Method for detecting ATP (adenosine triphosphate) and hydrolysate thereof

Abstract

The invention discloses a method for detecting ATP (adenosine triphosphate) and hydrolysate thereof, a novel fluorescent sensor array is constructed by the method, and the array is composed of three high-stability zirconium porphyrin luminescent metal organic frameworks (LMOFs), specifically including PCN-221, PCN-222 and PCN-224. The three LMOFs can interact with adenosine triphosphate (ATP) and hydrolysates thereof to different extents, and a Zr-O-P bond is formed to inhibit a charge transfer process from a porphyrin luminescent ligand to metal zirconium, so that the fluorescence of the LMOFs is remarkably enhanced. The fluorescence enhancement degrees of the LMOFs are different due to differences in the number and steric hindrance effects of ATP and different hydrolysates ADP, AMP, PPi and Pi phosphate groups, so that the five hydrolysates are accurately distinguished. Experimental data show that the LMOFs sensor array not only can distinguish and recognize ATP and the ATP hydrolysate in a complex biological environment, but also can monitor the ATP hydrolysis process in real time, and a promising method is provided for diagnosis of diseases related to the ATP hydrolysate and detection of the biological process.

Description

Technical Field

[0001] The present invention relates to a method for distinguishing and detecting ATP and its hydrolysis products based on a luminescent metal organic framework sensor array, and belongs to the technical field of fluorescence sensing. Background Art

[0002] Adenosine triphosphate (ATP) is the primary energy transfer molecule within cells. It is hydrolyzed enzymatically to produce adenosine diphosphate (ADP), adenosine monophosphate (AMP), pyrophosphate (PPi), and inorganic phosphate (Pi). It plays important physiological roles in cellular metabolism, energy transfer, and signal transduction. Changes in the concentration of ATP and its hydrolysis products are often closely associated with the development of various diseases, such as cardiovascular disease, neurodegenerative diseases, and metabolic disorders. Therefore, accurately distinguishing and detecting ATP and its hydrolysis products is crucial for understanding the mechanisms of energy metabolism in vivo and for disease diagnosis.

[0003] Fluorescence analysis has been widely used in biomolecule detection due to its high sensitivity, rapid response, and ease of operation. Luminescent metal-organic frameworks (LMOFs) are a class of materials with fluorescent properties. Their high specific surface area, adjustable structure, and stable optical properties make them an ideal platform for detecting biomolecules. For the detection of ATP and its hydrolysis products, LMOFs can achieve detection by specifically interacting with target molecules, causing changes in the fluorescence signal. However, most current fluorescence sensors follow a "key-lock" model, meaning that one LMOF can only detect one analyte. While these methods perform well in terms of sensitivity and selectivity, the simultaneous presence of ADP, AMP, PPi, and Pi during the ATP hydrolysis process in vivo poses significant limitations for single fluorescence sensors when detecting analytes with similar molecular structures, such as ATP and its hydrolysis products. This makes it difficult to achieve efficient and accurate differential detection of multiple targets in complex systems.

[0004] A fluorescence sensor array is a detection system that combines multiple luminescent materials to produce a "fingerprint response" to target molecules. Each luminescent material responds to multiple analytes. By collecting and statistically analyzing the cross-response signals of varying degrees, high-throughput identification of analytes with similar structures and properties is achieved. Currently, LMOF-based fluorescence sensor arrays have shown great potential in the identification of various substances, including proteins, antibiotics, metal ions, and pesticides. However, the differential identification and detection of ATP and its hydrolysis products has been rarely reported. Summary of the Invention

