Double-ligand Eu-MOF and application thereof in detection of nicotinamide mononucleotide

Abstract

The invention discloses a double-ligand Eu-MOF and application thereof in detection of nicotinamide mononucleotide, and belongs to the technical field of analysis and detection. The preparation method of Eu-TATB-BDC-NH2 comprises the following steps: dissolving 2, 4, 6-tri (4-carboxyphenyl)-1, 3, 5-triazine, 2-aminoterephthalic acid and europium nitrate hexahydrate in a mixed solution of water and N, N-dimethylformamide (DMF), adding nitric acid to adjust the pH value, ultrasonically dispersing uniformly, reacting in a high-pressure reaction kettle (polytetrafluoroethylene lining), cooling, centrifuging, washing and drying to obtain pink solid Eu-TATB-BDC-NH2; when the Eu-MOF is used for detecting the NMN, the method is simple, convenient, rapid and high in sensitivity, and a new strategy is provided for detecting the NMN in an actual sample.

Description

Technical Field

[0001] The invention relates to a dual-ligand Eu-MOF and application thereof in detecting nicotinamide mononucleotide, belonging to the technical field of analysis and detection. Background Art

[0002] Nicotinamide mononucleotide (NMN) has received extensive attention and research due to its potential anti-aging ability and medicinal value. At present, the market size of health foods with NMN as the main ingredient is gradually expanding, and its price remains high. However, some unscrupulous businesses falsely advertise the content of NMN in order to make huge profits, greatly damaging the legitimate rights and interests of consumers. Therefore, the detection of NMN in health foods is of great significance to market supervision and maintenance of market order.

[0003] At present, the main analytical techniques used to detect NMN include fluorescence, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. High performance liquid chromatography and liquid chromatography-mass spectrometry are sensitive and accurate, but they require professional operation and are expensive. The fluorescence method is favored by R&D personnel because of its simple operation, fast response, and low detection cost. However, traditional fluorescence sensors only have a single fluorescence emission signal that is easily interfered by environmental conditions and is difficult to apply in practice.

[0004] Ratiometric fluorescence sensors can reduce the errors caused by the environment and equipment, improve the stability of the sensor, and have greater application value by adding a stable fluorescence source as an internal reference. In the construction of ratiometric fluorescence sensors, metal organic frameworks (MOFs) have been widely studied due to their diverse luminescence mechanisms, including metal center luminescence (MC), ligand center luminescence (LC), guest substance luminescence, and excellent structural properties. Researchers have used MOFs to construct ratiometric fluorescence sensors for metal ions, small molecules, etc. However, these sensors often require the addition of guest substances, the synthesis steps are complicated, and there are no sensors for detecting NMN.

[0005] Therefore, it is necessary to develop a simple method to synthesize MOFs to construct a ratiometric fluorescence sensor for detecting NMN.

[0006] The use of lanthanide metals can simplify the synthesis steps of MOFs. Lanthanide metal organic frameworks (Ln-MOFs) are MOFs with lanthanide metals as metal nodes. Their unique metal center luminescence (MC) properties can be used as another luminescence center in addition to the ligand (LC) of MOFs, thereby replacing the guest substance. Eu-MOF, as a member of Ln-MOF, has shown great advantages in applications related to luminescence. Generally speaking, Ln 3+ The luminescence intensity of the ion depends on the ligand and Ln 3+ The use of strong absorbing ligands can improve the energy transfer efficiency and thus enhance the Ln 3+However, the auxiliary pigment functional groups such as -NH2 and -OH will reduce the energy level of the ligand and cannot sensitize Ln 3+ ions. Therefore, single-ligand lanthanide metal organic frameworks often cannot achieve the ligand and Ln 3+ Simultaneous strong emission of ions. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a dual ligand Eu-MOF and its application in detecting nicotinamide mononucleotide. A fluorescent sensor Eu-MOF is constructed by dual ligand 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (H3TATB), 2-aminoterephthalic acid (NH2-BDC) and europium nitrate hexahydrate. The MOF has blue and red fluorescence emission peaks at 424nm and 622nm respectively under 365nm excitation. In the presence of copper ions, the copper ions bind to the amino groups on the MOF, resulting in quenching of the blue emission peak at 424nm. When NMN is added, due to the stronger binding force with copper ions, competition occurs, thereby restoring the blue fluorescence of MOF at 424nm. During this process, the red fluorescence at 622nm remains basically constant. At the same time, the ratio of the fluorescence intensity at 424nm and 622nm (F 424 / F 622 ) is linearly correlated with the concentration of NMN, achieving the purpose of detecting NMN. The detection method is simple and fast, and no organic reagents are required during the detection process; the dual-ligand Eu-MOF (Eu-TATB-BDC-NH2) was synthesized by a one-step hydrothermal method and applied to the construction of NMN sensors.

