Double-emission-ratio fluorescence sensor as well as preparation method and application thereof

Abstract

The invention discloses a dual-emission-ratio fluorescence sensor and a preparation method and application thereof, and relates to the technical field of metal organic framework fluorescence sensing, the dual-emission-ratio fluorescence sensor comprises a europium metal organic framework material: EuUPMA, the EuUPMA is formed by coordination self-assembly of Eu < 3 + >, 2-hydroxyterephthalic acid (H2BDC-OH) and 1, 2, 4, 5-benzenetetracarboxylic acid (PMA), and the EuUPMA is prepared from Eu < 3 + >, 2-hydroxyterephthalic acid (H2BDC-OH) and 1, 2, 4, 5-benzenetetracarboxylic acid (PMA). EuUPMA respectively generates characteristic fluorescence emission of H2BDC-OH ligand and Eu < 3 + > at 428nm and 614nm, phosphate radical (Pi) generated by pyrophosphate (PPi) hydrolysis catalyzed by ALP is combined with Eu < 3 + >, so that EuUPMA generates ratio fluorescence response, and the fluorescence intensity ratio (I428 / I614) and the concentration of ALP are dynamically changed for realizing ALP quantitative detection; the EuUPMA with the dual-emission characteristic is synthesized, the method is easy to operate, high in sensitivity and good in stability, and the EuUPMA can be used for high-sensitivity accurate detection of ALP in human serum so as to solve the problems existing in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of metal-organic framework fluorescence sensing, and in particular to a highly efficient detection of alkaline phosphatase (ALP) activity using a europium-doped UiO-66 type dual-emission lanthanide metal-organic framework (EuUPMA) ratio fluorescence sensor. Background Art

[0002] Alkaline phosphatase (ALP) is an important dephosphorylating hydrolase that is widely distributed in tissues such as the liver, bone, and intestine and participates in physiological processes such as bone mineralization, cell differentiation, and signal transduction by catalyzing the hydrolysis of phosphate esters. Abnormal expression of ALP is closely related to various diseases such as osteomalacia, diabetes, liver dysfunction, Wilson's disease, and cancer. For example, an increase in serum ALP activity is an important indicator for diagnosing primary bone cancer and liver metastases, and salivary ALP levels can non-invasively evaluate the progression of periodontitis. Therefore, the development of highly sensitive, highly accurate, and anti-interference ALP detection methods is of great significance for early disease diagnosis and treatment monitoring. Currently, ALP detection methods mainly include high-performance liquid chromatography, surface-enhanced Raman spectroscopy, colorimetry, electrochemistry, and fluorescence methods. Among them, the fluorescence method has attracted much attention due to its advantages such as simple operation, rapid response, and high sensitivity. Nevertheless, most fluorescence sensing methods mainly rely on the change of a single fluorescence signal as the detection method for target molecules, which is often easily affected by environmental interference (such as light source fluctuations and probe concentration differences), resulting in insufficient detection stability and possible false positive signals, thus affecting the accuracy of target detection and limiting its application in complex actual samples. In contrast, ratio-type fluorescence sensors can perform self-calibration by setting an internal reference and use the ratio of the intensities of different fluorescence emission peaks as the signal output unit, which can reduce the influence of external factors on target detection and improve detection accuracy, becoming a research hotspot in recent years.

[0003] Metal-organic frameworks (MOFs) have been widely used in the field of fluorescence sensing due to their large specific surface area, functional group diversity, and highly adjustable structure. Among them, lanthanide metal-organic frameworks (LnMOFs) have attracted much attention due to their large Stokes shift, long luminescence lifetime, and narrow emission bandwidth, especially dual-emission fluorescence LnMOFs. Although there have been reports on the use of related LnMOFs for ALP detection, the detection mechanism based on the pore size of LnMOFs or the interaction between ALP and organic ligand functional groups may have problems of insufficient selectivity, which may reduce the accuracy and reliability of ALP detection. Utilizing the dephosphorylation reaction of ALP specifically catalyzing the hydrolysis of pyrophosphate (PPi) to generate phosphate (Pi) can significantly improve the selectivity of the ALP sensor and has more advantages for the detection application of ALP in complex sample systems.

