A fluorescence / colorimetric dual-mode sensor for detecting captopril and preparation method and application thereof

Abstract

The application discloses a fluorescent / colorimetric dual-mode sensor for detecting captopril and a preparation method and application thereof, and the fluorescent / colorimetric dual-mode sensor comprises ZrFe MOF, hydrogen peroxide and 3,3',5,5'-tetramethyl benzidine; and the ZrFe MOF is self-assembled from zirconium metal ions, iron metal ions and organic ligands of diamino terephthalic acid. The application adopts a hydrothermal method to synthesize a bimetallic ZrFe MOF nanomaterial which has excellent peroxidase activity and stable fluorescent characteristics. By utilizing the inhibitory effect of captopril on the ZrFe MOF catalytic TMB-H2O2 color reaction and the characteristic that captopril can significantly enhance the blue fluorescence of the ZrFe MOF, the fluorescent / colorimetric dual-mode sensing of captopril is successfully realized.

Description

Technical Field

[0001] This invention relates to the field of fluorescence / colorimetric dual-mode sensor technology, and more particularly to a fluorescence / colorimetric dual-mode sensor for detecting captopril, its preparation method, and its application. Background Technology

[0002] Captopril (CP) is the first orally effective angiotensin-converting enzyme inhibitor (ACEI). Due to its significant blood pressure-lowering effect, it is widely used clinically to treat hypertension, heart failure, and left ventricular dysfunction after myocardial infarction. However, driven by profit, some unscrupulous manufacturers illegally add captopril to functional foods claiming to have blood pressure-lowering functions to create the illusion of "rapid effect" and "significant efficacy." This unregulated illegal addition leads to consumers unknowingly ingesting drug components for extended periods, potentially inducing various toxic side effects such as persistent dry cough, hypotension, loss of taste, and rashes. In severe cases, it can even cause acute kidney injury, hyperkalemia, and shock. Furthermore, this practice masks the true effective ingredients of the product, interfering with or delaying consumers' access to proper medical treatment, thus causing irreversible health damage. Currently, the detection of illegal additives such as captopril mainly relies on high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods using large-scale laboratory instruments. While these methods offer high accuracy and sensitivity, they generally suffer from limitations such as expensive equipment, complex operation, long testing cycles, and high operator skill requirements, making it difficult to meet the practical needs of rapid on-site screening, market sampling, and home self-testing at the grassroots regulatory level. Therefore, developing a simple, rapid, visual, and low-cost on-site captopril testing method is of great practical significance for improving regulatory efficiency and effectively protecting public health. Summary of the Invention

[0003] Objectives of the Invention: The first objective of this invention is to provide a highly sensitive and rapid fluorescence / colorimetric dual-mode sensor for detecting captopril; the second objective of this invention is to provide a method for preparing the fluorescence / colorimetric dual-mode sensor; and the third objective of this invention is to provide applications of the fluorescence / colorimetric dual-mode sensor.

[0004] To achieve the aforementioned primary objective, the technical solution of the fluorescence / colorimetric dual-mode sensor provided by this invention is as follows:

[0005] The fluorescence / colorimetric dual-mode sensor of the present invention comprises ZrFe MOF, hydrogen peroxide (H2O2), and a chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB); the ZrFe MOF is formed by zirconium metal ions, iron metal ions, and the organic ligand diaminoterephthalic acid.

[0006] The ZrFe MOF has an octahedral structure and a particle size of 95~105 nm.

[0007] To achieve the second objective mentioned above, the technical solution for fabricating a fluorescence / colorimetric dual-mode sensor provided by this invention is as follows:

[0008] The method for preparing the fluorescence / colorimetric dual-mode sensor of the present invention includes a method for preparing ZrFe MOF, wherein the method for preparing ZrFe MOF involves dissolving zirconium metal salt, iron metal salt, diaminoterephthalic acid and acetic acid in an organic solvent, carrying out a hydrothermal reaction, and collecting the precipitate, which is ZrFe MOF.

[0009] The organic solvent is N,N-dimethylformamide (DMF).

[0010] After collecting the precipitate, the precipitate is washed with N,N-dimethylformamide and ethanol, and then dried to obtain ZrFe MOF.

[0011] To achieve the aforementioned third objective, the technical solution for the application of the fluorescence / colorimetric dual-mode sensor provided by this invention is as follows:

[0012] The fluorescence / colorimetric dual-mode sensor described in this invention can be used in captopril detection.

[0013] Specifically, the present invention provides a captopril detection method utilizing the above-mentioned fluorescence / colorimetric dual-mode sensor, comprising the following steps:

[0014] (1) Take captopril solutions of varying concentrations and mix them with ZrFe MOF respectively. After incubation, the test solution is obtained and its fluorescence intensity is measured and recorded as F. i Simultaneously, water was used as a blank sample and mixed with ZrFe MOF. After incubation, a blank sample test solution was obtained, and its fluorescence intensity was measured and recorded as F0. A graph was constructed with captopril concentration as the x-axis and fluorescence intensity F0 as the y-axis. i -F0 is the fluorescence standard curve on the ordinate;

[0015] (2) Captopril solutions of varying concentrations were mixed with ZrFeMOF, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine, respectively. After incubation, the test solution was obtained, and its ultraviolet absorbance was measured and recorded as A. i Simultaneously, water was used as a blank sample and mixed with ZrFe MOF, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine. After incubation, a blank sample test solution was obtained, and its UV absorbance was measured and denoted as A0. A graph was constructed with captopril concentration as the x-axis and UV absorbance A0 as the y-axis. i - A0 is the colorimetric standard curve on the ordinate;

[0016] (3) Mix the sample to be tested with ZrFe MOF, incubate the reaction to obtain the test solution, measure its fluorescence intensity, subtract F0 from the measured fluorescence intensity, and substitute it into the fluorescence standard curve constructed in step (1) to calculate the concentration of captopril in the sample to be tested.

