A gold nanocluster fluorescent sensing composite system, a preparation method and application thereof

Abstract

This invention discloses a gold nanocluster fluorescent sensing composite system, its preparation method, and its applications. Using ATT as both a protective and reducing agent, this invention synthesizes highly fluorescent gold nanoclusters ATT-AuNCs in one step; Eu is then added... 3+ A gold nanocluster fluorescence sensing composite system was constructed for high-sensitivity detection of TCs. Eu 3+ The fluorescence of ATT-AuNCs can be quenched. When TCs are added again, they competitively block Eu. 3+ The interaction with ATT-AuNCs restores the fluorescence of ATT-AuNCs, enabling the specific detection of TCs. This invention provides a novel technical approach for TC detection using a gold nanocluster fluorescent composite system. The preparation process is simple, the response speed is fast, and the anti-interference ability is strong.

Description

Technical Field

[0001] This invention relates to the field of nanotechnology, and in particular to a gold nanocluster fluorescent sensing composite system, its preparation method, and its applications. Background Technology

[0002] Antibiotics are drugs that inhibit or kill microorganisms by interfering with their physiological metabolic processes. Tetracycline antibiotics (TCs) are among the most widely used. TCs mainly include oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DOX). They possess broad-spectrum antibacterial activity, are stable, and inexpensive, and are commonly used to treat human and animal diseases. However, with the overuse of antibiotics, large amounts of incompletely metabolized TCs enter the environment and gradually accumulate in plants and animals. Studies have shown that vegetables can absorb TCs from irrigation water and accumulate in their edible parts. TCs in surface water can enter aquatic products such as fish and shrimp, eventually entering the human body through the food chain, potentially causing allergic reactions, liver failure, and even intestinal diseases, seriously endangering human health. Therefore, effective detection of antibiotics is crucial.

[0003] High-performance liquid chromatography-mass spectrometry (HPLC-MS) is the most commonly used traditional method for the detection of TCs. HPLC-MS technology can be used to quantitatively detect antibiotics such as tetracycline, chlortetracycline, and oxytetracycline in soil after extraction. However, this involves complex and time-consuming sample pretreatment, expensive equipment, and professional operation, which cannot meet the application needs of rapid on-site detection.

[0004] To overcome the limitations of traditional detection methods in terms of operational complexity, analysis cycle, and equipment dependence, a series of novel rapid detection technologies have emerged, including colorimetric methods, fluorescence methods, electrochemical methods, and surface-enhanced Raman scattering. Among them, fluorescence sensing technology has attracted much attention for its high sensitivity, good selectivity, and convenient potential for on-site application in the rapid detection of tetracycline antibiotics. One method involves the self-assembly of guanylic acid, luminol, and europium ions to form a blue fluorescent complex. This complex is then mixed with sodium alginate and cross-linked with calcium chloride to prepare fluorescent hydrogel microspheres. Upon the addition of tetracycline, the intrinsic red fluorescence of europium ions is excited, while the blue fluorescence of the fluorescent complex is quenched due to the internal filtration effect. Quantitative detection of tetracycline can be achieved through ratiometric fluorescence changes and visual color differences, with a detection limit of 5 μg / kg. This method has a fast response and good sensitivity and specificity; however, the particle size difference of the hydrogel microspheres may cause slight variations in the fluorescence signal intensity, affecting the accuracy of the detection results.

[0005] Gold nanoclusters (AuNCs), as a novel fluorescent nanomaterial, possess unique advantages such as quantum size effect, tunable fluorescence, excellent biocompatibility, and large Stokes shift, and have made breakthrough progress in the detection of tetracycline analogs in recent years. However, they also have drawbacks, including cumbersome synthesis steps leading to excessive synthesis time and insufficient sensitivity for detecting tetracycline analogs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a gold nanocluster fluorescence sensing composite system for the detection of antibiotics.