[0005] The present invention provides a method for detecting ATP and its hydrolysis products. This method first constructs a novel fluorescent sensor array composed of three highly stable zirconium porphyrin luminescent metal-organic frameworks (LMOFs): PCN-221, PCN-222, and PCN-224. This array is used to detect ATP and its hydrolysis products. Because ATP, ADP, AMP, PPi, and Pi have different numbers of phosphate groups, molecular sizes, and steric effects, they bind to these three LMOFs to varying degrees via Zr-OP bonds, generating distinguishable fluorescent signal responses. Statistical analysis and processing of these fluorescent signals enables efficient differentiation and detection of ATP and its hydrolysis products, effectively overcoming the limitations of single sensors.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A method for detecting ATP and its hydrolysis products comprises the following steps: (1) Synthesis of luminescent metal-organic framework materials PCN-221, PCN-222 and PCN-224; (2) Constructing a fluorescence sensor array: In a 96-well plate, the three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 dispersions described in step (1) were transferred to the 6×6 areas of the 96-well plate, and different concentrations of ATP, ADP, AMP, PPi, Pi and blank group solutions were added to the corresponding wells within the concentration range of 1 μM~1 mM. Six parallel experiments were performed, and the solution was diluted to 200 μL after incubation for 30 min. The fluorescence intensity of the solution in each well was measured with an enzyme-linked microplate reader to obtain the fluorescence sensor array detection data; the detection range and detection limit of the fluorescence sensor array were determined; (3) Data analysis: using fluorescence enhancement factor (FF 0 ) / F 0 Describe the fluorescence responses of PCN-221, PCN-222, and PCN-224 to different ATP hydrolysis products, where F and F 0 The fluorescence intensity of the material with or without ATP hydrolysis products was obtained by principal component analysis (PCA) and hierarchical cluster analysis (HCA). (FF 0 ) / F 0 The data were processed to verify the detection capability of the constructed fluorescence sensor array for ATP hydrolysis products; (4) Detection of ATP and its hydrolysis products in biological samples: Take the biological sample to be tested, and transfer the dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 to the 6×6 areas of the 96-well plate respectively. Add ATP, ADP, AMP, PPi, Pi and blank group solutions containing the biological sample to be tested to the corresponding wells, incubate for 30 min and then dilute to 200 μL. Use an enzyme-labeled instrument to measure the fluorescence intensity of the solution in each well to obtain the fluorescence sensor array detection data in the biological sample. Use principal component analysis (PCA) to analyze the obtained fluorescence intensity. (FF 0 ) / F 0 The data is processed to complete the detection of ATP and its hydrolysis products.

[0007] In the above step (1), the synthesis method of PCN-221 is as follows: 0.013 mmol TCPP and 0.043 mmol ZrCl4 are dissolved in 2 mL of DMF, and 350 μL of acetic acid is added. The mixture is transferred to a high-pressure reactor, reacted at 120 °C for 12 h, and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60 °C to obtain PCN-221.

[0008] In the above step (1), the synthesis method of PCN-222 is as follows: 0.013 mmol TCPP and 0.11 mmol ZrOCl2·8H2O are dissolved in 3.3 mL DMF, and then 0.15 mL trifluoroacetic acid is added and transferred to a high-pressure reactor. The reaction is carried out at 120°C for 24 h and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60°C to obtain PCN-222.

[0009] In the above step (1), the synthesis method of PCN-224 is as follows: 0.013 mmol TCPP, 0.13 mmol ZrCl4 and 3.3 mmol benzoic acid are mixed in 4 mL of DMF, the mixture is transferred to a high-pressure reactor, reacted at 120 °C for 24 h, and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60 °C to obtain PCN-224.

[0010] In the above step (2), the concentration of the dispersion of the luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 is 40~60 μg∙mL -1 .

[0011] In the above step (2), the excitation wavelength of the luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 is λex=450 nm, and the emission wavelength is λem=654 nm.

[0012] In the above step (2), the concentrations of ATP, ADP, AMP, PPi, and Pi were 1 mM, 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, and 1 µM.

[0013] In the above step (2), the present invention also evaluates the anti-interference ability of the sensor array, specifically by: in a 96-well plate, a concentration of 40-60 μg∙mL -1 The dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 were transferred to 13×6 areas of 96-well plate, and 8 interfering anions CO3 2- 、HCO3 - 、SO4 2- 、SO3 2- 、NO3 - 、NO2 - 、Cl - 、Ac - The solutions of ATP, ADP, AMP, PPi, and Pi, the five ATP hydrolysis products, were added to the corresponding wells and incubated for 30 min before being diluted to 200 µL. The fluorescence intensity of the solution in each well was measured using a microplate reader. The data were processed using the method in step (3). The concentrations of the eight interfering anions were all 100 µM, and the concentrations of the five ATP hydrolysis products were all 50 µM.