[0004] To solve the above problems, we provide a dual-emission ratiometric fluorescence sensor to address the issues mentioned above. Summary of the Invention

[0005] The object of the present invention is to provide a dual-emission ratiometric fluorescence sensor and its preparation method and application. Specifically, an ALP ratiometric fluorescence sensor EuUPMA based on dual-emission lanthanide metal-organic framework materials is provided. Through ligand regulation and lanthanide ion post-modification strategies, EuUPMA with dual-emission characteristics is designed and synthesized. A detection mechanism based on Pi-induced proportional fluorescence response is established to achieve efficient analysis of ALP activity. This method is simple to operate, highly sensitive, and has good stability, and can be used for highly sensitive and accurate detection of ALP in human serum to solve the problems existing in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A dual-emission ratiometric fluorescence sensor, comprising europium metal-organic framework material: EuUPMA, wherein the EuUPMA is formed by the coordination self-assembly of Eu 3+ , 2-hydroxyterephthalic acid (H 2 BDC-OH) and 1,2,4,5-benzenetetracarboxylic acid (PMA). The EuUPMA produces characteristic fluorescence emissions of H 2 BDC-OH ligand and Eu 3+ at 428 nm and 614 nm respectively. The phosphate (Pi) generated by the hydrolysis of pyrophosphate (PPi) catalyzed by ALP combines with Eu 3+ to cause a ratiometric fluorescence response of EuUPMA, and the fluorescence intensity ratio (I 428 / I 614 ) changes dynamically with the ALP concentration for realizing the quantitative detection of ALP.

[0008] Furthermore, a preparation method of a dual-emission ratiometric fluorescence sensor specifically includes the following steps:

[0009] Step 1: Disperse zirconium chloride (ZrCl 4 ), 1,2,4,5-benzenetetracarboxylic acid (PMA) and 2-hydroxyterephthalic acid (H 2 BDC-OH) in DMF, stir until completely dissolved, and then add concentrated hydrochloric acid and stir.

[0010] Step 2: Transfer the mixed solution of Step 1 to a reaction kettle for solvothermal reaction. After the reaction is completed, centrifuge to collect the solid product, and wash and dry to obtain UiO-66-PMA material.

[0011] Step 3: Disperse the UiO-66-PMA obtained in Step 2 in Eu(NO3 ) 3 6H 2 O aqueous solution, and then stirred for reaction, centrifuged for washing and dried to obtain the EuUPMA fluorescent sensor.

[0012] Another object of the present invention is to provide: an application method of a dual emission ratio fluorescence sensor in ALP detection, specifically comprising the following method: 50 μL Tris-HCl buffer (500 mM, pH 7.0), 50 μL 100 mM MgCl 2 , 33 μL 2 mMPPi, 10 μL ALP with different enzyme activities and 807 μL ultrapure water were added to the centrifuge tube in sequence, mixed thoroughly and incubated at 37°C for 1 hour. Subsequently, 50 μL of the EuUPMA dispersion was added to the above solution, and the reaction was continued at 37°C for 1 hour. The fluorescence spectrum was measured and the ratio of the fluorescence intensity at 428 nm to 614 nm (I 428 / I 614 ) and the concentration of ALP, and the concentration of ALP in the test solution was calculated based on the standard curve.

[0013] Furthermore, the concentration of the EuUPMA dispersion is 1 mg / mL, the excitation wavelength of the fluorescence emission spectrum is 270 nm, the emission wavelength range is 350 nm to 725 nm, the slit is 4 nm, the detection range is 1 to 500 U / L, and the detection limit is 0.6 U / L.

[0014] Another object of the present invention is to provide: an application method of a dual emission ratio fluorescence sensor in the determination of ALP enzyme activity inhibition rate, specifically comprising the following method: 50 μL Tris-HCl buffer (500 mM, pH 7.0), 50 μL 100 mM MgCl 2 , 10 μL 100 U / mL ALP, 33 μL 2 mM PPi and different concentrations of Na 3 VO 4 After sufficient reaction, add 50 μL of EuUPMA dispersion and dilute to 1 mL with ultrapure water. After the reaction in the dark, measure its fluorescence spectrum and calculate IC 50 value.