[0007] This invention provides a gold nanocluster fluorescence sensing composite system, wherein the fluorescence sensing composite system is ATT-AuNCs / Eu 3+ The ATT-AuNCs / Eu 3+ Gold nanoclusters modified with 6-aza-2-thiothymidine (ATT-AuNCs) and europium ions (Eu) 3+ The composition is as follows: ATT-AuNCs emit yellow-green fluorescence, and Ce is added to ATT-AuNCs. 3+ Er 3+ Gd 3+ La 3+ 、Tb 3+ Tm 3+ Eu 3+ and Nd 3+ Subsequently, these rare earth ions all caused varying degrees of change in the fluorescence intensity of ATT-AuNCs, accompanied by a significant redshift in emission. Only Eu... 3+ The emission peak position remained unchanged, while the fluorescence underwent significant quenching, indicating that ATT-AuNCs inhibited Eu... 3+ It has a high degree of selectivity.

[0008] Furthermore, the preparation method of the gold nanocluster fluorescent sensing composite system includes the following steps: adding ATT dissolved in NaOH to a HAuCl4 solution, stirring at room temperature in the dark, filtering to obtain an ATT-AuNCs solution, and then reacting it with Eu... 3+ Mixing yields ATT-AuNCs / Eu 3+ The ATT is 6-aza-2-thiothymine.

[0009] Furthermore, the ATT-AuNCs and the Eu 3+ The reaction time is >30s.

[0010] The present invention also provides an application of a gold nanocluster fluorescence sensing composite system in the detection of tetracycline analogs (TCs).

[0011] Furthermore, the TCs are any one of tetracycline, chlortetracycline, oxytetracycline, and doxycycline, and the tetracycline, chlortetracycline, and oxytetracycline are related to ATT-AuNCs / Eu 3+ After the solutions are mixed, ATT-AuNCs / Eu 3+ The fluorescence intensity of the antibodies gradually increases with the increase of antibiotic concentration.

[0012] Furthermore, the detection limits for tetracycline, chlortetracycline, and oxytetracycline are 63 μg / L, 34 μg / L, and 18 μg / L, respectively.

[0013] Furthermore, the application includes the following steps:

[0014] S1: ATT-AuNCs / Eu are obtained according to the preparation method described above. 3+ ; S2: Prepare TCs solutions with concentration gradients, and react them with ATT-AuNCs / Eu... 3+ The solution was mixed and oscillated for reaction. The fluorescence spectrum and intensity of the system were measured in the 450-650 nm range using an excitation wavelength of 360-420 nm. The fluorescence recovery efficiency was evaluated using (F-F0) / F0, and a standard curve was established between (F-F0) / F0 and TCs concentration. F and F0 represent the ATT-AuNCs / Eu ratio in the presence and absence of TCs, respectively. 3+ fluorescence intensity; S3: Mix the water sample to be tested with ATT-AuNCs / Eu 3+ The solution was mixed and shaken to react. The fluorescence spectrum and fluorescence intensity of the system in the range of 450-650 nm were measured with an excitation wavelength of 360-420 nm. (F-F0) / F0 was calculated. The concentration of TCs in the sample was calculated based on the TCs standard curve obtained in S2.

[0015] Furthermore, F and F0 represent the ATT-AuNCs / Eu values ​​when TCs are present and absent at the maximum emission wavelength, respectively. 3+ The fluorescence intensity. In some embodiments, F and F0 represent the ATT-AuNCs / Eu ratio at 529 nm with and without TCs under 365 nm excitation light. 3+ The fluorescence intensity.

[0016] Furthermore, the oscillation reaction time described in steps S2 and S3 is >30 s, ATT-AuNCs / Eu 3+ The sensing system can obtain a good response to tetracycline in 30 seconds at room temperature.