[0017] (4) Mix the sample to be tested with ZrFe MOF, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, incubate the reaction to obtain the test solution, measure its ultraviolet absorbance, subtract A0 from the measured ultraviolet absorbance, and substitute it into the colorimetric standard curve constructed in step (2) to calculate the concentration of captopril in the sample to be tested.

[0018] In steps (1) and (3), the incubation time is 15-50 min and the incubation temperature is 25°C-70°C; in steps (2) and (4), the incubation time is 20-150 min and the incubation temperature is 25°C-45°C.

[0019] In steps (1) and (3), the fluorescence intensity is measured under the following conditions: the fluorescence intensity at 460 nm is measured, the excitation wavelength is 350 nm, and the fluorescence spectrum range is 400~550 nm.

[0020] In steps (2) and (4), the absorbance at 652 nm is measured, and the absorption spectrum range is 550~750 nm.

[0021] This invention utilizes a hydrothermal method, employing diaminoterephthalic acid (H2BDC-NH2) as the organic ligand, to simultaneously introduce Fe(III) and Zr(IV) metal centers, successfully preparing a ZrFe MOF possessing both strong peroxidase activity and stable fluorescence properties. The core sensing mechanism of this sensor is as follows: On one hand, the thiol group (-SH) in the captopril molecule effectively inhibits the ZrFe MOF-catalyzed TMB-H2O2 reaction, generating the blue oxidation product oxTMB, thus achieving colorimetric signal detection ("signal off"). On the other hand, the carboxyl group (-COOH) of captopril specifically interacts with the amino group (-NH2) on the MOF ligand, forming an amide bond, thereby restricting intramolecular rotation of the ligand and significantly enhancing the blue fluorescence of the ZrFe MOF itself, achieving fluorescence signal detection ("signal on"). This unique "fluorescence enhancement-colorimetric inhibition" dual-response mechanism originates from the independent regulation of different material properties by the analyte, effectively avoiding signal crosstalk. To further meet the needs of different scenarios, we have further constructed a smartphone detection platform by converting colorimetric signals into RGB values, enabling visualized detection of target objects and providing broad application prospects for rapid on-site screening and portable analysis of illegal additives in health foods.

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: A bimetallic ZrFe MOF nanomaterial with both excellent peroxidase activity and stable fluorescence properties was synthesized using a hydrothermal method. Utilizing the inhibitory effect of captopril on the TMB-H2O2 colorimetric reaction catalyzed by ZrFe MOF and the property that captopril can significantly enhance the blue fluorescence of ZrFe MOF (emission enhancement mechanism due to molecular rotational restriction), a dual-mode fluorescence / colorimetric sensing of captopril was successfully achieved. After optimization, the fluorescence method showed a good linear relationship with the captopril concentration in the range of 1.95–250 µM (R0). 2 =0.9913), with a detection limit as low as 0.33 µM; the colorimetric method showed a good linear relationship with captopril concentration in the range of 0.3–30 µM (R0.9913). 2 =0.9959), with a detection limit as low as 0.22µM. Based on this, and further combined with smartphone RGB value conversion, rapid and visualized detection of captopril was achieved. Within the concentration range of 0.83–20µM, the (R+G) / B value showed a good linear relationship with captopril concentration, with a detection limit as low as 0.27µM, providing a feasible solution for on-site screening of illegal additives in functional foods. Attached Figure Description

[0023] Figure 1 This is a scanning electron microscope image of ZrFe MOF.

[0024] Figure 2 This is a transmission electron microscope image of ZrFe MOF.

[0025] Figure 3 This is a Captopril visualization detection based on RGB colorimetric analysis of smartphones.

[0026] Figure 4 This is a schematic diagram of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1: Preparation of ZrFe MOF

[0029] The preparation steps of ZrFe MOF are as follows: ZrCl4 (0.27 M, 5 mL), FeCl3·6H2O (0.13 M, 5 mL), 2-aminoterephthalic acid (H2BDC-NH2, 0.27 M, 10 mL), and acetic acid (HAC, 5 mL) were dissolved in 5 mL of N,N-dimethylformamide (DMF) solution. After magnetic stirring for 30 min, the mixture was transferred to a stainless steel autoclave lined with Teflon and reacted at 120 °C for 12 h. The precipitate was collected by centrifugation and washed several times with DMF and ethanol. Finally, the product was dried overnight in a vacuum oven at 60 °C to obtain a brownish-yellow dry powder of ZrFe MOF.

