Holmium oxide enhanced silver quantum dot nano-enzyme and application thereof in detection of amoxicillin

Abstract

The invention discloses a holmium oxide enhanced silver quantum dot nano-enzyme and application thereof in detection of amoxicillin. The nano-enzyme is prepared by adopting a one-pot synthesis method, is a nanosphere with the diameter of 3.30 + / -0.66 nm, and has excellent mimic enzyme activity and intrinsic fluorescence property. Based on the regulation effect of amoxicillin on the catalytic activity of the nano-enzyme and the enhancement effect of amoxicillin on fluorescence, a method for detecting antibiotic amoxicillin residues in agricultural products through multiple channels of UV-vis spectrum, RGB colorimetry and fluorescence spectrum is established. The method is easy and convenient to operate, high in sensitivity and good in selectivity, is suitable for rapid detection of amoxicillin in water samples, soil, agricultural products, agricultural and sideline products and drugs, and can achieve on-site fluorescence imaging of amoxicillin residues in living samples, the multi-channel detection limits of UV-vis spectrum, RGB colorimetric and fluorescence spectrum are 6.91 * 10 <-8 > M, 1.18 * 10 <-7 > M and 3.56 * 10 <-9 > M respectively, and the method is suitable for rapid detection of amoxicillin residues in living samples. The method has a good application prospect in the fields of environmental monitoring and food safety.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution monitoring, specifically relating to a holmium oxide-enhanced silver quantum dot nanozyme and its application in the detection of amoxicillin. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Antibiotics are two of the most common chemical agents in nature used to prevent diseases and promote health in organisms (including humans). However, the overuse of antibiotics has led to a series of adverse effects on humans. For example, amoxicillin (AMX), a commonly used broad-spectrum antibiotic, has a variety of side effects, including headache, vomiting, allergic reactions, thrombocytopenia, convulsions, meningitis, and vasculitis. Therefore, researchers are dedicated to developing efficient sensors to monitor AMX and prevent its contamination. Currently, conventional methods for AMX detection mainly rely on instrumental analysis techniques such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Although these methods offer high sensitivity and accuracy, they rely on expensive, large-scale instruments, require complex sample pretreatment and specialized operators, making it difficult to achieve on-site, rapid, and real-time monitoring. Furthermore, most existing detection methods rely on sophisticated instruments or require skilled operators and are susceptible to interference in complex biological matrices, severely limiting their application in in vivo, real-time sensing.

[0004] Therefore, it is necessary to develop an effective multi-channel sensing method to provide a potential pathway for high-performance sensors used in actual field pollution monitoring, which is of great significance for protecting the environment and maintaining human health. Summary of the Invention The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0005] To address the shortcomings of existing pollution detection technologies, the first objective of this invention is to provide a holmium oxide-enhanced silver quantum dot nanozyme (Ho2O3-Ag QDs).

[0006] A second objective of this invention is to provide the application of the holmium oxide-enhanced silver quantum dot nanozyme in the detection of amoxicillin.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A holmium oxide-enhanced silver quantum dot nanozyme, spherical in shape with a diameter of 3.30 ± 0.66 nm, exhibits excellent enzyme-mimicking catalytic activity (the Michaelis constant (Km) for catalyzing TMB and H2O2 is [not specified in the original text]. m The value is 0.095 mM / 9.06×10. -8 M·s -1 Maximum reaction rate (V max The value is 0.599 mM / 2.69 × 10⁻⁶. -7 M·s -1 ), intrinsic fluorescence properties (excitation and emission wavelengths of 355 nm and 459 nm, respectively) and stability (zeta potential of -32.69 mV).

[0008] A method for preparing the holmium oxide-enhanced silver quantum dot nanozyme follows the synthesis steps of a previous national invention patent (CN202410880132.1), involving the addition of 0.01 mmol of rare earth Ho(NO3)3. The optimal preparation method was achieved by replacing Bi(NO3)3 with 5H2O.

[0009] Application of a holmium oxide-enhanced silver quantum dot nanozyme in the detection of amoxicillin concentration.

[0010] Furthermore, the method for detecting the concentration of amoxicillin is UV-vis, RGB, and fluorescence assay.

[0011] Furthermore, the UV-vis and RGB colorimetric determination method includes the following steps: (1) Dissolve the holmium oxide-enhanced silver quantum dot nanozyme in triple-distilled water to prepare a Ho2O3-Ag QDs standard solution with a concentration of 0.05 mg / mL; (2) Dissolve 3,3',5,5'-tetramethylbenzidine (TMB) in dimethyl sulfoxide (DMSO) to prepare a TMB standard solution with a concentration of 1.5 mmol / L; (3) Dissolve 30% H2O2 in triple-distilled water to prepare a 3% H2O2 standard solution; (4) Mix 600 μL of Ho2O3-Ag QDs standard solution obtained in step (1), 200 μL of citrate-phosphate buffer solution with pH 4.0, 200 μL of TMB standard solution obtained in step (2), 350 μL of H2O2 standard solution obtained in step (3), and 200 μL of AMX test solution, and bring the volume to 3.0 mL with purified water. After aging at room temperature for 30 minutes, measure the UV-Vis absorbance A at 652 nm using a UV spectrometer. 652Meanwhile, using a home smartphone, we took color photos of the samples and used a smartphone app to analyze the RGB color values ​​of the photos and calculate the (R+G) / B value. (5) Mix 600 μL of the Ho2O3-Ag QDs standard solution obtained in step (1), 200 μL of citrate-phosphate buffer solution with pH 4.0, 200 μL of the TMB standard solution obtained in step (2), and 350 μL of the H2O2 standard solution obtained in step (3), and bring the volume to 3.0 mL with purified water. Measure the absorbance A of the system at 652 nm wavelength at room temperature. 0 652 And [(R+G) / B] 0 Numerical value; (6) Take A obtained in step (5) 652 And A obtained in step (6) 0 652 Substitute into the formula △ A 652 = A 652 - A 0 652 Calculations yielded △ A 652 , will the △ A 652 Substitute into the linear regression equation △ A 652 = 1.39×10 -4 c AMX - 1.34×10 -2 (c) AMX The concentration of AMX in the sample is calculated by using the concentration of amoxicillin (R+G) / B; simultaneously, the [(R+G) / B] is calculated. 0 Substitute into the formula △ (R+G) / B = (R+G) / B - [(R+G) / B] 0 Calculations yielded △ (R+G) / B; The above △ Substituting (R+G) / B into the linear regression equation △ (R+G) / B=1.85×10 -4 c AMX +3.11×10 -2 The concentration of AMX in the sample was calculated.

