Curcumin ratio type fluorescent sensor fluorescent probe and curcumin and derivative detection method and device

Abstract

The application provides a curcumin ratio type fluorescent sensor fluorescent probe and a curcumin and derivative detection method and device. n The btec is a 1,2,4,5-benzene tetraformate ligand. The curcumin ratio type fluorescent sensor fluorescent probe is applied to curcumin detection, has super-high sensitivity and selectivity, a detection limit reaches nanomolar level (10 ‑9 M order of magnitude), a response time is short to dozens of seconds, has strong anti-interference ability and can be used repeatedly.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical chemistry and material science, and particularly relates to a curcumin ratio type fluorescent sensor fluorescent probe based on a metal organic framework (MOF) and a curcumin and derivative detection method and device. BACKGROUND

[0002] Curcumin (CUR) is a natural polyphenolic bioactive substance existing in the rhizome of turmeric, and has significant pharmacological effects. Existing studies have shown that curcumin has unique biological activities in anti-inflammatory, antioxidant, anti-tumor, antibacterial and neuroprotective aspects, and is one of the important candidate molecules in the field of natural medicine research. For example, curcumin can reduce the production of inflammatory mediators by inhibiting key signaling pathways such as nuclear factor kappa B (NF-κB), thereby playing an anti-inflammatory and disease relieving role. In addition to the medicinal value, curcumin is also widely used in the food industry as a natural pigment, food additive, and in the cosmetics industry as a core active ingredient with antioxidant function. Its safety and natural source make it a highly demanded functional raw material on the market.

[0003] However, the use of curcumin is not completely risk-free. A large number of studies have shown that excessive intake of curcumin can cause gastrointestinal discomfort, nausea, liver dysfunction and other adverse reactions. Therefore, international food safety agencies have set strict limits on the amount of curcumin used. The European Union has set the maximum daily intake of curcumin at 3 mg / kg of body weight; the Chinese national standard (GB 2760-2014) also sets maximum limits for the addition of curcumin in different food categories, such as 200 mg / kg in candies and 100 mg / kg in soy sauce. It is worth noting that due to the huge market demand, some unscrupulous enterprises adulterate inferior pigments or use non-standard processes, resulting in adulteration of curcumin products, which poses a serious threat to consumer health and the standardized development of the industry. Therefore, it is of great practical significance to develop a rapid, accurate and suitable on-site detection method for curcumin analysis.

[0004] Currently, the detection methods of curcumin mainly include thin layer chromatography, ultraviolet-visible spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis and electrochemical detection, etc. Although these methods have high sensitivity and reliability, they also have obvious shortcomings: expensive instruments and equipment, complex sample pretreatment, time-consuming detection process, high technical requirements for operators, etc. Especially in the actual needs of food production line supervision, market circulation supervision or on-site rapid screening, these methods are difficult to realize real-time, portable and efficient detection.

[0005] Metal-Organic Frameworks (MOFs) as a new type of porous crystalline material, due to its highly adjustable structure, excellent optical performance and rich functionalization possibility, gradually shows great potential in the field of fluorescence sensing. However, there is no literature report on the use of luminescent MOFs to construct a fluorescence sensing system for curcumin, an important food active ingredient. This indicates that the existing technology has not covered the demand gap of curcumin detection in the aspects of "portability, low cost, high selectivity, high sensitivity, visual interpretation" and the like. SUMMARY

[0006] Therefore, the present application aims to overcome the deficiencies of the prior art in curcumin detection, and provides a curcumin ratio type fluorescence sensor fluorescence probe, and a curcumin and its derivative detection method and device.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: a curcumin ratio type fluorescence sensor fluorescence probe, characterized in that the fluorescence probe material is a zinc metal organic complex [Zn2(btec)(H2O)2] n , wherein the btec is 1,2,4,5-benzene tetracarboxylate ligand, and n of the complex generally refers to the coordination number, that is, the coordination number of the central ion is 2, 4, 6 or 8.

