase in fluorescence intensity of probe (I) after adding norfloxacin. This indicates that probe (I) has good selectivity for norfloxacin.

[0063] Specific embodiment four: Preparation of a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin.

[0064] 1. A molecularly imprinted modified iridium probe fluorescent sensor (Ir-NOR@MIP-CdSe@ZnS) was synthesized using a reverse microemulsion method, including the following steps:

[0065] Step 1: An oil phase system was constructed using 8.0 mL of cyclohexane (porogen) and 2.0 mL of Triton X-100 (surfactant). After forming a homogeneous dispersion by magnetic stirring at 400 r / min for 10 min, 50 μL of probe (I) solution with a concentration of 1 mg / mL, 60 μL of crosslinking agent tetraethoxysilane (TEOS), and 200 μL of ammonia initiator with a concentration of 25 wt% were injected sequentially. The pre-crosslinking reaction was carried out by magnetic stirring at 400 r / min for 2 h. After the reaction was completed, a microemulsion system was obtained.

[0066] Step 2: Add 300 L of 10 mg / mL template molecule norfloxacin (solvent DMSO) and 54 μL of functional monomer 3-aminopropyltriethoxysilane (APTES) to 200 L of cyclohexane medium, and stir magnetically at 400 r / min for 2 h to form a stable complex.

[0067] Step 3: After adding the complex to the microemulsion system, in-situ polymerization was carried out under light-protected conditions with magnetic stirring at 400 r / min for 12 h. After the reaction was completed, 10 mL of acetone was added to demulsify and terminate the reaction. The precipitate was collected by centrifugation (8.5 × 10³ g, 10 min). The precipitate was washed with ultrapure water (10 mL × 3 times) to remove residual monomers. Then, multiple rounds of ultrasonic elution were performed using an ethanol-acetonitrile mixture (made by mixing ethanol and acetonitrile in a volume ratio of 8:2) (45 min / time, 10 mL / round) until LC-UV detection confirmed that the template molecules were completely removed, and Ir-NOR@MIP was obtained.

[0068] 2. The synthesis of the control group Ir-NOR@NIP is the same as the preparation method of Ir-NOR@MIP described above, except that in step 3, an equal volume of cyclohexane is used instead of norfloxacin template.

[0069] 3. The synthesis of Ir-NOR@MIP-CdSe@ZnS is the same as the preparation method of Ir-NOR@MIP described above. The difference is that after the complex is transferred to the microemulsion system in step 3, in-situ polymerization is carried out under light-protected conditions with magnetic stirring at 400 r / min for 10 h. Then, CdSe@ZnS quantum dots are added and magnetic stirring is carried out at 400 r / min for 2 h. After the reaction is completed, 10 mL of acetone is added to demulsify and terminate the reaction. The elution steps after the reaction are the same as those in step 3 above.

[0070] Optimization of the preparation method of molecularly imprinted modified iridium probe fluorescent sensor in specific embodiments five and four.

[0071] The fluorescence enhancement efficiency of the molecularly imprinted iridium probe fluorescent sensor was quantified using the Stern-Volmer equation:

[0072] f0 / f=1+Ksv[Q],

[0073] Where f0 represents the fluorescence intensity of Ir-NOR@MIP-CdSe@ZnS without the template molecule, and is taken as I0442 / I0630. I0442 and I0630 correspond to the fluorescence intensities of the sensor at wavelengths of 442 nm and 630 nm, respectively, without the template molecule. f represents the fluorescence intensity of Ir-NOR@MIP-CdSe@ZnS after incubation with the template molecule for 5 minutes, and is taken as I442 / I630. I442 and I630 correspond to the fluorescence intensities of the sensor at wavelengths of 442 nm and 630 nm, respectively, after the reaction with the template molecule. [Q] is the concentration of the antibiotic norfloxacin. The imprinting factor (abbreviated as IF) is the ratio of the Ksv value of Ir-NOR@MIP-CdSe@ZnS to the Ksv value of Ir-NOR@NIP-CdSe@ZnS.

[0074] Determining the optimal dosage of crosslinking agent:

[0075] The amount of crosslinking agent TEOS in step 1 of specific embodiment 4 was set to 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, and 80 μL, respectively. Different Ir-NOR@MIP-CdSe@ZnS were prepared according to the method of specific embodiment 4. The f / f0 ratio of each group of Ir-NOR@MIP-CdSe@ZnS was measured, and the imprinting factor IF value was calculated.