[0012] Furthermore, the fluorescence assay employs the following steps: (1) Dissolve the Ho2O3-Ag QDs in triple-distilled water to prepare a Ho2O3-Ag QDs standard solution with a concentration of 0.05 mg / mL; (2) Mix 100 μL of the Ho2O3-Ag QDs standard solution obtained in step (1), 200 μL of phosphate buffer solution with pH 8.0, and 200 μL of AMX test solution, and make up to 3.0 mL with purified water. Measure the fluorescence intensity F of the system at a wavelength of 459 nm. 459 ; (3) Mix 100 μL of the Ho2O3-Ag QDs standard solution obtained in step (1) with 200 μL of phosphate buffer solution with pH 8.0, and bring the volume to 3.0 mL with purified water. Measure the fluorescence intensity F of the system at a wavelength of 459 nm. 0 459 ; (4) Take the F obtained in step (2) 459 And F obtained in step (3) 0 459 Substitute into the formula △ F 459 =F 459 F 0 459 Calculations yielded △ F 459 , respectively the △F 459 Substitute the value into the linear regression equation △ F 459 = 4.75 c AMX + 299.84, the concentration of AMX in the sample is calculated.

[0013] Furthermore, the Ho2O3-Ag QDs are used to detect the concentration of AMX in water, soil, agricultural products, agricultural by-products or pharmaceuticals; and to perform on-site fluorescence imaging response for AMX residues in agricultural products, confirming that they are not affected by other interfering substances in the biological system.

[0014] The agricultural products mentioned are edible rice, straw, apples, cucumbers, live zebrafish, or live chive blossoms; the agricultural by-products mentioned are pork or milk.

[0015] Furthermore, when the Ho2O3-Ag QDs are used to detect the concentration of AMX in water, the preparation of the AMX test sample includes the following steps: randomly select the water sample to be tested, filter it three times with a microporous filter membrane with a pore size of 0.22 μm, and the filtrate is the AMX test solution; When the Ho2O3-Ag QDs are used to detect the concentration of AMX in pork, rice, or straw samples, the preparation of the AMX test solution includes the following steps: take pork, rice, or straw, crush them in triple-distilled water, mix thoroughly for 20 min, centrifuge at 11000 r / min for 10 min, and the resulting supernatant is the AMX test solution; the mass-to-volume ratio of the pork, rice, or straw to triple-distilled water is 20 g: 40 mL; When the Ho2O3-Ag QDs are used to detect the concentration of AMX in milk, the preparation of the AMX test solution includes the following steps: take milk, stir it evenly, centrifuge at 11000 r / min for 10 min, and the supernatant is the AMX test solution.

[0016] When the Ho2O3-Ag QDs are used to detect the concentration of AMX in soil, the preparation of the AMX test solution includes the following steps: randomly take soil samples, disperse them in triple-distilled water, stir continuously at room temperature for 24 h, filter three times with a microporous filter membrane with a pore size of 0.22 μm, and the filtrate is the AMX test solution; the mass-volume ratio of soil sample to triple-distilled water is 10 g: 40 mL; When the Ho2O3-Ag QDs are used to detect the concentration of AMX in apple or cucumber samples, the preparation of the AMX test solution includes the following steps: take an apple or cucumber, crush it in triple-distilled water, mix thoroughly for 20 min, centrifuge at 11000 r / min for 15 min, and the resulting supernatant is the AMX test solution; the apple or cucumber is weighed to be 50.0 g; When the Ho2O3-Ag QDs are used to detect the concentration of AMX in AMX capsules, the preparation of the AMX test solution includes the following steps: take the powder from the capsule, dissolve it in triple-distilled water, centrifuge at 11000 r / min for 10 min, and the resulting supernatant is the AMX test solution; the mass-to-volume ratio of the AMX capsule to the triple-distilled water is 5 mg: 10 mL; The fluorescence imaging experiment: Edible rice, live zebrafish, and live chive blossoms were placed in a solution with a concentration of 1.67 × 10⁻⁶. -3 The cells were cultured in an aqueous dispersion of Ho₂O₃-Ag QDs at a concentration of 0 mg / mL for 24 hours. Subsequently, concentrations of 0 mg / mL and 8.21 × 10⁻⁶ mg / mL were added. -7 1.91×10 -6 and 3.56×10 -6 The samples were incubated with AMX standard solution M for another 24 h. After washing the rice, live zebrafish, and live chive flower samples with sufficient water, fluorescence imaging was performed using a ZF-20A four-channel darkroom UV analyzer manufactured by Shanghai Yuezhong Instrument Co., Ltd., under excitation at a wavelength of 365 nm.

[0017] This invention designs and synthesizes a holmium oxide-enhanced silver quantum dot nanozyme material. The effects of pH, TMB dosage, H2O2 dosage, reaction time, and coexisting substances on UV-vis detection were discussed. The optimal UV-vis testing conditions were determined to be: 600 μL of 0.05 mg / mL Ho2O3-Ag QDs standard solution, 200 μL of pH 4.0 citrate-phosphate buffer solution, 200 μL of TMB, 350 μL of H2O2, and 1200 s at room temperature, resulting in the highest sensitivity. Under these conditions, interference from other common ions can be eliminated. The linear regression equation for colorimetric detection of AMX is: △ A 652 = 1.39×10 -4 c AMX -1.34×10 -2 (10 -9 mol·L -1 The linear range is 8.21~2740.00×10⁻⁶. -8 mol·L -1 R 2 The value was 0.9968, and the detection limit was 6.91 × 10⁻⁶. - 8 mol·L -1 The RSD is less than 3.9%.