[0008] The present invention provides a method for preparing a dual-ligand Eu-MOF, which specifically comprises the following steps:

[0009] 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, 2-aminoterephthalic acid, and europium nitrate hexahydrate (Eu(NO3)36H2O) are dissolved in a mixture of water and N,N-dimethylformamide (DMF), nitric acid is added to adjust the pH, ultrasonically dispersed uniformly, reacted in a high-pressure reactor (polytetrafluoroethylene lining), cooled, centrifuged, washed, and dried to obtain a dual-ligand Eu-MOF, Eu-TATB-BDC-NH2.

[0010] In one embodiment, the molar ratio of H3TATB, NH2-BDC, and Eu(NO3)3 6H2O is 0.5 to 1:2:2.

[0011] In one embodiment, water and DMF are mixed in a volume ratio of 2:3.

[0012] In one embodiment, the pH is adjusted to 3.0-5.0.

[0013] In one embodiment, the reaction temperature is 120-160° C., and the reaction time is 12-24 hours.

[0014] In one embodiment, the centrifugation condition is 8000-10000 r / min, and the time is 5-10 min.

[0015] In one embodiment, DMF and ethanol are used for washing.

[0016] In one embodiment, the drying conditions are: 60-80° C., and the drying time is 10-15 hours.

[0017] The present invention provides a dual-ligand Eu-MOF prepared by the above method.

[0018] The present invention provides a method for detecting NMN, which specifically comprises:

[0019] (1) Constructing a quantitative relationship model

[0020] The above-mentioned dual-ligand Eu-MOF was added into ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ , hydrochloric acid solution was added to adjust the pH, and different concentrations of NMN (0.005-2g / L) were added after ultrasonic homogenization. After incubation for a period of time, the fluorescence intensity of the mixed system at 424nm and 622nm was recorded at an excitation wavelength of 365nm, and the ratio of the fluorescence intensity at 424nm to 622nm (F 424 / F 622 ), with the concentration of NMN as the horizontal axis, F 424 / F 622 As the vertical axis, a quantitative relationship model is constructed;

[0021] (2) Determination of NMN content in the sample to be tested

[0022] The above-mentioned dual-ligand Eu-MOF was added into ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ , add hydrochloric acid solution to adjust the pH, add the sample to be tested after ultrasonic homogenization, incubate for a period of time, record the fluorescence intensity of the mixed system at 424nm and 622nm under an excitation wavelength of 365nm; and calculate the NMN content in the sample to be tested based on the quantitative relationship model constructed in step (1).

[0023] In one embodiment, the concentration of Eu-TATB-BDC-NH2 in the dispersed solution is 10-100 mg / L; preferably 25 mg / L.

[0024] In one embodiment, Cu 2+The concentration is 25-300 μmol / L; preferably 200 μmol / L.

[0025] In one embodiment, the pH value of the hydrochloric acid solution is adjusted to 4.0-10.0, preferably 6.0.

[0026] In one embodiment, the incubation time is 1 to 6 minutes, preferably 1 minute.