[0017] Furthermore, the Eu 3+ The concentration is 0-100 μM, preferably 30 μM, Eu 3+When the concentration is too high, the response of low concentrations of TC is weak, and the free Eu... 3+ There will be a greater chance of quenching the fluorescence of ATT-AuNCs, thus reducing the sensitivity of the assay. When Eu 3+ The fluorescence recovery efficiency (F-F0) / F0 is highest at a concentration of 30 μM.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The gold nanocluster fluorescent probe of the present invention is simple to prepare under mild conditions and can be prepared in one step at room temperature. (2) The ATT-AuNCs / Eu of the present invention 3+ It can react rapidly with TCs, and complete response is achieved after incubation at room temperature for 30 seconds, enabling rapid detection of TCs; (3) This invention is based on ATT-AuNCs / Eu 3+ The fluorescence intensity changes of the fluorescent probe enabled the sensitive detection of tetracycline analogs such as tetracycline, chlortetracycline, and oxytetracycline. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The fluorescent sensing probe ATT-AuNCs / Eu in this invention 3+ A schematic diagram illustrating the principle of TCs detection.

[0021] Figure 2 (A) is the fluorescence emission spectrum of ATT-AuNCs after adding different rare earth metal ions in Example 2 of the present invention; (B) is the normalized fluorescence emission spectrum of ATT-AuNCs after adding different ions; (C) is the fluorescence emission spectrum of ATT-AuNCs after adding a series of different concentrations of Eu. 3+ Fluorescence emission spectra of ATT-AuNCs; (D) represents the fluorescence quenching efficiency (F0-F) / F0 at 529 nm and Eu. 3+ A graph showing the relationship between concentrations.

[0022] Figure 3 In Embodiment 3 of this invention, ATT-AuNCs with added Eu 3+ Before and after fluorescence emission spectra and ATT-AuNCs and ATT-AuNCs / Eu 3+ Fluorescence response diagram to TC.

[0023] Figure 4 (A) is Eu in Embodiment 4 of the present invention. 3+ Concentration of ATT-AuNCs / Eu 3+ (A) Effect of fluorescence recovery efficiency (F-F0) / F0 on the sensing system; (B) Effect of tetracycline analog reaction time on ATT-AuNCs / Eu 3+ The effect of fluorescence recovery efficiency (F-F0) / F0 on the sensing system.

[0024] Figure 5 (A) is the ATT-AuNCs / Eu in Embodiment 5 of the present invention. 3+ Fluorescence emission spectra at different TC concentrations; (B) is a standard curve of fluorescence recovery efficiency (F-F0) / F0 at 529 nm versus TC concentration.

[0025] Figure 6 (A) is the ATT-AuNCs / Eu in Embodiment 5 of the present invention. 3+ Fluorescence emission spectra at different CTC concentrations; (B) is a standard curve of fluorescence recovery efficiency (F-F0) / F0 at 529 nm versus CTC concentration.

[0026] Figure 7 (A) is the ATT-AuNCs / Eu in Embodiment 5 of the present invention. 3+ Fluorescence emission spectra at different OTC concentrations; (B) is a standard curve of fluorescence recovery efficiency (F-F0) / F0 at 529 nm versus OTC concentration.

[0027] Figure 8 ATT-AuNCs / Eu in Embodiment 6 of the present invention 3+ Experimental diagrams of selectivity and anti-interference of TCs. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] Example 1: Preparation of ATT-AuNCs gold nanoclusters The preparation steps of 6-aza-2-thiothymidine (ATT) protected gold nanoclusters (ATT-AuNCs) are as follows: ATT solution (3 mL, 80 mM) dissolved in 0.2 M sodium hydroxide solution was added to tetrachloroauric acid (HAuCl4) solution (3 mL, 10 mg / mL), and stirred continuously at room temperature in the dark for 1 h to obtain a yellow crude ATT-AuNCs solution. The crude solution was then purified by filtration through a 0.22 μm nylon membrane, and the filtrate was collected to obtain the final ATT-AuNCs solution, which was stored at 4 °C in the dark for later use.