[0018] This invention, by investigating the effects of pH, reaction time, and coexisting substances on fluorescence detection, determined the optimal testing conditions: 100 μL of a 0.05 mg / mL Ho₂O₃-Ag QDs standard solution and 200 μL of a pH 8.0 mixed phosphate buffer solution, which resulted in the highest sensitivity. Under these conditions, interference from other common ions can be eliminated, and the linear regression equation for AMX fluorescence detection is as follows: △ F 459 = 4.75 c AMX + 299.84 (10 -9 mol·L -1 Linear range: 5.47~3560×10 -9 mol·L -1 R 2 The value was 0.9974, and the detection limit was 3.56 × 10⁻⁶. -9 mol·L -1 RSD is less than 4.1%.

[0019] The beneficial effects of this invention are: (1) The holmium oxide-enhanced silver quantum dot nanozyme prepared in this invention achieves synergistic enhancement of enzyme-mimicking activity and fluorescence performance of silver quantum dots through rare earth Ho2O3 doping, constructs a high-performance bifunctional nanomaterial, proposes a new method for multi-mode detection of AMX based on holmium oxide-enhanced silver quantum dot nanozyme catalysis, forms a multi-channel sensing platform, improves the reliability and applicability of detection, and the colorimetric method is visible to the naked eye, which is convenient for rapid on-site judgment.

[0020] (2) The method provided by this invention has good selectivity, strong resistance to common ion and biomolecular interference, and high sensitivity (LOD up to 10). -9 M), low detection limit (3.56×10). -9 (M), which can be directly used for the analysis of complex real samples, with high recovery rate and accurate and reliable results.

[0021] (3) This invention is the first to apply Ho2O3-Ag QDs to AMX fluorescence imaging of edible rice, live zebrafish and live chive flowers, realizing in-situ and visual monitoring of antibiotic residues in organisms, providing a new technical means for the live assessment of environmental pollution and on-site screening of food safety; solving two major problems in environmental science: in-situ behavior analysis of pollutants and rapid on-site detection in food safety, and providing a powerful, intuitive and highly promising technical platform.

[0022] (4) The entire preparation and testing process is simple to operate, low in cost, green and environmentally friendly, and has broad prospects for industrial application. Attached Figure Description

[0023] Figure 1 The image shows the structural characterization of the Ho2O3-Ag QDs nano-mimetic enzyme prepared in Example 1. Figure 1 a) is a TEM image. Figure 1 b) is the EDS plot. Figure 1 c) is the XRD pattern. Figure 1 d) is the FT-IR plot; Figure 2 The image shows the structural characterization of the Ho2O3-Ag QDs nano-mimetic enzyme prepared in Example 1. Figure 2 a) is a DLS diagram. Figure 2 b) is the fluorescence spectrum. Figure 2 c) is the zeta potential diagram. Figure 2 d) is the wide-angle X-ray photoelectron spectrum. Figure 2 e) is the high-resolution XPS spectrum of Ag 3d. Figure 2 f) is the high-resolution XPS spectrum of Ho 4d; Figure 3 The graph shows the effect of different experimental conditions on the catalytic oxidation of TMB by Ho2O3-Ag QDs prepared in Example 1. Figure 3 a) A graph showing the effect of different pH values ​​on the catalytic oxidation of TMB by Ho2O3-Ag QDs prepared in Example 1. Figure 3 b) A graph showing the effect of different TMB contents on the catalytic oxidation of TMB by Ho2O3-Ag QDs prepared in Example 1. Figure 3 c) A graph showing the effect of different H2O2 contents on the catalytic oxidation of TMB by Ho2O3-Ag QDs prepared in Example 1. Figure 3 d) is a graph showing the effect of different reaction times on the catalytic oxidation of TMB by Ho2O3-Ag QDs prepared in Example 1; Figure 4 The graph shows the synergistic effect and steady-state kinetics of the components of the Ho2O3-Ag QDs prepared in Example 1. Figure 4 a) UV-vis spectra of each component Figure 4 b) A 600-second time scan of each component. Figure 4 c) is a double reciprocal curve of reaction rate and TMB concentration. Figure 4 d) is a double reciprocal curve of reaction rate and H2O2 concentration; Figure 5 The steady-state kinetic curves and enzyme mechanism diagrams of the Ho2O3-Ag QDs prepared in Example 1 are shown below. Figure 5 a) shows the steady-state kinetic curves for the reaction rate and TMB concentration. Figure 5 b) shows the steady-state kinetic curves of the reaction rate and H2O2 concentration. Figure 5 c) UV-vis spectra of O2 and N2, Figure 5 d) UV-vis spectra of hydroxylamine hydrochloride and isopropanol; Figure 6 The UV-vis spectra, UV absorption intensity statistics, and linear relationship curves of the Ho2O3-Ag QDs-TMB-H2O2 system for colorimetric catalysis under common ion interferences are shown. Figure 6 (ac) represents the color, RGB values, and UV-vis spectrum of the Ho2O3-Ag QDs-TMB-H2O2 system catalytic colorimetric AMX under common ion interferences. Figure 6 (df) represents the UV-vis spectral titration curve, color, and RGB values ​​of the catalytic colorimetric AMX. Figure 6 gh) is △ A 652 , △ (R+G) / B and c AMX The linear relationship curve; Figure 7 The fluorescence intensity curves of Ho₂O₃-Ag QDs at different pH values ​​and the time response curve at pH 8.0 are shown. Figure 7a) Fluorescence intensity curves of Ho2O3-Ag QDs at 459 nm under different pH conditions; Figure 7 b) is the time response curve of Ho2O3-Ag QDs at 459 nm after adding AMX at pH 8.0; Figure 8 The spectrum, fluorescence absorption intensity statistics, and linear relationship diagram of Ho₂O₃-Ag QDs for AMX recognition under common ion interferences are shown. Figure 8 a) is the spectrum of common ion interference substances enhancing the fluorescence recognition of AMX by Ho2O3-Ag QDs. Figure 8 b) is a statistical graph of the AMX fluorescence absorption intensity of Ho2O3-Ag QDs under common ion interference. Figure 8 c) Fluorescence titration curves of Ho₂O₃-Ag QDs Figure 8 d) is △ F 459 With c AMX The linear relationship curve; Figure 9 Visualized fluorescence imaging responses of Ho2O3-Ag QDs to the antibiotic AMX when applied to edible rice, live chive flowers, and live zebrafish. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0025] Example 1 1. Preparation of Ho2O3-Ag QDs (1) Weigh 0.6 mmol of FA and dissolve it in 12.00 mL of 1% ammonia solution (pH 8.5) to obtain 0.05 mol / L FA ammonia solution; (2) Weigh 0.09 mmol AgNO3 and dissolve it in 9.00 mL of aqueous solution to obtain 0.01 mol / L AgNO3 aqueous solution; (3) Weigh 0.01 mmol Ho(NO3)3 5H2O was dissolved in 1.00 mL of aqueous solution to obtain a 0.01 mol / L aqueous solution of Ho(NO3)3; (4) Mix the solutions obtained in steps (1), (2) and (3) and stir for 12 h. Through the electrostatic interaction between the metal Ag and Ho cations and the negative charge or lone pair electrons on folic acid, Ag and Ho cations are adsorbed onto the surface of folic acid. Then add 1.00 mL of sodium borohydride solution with a concentration of 0.2 mol / L and mix thoroughly for 2 h. Centrifuge the reaction mixture at 9000 r / min for 6 min and wash it 3 times with triple-distilled water to obtain holmium oxide enhanced silver quantum dot nanozyme, named Ho2O3-Ag QDs.