[0030] Characterization of ZrFe MOF

[0031] In this embodiment, a ZrFe bimetallic MOF was synthesized via a solvothermal reaction, and the prepared ZrFe MOF was comprehensively characterized using various techniques. To clarify the morphological characteristics and elemental composition of the ZrFe MOF, SEM, TEM, and related energy dispersive spectroscopy analyses were performed sequentially. First, the microstructure of the material was observed using SEM. For example... Figure 1 As shown, the synthesized material exhibits a well-dispersed, regularly morphological octahedral structure. Further particle size analysis revealed an average particle size of approximately 96.8 nm and a narrow particle size distribution, demonstrating good uniformity. To further investigate its internal structure and elemental distribution, TEM and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analyses were performed. TEM images ( Figure 2 The octahedral outline and edge details of the material were clearly revealed. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and EDS elemental mapping images confirmed the presence of all five elements—C, N, O, Zr, and Fe—and their uniform distribution throughout the octahedral framework, providing direct evidence for the successful construction and uniform distribution of Zr / Fe bimetallic nodes. Furthermore, the ZrFe MOF was further characterized using an IRTracer-100 Fourier transform infrared spectroscopy system to explore its surface functional groups. The infrared spectrum of the H2BDC-NH2 ligand showed a strong characteristic peak at 1670 cm⁻¹ attributable to the stretching vibration of the carboxyl group (C=O), and a broad absorption band in the 2500–3650 cm⁻¹ range, corresponding to the stretching vibrations of the hydroxyl (O–H) and amino (N–H) groups due to intermolecular hydrogen bonding. Compared to the ligand, the infrared spectrum of the synthesized ZrFe MOF showed significant changes: the intensity of the C=O stretching vibration peak at 1670 cm⁻¹ was significantly weakened, while the broad absorption band in the 2500-3650 cm⁻¹ region broadened and its intensity decreased. These changes indicate that the carboxyl oxygen atom in the ligand interacts with the metal ion (ZrFeMOF). 4Coordination occurred in the Zr-O / N-H region, leading to changes in the electron cloud density and force constant of the C=O bond. The alteration of the O–H / N–H region may be related to changes in the intramolecular or skeletal hydrogen bonding environment after coordination, collectively confirming the successful coordination between the organic ligand and the metal ion. Furthermore, the triple absorption bands at 768, 664, and 482 cm⁻¹ are attributed to the stretching vibrations of the Zr–O–Zr bridging bond, while the characteristic peak at 567 cm⁻¹ corresponds to the stretching vibrations of the Fe–O–Fe bond, further supporting the formation of Zr–O and Fe–O metal cluster nodes.

[87] The XRD pattern of the ZrFe MOF showed sharp and clear characteristic diffraction peaks at 2θ = 7.4°, 8.5°, and 25.7°, indicating that the synthesized material has good crystallinity. Finally, the optical properties of the synthesized material were further investigated. The ZrFe MOF produced a maximum emission peak at 460 nm under 350 nm excitation. Its UV-Vis absorption spectrum showed two distinct absorption bands at 280 nm and 390 nm: the former can be attributed to the π→π* electronic transition of the ligand benzene ring skeleton, while the latter may originate from the n→π* transition related to amino or metal-ligand charge transfer (MLCT). Based on the above FTIR, XRD, and optical spectral analysis results, it can be confirmed that a ZrFe bimetallic MOF material with the expected structure, good crystallinity, and characteristic optical properties was successfully synthesized.

[0032] Example 2: Feasibility of Captopril Detection

[0033] Fluorescence detection mode: ZrFe MOF (500 µg / mL, 150 µL) and captopril solution (500 µM, 50 µL) were added to a 96-well plate. The solution was incubated at 60 °C for 15 min. After cooling to room temperature, the fluorescence intensity F at 460 nm was measured. i The excitation wavelength was 350 nm, and the fluorescence spectrum range was 400–550 nm. The fluorescence intensity measured using water as a blank sample was denoted as F0. i -F0 is used as the detection signal for quantitative analysis. The above experiment should be repeated at least 3 times.

[0034] UV detection mode: ZrFe MOF (500 µg / mL, 10 µL), captopril solution (30 µM, 50 µL), NaAc-HAc buffer (0.2 M, pH 3.5, 100 µL), H2O2 (100 mM, 20 µL), and 3,3',5,5'-tetramethylbenzidine (TMB, 5 mM, 20 µL) were added sequentially to a 96-well plate. After incubation at 37°C for 20 min, the absorbance A at 652 nm was measured. iThe absorption spectrum ranges from 550 to 750 nm. The absorbance measured using water as a blank sample is denoted as A0, and ΔA (ΔA = A - A0) is used as the detection signal for quantitative analysis. The above experiment should be repeated at least three times.