[0030] Example 2 ATT-AuNCs / Eu 3+ Construction of a composite sensing system Investigating Ce 3+ Er 3+ Gd 3+ La 3+ 、Tb 3+ Tm 3+ Eu 3+ and Nd 3+ Effect of different rare earth ion solutions on the fluorescence intensity of ATT-AuNCs. 50 μL of different rare earth ion solutions were added to 100 μL of water and 50 μL of ATT-AuNCs solution, including Eu... 3+ The concentration was 100 μM, the concentration of other rare earth ions was 1 mM, the excitation wavelength was λex = 365 nm, and the width of the excitation and emission slits was 1 nm. The fluorescence emission spectrum of the fluorescence system in the range of 450-650 nm was obtained.

[0031] Figure 2 The original fluorescence intensity map of A and Figure 2 Normalized fluorescence intensity of B shows that different metal ions (Ce) 3+ Er 3+ Gd 3+ La 3+ 、Tb 3+ Tm 3+ Eu 3+ and Nd 3+ After interaction with ATT-AuNCs, Eu 3+ The fluorescence quenching effect of ATT-AuNCs was most significant, while the quenching effect of other ions was relatively weak. This indicates that ATT-AuNCs have a strong quenching effect on Eu... 3+ They exhibit stronger selective interactions. Figure 2 C shows Eu 3+As the concentration of Eu increases (0, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 800, 1000 μM), the fluorescence intensity of ATT-AuNCs at 529 nm gradually decreases. (F0-F) / F0 is introduced here to evaluate the fluorescence quenching efficiency, where F and F0 are the Eu concentrations at 529 nm and the fluorescence intensity of AuNCs, respectively. 3+ Fluorescence intensity of ATT-AuNCs in and out of presence. Figure 2 As shown in D, within the concentration range of 0~20 μM, (F0-F) / F0 and Eu 3+ The concentration of Eu showed a good linear relationship. 3+ At a concentration of 200 μM, the fluorescence quenching efficiency reached approximately 95%. Further increases in Eu... 3+ The concentration and degree of fluorescence quenching of Eu did not change significantly. 3+ The superiority of ATT-AuNCs in modulating fluorescence intensity for subsequent sensing, therefore ATT-AuNCs / Eu 3+ It was used as a composite fluorescence system for subsequent experiments.

[0032] Example 3 ATT-AuNCs / Eu 3+ Feasibility analysis of TCs detection Using ATT-AuNCs as the fluorescent host, Eu was first introduced 3+ A fluorescence quenching system was constructed, and different concentrations of tetracycline antibiotics (TC) were added. The effect of TC on the fluorescence recovery of the system was analyzed by detecting changes in the fluorescence emission spectrum.

[0033] Fluorescence emission spectrum as follows Figure 3 As shown, at an excitation wavelength of 365 nm, the fluorescence of ATT-AuNCs remained essentially the same before and after the addition of 10 mg / L TC, indicating that there is no direct interaction between the TC solution alone and ATT-AuNCs. The addition of Eu... 3+ At this point, the fluorescence intensity of ATT-AuNCs at 529 nm decreased significantly. Adding 5 mg / L TC solution at this time significantly enhanced the fluorescence signal, indicating that TC put the fluorescence of the system in a "turn-on" state. When the TC concentration was 10 mg / L, the fluorescence intensity at 529 nm was almost completely restored. Therefore, it can be seen that ATT-AuNCs / Eu 3+ The fluorescence recovery efficiency is related to the TC concentration; as the TC concentration increases, the fluorescence recovery efficiency of the system increases accordingly. The above results indicate that, based on Eu... 3+ It is feasible to use mediated ATT-AuNCs for quantitative detection of TC.

[0034] Example 4 ATT-AuNCs / Eu3+ Optimization of TC detection conditions To obtain ATT-AuNCs / Eu 3+ To achieve the optimal sensitivity for TCs detection in the system, a series of Eu values ​​were set. 3+ Concentration and reaction time. ATT-AuNCs and Eu 3+ The solution volumes were 50 μL and 50 μL, respectively, and then 100 μL of TC solution was added, with a fixed concentration of 5 mg / L.