[0026] 2. Structural Characterization The structure of the Ho2O3-Ag QDs nanomimetic enzyme prepared above was characterized by TEM, DLS, EDS, XRD, FTIR, zeta potential and XPS.

[0027] Figure 1 The image shows the structural characterization of the Ho2O3-Ag QDs nano-mimetic enzyme prepared in Example 1. Figure 1 (a) The TEM image confirms that the Ho2O3-Ag QDs material has a morphology of spherical particles of about 3.30 nm, which indicates that folic acid and Ho2O3-Ag have been successfully combined. Figure 1 (b) The EDS diagram proves that C, N, O, Ag and Ho elements are present in Ho2O3-Ag QDs, and the molar ratio of Ag to Ho is 9:1, which is consistent with the ratio of feed amount; Figure 1 The inset in (b) shows that Ho₂O₃-Ag QDs have two interplanar spacings of 0.191 nm and 0.229 nm, which correspond to the (111) crystal plane of Ag NPs and the (440) crystal plane of Ho₂O₃ NPs, respectively. The XRD pattern of the composite material clearly shows the crystallinity and morphology of the Ho₂O₃-Ag QDs material, which has six characteristic peaks, such as... Figure 1 As shown in (c), the peaks at 2θ = 29.3°, 49.3° and 56.2° are from the (222), (440) and (145) crystal planes of Ho2O3NPs, while the peaks at 2θ = 38.1°, 64.4° and 77.9° are from the (111), (220) and (311) crystal planes of Ag NPs. This indicates that the proposed Ho2O3-AgQDs have excellent crystallinity and that Ho2O3-enhanced Ag quantum dots have been successfully constructed.

[0028] from Figure 1 (d) FT-IR analysis of FA and Ho2O3-Ag QDs shows that the FT-IR spectrum of free FA is characterized by high concentrations at 3533, 3422, 1693, 1608, 1483, and 1338 cm⁻¹. 1 Several characteristic peaks appeared, attributed to the stretching vibrations of OH, NH, C=O, and aromatic rings, respectively. In the FT-IR spectrum of Ho₂O₃-Ag QDs, the characteristic peak corresponding to the O–H / N–H bond stretching vibration shifted to 3391 cm⁻¹. - ¹, 1693 cm - The characteristic peak of the C=O bond disappears at ¹. In Ho₂O₃-Ag, there is strong coordination between the blank d orbitals of Ag or Ho atoms and the lone pairs or π electrons of the OH, NH, and C=O bonds in folic acid, as well as the aromatic ring. There is also strong coordination between the electron-rich O and N atoms in the OH, NH, and C=O bonds. Furthermore, the characteristic FT-IR peaks of the aromatic ring shift to 1594, 1503, and 1397 cm⁻¹, respectively. -1 Offset by -12, 20, and 59 cm -1 This indicates that a certain interaction also exists between the electron-deficient Ho2O3-Ag and the electron-rich aromatic ring.

[0029] Figure 2 The DLS plot in (a) further shows that the quantum dot material exhibits a normal distribution trend; Figure 2 The fluorescence spectrum in (b) demonstrates that the material itself possesses excellent fluorescence spectral properties, with an excitation peak at 355 nm and an emission peak at 459 nm, further confirming the nanoscale size of the quantum dots; Figure 2 As shown in (c), the zeta potential of the relevant components of the Ho₂O₃-Ag QDs material in aqueous solution is 8.75 mV for the silver holmium nanomaterial alone, while the zeta potential of the Ho₂O₃-Ag QDs material combined with folic acid is -32.69 mV. The absolute values ​​are significantly higher than those of the silver holmium nanomaterial alone, indicating sufficient electrostatic repulsion to alleviate the aggregation of Ho₂O₃-Ag QDs. Compared with pure Ho₂O₃-Ag NPs synthesized under similar conditions (repeating the synthesis steps of Ho₂O₃-Ag QDs, but replacing the relevant steps of the FA solution with pure water), the strong interaction between FA and Ho₂O₃-Ag quantum dots gives the Ho₂O₃-Ag QDs better stability. Figure 2 (d) XPS total spectrum confirmed that the prepared Ho2O3-Ag QDs material contained all feed elements Ag, Ho, C, N and O. Figure 2 The characteristic peaks at 374.29 eV and 368.29 eV in the Ag 3d high-resolution XPS spectrum of (e) correspond to Ag, respectively. 0 3D 3 / 2 and 3D 5 / 2 The track indicates the Ag feed. + It has been completely restored to Ag 0 . Figure 2In (f), the 161.80 eV in the Ho 4d high-resolution XPS spectrum is attributed to Ho. 3+ 4D 5 / 2 The track indicates the Ho used for feeding. 3+ Doping with folic acid-modified silver quantum dots improved the peroxidase-mimicking activity of Ho2O3-Ag QDs nanozymes.