[0014] A method for detecting ATP and its hydrolysis products, characterized by comprising the following steps: (1) Synthesis of luminescent metal-organic framework materials PCN-221, PCN-222 and PCN-224; (2) Constructing a fluorescence sensor array: In a 96-well plate, the three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 dispersions described in step (1) were transferred to the 6×6 areas of the 96-well plate, and different concentrations of ATP, ADP, AMP, PPi, Pi and blank group solutions were added to the corresponding wells within the concentration range of 1 μM~1 mM. Six parallel experiments were performed, and the solution was diluted to 200 μL after incubation for 30 min. The fluorescence intensity of the solution in each well was measured with an enzyme-linked microplate reader to obtain the fluorescence sensor array detection data; the detection range and detection limit of the fluorescence sensor array were determined; (3) Data analysis: using fluorescence enhancement factor (FF 0 ) / F0 Describe the fluorescence responses of PCN-221, PCN-222, and PCN-224 to different ATP hydrolysis products, where F and F 0 The fluorescence intensity of the material with or without ATP hydrolysis products was obtained by principal component analysis (PCA) and hierarchical cluster analysis (HCA). (FF 0 ) / F 0 The data were processed to verify the detection capability of the constructed fluorescence sensor array for ATP hydrolysis products; (4) Detection of ATP hydrolysis products in biological samples: Take 200 μL of 1 mM ATP solution, add 1560 μL of Tris-HCl buffer, 240 μL of 1.08 U·mL -1 The adenosine triphosphate diphosphatase (ATPase: ADPase) was added, with a ratio of 1:1. The reaction solution was thoroughly mixed and reacted at 37°C. Four identical reactant solutions were prepared according to the above method. The enzymes were removed by centrifugation at 0 min, 30 min, 60 min, and 120 min to stop the reaction. The reactant solutions at each time point were obtained. The reaction solutions at each time point were tested using a fluorescence sensor array. The obtained solutions were then analyzed using principal component analysis (PCA). (FF 0 ) / F 0 The data is processed to complete the detection of ATP and its hydrolysis products.

[0015] In the above step (2), the excitation wavelength of PCN-221, PCN-222 and PCN-224 is λex = 450 nm, the emission wavelength is λem = 654 nm, and the room temperature is 23 ± 5 °C.

[0016] Compared with the prior art, the advantages of the present invention are: the present invention provides a method for preparing a fluorescence sensor array composed of Zr-based porphyrin LMOFs, namely PCN-221, PCN-222 and PCN-224, and is used for high-throughput differential detection of ATP and its hydrolysis products. Due to the different degrees of binding between the Zr nodes in the three LMOFs and ATP, ADP, AMP, PPi and Pi, distinguishable fluorescence signal responses and unique fingerprints can be generated, achieving efficient differentiation and detection of ATP and its hydrolysis products, thereby effectively overcoming the limitations of a single detection method. Experiments have shown that the LMOFs sensor array of the present invention can not only efficiently and accurately detect ATP and its hydrolysis products in complex biological environments, such as fetal bovine serum FBS, but also effectively reflect the ATP hydrolysis process, and clearly display the conversion of ATP to ADP and AMP through changes in fluorescence signals. Therefore, it has great potential in the diagnosis of diseases related to ATP hydrolysis products and monitoring of biological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flow chart of the LMOFs fluorescence sensor array of the present invention; Figure 2 SEM and PXRD images of LMOFs. Figure 2 ac are SEM images of PCN-221, PCN-222 and PCN-224, respectively. Figure 2 df are the PXRD patterns of PCN-221, PCN-222, and PCN-224, respectively; Figure 3 FT-IR and fluorescence emission spectra of LMOFs. Figure 3 a is the FT-IR spectrum of PCN-221, PCN-222 and PCN-224, Figure 3 b is the fluorescence emission spectra of PCN-221, PCN-222 and PCN-224, and the inset is the fluorescence photos of PCN-221, PCN-222 and PCN-224 under UV light; Figure 4 (a, d) Fluorescence enhancement factors after adding different ATP hydrolysis products to the sensor array (FF 0 ) / F 0 Response plot, (b, e) PCA plot, (c, f) HCA plot; Figure 5 (a) Fluorescence enhancement factor when interfering anions are added to the sensor array (FF 0 ) / F 0 response plot, (b) PCA plot; Figure 6 Analytical performance diagram of the sensor array. Figure 6 ab are the sensor arrays after different ATP hydrolysis products containing FBS (a) (FF 0 ) / F 0 response plot, (b) PCA plot; Figure 6 cd are sensor arrays monitoring the ATP hydrolysis process: (c) (F- F 0 ) / F 0 Response plot, (d) PCA plot. Specific implementation methods

[0018] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: Preparation of Zr-based porphyrin LMOFs (1) PCN-221: 0.013 mmol TCPP and 0.043 mmol ZrCl4 were dissolved in 2 mL of DMF. 350 µL of acetic acid was added and the mixture was transferred to a high-pressure reactor. The mixture was reacted at 120 °C for 12 h and then cooled to room temperature. The purple product was separated by centrifugation and washed with DMF and ethanol. The product was dried in vacuo at 60 °C to obtain PCN-221.