[0035] Enzymatic kinetic analysis of ZrFe MOF

[0036] To investigate the peroxidase-like catalytic efficiency and substrate affinity of ZrFe MOF, steady-state kinetics were studied using TMB and H2O2 as substrates. The kinetic parameters obtained based on the Michaelis-Menten equation and the Lineweaver-Burk plot are listed in Table 1. The results show that when TMB is used as the substrate, the Michaelis constant (Km) of ZrFe MOF is 18.13 mM, significantly higher than that of HRP (Km = 0.43 mM). Compared to HRP, the binding affinity of ZrFe MOF to TMB molecules is weaker. When H2O2 is used as the substrate, the Km value of ZrFe MOF is 0.11 mM, significantly lower than that of HRP (Km = 3.70 mM). This indicates that ZrFe MOF has a very high affinity for H2O2 and can more effectively capture and activate H2O2 molecules. In summary, ZrFe MOF exhibits a unique kinetic characteristic of high affinity for H2O2 and low affinity for TMB. Its overall highly efficient peroxidase-like catalytic activity is likely primarily due to its excellent activation ability for H2O2. This can be attributed to the synergistic effect of the Fe and Zr bimetallic active centers in the material, which provides a superior catalytic interface for the adsorption and decomposition of H2O2.

[0037] Table 1 Michaelis constants (K) of ZrFe MOF and HRP m ) and maximum reaction rate (V max )

[0038]

[0039] Example 3

[0040] Optimization of Captopril Detection Conditions

[0041] To achieve more sensitive captopril detection, this embodiment aims to optimize experimental conditions such as reaction pH, ZrFe MOF concentration, incubation time, and incubation temperature. In optimizing the experimental conditions, only the condition to be optimized was changed as the sole variable; the remaining conditions were the same as in Example 2. For the fluorescence detection mode, the detection system (F...) under this variable was examined. iThe optimal condition was determined by the maximum value of ΔA / A0 under these conditions. The final concentration of captopril was 500 µM. The pH range was 3–11, with specific test points at pH = 3, 5, 7, 9, and 11. The concentration range of ZrFeMOF was 0–2 mg / mL, with specific test points at 0, 0.5, 1, 1.5, and 2 mg / mL. The reaction time range was 0–50 min, with specific test points at 0, 5, 10, 20, 30, 40, and 50 min. The reaction temperature range was 25°C–70°C, with specific test points at 25, 37, 45, 60, and 70°C. For UV detection mode, the optimal condition was determined by the maximum value of ΔA / A0 under these variables. Where ΔA = A 652 -A0, the absorbance at 652 nm obtained using ultrapure water as a blank is designated as A0. The final concentration of captopril was 10 µM. The pH range was 3–11, with specific test points at 3, 5, 7, 9, and 11. The concentration range of ZrFeMOF was 0.06–2 mg / mL, with specific test points at 0.01, 0.06, 1, 1.5, and 2 mg / mL. The reaction time range was 0–50 min, with specific test points at 0, 5, 10, 20, 30, 40, and 50 min. The reaction temperature range was 25°C–70°C, with specific test points at 25, 37, 45, 60, and 70°C. All experiments were repeated at least three times.

[0042] Optimization of conditions for captopril detection

[0043] To obtain more accurate and sensitive detection results, the experimental conditions for fluorescence and colorimetric detection were further optimized. Similar to natural horseradish peroxidase (HRP), the enzymatic catalytic activity of ZrFe MOF is related to the pH of the NaAc-HAc buffer. As the pH increases, the enzymatic catalytic activity of ZrFe MOF first increases and then gradually decreases, reaching its peak at pH 3.5. Therefore, in this embodiment, a NaAc-HAc buffer at pH 3.5 was selected as the optimal reaction condition for evaluating the peroxidase-like activity of ZrFe MOF.

[0044] The fluorescence of ZrFe MOF mainly originates from its organic ligand (H2BDC-NH2), and its signal is extremely sensitive to the pH of the solution. Within the pH range of 3-11, the fluorescence intensity change rate (F / F0, where F0 and F represent the fluorescence intensity before and after the addition of captopril, respectively) was used as the sensing response signal. F / F0 reached its maximum at pH 7, indicating that the fluorescence signal of ZrFe MOF responded most significantly to the target analyte captopril under these conditions. This may be because a neutral environment is most conducive to maintaining the structural stability of the MOF and its effective interactions with the target analyte (such as electrostatic interactions and hydrogen bonding). Therefore, a buffer solution with pH 7.5 was used in subsequent experiments. Furthermore, as the ZrFe MOF concentration increased to 500 μg / mL, F / F0 increased significantly. However, when the ZrFe MOF concentration continued to increase to 2000 μg / mL, F / F0 decreased instead. This may be because excessively high ZrFe MOF concentrations cause greater interference, leading to a decrease in the detection signal. Incubation time affects the completeness of the enzymatic reaction. After 15 minutes of incubation, the detection signal hardly increases anymore. To shorten the analysis cycle and improve detection efficiency as much as possible, the optimal incubation time was set to 15 minutes. The effect of incubation temperature on the fluorescence detection signal was investigated. The fluorescence detection signal F / F0 reached its maximum value at an incubation temperature of 60 °C. In summary, the optimal experimental conditions for the fluorescence detection mode are pH 5–9, ZrFeMOF concentration of 500–1500 μg / mL, incubation time of 15–50 minutes, and incubation temperature of 25–70 °C. The optimal experimental conditions for the fluorescence detection mode were finally determined to be pH 7, ZrFeMOF concentration of 500 μg / mL, incubation time of 15 minutes, and incubation temperature of 60 °C.