[0030] Example 2 1. The enzyme-mimicking catalytic activity and AMX sensing performance of the Ho2O3-Ag QDs prepared above were tested using the following steps: (1) Weigh 5.00 mg Ho2O3-Ag QDs and dissolve them in 100.0 mL of triple-distilled water to prepare a solution with a concentration of 0.05 mg / mL Ho2O3-Ag QDs. 3+ Enhanced silver quantum dot nanozyme standard solution; (2) Weigh 2.74 mmol AMX, dissolve in 1000.0 mL of triple-distilled water to prepare a standard solution of 2.74 mmol / L AMX; weigh 1.5 mmol TMB, dissolve in 1000.0 mL of DMSO to prepare a standard solution of 1.5 mM TMB; measure 100.0 mL of 30% H2O2, dissolve in 900.0 mL of triple-distilled water to prepare a standard solution of 3% H2O2. (3) To make the experimental results more reasonable, before studying the catalytic activity of Ho2O3-Ag QDs simulating peroxidase, the reaction time (0~3600 s), pH (2.6~6.0), and c were adjusted. TMB (c) TMB The concentration of TMB (0.025~0.125 mM) and c H2O2 (c) H2O2 The conditions, including the concentration of H2O2 (0.017~0.13 M), were optimized, and the results are as follows: Figure 3 As shown in (ad), the results indicate that the optimal experimental conditions for the Ho2O3-Ag QDs enzyme mimicry are 0.10 mM TMB, 0.117 M H2O2, and pH 4.0, with aging at 25℃ for 1200 s. Under these optimal conditions, the enzyme mimicry activities of FA, Ho2O3-Ag NPs, and Ho2O3-Ag QDs were compared. Figure 4 As shown in (a), among the four test systems with conventional RGB values, the Ho2O3-Ag QDs-TMB-H2O2 system has the highest A value. 652 Both the color depth and the maximum or deepest value are achieved. Furthermore, within 600 seconds, system A... 652Its growth rate was also the fastest, rising from an initial 0.117 au to 0.467 au ( Figure 4 (b) This indicates that Ho2O3-Ag QDs have excellent synergistic catalytic enhancement capabilities, which can accelerate the redox reaction of TMB and H2O2.

[0031] 2. Investigation of enzyme kinetic parameters simulated by Ho2O3-Ag QDs: like Figure 4 (c) and 4(d) recorded the absorption intensity of the UV absorption peak at 652 nm over 600 s under varying concentrations of TMB and H2O2, respectively. It was found that the double reciprocal lines corresponding to different concentrations of TMB or H2O2 were parallel to each other, indicating that the catalytic kinetic model for the redox reaction of TMB and H2O2 promoted by Ho2O3-Ag QDs is the classic ping-pong model. Based on the Michaelis-Menten equation, the corresponding initial reaction rate v was calculated, and the relationship between the reaction rate and the concentrations of TMB and H2O2 (vc) was plotted. TMB and VC H2O2 And the double reciprocal curves ((1 / v) - (1 / c)) of the reaction rate reciprocal versus the reciprocal of TMB concentration and H2O2 concentration, plotted according to the Lineweaver-Burk equation. TMB ) and (1 / v)-(1 / c H2O2 )),like Figure 5 (a) and Figure 5 (b) shows); then the Michaelis constant (K) for Ho2O3-Ag QDs catalyzing TMB and H2O2 was calculated. m The value is 0.095 mM / 9.06×10. -8 M·s -1 Maximum reaction rate (V max The value is 0.599 mM / 2.69 × 10⁻⁶. -7 M·s -1 .

[0032] 3. Investigation into the enzyme-mimicking catalytic mechanism of Ho2O3-Ag QDs Isopropanol or hydroxylamine hydrochloride (common OH and O2 scavengers, respectively) were added to the constructed Ho2O3-Ag QDs-TMB-H2O2 system under O2 or N2 conditions. Figure 5 As shown in (c) and 5(d), the presence of hydroxylamine hydrochloride, O2, or N2 affects A 652 Almost no effect; while isopropanol can cause A 652The concentration of ·OH decreased significantly from 0.514 au to 0.061 au, and the system color changed from dark blue to colorless. This indicates that ·OH preferentially undergoes a rapid hydrogen extraction reaction with isopropanol, generating a relatively stable free radical, which is then "consumed" and cannot react with TMB. These results fully confirm that ·OH is an indispensable active species for the direct oxidation of TMB in the catalytic reaction. Ho₂O₃-Ag QDs accelerate the redox reaction of TMB with H₂O₂ following a peroxidase-like mechanism, catalyzing the redox reaction of TMB with H₂O₂ using the classic ping-pong model.

[0033] 4. Selectivity of AMX by catalytic colorimetric detection: Add 600 μL of 0.05 mg / mL Ho2O3-Ag QDs standard solution, 200 μL of pH 4.0 citrate-phosphate buffer solution, 200 μL of TMB prepared in step (2), and 350 μL of H2O2 prepared in step (2) to a 3 mL volumetric flask. (Note: The original text contains some inconsistencies and unclear grammatical structures. A more accurate translation would require the full context.) -5 AMX and other interfering ions were diluted to 3 mL with purified water, mixed thoroughly, and aged at room temperature for 30 min. The UV-vis spectra of the sample were measured from 300 to 800 nm, and the color changes were observed.