[0027] The present invention also provides the application of the above-mentioned dual ligand Eu-MOF in detecting the NMN content in food and health products.

[0028] Beneficial effects:

[0029] (1) The present invention synthesizes a Eu-MOF through a dual ligand strategy, which has blue and red fluorescence emissions under 365nm fluorescence excitation; and 2+ When NMN is present, the blue fluorescence of Eu-MOF is quenched due to static quenching; after adding NMN, the blue fluorescence is restored due to competition, and the red fluorescence remains unchanged during this process. The response time of this method is 1 min, and no organic reagents are required during the detection process. A sensitive, fast and simple NMN ratio fluorescence sensor was established.

[0030] (2) The method for detecting NMN established by the present invention has a linear range of 5 to 2000 mg / L and a detection limit of 0.1 mg / L (S / N=3). The results of the HPLC in the detection of actual samples are consistent, and in the spike recovery experiment, the spike recovery rate is 92.2% to 103.8%, and the relative standard deviation is 1.5% to 3.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the process of nicotinamide mononucleotide ratio fluorescence sensing based on dual-ligand Eu-MOF of the present invention;

[0032] Figure 2 The environmental scanning electron microscope images of Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention; A is a 7.0k scanning electron microscope image; B is a 25k scanning electron microscope image;

[0033] Figure 3 The X-ray diffraction pattern and Fourier transform infrared spectrum of Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention; A is the X-ray diffraction pattern; B is the Fourier transform infrared spectrum;

[0034] Figure 4 This is the X-ray photoelectron spectrum of Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention; A is the full spectrum, B is Eu, C is N, and D is C;

[0035] Figure 5 The fluorescence spectrum performance of Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention is compared with the fluorescence spectra of Eu-TATB-BDC-NH2, Eu-TATB and Eu-BDC-NH2;

[0036] Figure 6 Schematic diagram of the feasibility of Eu-TATB-BDC-NH2 detection prepared in Example 1 of the present invention; when Cu 2+ and fluorescence spectrum changes of NMN;

[0037] Figure 7 Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention is added with Cu 2+ and the fluorescence lifetime changes of NMN;

[0038] Figure 8 Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention is added with Cu 2+ UV absorption spectra before and after, as well as excitation and emission spectra;

[0039] Fig. 9 Eu-TATB-BDC-NH2 prepared in Example 1 of the present invention is added with Cu 2+ N1s X-ray photoelectron spectra before and after;

[0040] Fig.10 Eu-TATB-BDC-NH2 / Cu 2+ Cu 2p X-ray photoelectron spectra before and after adding NMN, A is Eu-TATB-BDC-NH2 / Cu 2+ Cu 2p X-ray photoelectron spectrum before adding NMN, B is.Eu-TATB-BDC-NH2 / Cu 2+ Cu 2p X-ray photoelectron spectrum after adding NMN;

[0041] Fig.11 This is the optimization diagram of the detection conditions of the NMN sensor; A is the Cu 2+ A is the optimization of concentration; B is the optimization of sensor reaction pH; C is the optimization of Eu-TATB-BDC-NH2 concentration in the sensor; D is the optimization of the reaction time of adding NMN to the sensor;

[0042] Fig.12 The fluorescence spectra and quantitative relationship diagram constructed in NMN detection; A is the fluorescence spectra at different concentrations; B is the constructed quantitative relationship diagram;

[0043] Fig.13The selectivity and anti-interference of the detection method of the present invention, A is selectivity, B is anti-interference;

[0044] Fig.14 The stability of the sensor of the present invention after being stored in water for different time periods. DETAILED DESCRIPTION

[0045] Example 1

[0046] The preparation method of Eu-TATB-BDC-NH2 specifically comprises the following steps:

[0047] 1.4mmol Eu(NO3)3 6H2O, 1.4mmol NH2-BDC, 0.46mmol H3TATB, and 300μL nitric acid were ultrasonically dispersed in a mixture of 15mL DMF and water (water and DMF were mixed in a volume ratio of 2:3) and the pH was 4.0. The mixture was reacted at 160°C for 12h in a 50mL high-pressure reactor (polytetrafluoroethylene lining). After the reaction was completed, it was naturally cooled to room temperature and centrifuged (8000r / min, 5min) to obtain the precipitate, which was washed several times with DMF and ethanol, and then dried in vacuo at 60°C for 12h to obtain a pink solid powder, namely Eu-TATB-BDC-NH2.