[0008] The curcumin ratio type fluorescence sensor fluorescence probe of the present application is further described above, and the zinc metal organic complex [Zn2(btec)(H2O)2] n has a three-dimensional coordination polymer crystal structure, and has a three-dimensional pore network composed of binuclear Zn 2+ cluster and rigid polycarboxylic acid ligand; the material emits bright blue fluorescence (emission peak about 426nm) under ultraviolet excitation, and has good thermal stability.

[0009] The curcumin ratio type fluorescence sensor fluorescence probe of the present application is further described above, and the zinc metal organic complex [Zn2(btec)(H2O)2] n is obtained by the following method: dissolving Zn(NO3)2 and H4btec in a mixed solvent of water and acetonitrile, stirring at room temperature, heating reaction, and programmed cooling crystallization to obtain the crystal product.

[0010] The curcumin ratio type fluorescence sensor fluorescence probe of the present application is further described above, and the molar ratio of Zn(NO3)2 to H4btec is 2:1, the reaction temperature is 100-120℃, and the reaction time is 60-80 hours. Preferably, heating reaction in an oven at 110℃ for 72 hours, and then programmed cooling to room temperature at a rate of 5℃ per hour to crystallize.

[0011] The present invention also provides a method for detecting curcumin and its derivatives, comprising the curcumin ratiometric fluorescent sensor and fluorescent probe described in any one of the above-mentioned methods.

[0012] The present invention further describes a method for detecting curcumin and its derivatives as described above. Specifically, the curcumin ratiometric fluorescent sensor fluorescent probe material is dispersed in the liquid sample to be tested and excited by a near-ultraviolet light source. The change in the ratio of fluorescence quenching of the fluorescent probe material to fluorescence enhancement of curcumin enables curcumin detection.

[0013] The present invention further refines the method for detecting curcumin and its derivatives as described above by establishing a fluorescence ratio signal based on the intrinsic emission of the fluorescent probe material at 426 nm and the emission of the analyte curcumin at approximately 538 nm. When detecting curcumin, the emission wavelengths are set to 426 nm and 538 nm, and the fluorescence intensity ratio I is used to determine the fluorescence ratio. 538 / I 426 Quantitative analysis was performed based on a linear relationship with curcumin concentration, such as... Figure 3 As shown in (c), the linear equation is I. 538 / I 426 = 1.50×10 6 [CUR] +0.1352, passing the detection limit formula 3σ / K (K=1.50 × 10⁻⁶). 6 Calculated (σ represents the standard deviation of the target substance in the sample), the detection limit of CUR is 4.68 × 10⁻⁶. -9 M, linear range is 10 -9 M to 2×10 -7 M.

[0014] The present invention provides a method for detecting curcumin and its derivatives as described above, wherein the excitation wavelength of the near-ultraviolet light source is 328 nm.

[0015] During detection, the fluorescent probe of the curcumin ratiometric fluorescence sensor is mixed with the sample in suspension and excited using a near-UV light source (approximately 328 nm). Without curcumin, the probe emits strong blue fluorescence at 426 nm. When curcumin is present in the sample, the 426 nm emission of the probe is quenched, while curcumin produces strong green fluorescence at 538 nm. As the curcumin concentration increases, the fluorescence intensity at 426 nm gradually decreases while that at 538 nm gradually increases, thus forming Ig. 538 / I 426 The ratio signal; where at 10 -9 ~2×10 -7 In the low concentration range of M, the fluorescence intensity ratio I 538 / I 426 It showed a good linear relationship with curcumin concentration (correlation coefficient R² = 0.992); the detection limit calculated using the 3σ / K method was as low as 4.68 × 10⁻⁶.-9 M.

[0016] Experiments show that the fluorescent detection of the present application can complete the response in about 30 seconds, showing excellent rapidity and selectivity: in a system containing various food additive interferents, only curcumin causes significant enhancement of the probe's 538nm emission and the ratio signal is as high as more than 20 times, while other substances hardly affect the fluorescence of the probe. 538 / I 426 The ratio does not obviously attenuate, and the PXRD (X-ray diffraction) results show that the crystal structure remains stable before and after recycling.