[0035] When Eu 3+ When the concentration is too high, the response of low concentrations of TC is weak, and the free Eu... 3+ There is a greater chance that the fluorescence of ATT-AuNCs will be quenched, thus reducing the assay sensitivity; therefore, only Eu will be discussed. 3+ The degree of response of the solution concentration to TC in the range of 0-100 μM. Results are as follows... Figure 4 As shown in A, Eu 3+ The optimal concentration is 30 μM, at which the fluorescence recovery efficiency (F-F0) / F0 is the highest.

[0036] Further investigation into the effect of reaction time on ATT-AuNCs / Eu 3+ The effect of fluorescence, the results are as follows Figure 4 As shown in B, TC and Eu 3+ The reaction rate was relatively fast. After mixing for 30 s, the fluorescence recovery efficiency (F-F0) / F0 of the system reached a stable state, and no significant changes were observed thereafter. Therefore, the optimal reaction time was 30 s.

[0037] Example 5: Establishment of the TCs Standard Curve Under optimal detection conditions, the effects of different concentrations of TC solution on ATT-AuNCs / Eu were investigated. 3+ Fluorescence intensity at 529 nm after solution mixing.

[0038] Figure 5 As shown in Figure A, with the increase of TC concentration, the fluorescence gradually increases, and the fluorescence recovery efficiency (F-F0) / F0 continuously increases. When the TC concentration is 10 mg / L, it tends to stabilize, and the fluorescence is basically completely recovered. Figure 5 As shown in Figure B, within the ranges of 0–1 mg / L and 1–10 mg / L, (F–F0) / F0 exhibits a good linear relationship with TC concentration, with the linear equations being (F–F0) / F0 = 0.36289C – 0.00758 (R0). 2 =0.997) and (F-F0) / F0=0.10977C+0.20317(R 2=0.996), according to the formula for calculating the limit of detection (LOD) = 3σ / k, (where LOD is the limit of detection, σ is the standard deviation of the blank measurement, and k is the slope of the linear regression equation), the LOD of tetracycline is calculated to be 63 μg / L.

[0039] For different concentrations of chlortetracycline (CTC) and oxytetracycline (OTC). Figure 6 and Figure 7 The results showed a response trend consistent with that of tetracycline (TC): as the content of CTC and OTC increased, the fluorescence intensity of ATT-AuNCs gradually increased, and the limits of detection (LOD) of the two were as low as 34 μg / L and 18 μg / L, respectively.

[0040] Example 6 ATT-AuNCs / Eu 3+ Selectivity and robustness analysis To evaluate ATT-AuNCs / Eu 3+ To assess selectivity and interference resistance, amoxicillin (AMX), erythromycin (ERY), chloramphenicol (CPL), norfloxacin (NOR), ofloxacin (OFL), ciprofloxacin (CIP), and vancomycin (VAN) were selected as interfering agents to investigate the ATT-AuNCs / Eu ratio. 3+ The detection stability of TCs in the presence of interfering agents was improved when the concentration of interfering antibiotics was twice that of TCs.

[0041] Figure 8 The results showed that after adding antibiotics other than TCs, the fluorescence intensity of ATT-AuNCs at 529 nm did not change significantly in the sensing system, indicating that the presence of interfering substances had little impact on the normal detection of TCs. This suggests that ATT-AuNCs / Eu 3+ The fluorescence sensing method for detecting TCs has good specificity and anti-interference ability.

[0042] Example 7: Analysis of Actual Samples Different concentrations of TCs solutions were added to real water samples for detection, as shown in Table 1. The recoveries ranged from 90.2% to 105.4%, and the relative standard deviations (RSDs) ranged from 0.3% to 14.0%, which demonstrates that the sensor has good performance in detecting TCs in real samples.