[0076] The results are as follows Figure 4 As shown, the amount of TEOS affects the shell thickness of the polymer outer layer. The amount of TEOS added affects the fluorescence efficiency of the polymer. With the increase of crosslinking agent, the f / f0 ratio of Ir-NOR@MIP-CdSe@ZnS gradually increases. When the amount of TEOS is too large, the f / f0 ratio of Ir-NOR@NIP-CdSe@ZnS still gradually increases, possibly because excessive TEOS leads to an excessively large silicon sphere area, increasing non-characteristic recognition. Therefore, the fluorescence response is optimal when the amount of TEOS is 50 μL.

[0077] 2. Determination of the optimal ratio of template molecule to functional monomer

[0078] In step 2 of specific embodiment four, norfloxacin and APTES were set to a molar ratio of 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12, respectively. Different Ir-NOR@MIP-CdSe@ZnS were prepared according to the method of specific embodiment four. The f / f0 ratio of each group of Ir-NOR@MIP-CdSe@ZnS was measured, and the imprinting factor IF value was calculated.

[0079] The results are as follows Figure 5As shown, functional monomers affect the performance of the polymer's recognition sites. Insufficient APTES leads to a reduction in the number of functional groups binding to norfloxacin, while excessive APTES affects the polymer's porosity and specific surface area, thus impacting the formation of imprinted cavities. When the molar ratio of norfloxacin to APTES is 1:8, the imprinting factor IF value is maximized at 4.29, while the sensitizing intensity f / f0 of Ir-NOR@NIP-CdSe@ZnS is minimized. Therefore, a molar ratio of norfloxacin to APTES of 1:8 is selected as the optimal ratio.

[0080] Specific Implementation Example 6: Selectivity and Competitiveness Analysis of Norfloxacin with Ir-NOR@MIP-CdSe@ZnS.

[0081] To evaluate the selectivity and competitive characteristics of the Ir-NOR@MIP-CdSe@ZnS sensor for norfloxacin detection, structural analogs of norfloxacin were selected as control analytes: lomefloxacin (LFX), pefloxacin (PFX), ofloxacin (OFX), enrofloxacin (ENR), gatifloxacin (GAT), enoxacin (ENO), moxifloxacin (MXF), sparfloxacin (SPA), and ciprofloxacin (CIP). The concentration of the template molecule norfloxacin was 25.0 mg / L, and the concentration of the structural analogs was 50.0 mg / L.

[0082] Ir-NOR@MIP-CdSe@ZnS was dissolved in dimethyl sulfoxide to obtain a 10 mg / L Ir-NOR@MIP-CdSe@ZnS solution. 900 μL of the Ir-NOR@MIP-CdSe@ZnS solution was mixed with 100 μL of the test solution and added to a cuvette. After reacting for 30 s, based on the dual-channel ratiometric fluorescence detection principle, a multi-functional microplate reader (excitation wavelength 360 nm, slit width 5 nm) was used to simultaneously capture the emission spectral intensity of the Ir-NOR@MIP-CdSe@ZnS sensor at wavelengths of 442 nm and 630 nm. The fluorescence enhancement intensity f / f0 and the molecular imprinting factor IF value were calculated.

[0083] The results are as follows Figure 6 As shown in Figure A, the surface of Ir-NOR@MIP-CdSe@ZnS contains specific recognition sites for norfloxacin. For structural analogs, the size and shape of the imprint do not match these sites. Ir-NOR@MIP-CdSe@ZnS effectively reduces interference from CIP and other similar compounds. The IF value of Ir-NOR@MIP-CdSe@ZnS for norfloxacin is as high as 1.68, which is significantly higher than the IF value of CIP (0.71). In competitive experiments, such as... Figure 6As shown in Figure B, the results indicate that the addition of structural analogs such as CIP did not affect the specific response sensitivity between Ir-NOR@MIP-CdSe@ZnS and norfloxacin. In other words, the presence of the molecular imprinted layer enables Ir-NOR@MIP-CdSe@ZnS to exhibit good selective recognition ability for norfloxacin.

[0084] Specific Example 7: Sensitivity Analysis of Ir-NOR@MIP-CdSe@ZnS to Norfloxacin

[0085] This study investigated the fluorescence sensitization intensity f / f0 of norfloxacin at different concentrations (0.01–15.0 mg / L) and explored its linear relationship with NOR concentration to establish a corresponding standard curve. The linear range and precision were verified by plotting the standard curve and calculating the regression equation.

[0086] Experimental Methods: Norfloxacin standard solutions with concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 30 mg / L, 50 mg / L, and 100 mg / L were prepared. 10 μL of each norfloxacin standard solution was added to 990 μL of Ir-NOR@MIP-CdSe@ZnS solution (pH=7, 10 mg / L). The reaction was allowed to proceed for 5 min. The emission spectra of the Ir-NOR@MIP-CdSe@ZnS solutions with different concentrations of norfloxacin were measured at wavelengths of 442 nm and 630 nm. The fluorescence sensitization intensity f / f0 was calculated. Results are as follows: Figure 7 As shown, the fluorescence intensity changes linearly with increasing norfloxacin concentration, with the linear formula being Y = (-0.12837)X + 1.9976, and the linear relationship being R. 2 =0.997, detection limit is 0.32 μg / L.