[0015] Furthermore, different concentrations of Na 3 VO 4 The post-reaction condition was incubated at 37°C for 30 minutes, and the light-protected reaction condition was incubated at 37°C for 1 hour. 50 The value is 88.165 μM.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses 2-hydroxyterephthalic acid (H 2BDC-OH) and 1,2,4,5-benzenetetracarboxylic acid (PMA) as organic ligands, and Zr 4+ The UiO-66-PMA skeleton was formed and further doped with europium ions (Eu 3+ ), a dual-emission EuUPMA was prepared, which produces H at 428nm and 614nm respectively. 2 BDC-OH ligand and Eu 3+ When ALP specifically catalyzes the hydrolysis of PPi to generate Pi, Pi reacts with Eu 3+ Binding leads to enhanced fluorescence emission of the ligand and Eu 3+ The fluorescence of ALP is significantly weakened, so that ALP can be quantified by the change of fluorescence intensity ratio. In addition, the effect of inhibitors on ALP enzyme activity can be evaluated by enzyme inhibition experiments, which is expected to provide technical support for the screening of ALP inhibitors and related biomedical research. Compared with traditional technologies, the specific beneficial effects of the present invention include:

[0017] 1. The EuUPMA fluorescent sensor prepared by the present invention utilizes ALP to specifically catalyze the hydrolysis of PPi to generate Pi-induced I 428 / I 614 The change in ratio enables highly accurate analysis of ALP activity. This technology performs self-calibration through changes in the intensity of different fluorescence peaks, thereby effectively eliminating the effects of probe concentration differences and environmental interference on the detection results.

[0018] 2. The EuUPMA fluorescence sensor prepared by the present invention has a linear detection range of 1 to 500 U / L, and a detection limit as low as 0.6 U / L; it has excellent selectivity and anti-interference ability for the detection of actual samples containing potential interferents (such as papain, glucose oxidase, nuclease, trypsin and amino acids, etc.), and can meet the detection needs of complex samples.

[0019] 3. The EuUPMA fluorescent sensor prepared by the present invention can be used for the rapid detection of ALP activity in human serum (spiked recovery rate 92.17% to 108.67%), and supports the evaluation of inhibitor efficacy (such as Na 3 VO 4 IC 50 =88.165 μM), providing a tool for screening ALP-related inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Powder X-ray diffraction (PXRD) pattern of UiO-66-PMA and EuUPMA (A); X-ray photoelectron spectroscopy (XPS) pattern (B); O 1s XPS pattern (C); Fourier transform-infrared (FT-IR) spectrum (D);

[0021] Figure 2 :(A) Fluorescence spectra of EuUPMA with different concentrations of ALP added; (B) Relationship between the fluorescence intensity ratio (I 428 / I 614 ) at 428 nm and 614 nm and the concentration of ALP. The inset is the linear fitting graph of I 428 / I 614 versus the concentration of ALP;

[0022] Figure 3 : Selectivity and anti-interference ability of EuUPMA ratiometric fluorescence detection for ALP;

[0023] Figure 4 : (A) Fluorescence response spectra of the ALP ratiometric fluorescence sensor after adding different concentrations of Na 3 VO 4 ; (B) Relationship curve of the inhibition rate of Na 3 VO 4 on ALP activity with the change of its concentration. Detailed implementation manners