[0043] Table 1. Measurement results of TCs in actual water samples (n=3)

[0044] Comparative Example 1: Based on His-AuNCs / Eu 3+ Detection of TC concentration Using L-histidine-modified AuNCs (His-AuNCs) as fluorescent probes, His-AuNCs exhibited strong fluorescence at 475 nm without TC under 375 nm excitation (F). 475 Eu 3+ It exhibits weak fluorescence at 620 nm (F 620 The presence of TC quenches the fluorescence of His-AuNCs, while simultaneously enhancing the fluorescence of the EuTC complex via the aid of His-AuNCs, as indicated by the ratio signal F. 620 / F 475 The sensor achieves TC detection. After optimization, the detection linear range is 10 nM–60 μM, with a detection limit of 4 nM. This sensor has been successfully applied to tetracycline analysis in spiked samples, but the reaction time is relatively long, at 10 min.

[0045] Comparative Example 2: Gold Nanoclusters Based on DNA Templates / Eu 3+ Detecting TC concentration Using DNA template gold nanoclusters for Eu 3+ - This probe is an enhancer for the tetracycline (TC) complex (EuTC) fluorescence enhancement system, used to detect TC concentration. It exhibits linearity between 10 nM and 5 µM, with a LOD of 4 nM. However, the DNA used in its preparation is expensive, and the probe synthesis takes 12 hours.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gold nanocluster fluorescence sensing composite system, characterized in that, The fluorescence sensing composite system is ATT-AuNCs / Eu 3+ The ATT-AuNCs / Eu 3+ It consists of gold nanoclusters modified with 6-aza-2-thiothymidine and europium ions.

2. The preparation method of the gold nanocluster fluorescence sensing composite system according to claim 1, characterized in that, The steps include: adding ATT dissolved in NaOH to a HAuCl4 solution, stirring at room temperature in the dark, filtering to obtain an ATT-AuNCs solution, and then reacting it with Eu... 3+ Mixing yields ATT-AuNCs / Eu 3+ The ATT is 6-aza-2-thiothymine.

3. The preparation method according to claim 2, characterized in that, The ATT-AuNCs and the Eu 3+ The reaction time is >30s.

4. The application of the gold nanocluster fluorescence sensing composite system according to claim 1 in the detection of tetracycline analogs (TCs).

5. The application according to claim 4, characterized in that, The TCs are any one of tetracycline, chlortetracycline, oxytetracycline, and doxycycline.

6. The application according to claim 5, characterized in that, The detection limits for tetracycline, chlortetracycline, and oxytetracycline were 63 μg / L, 34 μg / L, and 18 μg / L, respectively.

7. The application according to claim 4, characterized in that, Includes the following steps: S1: ATT-AuNCs / Eu obtained by the preparation method according to claim 2 3+ ; S2: Prepare TCs solutions with concentration gradients, and react them with ATT-AuNCs / Eu... 3+ The solution was mixed and oscillated for reaction. The fluorescence spectrum and intensity of the system were measured in the 450-650 nm range using an excitation wavelength of 360-420 nm. The fluorescence recovery efficiency was evaluated using (F-F0) / F0, and a standard curve was established between (F-F0) / F0 and TCs concentration. F and F0 represent the ATT-AuNCs / Eu ratio in the presence and absence of TCs, respectively. 3+ fluorescence intensity; S3: Mix the water sample to be tested with ATT-AuNCs / Eu 3+ The solution was mixed and shaken to react. The fluorescence spectrum and fluorescence intensity of the system in the range of 450-650 nm were measured with an excitation wavelength of 360-420 nm. (F-F0) / F0 was calculated. The concentration of TCs in the sample was calculated based on the TCs standard curve obtained in S2.

8. The application according to claim 7, characterized in that, F and F0 represent the presence and absence of TCs at the maximum emission wavelength, respectively, for ATT-AuNCs / Eu 3+ The fluorescence intensity.

9. The application according to claim 7, characterized in that, The oscillation reaction time described in steps S2 and S3 is >30s.

10. The application according to claim 7, characterized in that, The Eu 3+ The concentration is 0-100 μM.

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