[0087] Specific Implementation Example 8: Norfloxacin in actual samples was determined using the optimal conditions and method of Specific Implementation Example 7.

[0088] Seawater sample pretreatment: Seawater samples were filtered through a 0.22 μm membrane to remove particles and microorganisms, yielding 100 μL of test solution, which was then stored at 4℃.

[0089] Solid sample pretreatment: 5 g of fish, shrimp, and shellfish samples were accurately weighed, vortexed, and then extracted with 10 mL of acetonitrile using ultrasound. The samples were then subjected to an 8.5 × 10⁻⁶ ppm filtration process. 3 The supernatant was obtained by centrifugation for 10 min. Subsequently, it was defatted using acetonitrile-saturated n-hexane liquid-liquid partitioning, concentrated and dried by rotary evaporation, and then reconstituted with 10 mL of ethanol. Finally, it was subjected to 8.5 × 10⁻⁶ centrifugation. 3Centrifuge for 10 min, and dissolve the precipitate in a water-ethanol solvent system to obtain 100 μL of test solution, wherein the volume fraction of water in the water-ethanol solvent system is 0-50%.

[0090] 900 μL of 10 mg / L Ir-NOR@MIP-CdSe@ZnS solution was mixed with 100 μL of the test solution and added to a cuvette. After reacting for 30 s, based on the dual-channel ratiometric fluorescence detection principle, the emission spectral intensity of the Ir-NOR@MIP-CdSe@ZnS sensor at wavelengths of 442 nm and 630 nm was simultaneously captured using a multifunctional microplate reader (excitation wavelength 360 nm, slit width 5 nm). The fluorescence enhancement intensity f / f0 was calculated, and the results are shown in Table 1.

[0091] Table 1. Recovery and RSD of norfloxacin in seawater and aquatic animals determined by Ir-NOR@MIP-CdSe@ZnS (n=3)

[0092]

[0093] The recovery and standard deviation of norfloxacin were determined using the standard addition method. Four blank spiking concentrations (0.15 mg / L, 0.3 mg / L, 0.6 mg / L, and 1.5 mg / L) were selected in the optimal response system in seawater and aquatic products. The relative recoveries of norfloxacin ranged from 88% to 110%. Compared with the current method (GB31656.3-2021, 2.5 μg / kg), this method has a lower limit of detection and fully complies with the "National Food Safety Standard Maximum Residue Limits for 41 Veterinary Drugs in Food" (GB31650.1-2022, 2 μg / kg).

[0094] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. An iridium metal complex fluorescent probe for detecting norfloxacin, characterized in that... The chemical structural formula of the iridium metal complex is shown in formula (I). （I）。 2. A method for preparing an iridium metal complex fluorescent probe for detecting norfloxacin as described in claim 1, characterized in that... Includes the following steps: Step 1: Iridium trichloride hydrate and 2-phenylquinoline were dissolved in 2-ethoxyethanol and ultrapure water and reacted to obtain an orange crude compound with the chemical structure shown in formula (II). （Ⅱ）； Step 2: The orange crude compound, 4,4'-(1,10-phenanthroline-2,9-diyl)diphenylamine, and anhydrous sodium carbonate were dissolved in 2-ethoxyethanol and reacted under heating. Then, NH4PF6 was added and stirred. Silica gel column chromatography was performed using cyclohexane-ethyl acetate as the eluent to obtain an iridium metal complex fluorescent probe for the detection of norfloxacin. Its chemical structure is shown in formula (I). （I）。 3. A method for detecting norfloxacin using an iridium metal complex fluorescent probe as described in claim 1, wherein this method is not intended for disease diagnosis or treatment, characterized in that... Includes the following steps: Step 1: Dissolve the iridium metal complex fluorescent probe in dimethyl sulfoxide to prepare a 5-30 mg / L iridium metal complex fluorescent probe solution; Step 2: Using an ELISA reader, set the excitation wavelength to 360 nm, the scanning range to 400-700 nm, and the slit width to 5 nm. Measure the fluorescence intensity of the iridium metal complex fluorescent probe solution at 495 nm and record it as F0. Add the sample solution to be tested to the iridium metal complex fluorescent probe solution at a volume ratio of 1:80-120 to obtain a mixture. After reacting for 4-6 min, measure the fluorescence intensity of the mixture at 495 nm and record it as F. Calculate the fluorescence enhancement intensity F / F0. Step 3: Calculate the concentration of norfloxacin in the sample solution based on the linear relationship between norfloxacin concentration and fluorescence sensitization intensity F / F0.