[0020] (2) PCN-222: Dissolve 0.013 mmol TCPP and 0.11 mmol ZrOCl2·8H2O in 3.3 mL DMF, add 0.15 mL trifluoroacetic acid, and transfer to an autoclave for reaction at 120°C for 24 h. After the reaction, cool to room temperature, separate the purple product by centrifugation, wash with DMF and ethanol, and dry in vacuo at 60°C to obtain PCN-222.

[0021] (3) PCN-224: 0.013 mmol TCPP, 0.13 mmol ZrCl4, and 3.3 mmol benzoic acid were mixed in 4 mL of DMF. The mixture was transferred to an autoclave and reacted at 120 °C for 24 h. After the reaction, the mixture was cooled to room temperature, and the purple product was separated by centrifugation and washed with DMF and ethanol. It was then dried in vacuo at 60 °C to obtain PCN-224.

[0022] The three synthesized PCNs were characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR) and fluorescence intensity analysis using a microplate reader. The results are as follows: like Figure 2ac are SEM images of PCN-221, PCN-222, and PCN-224. PCN-221 is a cubic structure of uniform size, about 100-400 nm in size, PCN-222 is a spindle-shaped structure of about 320 × 160 nm in size, and PCN-224 is an ellipsoid with wrinkles on the surface, about 1 μm in size. Figure 2 df are the PXRD patterns of PCN-221, PCN-222 and PCN-224, respectively. The sharp diffraction peaks of the three are highly consistent with the standard simulation patterns, confirming the successful synthesis of the three PCNs.

[0023] like Figure 3 a is the FT-IR spectrum of TCPP, PCN-221, PCN-222 and PCN-224. The results show that the 3440 cm -1 is the OH vibration of TCPP, 1660 cm -1 C=O vibration of TCPP, 1598 cm -1 C=C vibration of TCPP, 1415 cm -1 C–N vibration of TCPP, 1269 cm -1 is the C–OH asymmetric stretching vibration of TCPP; in PCNs, 1269 cm -1 The peak at 650 cm is weakened or even disappears, and a new peak at 650 cm is added. -1 The peak is attributed to the Zr-O-Zr rocking vibration, which indicates that Zr is successfully coordinated with the -COOH in TCPP.

[0024] like Figure 3 b is the fluorescence emission spectra of TCPP, PCN-221, PCN-222 and PCN-224. Under an excitation wavelength of 450 nm, TCPP, PCN-221, PCN-222 and PCN-224 show characteristic emission peaks at 654 nm. The emission peak position of PCNs remains unchanged compared with TCPP, indicating that the binding of Zr to TCPP does not change its fluorescence properties. However, the fluorescence intensity of PCNs is lower than that of TCPP, which is attributed to the formation of coordination bonds between the lone pair electrons of the TCPP carboxyl group and Zr (IV), resulting in ligand-to-metal charge transfer (LMCT). The inset shows that all three PCNs have obvious fluorescence under ultraviolet light.

[0025] Example 2: Construction of Zr-based porphyrin LMOFs fluorescence sensor array In a 96-well plate, dispersions of three luminescent metal-organic framework materials, PCN-221, PCN-222, and PCN-224, were transferred to 6×6 areas of the 96-well plate. Different concentrations of ATP, ADP, AMP, PPi, Pi, and a blank solution were added to the corresponding wells within the concentration range of 1 µM to 1 mM. Six parallel experiments were performed. After incubation for 30 minutes, the solution was diluted to 200 µL. The fluorescence intensity of the solution in each well was measured using a microplate reader to obtain fluorescence sensor array detection data. The concentrations of the three PCNs were all 40-60 µg·mL -1 The concentrations of ATP, ADP, AMP, PPi, and Pi were 1 mM, 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, and 1 µM.