[0048] Characterization of Eu-TATB-BDC-NH2:

[0049] The Eu-TATB-BDC-NH2 was characterized by scanning electron microscopy (SEM). Figure 2 As shown in Figure 2, Eu-TATB-BDC-NH2 is a fiber rod structure. The powder X-ray diffraction (PXRD) pattern of Eu-TATB-BDC-NH2 is shown in Figure 2. Figure 3 A. The diffraction peaks of Eu-TATB-BDC-NH2 are different from those of Eu-BDC-NH2 (the preparation method is consistent with that of Example 1, the only difference is that the ligand H3TATB is omitted) and Eu-TATB (the preparation method is consistent with that of Example 1, the only difference is that the ligand NH2-BDC is omitted), indicating that the structure of the dual ligand changes the crystal structure of MOF, but the crystallinity is good.

[0050] like Figure 3 As shown in Figure B, the chemical structures of H3TATB, NH2-BDC, Eu-BDC-NH2, Eu-TATB and Eu-TATB-BDC-NH2 were characterized by infrared spectroscopy at 2500-3000cm -1 The broad band at 880–692 cm in the FT-IR spectrum disappears from Eu-TATB-BDC-NH2, so H3TATB and BDC-NH2 are completely deprotonated in MOF. -1The peak is attributed to the CH bending vibration in benzene. 1017cm -1 It is the characteristic emission peak of H3TATB. 1270cm -1 The peak at 1500-1700cm is the stretching vibration of -COO-. -1 The peaks between 3479cm and 3479cm come from the benzene ring or triazine ring. -1 and 3345cm -1 The peak at is attributed to the stretching vibration peak of NH, indicating that the amino group has been effectively integrated into the MOF.

[0051] The elemental and chemical composition of Eu-TATB-BDC-NH2 was analyzed by X-ray photoelectron spectroscopy (XPS): Figure 4 As shown in A: The coexistence of C, N, O and Eu elements in Eu-TATB-BDC-NH2 is confirmed again. The high-resolution spectrum of Eu 3d is decomposed into five peaks ( Figure 4 B). The peak at 1142.9 eV is the satellite signal (abbreviated as "Sat"). The two peaks at 1163.9 eV and 1134.6 eV are attributed to Eu 3+ The peaks at 1155.6eV and 1124.9eV indicate the presence of a small amount of Eu 2+ , these peaks originate from the surface layer, which is called the "surface valence transition" phenomenon. Figure 4 In C, the N 1s high-resolution spectrum has two peaks, the peak at 399.9 eV is related to the NH bond, and the peak at 398.8 eV comes from the CN bond. Figure 4 D), there are three split peaks at 288.1eV, 286.2eV and 284.4eV. They correspond to the carboxylic acid carbon, CN bond and alkyl carbon component of benzene ring respectively. The above results indicate that Eu-BTC-BDC-NH2 was successfully prepared.

[0052] Study on the spectral properties of Eu-BTC-BDC-NH2:

[0053] like Figure 5 As shown, under the 365nm excitation spectrum, the fluorescence spectrum of Eu-TATB-BDC-NH2 shows four different emission peaks, located at 424nm, 595nm, 622nm and 703nm. Among them, 424nm is attributed to the energy release of BDC-NH2, 595nm, 622nm and 703nm are due to the energy release of Eu 3+ The characteristic emission of H3TATB is compared with the characteristic emission peaks of Eu-BDC-NH2 and Eu-TATB, indicating that the addition of H3TATB realizes the antenna effect to sensitize Eu 3+ , demonstrating the success of the dual-ligand strategy.