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] Based on the change of a single fluorescence signal as the detection method of the target molecule, it is often easily interfered by environmental or instrument factors, and may generate false positive signals, thus affecting the accuracy of target detection and limiting its application in complex actual samples; the ratiometric fluorescence sensor uses the ratio of the intensities of different fluorescence emission peaks as the signal output unit, which can reduce the influence of external factors on target detection and improve the detection accuracy. Although there are currently relevant reports on the use of LnMOFs for ALP detection, the detection mechanism based on the pore size of LnMOFs or the interaction between ALP and the functional groups of organic ligands may have problems of insufficient selectivity, thereby reducing the precision and reliability of ALP detection. Therefore, the present invention utilizes the dephosphorylation reaction of ALP specifically catalyzing the hydrolysis of PPi to generate Pi, and constructs a ratiometric fluorescence sensor based on dual-emission EuUPMA for highly sensitive and highly selective detection of ALP activity. The ratiometric fluorescence sensor is induced by Pi generated by ALP-catalyzed PPi hydrolysis to cause different responses of the fluorescence peak of the H 2 BDC-OH ligand at 428 nm and the characteristic fluorescence emission of Eu 3+ at 614 nm. With the fluorescence intensity ratio I 428 / I614 Quantifying the ALP activity can significantly improve the accuracy of this strategy. This sensor has high sensitivity, high selectivity and anti-interference ability, and can be applied to the monitoring of ALP activity in human serum and the evaluation of inhibitor efficacy. Based on this, the present invention provides a ratiometric fluorescence sensor for highly efficient and highly accurate analysis and detection of ALP and its inhibitors.

[0026] Example 1:

[0027] Preparation of EuUPMA Fluorescence Sensor

[0028] 1. Disperse zirconium chloride (ZrCl 4 ), 1,2,4,5-benzenetetracarboxylic acid (PMA) and 2-hydroxyterephthalic acid (H 2 BDC-OH) in DMF, stir until completely dissolved, and then add concentrated hydrochloric acid and stir;

[0029] 2. Transfer the mixed solution in step (1) to a reaction kettle for solvothermal reaction. After the reaction is completed, centrifuge to collect the solid product, and obtain UiO-66-PMA material after washing and drying;

[0030] 3. Disperse the UiO-66-PMA obtained in step (2) in an aqueous solution of Eu(NO 3 ) 3 ·6H 2 O, and prepare the EuUPMA fluorescence sensor through stirring reaction, centrifugal washing and drying.

[0031] The powder X-ray diffraction (PXRD) of UiO-66-PMA and EuUPMA prepared in Example 1 is as Figure 1 shown in A. The characteristic diffraction peaks of both of them are highly consistent with those of the simulated UiO-66 single crystal, indicating that the material has high crystallinity and the post-doping of Eu 3+ does not change the crystal structure of the material. From the analysis results of X-ray photoelectron spectroscopy (XPS), it can be seen that compared with UiO-66-PMA, in addition to the characteristic peaks of C, N, and O, Eu3d characteristic peaks also appear at 1135.1 eV and 1164.9 eV in EuUPMA. At the same time, the characteristic peak of O1s shifts from 531.47 eV to 531.54 eV ( Figure 1 B and 1C), proving that Eu 3+ is successfully doped through the coordination interaction with the free carboxyl groups in UiO-66-PMA. In addition, as Figure 1 shown in D Fourier transform-infrared spectroscopy (FT-IR), UiO-66-PMA shows a characteristic absorption peak of carbonyl at 1710 cm -1 , while this characteristic absorption peak weakens in EuUPMA, indicating that Eu 3+Successfully coordinated with the carboxylic acid in UiO-66-PMA to form EuUPMA.

[0032] Method for ratiometric fluorescence sensing of ALP activity based on EuUPMA

[0033] Disperse the EuUPMA sensor prepared in Example 1 in Tris-HCl buffer (pH 7.0), mix it with different concentrations of ALP (0, 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 U / mL), after reacting at 37 °C for 1 hour, measure the fluorescence spectrum (excitation wavelength 270 nm, emission wavelength 350 - 725 nm). Using I 428 / I 614 The ratio as the ordinate and the ALP concentration as the abscissa (U / mL) to establish a standard curve.

[0034] As Figure 2 shown in 428 / I 614 The ratio shows a good linear correlation with the ALP concentration y = 1.382 + 5.182x (R 2 = 0.997), linear range: 1 - 500 U / L, detection limit: 0.6 U / L. It indicates that this ratiometric fluorescence sensor can achieve highly sensitive detection of ALP activity.