[0034] like Figure 6 As shown in (ac), except for AMX, all tested substances at 2x concentration of interfering substances (Na) + Mg 2+ Ba 2+ CO3 2- SO4 2- Cl - Threonine (Thr), histidine (His), valine (Val), glycine (Gly), serine (Ser), glutamic acid (Glu), ampicillin (Amp), penicillin sodium (PenG), and 2.00 × 10 -5 The presence of g / mL BSA had no significant effect on the color, RGB values, or UV-vis spectrum of the Ho2O3-Ag QDs-TMB-H2O2 system. Only the presence of 2.74 × 10 g / mL BSA in the system... -5 When M is AMX, A 652 As the concentration decreased from 0.562 au to 0.176 au, the system color changed from dark blue to light blue, and the RGB values ​​changed significantly. This indicates that the constructed Ho2O3-Ag QDs-TMB-H2O2 system exhibits high selectivity in achieving UV-vis and RGB recognition of AMX by mimicking peroxidase activity.

[0035] Example 4 Quantitative parameters for AMX detection using Ho2O3-Ag QDs-catalyzed UV-vis and RGB methods: To determine the quantitative analytical parameters for the Ho₂O₃-Ag QDs colorimetric detection of AMX and lay the foundation for its subsequent practical application, UV-vis spectral titration experiments and color change comparison experiments were conducted on different concentrations of AMX. AMX It is 8.21×10 -8 M to 2.74×10 -5 Within the range of M, UV-vis spectral titration experiments were conducted, and the corresponding color and RGB value changes were recorded. For example... Figure 6 As shown in (df), with c AMX The increase of A 652 The system's color and RGB values ​​both exhibit regular changes: A 652 As the concentration gradually decreases, the system changes from dark blue to light blue, exhibiting a significant subtractive color effect. △A 652 With c AMX The linear relationship is △ A 652 =1.39×10 -4 c AMX -1.34×10 -2 (8.21×10 -8 ~2.74×10 -5 M, R 2 =0.9968, Figure 6 (g); △ (R+G) / B and c AMX The linear relationship is △ (R+G) / B=1.85×10 -4 c AMX +3.11×10 -2 (1.37×10 -7 ~2.74×10 - 5 M, R 2 =0.9959, Figure 6 (h). The corresponding detection limits for the two are 6.91 × 10⁻⁶ and 6.91 × 10⁻⁶, respectively. -8 M (S / N=3) and 1.18×10 -7 M (S / N=3). The results show that Ho2O3-Ag QDs is an excellent catalytic colorimetric sensor for the quantitative detection of AMX.

[0036] Example 5 1. Optimization of AMX fluorescence detection: From Figure 7 As can be seen from (a) and (b), the fluorescence enhancement is greatest when AMX is added at pH 8.0, and AMX can be detected in 10 minutes with good stability.

[0037] 2. Selectivity of AMX fluorescence detection: Add 100 μL of 0.05 mg / mL Ho₂O₃-Ag QDs standard solution, 200 μL of pH 8.0 phosphate buffer solution, and 3.56 × 10⁻⁶ mg / mL sodium hydroxide solution to a 3 mL volumetric flask. -6 AMX and other interfering ions at mol / L were diluted to 3 mL with purified water, mixed thoroughly, and aged at room temperature for 10 min. The fluorescence emission spectra were then measured. The results are as follows: Figure 8 As shown in (a), all substances with a 2x concentration (Na) + Mg 2+ Ba 2+ CO3 2- SO4 2- Cl - Threonine (Thr), histidine (His), valine (Val), glycine (Gly), serine (Ser), glutamic acid (Glu), ampicillin (Amp), penicillin sodium (PenG), and 2.60 × 10 -6 In the presence of BSA (g / mL), Ho₂O₃-Ag QDs material exhibits enhanced fluorescence intensity at 459 nm and enhanced color under fluorescent light only when AMX is present. This indicates that Ho₂O₃-Ag QDs material has a unique fluorescence enhancement response to AMX, while other common ions show no significant changes.

[0038] Based on this, selective experiments were further conducted under conditions where AMX coexists with other interfering substances. Figure 8 (b) shows that when AMX coexists with other interfering components, F 459 The change was very small, within 5% error, which indicates that the coexisting interfering substances had virtually no impact on the fluorescence detection of AMX.

[0039] 3. Quantitative parameters for Ho2O3-Ag QDs fluorescence detection of AMX: In order to determine the quantitative analytical parameters for Ho2O3-Ag QDs fluorescence detection of AMX and lay the foundation for the subsequent practical application of quantitative detection of AMX, fluorescence spectroscopic titration experiments were conducted on different concentrations of AMX. Figure 8 (c) The results show that the absorption intensity of Ho₂O₃-Ag QDs at 459 nm gradually increases with the gradual increase of AMX concentration, accompanied by a deepening of color. From Figure 8 (d) Further observation shows that the AMX concentration is 5.47 × 10⁻⁶. -9 Up to 3.56×10 -6 Within the range of M, △ F 459 (F) 0 459 F 459 ) and c AMX They exhibit a good linear relationship, and the linear equations are as follows: △ F 459 = 4.75 c AMX + 299.84 (5.47×10 -9 ~3.56×10 -6 M, R 2 = 0.9960, Figure 8 d) The detection limit for AMX by fluorescence detection is 3.56 × 10⁻⁶. -9 M (S / N=3). Ho2O3-Ag QDs are also an excellent fluorescent sensor for the quantitative detection of AMX.

[0040] Example 6 The application of AMX in real samples using UV-vis, fluorescence, and RGB multi-channel detection.

[0041] 1. Preparation of the sample solution to be tested: Tap water and Yihe River water were randomly sampled from Laboratory B528 of Qufu Normal University and the Yihe River in Qufu City, respectively. Both were filtered three times through a 0.22 μm microporous membrane for subsequent testing.

[0042] Cucumbers, apples, rice, milk, and live zebrafish were purchased from a supermarket near Qufu Normal University; live chive blossoms, straw, and soil were randomly sourced from a farm near Qufu Normal University.

[0043] Weigh 20.0 g of pork, rice, or straw and place them in a juicer. Add 40.0 mL of water and mix homogenously for 20 minutes. Centrifuge the resulting mixture at 11000 r / min for 10 minutes, collect the supernatant, and use it for subsequent analysis.