[0054] Eu-BTC-BDC-NH2 / Cu 2+ Detection feasibility and mechanism exploration:

[0055] In order to explore the Eu-BTC-BDC-NH2 / Cu 2+ The mechanism of the fluorescence response of NMN was first investigated by 2+ and the fluorescence spectra in the case of NMN, such as Figure 6 . Add 200 μmol / L Cu 2+ After that, the fluorescence at 424 nm was quenched by about 80%, which may be due to the Cu 2+ The interaction between Eu 3+ The emission intensity at 622 nm was not significantly affected. Next, we studied the 2+ After adding NMN, due to the presence of more Cu 2+ The fluorescence intensity at 424 nm was observed to be restored and linear with the concentration of NMN. 3+ The 622 nm emission intensity remained unaffected, indicating that our ratiometric fluorescence sensor strategy is feasible.

[0056] In order to analyze the fluorescence quenching and recovery mechanism, a series of experiments were carried out, including fluorescence lifetime, UV-visible absorption spectrum, fluorescence spectrum, XPS, etc. Figure 7 The fluorescence lifetime of Eu-TATB-BDC-NH2 is 15.12 ns. 2+ The fluorescence lifetime of the post-ion system is 14.22ns, with no obvious change. Figure 8 Medium Cu 2+ The ultraviolet absorption spectrum of ions does not overlap with the excitation and emission spectra of Eu-TATB-BDC-NH2. 2+ After the addition of ions, the ultraviolet absorption peak of Eu-TATB-BDC-NH2 changed significantly, proving that a new complex was formed. The fluorescence quenching mechanism of Eu-TATB-BDC-NH2 is a single static quenching effect; Fig. 9 N1s narrow scan XPS spectrum of Eu-TATB-BDC-NH2 in Cu 2+ After addition, the voltage shifted from 398.9eV to 399.9eV, indicating that Cu 2+ Coordinated with nitrogen atoms. 2+Fluorescence signal recovery, XPS narrow scan spectrum was used to characterize Eu-TATB-BDC-NH2 / Cu before and after adding NMN 2+ The Cu 2p orbital binding energy changes. Fig.10 As shown, after adding NMN, Eu-TATB-BDC-NH2 / Cu 2+ The Cu 2p1 / 2 binding energy increases from 953.7eV to 954.5eV, indicating that Cu 2+ The interaction with NMN was greater than that with Eu-TATB-BDC-NH2, supporting the selection of Cu 2+ strategy as a fluorescence quencher.

[0057] Example 2

[0058] A novel Eu-TATB-BDC-NH2 / Cu 2+ A method for determining NMN content, the method comprising the following:

[0059] (1) Construction of quantitative relationship model

[0060] 2.5 mg Eu-TATB-BDC-NH2 was added to 100 mL ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ (200 μmol / L), the pH value of the hydrochloric acid solution was adjusted to 6.0, and different concentrations of NMN (0.005-2 g / L) were added after ultrasonic homogenization. After incubation for 1 min, the fluorescence intensity of Eu-TATB-BDC-NH2 at 424 nm and 622 nm was recorded at an excitation wavelength of 365 nm. The ratio of the fluorescence intensity at 424 nm to 622 nm (F 424 / F 622 ), and construct the standard curve of NMN.

[0061] With NMN concentration as the horizontal axis, F 424 / F 622 Construct a quantitative relationship model for the ordinate:

[0062] The quantitative relationship model of NMN concentration in the range of 0.005-0.3 g / L is: Y1=1.7124X+0.2828, R 2 =0.987, X represents NMN concentration; detection limit is 0.1 mg / L (S / N=3);

[0063] The quantitative relationship model of NMN concentration in the range of 0.3-2 g / L is: Y2=0.6861X+0.3246, R 2 =0.998;

[0064] (2) Determination of NMN content in the sample to be tested

[0065] 2.5 mg Eu-TATB-BDC-NH2 was added to 100 mL ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ (200 μmol / L), adjust the pH value of hydrochloric acid solution to 6.0, add 100 μL of the sample to be tested after ultrasonic homogenization, incubate for 1 minute, and record the fluorescence intensity of Eu-TATB-BDC-NH2 at 424nm and 622nm under 365nm excitation wavelength. Calculate the ratio of the fluorescence intensity at 424nm to 622nm (F 424 / F 622 ); and according to the quantitative relationship model constructed in step (1), calculate the content of NMN in the sample to be tested.