[0017] The present application also provides a curcumin and derivative detection device, comprising a detection device body, an excitation wavelength adjustable light source and an intelligent spectrum or image acquisition system, a cuvette groove is arranged on the detection device body, the light source corresponds to the cuvette groove, and the intelligent spectrum or image acquisition system analyzes the fluorescence color change (B / G ratio) to output the curcumin concentration.

[0018] The curcumin and derivative detection device of the present application is as described above, the detection device body is made of ABS polymer, i.e. 3D printing technology of ABS resin (acrylonitrile-butadiene-styrene copolymer), and has the light source and cuvette mounting position.

[0019] The present application provides a three-dimensional metal organic framework material [Zn2(btec)(H2O)2] n The material not only has stable and strong luminescence performance under ultraviolet excitation conditions, but also has high structural stability, mild preparation conditions, low cost, good cycle stability and anti-interference ability; more importantly, the present application first finds that the material exhibits highly selective fluorescence response to curcumin by using fluorescence resonance energy transfer and photoinduced electron transfer mechanisms, and can realize ultra-high sensitive detection of nanomolar (4.68x10 -9 M) through ratio-type fluorescence change, and has the advantages of fast response speed (about 30 seconds), strong anti-interference ability, recyclability and the like.

[0020] The present application utilizes a portable cuvette device made by 3D printing technology and an automatic identification real-time quantitative analysis system to construct a portable intelligent detection platform which can be operated on site, does not require professional personnel, can observe color changes by naked eyes and automatically output concentration; the platform not only simplifies the detection process and improves the detection efficiency, but also provides a new technical path for on-site detection of food safety supervision and related industries.The system includes three stages: calibration, RGB signal processing and data output, specifically including a calibration module for minimizing errors caused by external influences, for using two known concentrations within a linear concentration range for calibration and linear equation calculation; an RGB signal processing module for identifying and analyzing RGB values after the user uploads a photo of a film of two known analyte concentrations, so as to digitize the image; a data output module for the user to select a linear equation (R, G, B, R / B, B / R, R / G, G / R, B / G, G / B) and an output type (color ratio or log color ratio), input the corresponding concentration in the specified standard part, and identify the linear equation that can be calculated; and an identification and analysis module for identifying and calculating the concentration of the analyzed substance after inputting a film photo of an unknown concentration.

[0021] The present application first applies a luminescent coordination polymer [Zn2(btec)(H2O)2] n to curcumin detection, and provides a simple and rapid new strategy for ratio fluorescence sensing.Compared with the prior art, the curcumin ratio fluorescence sensor fluorescent probe of the present application has ultra-high sensitivity and selectivity to curcumin, with a detection limit reaching nanomolar level (10 -9 M order), a short response time of tens of seconds, strong anti-interference ability and multiple recycling use.The automatic identification real-time quantitative analysis system applies a smart phone terminal, realizes on-site sampling and measurement, greatly simplifies the detection process and reduces the cost; the system does not require complicated sample pretreatment and large instruments, and can accurately determine the curcumin content in a complex matrix, and has a wide application prospect in the fields of food safety and drug quality control.In addition, since curcumin derivatives and curcumin have similarities in molecular skeleton, conjugated system and spectral response, the detection method described in the present application can also be used for identification and quantitative detection of curcumin derivatives; by establishing a calibration curve of the corresponding ratio signal and concentration, the extended detection application of different curcumin derivatives can be realized.

[0022] The present application adopts a zinc-based metal organic framework material [Zn2(btec)(H2O)2] n The luminescence of the present application mainly comes from ligand-related emission and its modulation effect in a metal coordination environment, therefore, the fluorescent probe of the present application replaces Zn 2+and keep the isomorphic material of similar coordination topology also belong to the equivalent replacement of the present application, namely, under the premise of not changing the main light path of the probe material, the above-mentioned other transition metal ions replace Zn 2+ The transition metal-polycarboxylic acid ligand coordination polymers formed are isomorphic or similar structures, which can also be expected to achieve the ratio response of the fluorescence quenching / enhancement triggered by curcumin, thereby being used for high-sensitivity detection of curcumin and also included in the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The crystal structure of the curcumin ratio-type fluorescence sensor fluorescence probe of the present application, (a) coordination environment of the complex Zn ion, (b) polyhedral graph of the binuclear unit, (c) one-dimensional binuclear chain structure along the c-axis, and (d) three-dimensional framework view along the c-axis;