[0045] The colorimetric signal of ZrFe MOF primarily derives from its peroxidase-like activity, which catalyzes the color change of TMB and is highly sensitive to pH. Within the pH range of 3-11, ΔA / A0 reaches its maximum at pH 7. Furthermore, ΔA / A0 increases significantly with increasing ZrFeMOF concentration to 500 μg / mL. However, when the concentration is further increased to 2000 μg / mL, ΔA / A0 unexpectedly decreases, possibly due to background interference from high ZrFe MOF concentrations or increased nanoparticle aggregation, thus reducing the detection signal. Incubation time affects the completeness of the enzyme reaction; after 20 min, the detection signal shows almost no increase. To improve detection efficiency while minimizing the analysis cycle, the optimal incubation time is 20 minutes. The ΔA / A0 value is highest at an incubation temperature of 37°C. Therefore, the optimal experimental conditions for colorimetric detection of captopril are an incubation time of 20–50 min and an incubation temperature of 25–45°C; the optimal conditions for colorimetric detection of captopril are determined to be pH 7, ZrFe MOF concentration of 500 μg / mL, incubation time of 20 min, and incubation temperature of 37°C.

[0046] Example 4

[0047] Linearity and specificity

[0048] Fluorescence detection mode: ZrFe MOF (500 µg / mL, 150 µL) and 50 µL of captopril standard solutions of different concentrations (0, 2, 3, 4, 8, 16, 31, 62, 100, 150, 250 µM) were incubated at 60 ℃ for 15 min, then removed and cooled to room temperature. The fluorescence intensity F at 460 nm was measured. i The excitation wavelength was 350 nm, and the fluorescence spectrum range was 400–550 nm. The fluorescence intensity of the blank sample was F0, and F... i -F0 was used for quantitative analysis. To evaluate the specificity of the fluorescence method, K was selected. + ,Br - Mg 2+ Ca 2+ Na + Common ions, amino acids, and biomolecules such as alanine (Ala), phenylalanine (Phe), L-leucine (L-Leu), cysteine ​​(Cys), and glucose were used to investigate specificity. All interfering substances were at a concentration of 500 μg / mL (approximately 10 times that of captopril), and captopril was at a concentration of 250 µM. The experiment was repeated three times (n=3).

[0049] Colorimetric detection mode: ZrFe MOF (500 µg / mL, 10 µL) and 50 µL of captopril standard solutions of different concentrations (0.3, 1.5, 5, 10, 20, 30 µM), H2O2 (100 mM, 20 µL), and TMB (5 mM, 20 µL) were added to NaAc-HAc buffer (0.2 M, pH 3.5, 100 µL). After incubation at 37°C for 20 min in a shaker, the absorbance A at 652 nm was measured. i The absorption spectrum range is 550~750nm. The absorbance of the blank group is A0, and ΔA (ΔA=A) is calculated. i - A0) was used as the detection signal in the captopril colorimetric method for quantitative analysis. To evaluate the specificity of the colorimetric method, K was selected. + ,Br - Mg 2+ Ca 2+ Na + Common ions, amino acids, and biomolecules such as alanine (Ala), phenylalanine (Phe), L-leucine (L-Leu), and glucose (GLucose) were used to investigate specificity. All interfering substances were concentrated at 25 μg / mL (approximately 10 times that of captopril), and captopril was at a concentration of 10 µM. The experiment was repeated three times (n=3).

[0050] Linearity and specificity of fluorescence detection modes

[0051] Under optimal experimental conditions, the linearity and specificity of ZrFe MOF for the fluorescence pattern detection of captopril were investigated. With increasing captopril concentration, the fluorescence intensity at 460 nm continuously increased, exhibiting a good linear relationship in the range of 1.95–250 µM (F-F0 = 2.756C-13.68, R0). 2 =0.9913), and the detection limit was estimated to be 0.33 µM according to the 3σ / K rule. To evaluate the practicality of the constructed ZrFe MOF fluorescence sensing system, its selectivity, anti-interference ability, and long-term stability for captopril detection were systematically investigated. First, the high selectivity of this method was verified. It was effective in detecting various potentially coexisting substances (including common metal ions Na⁺, K⁺, and Ca²⁺). 2 ⁺、Mg 2In the presence of ZrFe MOF (containing ⁺, Br⁻ anion, amino acids alanine, phenylalanine, leucine, cysteine, and glucose), the fluorescence intensity of the system significantly increased only upon the addition of the target compound captopril, while signal changes caused by other substances were negligible. This indicates that ZrFe MOF has a specific response to captopril. Secondly, its anti-interference ability in complex matrices was further evaluated. Even when the aforementioned interfering substances were added to the detection system along with captopril, its fluorescence intensity showed no significant difference compared to the control group containing only captopril (p < 0.001). This confirms that the sensing method has good anti-interference performance in actual sample detection. Finally, the stability of the sensing material and method was investigated. Material stability is fundamental to sensing performance. After storing the ZrFe MOF dispersion at 4°C in the dark for 14 days, its intrinsic fluorescence intensity did not show significant attenuation, indicating that the material itself possesses excellent storage stability. Furthermore, the long-term reliability of the complete detection method was evaluated. When the same batch of material was stored for different periods to detect a fixed concentration of captopril (125µM), the fluorescence signal remained essentially unchanged. This demonstrates that the entire detection system exhibits stable performance and high reproducibility during long-term storage. In summary, the ZrFe MOF fluorescence sensing system for captopril detection not only possesses high selectivity and strong anti-interference capability but also exhibits excellent long-term stability. In conclusion, the superior performance of the ZrFe MOF fluorescence sensing system constructed in this embodiment in terms of selectivity, stability, and anti-interference indicates its strong potential for practical applications, laying a solid foundation for the development of reliable methods for on-site or point-of-care detection of captopril.