[0066] Example 3

[0067] Optimization of detection system conditions

[0068] Referring to the determination method in Example 2, 200 mg / L NMN was used as the determination sample, and the Cu content of the detection mixed system was adjusted. 2+ Detection conditions such as concentration, pH value, Eu-TATB-BDC-NH2 concentration and reaction time;

[0069] Cu 2+ Optimization of the concentration is critical to the function of the sensor. 2+ Insufficient concentration will lead to incomplete fluorescence quenching of Eu-TATB-BDC-NH2, which may affect the subsequent fluorescence onset. Fig.11 As shown in A, with Cu 2+ As the concentration increased from 0 to 300 μmol / L, the fluorescence intensity of Eu-TATB-BDC-NH2 at 424 nm gradually decreased. 2+ When the concentration was 200 μmol / L, the fluorescence intensity decreased by about 80%. 2+ It may interfere with the fluorescence recovery after adding NMN. Therefore, in subsequent determinations, Cu 2+ The concentration is 200μmol / L.

[0070] pH will affect the skeleton structure of MOF and the state of the target in the detection system, thus affecting Eu-TATB-BDC-NH2 / Cu 2+ The detection sensitivity of Eu-TATB-BDC-NH2 / Cu was investigated in the pH range of 4.0 to 10.0. 2+ The fluorescence intensity ratio at 424 and 622 nm (F 424 / F 622 ).like Fig.11As shown in B, when the pH increased from 4.0 to 6.0, the F 424 / F 622 The difference (△F 424 / F 622 ) gradually increases. When the pH is greater than 6.0, △F 424 / F 622 At the same time, F 424 / F 622 The pH value was larger at pH 6.0, so pH 6.0 was selected as the pH condition for subsequent experiments.

[0071] The concentration of the probe directly affects the intensity of fluorescence, thus affecting the sensitivity and accuracy of the sensor. 2+ The fluorescence intensity ratio F 424 / F 622 Changes with Eu-TATB-BDC-NH2 concentration of 10, 25, 50, 75, 100 mg / L ( Fig.11 C), with the increase of Eu-TATB-BDC-NH2 concentration, F 424 / F 622 When the concentration of Eu-TATB-BDC-NH2 is 25 mg / L, △F 424 / F 622 However, when the concentration of Eu-TATB-BDC-NH2 exceeds 25 mg / L, △F 424 / F 622 The value decreased and tended to be stable, indicating that NMN and Eu-TATB-BDC-NH2 / Cu 2+ The interaction was more significant at 25 mg / L.

[0072] like Fig.11 D shows that the addition of NMN to Eu-TATB-BDC-NH2 / Cu 2+ The fluorescence intensity changes with time, and it is found that after adding NMN, Eu-TATB-BDC-NH2 / Cu 2+ The fluorescence intensity changes rapidly and tends to be stable, so 1 min is selected as the incubation time for subsequent experiments.

[0073] Selective experiment

[0074] Select a range of common ions, including metal ions (Ca 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD +Precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu and resveratrol Res were used for control experiments, including:

[0075] 2.5 mg Eu-TATB-BDC-NH2 was added to 100 mL ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ (200 μmol / L), the pH value of the hydrochloric acid solution was adjusted to 6.0, and after ultrasonic homogenization, NMN and metal ions (Ca) at a concentration of 200 mg / L were added respectively. 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD + Precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu, and resveratrol Res were incubated for 1 min, and the fluorescence intensity of Eu-TATB-BDC-NH2 at 424 nm and 622 nm was recorded at an excitation wavelength of 365 nm. The ratio of the fluorescence intensity at 424 nm to that at 622 nm (F 424 / F 622 ).