[0024] Figure 2 The characterization data of the curcumin ratio-type fluorescence sensor fluorescence probe material of the present application, (a) PXRD (X-ray diffraction), (b) thermogravimetric analysis, and (c) infrared spectrum;

[0025] Figure 3 The fluorescence response and linear relationship diagram of the curcumin ratio-type fluorescence sensor fluorescence probe material of the present application applied to curcumin detection, (a) emission spectrum of the complex under different concentrations of curcumin, (b) CIE diagram of the complex under different concentrations of curcumin, (c) CUR in the concentration range of 10 -9 to 2×10 -7 M, I 538 / I 426 of the complex and CUR linear relationship diagram;

[0026] Figure 4 The fluorescence response and time response and anti-interference diagram of the curcumin ratio-type fluorescence sensor fluorescence probe material of the present application applied to curcumin detection, (a) luminescence intensity of the complex under addition of different food additives, (b) response time of the complex after addition of LMFX solution, (c) fluorescence spectra of the complex + CUR and CUR (λex = 328 nm), and (d) anti-interference experiment of the complex for detecting CUR in the presence of other food additives; 538 / I 426 of the complex and CUR linear relationship diagram;

[0027] Figure 5 The cycle stability of the curcumin ratio-type fluorescence sensor fluorescence probe of the present application, (a) schematic diagram of the influence of the cycle number on the complex during detection of CUR, and (b) X-ray powder diffraction pattern of the complex measured after one, three and five cycles of use;

[0028] Figure 6Figure 1 is a schematic diagram of FRET / PET mechanism for mechanism analysis of the curcumin ratiometric fluorescent sensor fluorescent probe, (a) UV spectra of the complex and various food additives, (b) excitation and emission spectra of CUR. DETAILED DESCRIPTION

[0029] Example 1

[0030] The curcumin ratiometric fluorescent sensor fluorescent probe can be prepared by a solvothermal method. Specifically, 0.060 g of zinc nitrate (Zn(NO3)2) and 0.024 g of tetracarboxylic acid ligand (H4btec) are dissolved in a mixed solvent, which is 5 mL of deionized water and 5 mL of acetonitrile, in a 25 mL polytetrafluoroethylene-lined reaction kettle, and then magnetically stirred at room temperature for 30 minutes. Then the reaction is heated in an oven at 110°C for 72 hours, and then cooled to room temperature at a rate of 5°C per hour. The generated crystal product is filtered, washed with water three times, and dried to obtain blue luminescent complex crystals, as shown in Figure 2 Figure 1 (a), which shows that the characteristic vibration peak of carboxylate appears at 1612 cm -1 and 1380 cm -1 , proving that the ligand has been deprotonated and coordinated with Zn 2+ , as shown in Figure 2 Figure 1 (c).The MOF crystal structure obtained from the crystallographic data of the complex is characterized by single crystal XRD and belongs to monoclinic space group 21 / c, and the specific crystallographic parameters are shown in Table 1, proving that the target complex is successfully obtained, as shown in P Figures 1 (a)-(d). Figure 1

[0031] Table 1. Crystallographic parameters of the curcumin ratiometric fluorescent sensor fluorescent probe

[0032]

[0033] Example 2

[0034] Fluorescence ratio detection of curcumin content in solution: 2.0 mg of the curcumin ratio-type fluorescent sensor fluorescent probe material crystal of Example 1 was weighed, finely ground and dispersed in 2.7 mL of deionized water to prepare a stable suspension by ultrasonic treatment for 30 minutes; 2.7 mL of the probe suspension was added to a fluorescence spectrophotometer cuvette, and the fluorescence emission spectrum was measured using an excitation wavelength of 328 nm, and an intense background emission peak (curcumin ratio-type fluorescent sensor fluorescent probe material) was observed at 426 nm; then a certain concentration of curcumin (CUR) standard solution was added dropwise to the cuvette, and the fluorescence spectrum change was continuously monitored. When the concentration of CUR gradually increased, the emission intensity of the curcumin ratio-type fluorescent sensor fluorescent probe material of the application at 426 nm was significantly weakened, and a new emission peak appeared at about 538 nm and the intensity gradually increased, and the fluorescence color of the solution gradually changed from blue to green.