[0052] Example 5

[0053] stability

[0054] Furthermore, to evaluate the stability of the fluorescence sensor, this embodiment measures the signal change (F) generated by the fluorescence sensor recognizing captopril. i The fluorescence intensity of ZrFe MOF (500 µg / mL) was investigated over a 14-day period. The specific experimental steps were as follows: ZrFe MOF was stored at 4℃. At different days, 50 µL of captopril (125 µM, 150 µL) was added, and the mixture was incubated at 60 °C for 15 min. After cooling to room temperature and mixing, the fluorescence intensity at 460 nm was measured, with an excitation wavelength of 350 nm. A blank experimental group was set up; the fluorescence intensity at 460 nm of the blank sample (using 150 µL of pure water instead of captopril) was defined as F0. i -F0 is used to examine the stability of the fluorescent probe signal. The above experiment should be repeated at least 3 times (n=3).

[0055] Furthermore, to evaluate the stability of the colorimetric sensor using this method, this embodiment investigates the effect of captopril on suppressing the colorimetric reaction of the TMB-H2O2 system, which leads to a change in the colorimetric signal ΔA.652 The changes were investigated over a 14-day period. The specific experimental procedures were as follows: ZrFe MOF (500 µg / mL) was stored at 4℃. At different times, 10 µL of the solution was taken and added sequentially to captopril (2 µM, 50 µL), NaAc-HAc buffer (0.2 M, pH 3.5, 100 µL), H2O2 (100 mM, 20 µL), and TMB (5 mM, 20 µL). After reacting at 37℃ for 20 min, the absorbance at 652 nm was measured. A blank experimental group was set up, using the UV channel signal response on the first day as a reference to investigate the relative stability of the colorimetric probe signal. The above experiment was repeated at least three times (n = 3).

[0056] Linearity and specificity of colorimetric detection modes

[0057] Under optimal colorimetric detection conditions, the linear response of the ZrFe MOF-based colorimetric sensing system to captopril detection was evaluated. As the captopril concentration gradually increased, the intensity of the characteristic absorption peak at 652 nm continuously decreased, indicating a concentration-dependent effect. The absorbance change (ΔA) showed a good linear relationship with captopril concentration in the range of 0.3–30 µM. The linear regression equation was ΔA = 0.00974 C + 0.02873, with a correlation coefficient R0. 2 =0.9959. The limit of detection (LOD) calculated based on 3σ / k is 0.22 µM. These results indicate that the method has a wide linear range and a low LOD, making it suitable for the quantitative analysis of captopril. To investigate the specificity of the established colorimetric method for captopril detection, the effects of various potential coexisting substances (including inorganic ions, amino acids, and glucose) on the sensing system were evaluated. When the concentration of interfering substances was 10 times that of captopril, only captopril caused a significant change in the absorbance signal; even when interfering substances coexisted with captopril, the colorimetric response signal was not significantly affected. These results demonstrate that the colorimetric method has good selectivity and anti-interference ability for captopril detection. Furthermore, the storage stability of ZrFe MOF peroxidase (POD) activity was further investigated. After 14 days of storage at room temperature in the dark, the POD-like activity of ZrFe MOF remained essentially unchanged; when used for captopril detection, the resulting colorimetric signal showed no significant fluctuations over 14 days. These results demonstrate that the colorimetric method based on ZrFeMOF POD-like activity exhibits good stability over 14 days and can achieve accurate detection of captopril in complex samples.

[0058] Example 6

[0059] Detection of captopril in human serum

[0060] Fluorescence detection mode: Human serum was diluted 50-fold with ultrapure water, and 50 µL of captopril at different concentrations (12.5, 30, 50, 150, 200, 225 µM) was added. Then, ZrFe MOF (500 µg / mL, 150 µL) was added, and the mixture was incubated at 60 °C for 15 min. After incubation and cooling to room temperature, the fluorescence intensity at 460 nm was measured, with an excitation wavelength of 350 nm. The above experiment was repeated at least 3 times.

[0061] Colorimetric detection mode: Human serum was diluted 50-fold with ultrapure water, and 50 µL of captopril at different concentrations (0.6, 1, 5, 7.5, 10, 20, 30 µM), ZrFe MOF (500 µg / mL, 10 µL), 100 µL NaAc-HAc buffer (0.2 M, pH 3.5), 20 µL H2O2 (100 mM), and 20 µL TMB (5 mM) were added. The mixture was incubated in a shaker at 37 °C for 20 min, and the absorbance at 652 nm was measured. The above experiment was repeated at least 3 times.