[0026] Fluorescence enhancement factor (FF 0 ) / F 0 Describe the different fluorescence responses of PCNs to ATP hydrolysis products, among which F and F 0 The fluorescence intensity of the material with and without ATP hydrolysis products is obtained. (FF 0 ) / F 0 The data were processed using principal component analysis (PCA) and hierarchical cluster analysis (HCA) to detect ATP and its hydrolysis products. The results are as follows: To evaluate the detection range and detection limit of the sensor array, we tested ATP hydrolysis products at concentrations of 1 mM, 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, and 1 µM. The results showed that the array exhibited excellent discrimination between ATP, ADP, AMP, PPi, and Pi over a wide concentration range of 5 µM to 1 mM. However, when the hydrolysis product concentration was reduced to 1 µM, the signals of ATP, ADP, and AMP began to overlap, indicating that the sensor array is no longer suitable for use below 1 µM hydrolysis product concentration.

[0027] Taking 10 µM concentration as an example, six replicates were performed on three PCNs for five ATP hydrolysis products, yielding a total of 90 data points ( Figure 4 a) Different hydrolysis products showed unique fingerprints on the PCNs array. PCA analysis ( Figure 4 b) The five hydrolysis products ATP, ADP, AMP, PPi, and Pi are tightly clustered and separated from each other without overlap. HCA results ( Figure 4 c) The discriminative ability of the array was further confirmed, and all products were accurately identified in six parallel experiments.

[0028] The 1 µM concentration is illustrated. Six replicates were performed on three PCNs for five ATP hydrolysis products, yielding a total of 90 data points ( Figure 4 d), it can be seen that the signals between ATP, ADP and AMP begin to overlap ( Figure 4 e, f).

[0029] Example 3: Anti-interference experiment of Zr-based porphyrin LMOFs fluorescence sensor array In a 96-well plate, the concentration of 40-60 µg∙mL -1 The dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 were transferred to 13×6 areas of 96-well plate, and 8 interfering anions CO3 2- 、HCO3 - 、SO4 2- 、SO3 2- 、NO3 - 、NO2 - 、Cl - 、Ac - The 5 ATP hydrolysis product solutions were added to the corresponding wells, incubated for 30 min, and then diluted to 200 μL. The fluorescence intensity of the solution in each well was measured by a microplate reader to obtain the data of the fluorescence sensor array in the presence of interfering substances. The principal component analysis (PCA) was used to analyze the obtained data. (FF 0 ) / F 0 The data was processed to verify the anti-interference ability of the constructed fluorescence sensor array. The results are as follows: When there are 8 interfering anions ( Figure 5 ), ATP, ADP, AMP, PPi, and Pi can still be separated from the other eight anions and are independent of each other, highlighting the high anti-interference ability of the sensor array.

[0030] Example 4: Application of Zr-based porphyrin LMOFs fluorescence sensor array in biological samples (1) Take the biological sample to be tested, and transfer the dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 to the 6×6 area of ​​the 96-well plate respectively. Add ATP, ADP, AMP, PPi, Pi and blank group solutions containing the biological sample to be tested to the corresponding wells, incubate for 30 min and then dilute to 200 μL. Use a microplate reader to measure the fluorescence intensity of the solution in each well to obtain the detection data of the fluorescence sensor array in the biological sample. Use principal component analysis (PCA) to analyze the obtained fluorescence intensity. (F- F 0) / F 0 The data was processed to verify the ability of the constructed fluorescence sensor array to detect ATP and its hydrolysis products in biological samples. The results are as follows: like Figure 6 As shown in a and b, in the presence of fetal bovine serum (FBS), the sensor array still produces unique fluorescence responses to ATP, ADP, AMP, PPi, and Pi. Further PCA analysis not only clearly distinguishes the response patterns of FBS and the five ATP hydrolysis products, but also successfully separates the five hydrolysis products from each other.

[0031] (2) Take 200 μL of 1 mM ATP solution, add 1560 μL of Tris-HCl buffer solution, 240 μL of 1.08 U∙mL -1 The adenosine triphosphate diphosphatase (ATPase: ADPase) ratio was 1:1. The reaction solution was thoroughly mixed and reacted at 37°C. Four identical reaction solutions were prepared according to the above method. The enzymes were removed by centrifugation at 0 min, 30 min, 60 min, and 120 min to stop the reaction. The reaction solutions at each time point were obtained. The reaction solutions at each time point were tested using a fluorescence sensor array. The obtained solutions were then analyzed using principal component analysis (PCA). (FF 0 ) / F 0 The data was processed to complete the detection of ATP and its hydrolysis products. Furthermore, the standard samples ATP, ADP+Pi, and AMP+2Pi were tested using a fluorescence sensor array. The obtained data were compared with the reactant solutions at the above-mentioned time points to verify the ATP hydrolysis reaction process.