[0035] Example 2

[0036] Inhibitor Na 3 VO 4 Efficacy evaluation

[0037] For the ratiometric fluorescence sensor prepared in Example 1 used for screening ALP inhibitors, mix 50 μL of Tris-HCl buffer (500 mM, pH 7.0), 50 μL of 100 mM MgCl 2 , 10 μL of 100 U / mL ALP, 33 μL of 2 mM PPi with different concentrations of Na 3 VO 4 After sufficient reaction, add 50 μL of EuUPMA dispersion and make up the volume to 1 mL with ultrapure water. After reacting in the dark, measure its fluorescence spectrum and calculate the IC 50 value. The IC 50 value, that is, the concentration of Na 3 VO 4 required to reduce the ALP activity by 50%, is used to evaluate the inhibitory effect of Na 3 VO 4 . By plotting the relationship curve between the inhibition rate and the concentration of Na 3 VO 4 ([[]] Figure 4B), non-linear regression analysis was performed on it to calculate the IC 50 value of 88.165 μM. The above results indicate that the EuUPMA ratiometric fluorescence sensor can be used for efficient screening of ALP inhibitors.

[0038] Test Example 1

[0039] Using the change in the fluorescence intensity ratio (I 428 / I 614 ) of EuUPMA to quantitatively analyze ALP and verify the sensitivity of the EuUPMA sensor for ALP detection. The results are shown in Figure 2 .

[0040] As can be seen from Figure 2 B, the EuUPMA fluorescence sensor prepared in Example 1 can respond ratiometrically to low-concentration ALP, confirming that this ratiometric fluorescence sensing platform has the ability to highly sensitively detect ALP activity.

[0041] Test Example 2

[0042] Explore the selectivity of the EuUPMA fluorescence sensor prepared in Example 1 for ALP detection and its anti-interference ability in complex systems. As can be seen from Figure 3 , in addition to ALP, the I 428 / I 614 values after the reaction of other possible interfering substances (such as papain, glucose oxidase, endonuclease, trypsin, and amino acids, etc.) with EuUPMA are almost the same as those of the blank sample. In addition, when ALP coexists with the interferents, the ratiometric fluorescence response of EuUPMA to ALP is not significantly affected, indicating the high specificity and anti-interference ability of this sensor for ALP detection.

[0043] Test Example 3

[0044] Verify the performance of the EuUPMA fluorescence sensor prepared in Example 1 for ALP detection in human serum. Different concentrations of ALP solution were added to human serum samples by the standard addition method. After treatment with trichloroacetic acid, impurities were removed by centrifugation at 5000 rpm for 20 minutes. The obtained supernatant was diluted 2-fold and then detected using the sensor prepared in Example 1. The obtained results are shown in Table 1.

[0045] Table 1

[0046]

[0047] As can be seen from Table 1, the recovery rate of the EuUPMA fluorescence sensor for the detection of ALP in human serum was between 92.17% and 108.67%, and the relative standard deviation (RSD) was between 2.46% and 4.37%. The above results provide a reliable basis for the detection of ALP activity in actual samples using this ratio fluorescence sensor.

[0048] In summary, the ratio fluorescence sensor based on dual-emission EuUPMA of the present invention can detect ALP activity with high sensitivity, high selectivity, accuracy, and reliability. At the same time, this sensor can also be used for the screening of ALP inhibitors and has been successfully used for the determination of ALP content in human serum.

[0049] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Although this specification is described according to the embodiments, not every embodiment only contains one technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A dual emission ratio fluorescence sensor, comprising a europium metal organic framework material: EuUPMA, characterized in that: The EuUPMA consists of Eu 3+ , 2-hydroxyterephthalic acid (H2BDC-OH) and 1,2,4,5-benzenetetracarboxylic acid (PMA) are formed by coordination self-assembly, and the EuUPMA generates H2BDC-OH ligand and Eu at 428nm and 614nm, respectively. 3+ The characteristic fluorescence emission of ALP is generated by the hydrolysis of pyrophosphate (PPi) and the phosphate (Pi) reacts with Eu 3+ After binding, EuUPMA produces a ratiometric fluorescence response, and the fluorescence intensity ratio (I 428 / I 614 ) changes dynamically with ALP concentration and is used to achieve quantitative detection of ALP.