[0062] Detection of captopril in human serum

[0063] The established fluorescence-colorimetric dual-mode method was used to detect captopril levels in human serum. In fluorescence mode, the fluorescence signal increased in a concentration-dependent manner with increasing captopril concentration, exhibiting a good linear relationship within the concentration range of 12.5–225 µmol / L. The regression equation was Y = 9.2037C + 788.4171, and the correlation coefficient R0 was [value missing]. 2 =0.9944. The limit of detection (LOD) was calculated to be 3.7 µM according to the 3σ / K rule. To evaluate the precision and accuracy of this method, quality control samples with three concentrations (high (100 µM), medium (25 µM), and low (15 µM)) were analyzed. The results are shown in Table 2. The recoveries of captopril were 99.6%–108.6%, and the relative standard deviations (RSDs) were 2.8%–10.1%. In colorimetric mode, the absorbance change of the system (ΔA) was... 652 Similarly, the concentration of captopril increased with increasing concentration, showing a good linear relationship in the range of 0.625–30 µmol / L, with the regression equation being Y = 0.01049C + 0.16682, R0. 2 =0.9981. Method validation was performed using quality control samples of the same concentration, and the results showed a recovery rate of 97.6%–105.3% and an RSD of 2.0%–9.2%. These results indicate that the established fluorescence-colorimetric dual-mode method has advantages such as speed, accuracy, and high specificity, and is suitable for high-throughput analysis of captopril in biological samples.

[0064] Table 2. Accuracy and precision of fluorescence-colorimetric detection of captopril in human serum (n = 6)

[0065]

[0066] Example 7

[0067] Detection of captopril in health supplements

[0068] Fluorescence pattern determination: Captopril at different concentrations was added using the standard addition method to achieve final concentrations of 12.5, 30, 50, 150, 200, and 225 µM in the detection system. The mixture was ultrasonically mixed, and then ZrFe MOF (500 µg / mL, 150 µL) was added. The mixture was incubated at 60 °C for 15 min, cooled to room temperature, and the fluorescence intensity at 460 nm was measured. The excitation wavelength was 350 nm. The above experiment was repeated at least three times.

[0069] Colorimetric assay: Captopril at different concentrations was added using the standard addition method to achieve final concentrations of 0.3125, 0.625, 1.25, 5, 7.5, 10, 20, and 30 µM in the detection system. The mixture was ultrasonically mixed, and then ZrFe MOF (500 µg / mL, 10 µL), 100 µL NaAc-HAc buffer (0.2 M, pH 3.5), 20 µL H₂O₂ (100 mM), and 20 µL TMB (5 mM) were added sequentially. The mixture was incubated at 37°C for 20 min in a shaker, and the absorbance at 652 nm was measured. This experiment was repeated at least three times.

[0070] Detection of captopril in health supplements

[0071] To verify the accuracy and reliability of the established fluorescence-colorimetric dual-mode sensing strategy in actual sample detection, three commercially available functional capsules for assisting in lowering blood pressure (Apocynum venetum capsules, Eucommia ulmoides capsules, and Shuxin capsules) were selected as the actual sample matrix, and spiked recovery experiments were conducted. In fluorescence detection mode, the system showed good concentration response to captopril added to all three capsule samples. The fluorescence intensity gradually increased with increasing captopril concentration, exhibiting good linearity within the concentration range of 12.5–225 µmol / L. To further evaluate the precision and accuracy of this method, captopril standards at low (15 µmol / L), medium (25 µmol / L), and high (100 µmol / L) concentrations were added to the three capsule samples. The results are shown in Table 3. The recoveries in fluorescence mode ranged from 86.1% to 113.5%, with relative standard deviations (RSDs) of 1.9% to 16.0%. Similarly, in colorimetric detection mode, the absorbance change (ΔA) of the system gradually increases with increasing captopril concentration, exhibiting a good linear relationship within the concentration range of 0.3–30 µmol / L. Spiking recovery experiments at the same concentration level showed recoveries of 90.2%–111.6% and RSDs of 2.1%–8.9% in colorimetric mode. These results demonstrate that the constructed fluorescence-colorimetric dual-mode sensing strategy possesses good accuracy and precision in the detection of captopril in functional health products, and has the potential for practical application.

[0072] Table 3. Accuracy and precision of fluorescence-colorimetric method for detecting captopril in health supplement capsules (n=3)

[0073]

[0074] Example 8

[0075] Captopril Visual Detection

[0076] Based on the significant solution color changes of the ZrFeMOF colorimetric system, this study further explores its integration with smartphones, aiming to develop a portable, visual sensor suitable for rapid on-site detection of captopril. ] Experimental setup such as Figure 3 As shown: A 96-well plate containing the reaction solution was placed over a white LED light source, and color images of each well were captured using a smartphone. As the captopril concentration increased from 0 to 250 µM, the color of the reaction solution gradually changed from blue to light blue. The RGB values ​​of the images were extracted using ImageJ software, and the (R+G) / B value was used as the quantitative detection signal. The results showed that within the concentration range of 0.83–20 µM, the (R+G) / B value exhibited a good linear relationship with the captopril concentration, with a linear regression equation of Y = 0.01786X - 0.00161 and a correlation coefficient R0. 2=0.9961, and the limit of detection (LOD) is 0.27µM. This method exhibits a wide linear range and high sensitivity. This smartphone integration strategy provides a feasible solution for rapid on-site detection of captopril, demonstrating promising application prospects in the field of point-of-care testing.