[0032] During ATP hydrolysis, apyrase possesses both ATPase and ADPase activities, and the ratio of their activities determines the type of hydrolysis product. For example, in a 1:1 ratio, ATP is first converted to ADP and Pi by ATPase, and then to AMP and 2Pi by ADPase. To simulate the hydrolysis process, hydrolysis samples were monitored at regular intervals. Signals were collected using an LMOF sensor array and plotted on a PCA scatter plot along with standard samples of ATP, ADP + Pi, and AMP + 2Pi. The results are as follows: like Figure 6As shown in c and d, the data points in the PCA scatter plots follow the ATP hydrolysis reaction from left to right. At 0 min, the data points closely overlap with those of the ATP standard sample. Data collected within 60 min primarily reflect the first stage of hydrolysis, which is dominated by ATPase. The second stage of hydrolysis, dominated by ADPase, begins after 60 min. After 120 min, hydrolysis is complete, and the PCA cluster shifts toward the AMP and 2 Pi regions of the standard sample, indicating that ATP is completely hydrolyzed by 120 min.

[0033] The above embodiments are merely illustrative of several possible implementations of the present invention, and their detailed description should not be construed as limiting the scope of protection of the present invention. It should be emphasized that those skilled in the art are fully capable of making various changes and improvements without departing from the core concept of the present invention, and such changes and improvements are all within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting ATP and its hydrolysis products, characterized in that The following steps are involved: (1) Synthesis of luminescent metal-organic framework materials PCN-221, PCN-222 and PCN-224; (2) Constructing a fluorescence sensor array: In a 96-well plate, the three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 dispersions described in step (1) were transferred to the 6×6 areas of the 96-well plate respectively. Within the concentration range of 1 μM~1 mM, different concentrations of ATP, ADP, AMP, PPi, Pi and blank group solutions were added to the corresponding wells. Six parallel experiments were performed. After incubation for 30 minutes, the solution was diluted to 200 μL. The fluorescence intensity of the solution in each well was measured with an enzyme-linked microplate reader to obtain the fluorescence sensor array detection data; the detection range and detection limit of the fluorescence sensor array were determined; (3) Data analysis: using fluorescence enhancement factor (FF 0 ) / F 0 Describe the fluorescence responses of PCN-221, PCN-222, and PCN-224 to different ATP hydrolysis products, where F and F 0 The fluorescence intensity of the material with or without ATP hydrolysis products was obtained by principal component analysis (PCA) and hierarchical cluster analysis (HCA). (FF 0 ) / F 0 The data were processed to verify the detection capability of the constructed fluorescence sensor array for ATP hydrolysis products; (4) Detection of ATP and its hydrolysis products in biological samples: Take the biological sample to be tested, and transfer the dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 to the 6×6 areas of the 96-well plate respectively. Add ATP, ADP, AMP, PPi, Pi and blank group solutions containing the biological sample to be tested to the corresponding wells, incubate for 30 min and then dilute to 200 μL. Use an enzyme marker to measure the fluorescence intensity of the solution in each well to obtain the fluorescence sensor array detection data in the biological sample. Use principal component analysis (PCA) to analyze the obtained fluorescence intensity. (FF 0 ) / F 0 The data is processed to complete the detection of ATP and its hydrolysis products.

2. The method for detecting ATP and its hydrolyzate according to claim 1, wherein: In step (1), the synthesis method of PCN-221 is as follows: 0.013 mmol TCPP and 0.043 mmol ZrCl4 are dissolved in 2 mL of DMF, and 350 μL of acetic acid is added. The mixture is transferred to a high-pressure reactor, reacted at 120 °C for 12 h, and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60 °C to obtain PCN-221.

3. The method for detecting ATP and its hydrolyzate according to claim 1, wherein: In step (1), the synthesis method of PCN-222 is as follows: 0.013 mmol TCPP and 0.11 mmol ZrOCl2·8H2O are dissolved in 3.3 mL of DMF, and then 0.15 mL of trifluoroacetic acid is added and transferred to a high-pressure reactor. The reaction is carried out at 120 °C for 24 h and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60 °C to obtain PCN-222.