4. A method for preparing a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin, characterized in that... Includes the following steps: Step 1: After mixing cyclohexane and Triton X-100 to form a homogeneous dispersion, the probe (I) described in claim 1, the crosslinking agent tetraethoxysilane, and the ammonia initiator are injected sequentially for pre-crosslinking reaction to obtain a microemulsion system; Step 2: Add the template molecule norfloxacin and the functional monomer 3-aminopropyltriethoxysilane to a cyclohexane medium and mix to form a stable complex; Step 3: After adding the complex to the microemulsion system, in-situ polymerization was carried out under light-protected conditions. Then, CdSe@ZnS quantum dots were added for reaction. After the reaction was completed, acetone was added to demulsify and terminate the reaction. The precipitate was collected by centrifugation. The precipitate was washed with ultrapure water to remove residual monomers. Then, multiple rounds of ultrasonic elution were performed with an ethanol-acetonitrile mixture until LC-UV detection confirmed that the template molecules were completely removed, resulting in a molecularly imprinted modified iridium probe fluorescent sensor for detecting norfloxacin.

5. The method for preparing a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin according to claim 4, characterized in that... The specific steps are as follows: Step 1: Mix 6-10 mL of cyclohexane and 1-3 mL of Triton X-100, and stir magnetically at 300-500 r / min for 8-12 min to form a homogeneous dispersion. Then, sequentially inject 40-60 μL of probe (I) solution with a concentration of 0.5-2 mg / mL, 50-70 μL of crosslinking agent tetraethoxysilane, and 150-250 μL of ammonia initiator with a concentration of 20-30 wt%. After the pre-crosslinking reaction is carried out by stirring magnetically at 300-500 r / min for 1-3 h, a microemulsion system is obtained. Step 2: Add 200-400 L of the template molecule norfloxacin with a concentration of 5-20 mg / mL and 50-60 μL of the functional monomer 3-aminopropyltriethoxysilane to 100-300 L of cyclohexane medium, and stir magnetically at 300-500 r / min for 1-3 h to form a stable complex. Step 3: After adding the complex to the microemulsion system, in-situ polymerization was carried out under light-protected conditions with magnetic stirring at 300-500 r / min for 10-12 h. Then, CdSe@ZnS quantum dots were added and magnetic stirring was carried out at 300-500 r / min for 2-300-500 h. After the reaction was completed, 8-12 mL of acetone was added to demulsify and terminate the reaction. The precipitate was collected by centrifugation. The precipitate was washed with ultrapure water to remove residual monomers. Then, multiple rounds of ultrasonic elution were performed using an ethanol-acetonitrile mixture (ethanol and acetonitrile in a volume ratio of 8:2) until LC-UV detection confirmed that the template molecules were completely removed, resulting in a molecularly imprinted iridium probe fluorescent sensor for detecting norfloxacin.

6. A method for detecting norfloxacin using a molecularly imprinted modified iridium probe fluorescent sensor prepared according to claim 4 or 5, wherein the method is not intended for disease diagnosis or treatment, characterized in that... Includes the following steps: Step 1: Dissolve the molecularly imprinted iridium probe fluorescent sensor in dimethyl sulfoxide to prepare a 5-30 mg / L molecularly imprinted iridium probe fluorescent sensor solution; Step 2: Using an ELISA reader, set the excitation wavelength to 360 nm, the scanning range to 400-700 nm, and the slit width to 5 nm. Measure the fluorescence intensity of the molecularly imprinted modified iridium probe fluorescent sensor solution at wavelengths of 442 nm and 630 nm. Take the I0442 / I0630 value as the fluorescence intensity of Ir-NOR@MIP-CdSe@ZnS without the addition of template molecules, and record it as f0. Step 3: Add the sample solution to the molecularly imprinted iridium probe fluorescent sensor solution at a volume ratio of 1:80-120 to obtain a mixed solution. After reacting for 4-6 minutes, measure the fluorescence intensity of the mixed solution at wavelengths of 442 nm and 630 nm. Take I... 442 / I 630 The value is taken as the fluorescence intensity of the molecularly imprinted iridium probe fluorescent sensor after incubation with the template molecule, denoted as f, and the fluorescence enhancement intensity f / f0 is calculated. Step 4: Calculate the concentration of norfloxacin in the sample solution based on the linear relationship between norfloxacin concentration and fluorescence sensitization intensity f / f0.

Images