[0076] The results are as follows Fig.13 As shown in A, metal ions (Ca 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD + Effect of precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu and resveratrol Res on the synthesis of Eu-TATB-BDC-NH2 / Cu 2+ The fluorescence signal was not significantly affected, which indicates that the detection system of the present invention exhibits high selectivity for NMN.

[0077] Anti-interference test

[0078] Select a range of common ions, including metal ions (Ca 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD + Precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu and resveratrol Res were used for control experiments, including:

[0079] 2.5 mg Eu-TATB-BDC-NH2 was added to 100 mL ultrapure water, and a uniformly dispersed solution was obtained after ultrasonication. Then, Cu 2+ (200 μmol / L), the pH value of the hydrochloric acid solution was adjusted to 6.0, and after ultrasonic homogenization, 200 mg / L NMN was added, and then 200 mg / L metal ions (Ca 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD + Precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu and resveratrol Res, after incubation for 1 min, the fluorescence intensity of Eu-TATB-BDC-NH2 at 424nm and 622nm was recorded at an excitation wavelength of 365nm. The ratio of the fluorescence intensity at 424nm to 622nm (F 424 / F 622 ).

[0080] The results are as follows Fig.13 As shown in B, metal ions (Ca 2+ , K + Mg 2+ 、Na + , Fe 2+ , Fe 3+ ), NAD + Precursors (nicotinamide Nam, nicotinamide riboside NR), leucine Leu and resveratrol Res have little effect on the fluorescence changes of the detection system.

[0081] Stability test

[0082] Eu-TATB-BDC-NH2 / Cu 2+ The aqueous solution was stored at room temperature for 15 days for stability testing. Fig.14 It can be seen that Eu-TATB-BDC-NH2 / Cu 2+ Response to NMN within 15 days (F 424 / F 622 ) remains basically unchanged, proving that Eu-MOF has good water stability.

[0083] Example 4

[0084] Determination of NMN content in actual samples

[0085] The three NMN supplements were purchased from Tmall.

[0086] Take 20 NMN supplement capsules, remove the capsules and mix the powder evenly. Take 50 mg of powder sample and add it to 100 mL of water. After ultrasonication for 10 minutes, centrifuge (8000 r / min) for 5 minutes, take the supernatant, adjust the pH to 6.0 with HCl, and test. Add different concentrations (100 and 200 mg / L) of NMN standards to the sample, and use the test method of Example 2 for detection. Each sample is measured 3 times, and the recovery rate and precision (relative standard deviation) are calculated.

[0087] The results are shown in Table 1. The recovery rate was between 92.2% and 103.8%, and the relative standard deviation was between 1.5% and 3.7%. The results were basically consistent with those of HPLC, indicating that this method is feasible for the detection of NMN in health foods.

[0088] Table 1 Concentration (mg / L), recovery (%) and precision (%, n=3) of NMN in actual samples

[0089]

[0090] Comparative Example 1

[0091] NMN was determined by HPLC. For specific methods, please refer to the literature: Zhou, H., Liu, A., Hu, M., Zheng, X., Kuang, W., & Xie, Y. A Novel HPLC Method for Quality Inspection of NRK Biosynthesized beta-Nicotinamide Mononucleotide. J Chromatogr Sci, 62(3), 241-248. https: / / doi.org / 10.1093 / chromsci / bmac107.

[0092] Comparative Example 2

[0093] NMN was determined using HPLC-MS / MS. For specific methods, please refer to the literature: Yan, J., Sakamoto, T., Islam, A., Ping, Y., Oyama, S., Fuchino, H., Kawakami, H., Yoshimatsu, K., Kahyo, T., & Setou, M. Cinnamomum verum J. Presl Bark Contains High Contents of Nicotinamide Mononucleotide. Molecules, 27(20).