4. The method for detecting ATP and its hydrolyzate according to claim 1, wherein: In step (1), the synthesis method of PCN-224 is as follows: 0.013 mmol TCPP, 0.13 mmol ZrCl4 and 3.3 mmol benzoic acid are mixed in 4 mL of DMF, the mixture is transferred to a high-pressure reactor, reacted at 120 °C for 24 h, and then cooled to room temperature. The purple product is separated by centrifugation and washed with DMF and ethanol, and then dried in vacuo at 60 °C to obtain PCN-224.

5. The method for detecting ATP and its hydrolyzate according to claim 1, wherein: In step (2), the concentration of the dispersion of luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 is 40~60 μg∙mL -1 .

6. The method for detecting ATP and its hydrolyzate according to claim 1, wherein: In step (2), the excitation wavelength of the luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 is λex=450 nm, and the emission wavelength is λem=654 nm.

7. The method for detecting ATP and its hydrolysis products according to claim 1, wherein: In step (2), the concentrations of ATP, ADP, AMP, PPi, and Pi were 1 mM, 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, and 1 µM.

8. The method for detecting ATP and its hydrolysis products according to claim 1, wherein: In step (2), the anti-interference ability of the sensor array was also evaluated by: in a 96-well plate, a concentration of 40~60 μg∙mL -1 The dispersions of three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 were transferred to 13×6 areas of 96-well plate, and 8 interfering anions CO3 2- , HCO 3- 、SO4 2- 、SO3 2- 、NO 3- 、NO 2- 、Cl - 、Ac - The solutions of ATP, ADP, AMP, PPi, and Pi, the five ATP hydrolysis products, were added to the corresponding wells and incubated for 30 min before being diluted to 200 μL. The fluorescence intensity of the solution in each well was measured using a microplate reader. The data were processed using the method in step (3). The concentrations of the eight interfering anions were all 100 μM, and the concentrations of the five ATP hydrolysis products were all 50 μM.

9. A method for detecting ATP and its hydrolysis products, characterized in that The following steps are involved: (1) Synthesis of luminescent metal-organic framework materials PCN-221, PCN-222 and PCN-224; (2) Constructing a fluorescence sensor array: In a 96-well plate, the three luminescent metal organic framework materials PCN-221, PCN-222 and PCN-224 dispersions described in step (1) were transferred to the 6×6 areas of the 96-well plate respectively. Within the concentration range of 1 μM~1 mM, different concentrations of ATP, ADP, AMP, PPi, Pi and blank group solutions were added to the corresponding wells. Six parallel experiments were performed. After incubation for 30 minutes, the solution was diluted to 200 μL. The fluorescence intensity of the solution in each well was measured with an enzyme-linked microplate reader to obtain the fluorescence sensor array detection data; the detection range and detection limit of the fluorescence sensor array were determined; (3) Data analysis: using fluorescence enhancement factor (FF 0 ) / F 0 Describe the fluorescence responses of PCN-221, PCN-222, and PCN-224 to different ATP hydrolysis products, where F and F 0 The fluorescence intensity of the material with or without ATP hydrolysis products was obtained by principal component analysis (PCA) and hierarchical cluster analysis (HCA). (FF 0 ) / F 0 The data were processed to verify the detection capability of the constructed fluorescence sensor array for ATP hydrolysis products; (4) Detection of ATP hydrolysis products in biological samples: Take 200 μL of 1 mM ATP solution, add 1560 μL of Tris-HCl buffer, 240 μL of 1.08 U·mL -1 The adenosine triphosphate diphosphatase (ATPase: ADPase) was added, with a ratio of 1:

1. The reaction solution was thoroughly mixed and reacted at 37°C. Four identical reactant solutions were prepared according to the above method. The enzymes were removed by centrifugation at 0 min, 30 min, 60 min, and 120 min to stop the reaction. The reactant solutions at each time point were obtained. The reaction solutions at each time point were tested using a fluorescence sensor array. The obtained solutions were then analyzed using principal component analysis (PCA). (FF 0 ) / F 0 The data is processed to complete the detection of ATP and its hydrolysis products.

10. The method for detecting ATP and its hydrolysis products according to claim 9, characterized in that: In step (2), the excitation wavelength of PCN-221, PCN-222 and PCN-224 is λex = 450 nm, the emission wavelength is λem = 654 nm, and the room temperature is 23 ± 5 °C.

Images