2. A method for preparing a dual-emission ratio fluorescence sensor, according to the dual-emission ratio fluorescence sensor of claim 1, characterized in that: The specific steps include: Step 1: Disperse zirconium chloride (ZrCl4), 1,2,4,5-benzenetetracarboxylic acid (PMA) and 2-hydroxyterephthalic acid (H2BDC-OH) in DMF, stir until completely dissolved, then add concentrated hydrochloric acid and stir; Step 2: The mixed solution of step 1 is transferred to a reactor for solvothermal reaction. After the reaction is completed, the solid product is collected by centrifugation, and the UiO-66-PMA material is obtained by washing and drying. Step 3: The UiO-66-PMA obtained in step 2 is dispersed in an aqueous solution of Eu(NO3)3·6H2O, and the EuUPMA fluorescent sensor is prepared by stirring, reacting, centrifuging, washing and drying.

3. The method for preparing a dual emission ratio fluorescence sensor according to claim 2, characterized in that: The masses of ZrCl4, PMA, and H2BDC-OH in step 1 are 1.19 g (4.96 mmol), 1.01 g (3.96 mmol), and 0.364 g (2 mmol), respectively. The stirring time is 30 minutes. The volume of concentrated hydrochloric acid is 1 mL. The stirring time after adding concentrated hydrochloric acid is 10 minutes.

4. The method for preparing a dual-emission ratio fluorescence sensor according to claim 2, characterized in that: In step 2, the heating temperature of the solvent thermal reaction is 180° C., the time is 24 hours, the centrifugal speed is 8000 rpm, the centrifugal time is 5 minutes, the washing solvents are DMF and acetone, the drying temperature is 80° C., and the time is 10 hours.

5. The method for preparing a dual-emission ratio fluorescence sensor according to claim 2, characterized in that: In step 3, the mass of UiO-66-PMA is 0.30 g, the mass of Eu(NO3)3·6H2O is 1.34 g (3 mmol), and the volume of the aqueous solution is 30 mL.

6. A method for applying a dual-emission ratio fluorescence sensor in ALP detection, according to the dual-emission ratio fluorescence sensor of claim 1, characterized in that: The specific method includes the following steps: 50 μL Tris-HCl buffer (500 mM, pH 7.0), 50 μL 100 mM MgCl2, 33 μL 2 mM PPi, 10 μL ALP with different enzyme activities and 807 μL ultrapure water are sequentially added to a centrifuge tube, fully mixed and incubated at 37° C. for 1 hour, then 50 μL of the EuUPMA dispersion is added to the above solution, the reaction is continued at 37° C. for 1 hour, and the fluorescence spectrum is measured, and the ratio of the fluorescence intensity at 428 nm to that at 614 nm (I 428 / I 614 ) and the concentration of ALP, and the concentration of ALP in the test solution was calculated based on the standard curve.

7. According to the application method of a dual emission ratio fluorescence sensor in ALP detection according to claim 6, the concentration of the EuUPMA dispersion is 1 mg / mL, the excitation wavelength of the fluorescence emission spectrum is 270 nm, the emission wavelength range is 350 nm to 725 nm, the slit is 4 nm, the detection range is 1 to 500 U / L, and the detection limit is 0.6 U / L.

8. A method for applying a dual-emission ratio fluorescence sensor in determining the inhibition rate of ALP enzyme activity, according to the dual-emission ratio fluorescence sensor of claim 1, characterized in that: The specific method includes the following: 50 μL Tris-HCl buffer (500 mM, pH 7.0), 50 μL 100 mM MgCl2, 10 μL 100 U / mL ALP, 33 μL 2 mM PPi are fully reacted with different concentrations of Na3VO4, 50 μL EuUPMA dispersion is added, and the volume is fixed to 1 mL with ultrapure water. After the reaction is protected from light, the fluorescence spectrum is measured to calculate the IC 50 value.

9. The use of a dual emission ratio fluorescence sensor according to claim 8 in determining the inhibition rate of ALP enzyme activity, characterized in that: After adding different concentrations of Na3VO4, the reaction conditions were incubated at 37°C for 30 minutes, and the light-protected reaction conditions were incubated at 37°C for 1 hour. 50 The value is 88.165 μM.

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