[0094] Comparative Example 3

[0095] Use MIP@QDs probe to detect NMN. For specific methods, please refer to the literature: Xie, L., Zhang, M., Wu, Y., Xiong, H., & Hao, l. One-step synthesis of molecularly imprinted fluorescent sensors for highly selective detection of nicotinamide mononucleotide. Journal of Materials Science: Materials in Electronics, 35(4).

[0096] Comparative Example 4

[0097] NMN was detected using the NBD-B2 / Styryl-51F probe. For specific methods, please refer to the literature: Lee, HG, Hong, NK, & Chang, YT (2023). NMN sensor cocktail: selective sensing of nicotinamide mononucleotide over citric acid. Chem Commun (Camb), 59(61), 9372-9375.

[0098] Comparison of the present invention with the NMN sensing detection method in the prior art (Comparative Examples 1 to 4), the results are shown in Table 2:

[0099] Table 2 Comparison of NMN sensing detection methods in the present invention and the prior art

[0100]

[0101] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A dual-ligand Eu-MOF, characterized in that: The preparation method of the dual-ligand Eu-MOF comprises the following steps: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, 2-aminoterephthalic acid and europium nitrate hexahydrate are dissolved in a mixed solution of water and DMF, the pH is adjusted, the mixture is reacted in a high-pressure reactor after ultrasonic treatment, and the mixture is cooled, centrifuged, washed and dried to obtain a dual-ligand Eu-MOF.

2. A dual-ligand Eu-MOF according to claim 1, characterized in that: The molar ratio of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, 2-aminoterephthalic acid and europium nitrate hexahydrate is 0.5 to 1:2:

2.

3. A dual-ligand Eu-MOF according to claim 1, characterized in that: Water and DMF were mixed in a volume ratio of 2:

3.

4. A dual-ligand Eu-MOF according to claim 1, characterized in that: Adjust the pH value to 3.0-5.

0.

5. A dual-ligand Eu-MOF according to claim 1, characterized in that: The reaction temperature is 120-160°C and the reaction time is 12-24 hours.

6. A method for detecting NMN, characterized in that: The specific steps include: (1) Constructing a quantitative relationship model The dual-ligand Eu-MOF according to claim 1 is added to ultrapure water, and a uniformly dispersed solution is obtained after ultrasonication, and then Cu is added to the above solution. 2+ , adjust the pH, add different concentrations of NMN after ultrasound, incubate for a period of time, record the fluorescence intensity of the mixed system at 424nm and 622nm under the excitation wavelength of 365nm, and calculate the ratio of the fluorescence intensity at 424nm to 622nm F 424 / F 622 , with the concentration of NMN as the horizontal axis, F 424 / F 622 As the vertical axis, a quantitative relationship model is constructed; (2) Determination of NMN content in the sample to be tested The dual-ligand Eu-MOF according to claim 1 is added to ultrapure water, and a uniformly dispersed solution is obtained after ultrasonication, and then Cu is added to the above solution. 2+ , adjust the pH, add the sample to be tested after ultrasound, incubate for a period of time, and record the fluorescence intensity of the mixed system at 424nm and 622nm under an excitation wavelength of 365nm; And based on the quantitative relationship model constructed in step (1), calculate the NMN content in the sample to be tested.

7. A method for detecting NMN according to claim 6, characterized in that: The concentration of the dual-ligand Eu-MOF in the dispersed solution is 10 to 100 mg / L.

8. A method for detecting NMN according to claim 6, characterized in that: Cu 2+ The concentration is 25-300 μmol / L.

9. A method for detecting NMN according to claim 6, characterized in that: The pH value was adjusted to 4.0 to 10.0; the incubation time was 1 to 6 minutes.

10. Application of the dual-ligand Eu-MOF according to claims 1 to 5 in detecting the content of NMN in food and health products.

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