[0044] After the random milk was thoroughly stirred, it was centrifuged at 11,000 r / min for 10 min, and the supernatant was collected for subsequent detection.

[0045] Dissolve 5.0 mg of AMX capsule powder in 10.0 mL of water, then centrifuge at 11000 r / min for 10 min. Collect the supernatant for subsequent analysis. This AMX capsule was manufactured by CSPC Zhongnuo Pharmaceutical Co., Ltd.

[0046] Weigh 50.0 g of edible apple or cucumber and place it in a juicer, homogenize for 20 min. Centrifuge the resulting mixture at 11000 r / min for 15 min, collect the supernatant, and dilute it 30 times with water for subsequent analysis.

[0047] Weigh 10.0 g of soil into a beaker, add 40.0 mL of water, and stir continuously for 24 h. After filtering the mixture through a 0.22 μm microporous membrane, collect the filtrate for subsequent analysis.

[0048] 2. UV-vis and RGB detection methods: Add 600 μL of 0.05 mg / mL Ho2O3-Ag QDs dispersion, 200 μL of pH 4.0 citrate-phosphate buffer solution, 200 μL of TMB prepared in step (2), 350 μL of H2O2, and 200 μL of AMX standard solution or the sample solution prepared above to a 3 mL volumetric flask. Dilute to 3 mL with purified water, mix thoroughly, and age at room temperature for 30 min. Measure the UV-Vis spectra at 300-800 nm to obtain A... 652 And use a home smartphone to measure the corresponding RGB values.

[0049] Add 600 μL of 0.05 mg / mL Ho₂O₃-Ag QDs dispersion, 200 μL of pH 4.0 citrate-phosphate buffer solution, 200 μL of TMB prepared in step (2), and 350 μL of H₂O₂ to a 3 mL volumetric flask, respectively. Dilute to 3 mL with purified water, mix thoroughly, and age at room temperature for 30 min. Measure the UV-Vis spectra at 300-800 nm to obtain A. 0 652 And [(R+G) / B] 0 ; A 652 and A 0 652 Substitute into the formula △ A 652 = A 652 A 0 652 △A is calculated 652 ,Will △ A 652 Substitute into the linear regression equation △ A 652 = 1.39 × 10 -4 c AMX - 1.34×10 -2 The concentration of AMX in the sample was calculated; simultaneously, the [(R+G) / B] was obtained. 0 Substitute into the formula △ (R+G) / B = (R+G) / B - [(R+G) / B] 0 Calculations yielded △ (R+G) / B; The above △Substituting (R+G) / B into the linear regression equation △ (R+G) / B = 1.85 × 10 -4 c AMX + 3.11×10 -2 The concentration of AMX in the sample was calculated.

[0050] The standard deviation (RSD) and recovery rate (%) of AMX were determined using a standard spiked method, as shown in Table 1. The recoveries of AMX detected by enzyme-catalyzed UV-vis and RGB methods were 97.9%–103.7% and 96.9%–104.5%, respectively, with relative standard deviations of less than 3.9% and 4.6%, respectively. The recovery rate of fluorescence-responsive AMX was 95.4%–103.3%, with a relative standard deviation of no more than 4.1%.

[0051] 3. Fluorescence detection method: Add 100 μL of 0.05 mg / mL Ho₂O₃-Ag QDs dispersion, 200 μL of pH 8.0 mixed phosphate buffer solution, and 200 μL of AMX standard solution or environmental sample to a 3 mL volumetric flask, respectively. Dilute to 3 mL with purified water, mix thoroughly, and immediately measure the fluorescence spectrum at an excitation wavelength of 355 nm to obtain the F... 459 ; Add 100 μL of 0.05 mg / mL Ho₂O₃-Ag QDs dispersion and 200 μL of pH 8.0 mixed phosphate buffer solution to separate 3 mL volumetric flasks. Dilute to 3 mL with purified water, mix thoroughly, and immediately measure the fluorescence spectrum at an excitation wavelength of 355 nm to obtain the F... 0 459 ; F 459 and F 0 459 Substitute into the formula △ F 459 = F 459 F 0 459 △F was calculated. 459 , will the △ F 459 Substitute the value into the linear regression equation △ F 459 = 4.75 c AMX +299.84, the concentration of AMX in the sample was calculated. The results are shown in Table 1. In Table 1, the fluorescence detection RSD of AMX in the actual samples was less than 4.1%, and the recovery rate was between 95.4% and 103.3%.

[0052] To clarify the detection reliability of AMX multichannel UV-vis, RGB and fluorescence analysis, F-values ​​and t-values ​​were calculated for all obtained results and compared with the corresponding standard values.

[0053] As shown in Table 2, the calculated F-values ​​and t-values ​​are both less than their corresponding standard values ​​(6.39 and 2.31), respectively. This indicates that there are no significant systematic differences among the three detection methods, demonstrating statistical equivalence. This confirms that the UV-vis, RGB, and fluorescence analysis channels can all be used independently and reliably for AMX quantification, and the results can be cross-referenced and verified. This demonstrates that Ho2O3-Ag QDs are a reliable peroxidase-simulating sensor that can be used for multi-channel detection of AMX in real, complex samples.

[0054] Example 7 Visual fluorescence imaging experiments of the Ho2O3-Ag QDs against the antibiotic AMX in organisms such as edible rice, live chive flowers, and live zebrafish.

[0055] The fluorescence imaging experiment employs the following steps: Some edible rice, live zebrafish, and live chive blossoms were placed in a solution with a concentration of 1.67 × 10⁻⁶. -3 The cells were cultured in an aqueous dispersion of Ho₂O₃-Ag QDs at a concentration of 0 mg / mL for 24 hours. Subsequently, concentrations of 0 mg / mL and 8.21 × 10⁻⁶ mg / mL were added. -7 1.91×10 -6 and 3.56×10 -6 The samples were incubated with AMX standard solution M for another 24 hours. After washing with sufficient water, the rice, live zebrafish, and live chive flower samples were subjected to fluorescence imaging recording using a ZF-20A four-channel darkroom UV analyzer manufactured by Shanghai Yuezhong Instrument Co., Ltd., under excitation at a wavelength of 365 nm.