[0077] like Figure 4 As shown, this embodiment successfully constructed a fluorescence / colorimetric dual-mode sensing platform based on ZrFe MOF for rapid visual detection of captopril. A ZrFe MOF exhibiting both peroxidase-like activity and blue fluorescence was synthesized via a one-step hydrothermal method. The sensing mechanism utilizes the dual effects of captopril: the thiol group inhibits the MOF-catalyzed TMB-H2O2 colorimetric reaction, effectively "turning off" the colorimetric signal; the carboxyl group undergoes amidation with the amino groups on the MOF surface, restricting intramolecular rotation of the ligand and enhancing the fluorescence signal, thus "turning on" the fluorescence. This dual-response mechanism effectively avoids signal crosstalk. Under optimized conditions, the fluorescence mode exhibits good linearity in the range of 1.95–250 µM (R0). 2 =0.9913), detection limit 0.33µM; the colorimetric mode exhibits good linearity in the range of 0.3–30µM (R² = 0.9913). 2 =0.9959), with a detection limit of 0.22µM. Combined with smartphone RGB conversion, visual detection is achieved in the range of 0.83–20µM, with a detection limit of 0.27µM. This method exhibits excellent selectivity for captopril, with no significant response to common interfering substances. Good recovery rates were achieved in serum and health supplement spike recovery experiments, validating its applicability to complex matrices. In summary, the dual-mode platform constructed in this invention combines the high sensitivity of fluorescence methods with the visualization advantages of colorimetry, improves reliability through dual-signal self-calibration, and, combined with smartphone analysis, provides a simple and economical technical solution for rapid on-site screening of illegally added captopril in functional foods, possessing practical application value in grassroots supervision and home self-testing.

Claims

1. A fluorescence / colorimetric dual-mode sensor for detecting captopril, characterized in that, It includes ZrFe MOF, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine; the ZrFe MOF is formed from zirconium metal ions, iron metal ions, and the organic ligand diaminoterephthalic acid.

2. The fluorescence / colorimetric dual-mode sensor for detecting captopril according to claim 1, characterized in that, The ZrFe MOF has an octahedral structure.

3. A method for fabricating the fluorescence / colorimetric dual-mode sensor according to claim 1 or 2, characterized in that, The ZrFe MOF is prepared by dissolving zirconium metal salt, iron metal salt, diaminoterephthalic acid and acetic acid in an organic solvent, carrying out a hydrothermal reaction, and collecting the precipitate, which is ZrFe MOF.

4. The method for fabricating a fluorescence / colorimetric dual-mode sensor according to claim 3, characterized in that, The organic solvent is N,N-dimethylformamide.

5. The method for fabricating a fluorescence / colorimetric dual-mode sensor according to claim 3, characterized in that, The precipitate was washed with N,N-dimethylformamide and ethanol, and then dried to obtain ZrFe MOF.

6. The application of the fluorescence / colorimetric dual-mode sensor as described in claim 1 or 2 in captopril detection.

7. A captopril detection method using the fluorescence / colorimetric dual-mode sensor according to claim 1 or 2, characterized in that, Includes the following steps: (1) Take captopril solutions of varying concentrations and mix them with ZrFe MOF respectively. After incubation, the test solution is obtained and its fluorescence intensity is measured and recorded as F. i Simultaneously, water was used as a blank sample and mixed with ZrFe MOF. After incubation, a blank sample test solution was obtained, and its fluorescence intensity was measured and recorded as F0. A graph was constructed with captopril concentration as the x-axis and fluorescence intensity F0 as the y-axis. i -F0 is the fluorescence standard curve on the ordinate; (2) Captopril solutions of varying concentrations were mixed with ZrFeMOF, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine, respectively. After incubation, the test solution was obtained, and its ultraviolet absorbance was measured and recorded as A. i Simultaneously, water was used as a blank sample and mixed with ZrFe MOF, hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine. After incubation, a blank sample test solution was obtained, and its UV absorbance was measured and denoted as A0. A graph was constructed with captopril concentration as the x-axis and UV absorbance A0 as the y-axis. i - A0 is the colorimetric standard curve on the ordinate; (3) Mix the sample to be tested with ZrFe MOF, incubate the reaction to obtain the test solution, measure its fluorescence intensity, subtract F0 from the measured fluorescence intensity, and substitute it into the fluorescence standard curve constructed in step (1) to calculate the concentration of captopril in the sample to be tested. (4) Mix the sample to be tested with ZrFe MOF, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine, incubate the reaction to obtain the test solution, measure its ultraviolet absorbance, subtract A0 from the measured ultraviolet absorbance, and substitute it into the colorimetric standard curve constructed in step (2) to calculate the concentration of captopril in the sample to be tested.

8. The captopril detection method according to claim 7, characterized in that, In steps (1) and (3), the incubation time is 15-50 min and the incubation temperature is 25°C-70°C; in steps (2) and (4), the incubation time is 20-50 min and the incubation temperature is 25-45°C.

9. The captopril detection method according to claim 7, characterized in that, In steps (1) and (3), the fluorescence intensity is measured under the following conditions: the fluorescence intensity at 460 nm is measured, the excitation wavelength is 350 nm, and the fluorescence spectrum range is 400~550 nm.

10. The captopril detection method according to claim 7, characterized in that, In steps (2) and (4), the absorbance at 652 nm is measured, and the absorption spectrum range is 550~750 nm.

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