[0056] Comparative Example 1 The difference between this comparative example and Example 7 is that the edible rice, live zebrafish, and live chive flowers were cultured in water for only 24 hours, while the other conditions were the same as in Example 7.

[0057] Comparative Example 2 The difference between this comparative example and Example 7 is that edible rice, live zebrafish, and live chive blossoms were placed only in a 3.56×10⁻⁶ container. -6 M was cultured in an AMX aqueous solution for 24 h, with other conditions the same as in Example 7.

[0058] like Figure 9 As shown, only at 3.56×10 -6No fluorescence signal was detected in MAMX samples, including edible rice, live zebrafish, and live chive flowers aged in water for only 24 hours. (The last part, "1.67 × 10," appears to be an unrelated fragment and is omitted from the translation.) -3 Extremely weak fluorescence signals were observed in edible rice, live zebrafish, and live chive flowers aged for 24 hours in an aqueous dispersion of mg / mL Ho2O3-Ag QDs. Furthermore, when these organisms were first exposed to 1.67 × 10 mg / mL Ho2O3-Ag QDs, extremely weak fluorescence signals were observed. -3 After aging for 24 hours with mg / mL Ho2O3-Ag QDs, 8.21×10 mg / mL Ho2O3-Ag QDs were added. -7 M, 1.91×10 -6 M and 3.56×10 -6 As AMX in M ​​continues to age for 24 hours, its fluorescence signal intensity gradually increases. This phenomenon fully demonstrates that Ho2O3-Ag QDs can be used for fluorescence imaging detection of AMX content in complex living organisms, and the interference of the living organism environment on AMX imaging detection is minimal.

[0059] Table 1. UV-vis, RGB, and fluorescence detection results of actual AMX samples (n=5) a 0.01 mg·mL -1 Ho2O3-Ag QDs, pH=4.0; b 1.67×10 -3 mg·mL -1 Ho2O3-Ag QDs, pH=8.0; c Obtained by ultraviolet-visible spectroscopy (UV-vis) analysis; d Obtained from RGB analysis; e Actual value = value in table × 200 (diluted 200 times before testing); f Actual value = value in table × 10 (diluted 10 times before testing); g Actual value = value in table × 30 (diluted 30 times before testing); h Actual value = value in the table × 2 (diluted 2 times before testing).

[0060] Table 2. Comparison of AMX detection results by UV-vis, RGB and fluorescence methods (n=5) a P=0.95, f1=4, f2=4, F<6.39; b P=0.95, f=8, t<2.31 To verify the reliability of the multi-channel analysis methods based on UV-vis, RGB, and fluorescence for AMX detection, this study calculated the F-values ​​and t-values ​​for all experimental data and compared them with the corresponding standard values. As shown in Table 2, the obtained F-values ​​and t-values ​​were both lower than their corresponding standard values ​​(6.39 and 2.31), respectively. The results indicate that the detection precision of the UV-vis, RGB, and fluorescence analysis methods used in this study is good, and there is no significant difference among the three methods. Ho2O3-Ag QDs are a reliable peroxidase-simulating sensor that can be used for multi-channel detection of AMX in complex real-world samples.

[0061] The above description merely illustrates several embodiments of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make modifications, substitutions, and improvements without departing from the concept and scope of the present invention, and these all fall within the protection scope of the present invention. Therefore, the patent protection scope of the present invention should be determined by the described claims.

Claims

1. A holmium oxide-enhanced silver quantum dot nanozyme, characterized in that, The holmium oxide-enhanced silver quantum dot nanozyme is spherical with a diameter of 3.30 ± 0.66 nm.

2. The application of the holmium oxide-enhanced silver quantum dot nanozyme of claim 1 in the detection of amoxicillin, characterized in that, The application is for multi-channel qualitative or quantitative analysis of amoxicillin.

3. The application according to claim 2, characterized in that, The method for detecting amoxicillin is UV-vis assay, RGB assay, or fluorescence multichannel assay.

4. The application according to claim 3, characterized in that, Both the UV-vis and RGB measurement methods include the following steps: After mixing and reacting the holmium oxide-enhanced silver quantum dot nanozyme, 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide, the sample to be tested, and citrate-phosphate buffer, the absorbance value of the system in the UV-vis spectrum at 652 nm or the RGB value of the visible color photograph is measured, and the change in absorbance is analyzed. △ A 652 or the change in RGB values △ The linear relationship between (R+G) / B and amoxicillin concentration was used to calculate the amoxicillin content.

5. The application according to claim 4, characterized in that, The pH of the citrate-phosphate buffer solution is 4.0; the concentration of the holmium oxide-enhanced silver quantum dot nanozyme in the reaction system is 0.01 mg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine is 0.1 mmol / L; and the mass fraction of the hydrogen peroxide is 3%.

6. The application according to claim 3, characterized in that, The fluorescence assay employs the following steps: The holmium oxide-enhanced silver quantum dot nanozyme, the sample to be tested, and phosphate buffer are mixed; the fluorescence intensity of the system at 459 nm is measured; and the change in fluorescence intensity is analyzed. △ F 459 The linear relationship between the amoxicillin concentration and the amoxicillin concentration was used to calculate the amoxicillin content.

7. The application according to claim 6, characterized in that, The pH of the phosphate buffer solution is 8.

8. The application according to claim 2, characterized in that, The applications include detecting amoxicillin concentrations in water, soil, agricultural products, agricultural by-products, or pharmaceuticals.

9. The application according to claim 8, characterized in that, The agricultural products include edible rice, straw, apples, cucumbers, live zebrafish, or live chive blossoms; the agricultural by-products include pork or milk.

10. The application according to claim 2, characterized in that, The multi-channel qualitative analysis involved using holmium oxide-enhanced silver quantum dot nanozymes to perform fluorescence imaging analysis of amoxicillin residues in living organisms.

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