[0035] As shown in Figure 3 (a)-(c), the fluorescence spectrum changes of the curcumin ratio-type fluorescent sensor fluorescent probe material of the application under different CUR concentrations can be seen, I 538 continuously increases with the increase of CUR concentration, while I 426 continuously decreases; analysis shows that in the CUR concentration range of 10 -9 ~ 2 × 10 -7 M, I 538 / I 426 has a linear relationship with the CUR concentration, and the correlation coefficient R2=0.992; the detection limit calculated according to the 3σ principle is 4.68 × 10 -9 M, which shows extremely high sensitivity.

[0036] Further anti-interference experiments were carried out, as shown in Figure 4 (a)-(d), curcumin (target analyte) and typical food preservatives such as sodium dehydroacetate, sodium diacetate, sodium benzoate, potassium sorbate, sodium lactate, calcium propionate, propyl p-hydroxybenzoate, etc. were added to the suspension of the curcumin ratio-type fluorescent sensor fluorescent probe material, respectively, and the concentration of each substance was 1 × 10 -3 M; it was found that only curcumin could cause the fluorescence of the complex at 538 nm to be greatly enhanced and the fluorescence at 426 nm to be quenched, and the remaining interference substances only caused weak or almost imperceptible fluorescence changes, indicating that the probe has high specificity for curcumin. In addition, the fluorescence color change from blue to green can be completed in about 30 seconds and the signal is stable, and the response is rapid.

[0037] To verify the repeatability of the probe, the same batch of curcumin ratio-type fluorescent sensor fluorescent probe complex suspension was sequentially subjected to curcumin detection, washing and regeneration cycle test. As shown in Figure 5(a) shows that the fluorescence intensity ratio (538 nm / 426 nm) of the zinc complex in the initial state is only 0.045, and after the addition of curcumin, the ratio can be increased to 17, showing a significant color change. After 5 repeated cycles, the fluorescence intensity of the curcumin ratio type fluorescence sensor probe material of the present application and I 538 / I 426 The ratio almost has no attenuation, and the performance remains stable, indicating that the zinc complex has good cyclic detection performance. As shown in Figure 5 (b) shows that the PXRD (X-ray diffraction) spectrum is consistent with the fresh sample, indicating that the probe material is stable in structure during the cyclic detection process and can be recycled multiple times. As shown in Figure 6 (a) shows that by comparing the emission spectrum of the complex with the ultraviolet spectrum of a variety of food additives, the ultraviolet absorption spectrum of CUR is at 430 nm, and the excitation of CUR is at 431 nm, while the emission of the complex is at 426 nm. As shown in Figure 6 (b) shows that the absorption spectrum and the excitation spectrum of CUR have a large overlap with the emission spectrum of the complex; this overlap indicates that there is a FRET (Förster resonance energy transfer) process between the emission of the complex and the absorption of CUR.

[0038] The embodiment of the present application can be detected by using a curcumin and derivative detection device, which can include a detection device body, an excitation wavelength adjustable light source, and an intelligent spectrum or image acquisition system. A cuvette groove, a light source mounting position, and an intelligent spectrum or image acquisition system mounting position are provided on the detection device body. The light source corresponds to the cuvette groove, and the intelligent spectrum or image acquisition system analyzes the fluorescence color change (B / G ratio) to output the curcumin concentration. Specifically, a portable fluorescence detection device prepared by using 3D printing technology can be used, the material is ABS engineering plastic, the light source is a high-intensity UV LED (365 nm), the sample pool slot, and the position of the fixed intelligent mobile phone; the structural design of the device allows the distance between the light source and the sample, the sample and the camera to be adjusted to obtain the best excitation and shooting effect.

[0039] The application also provides an automatic identification real-time quantitative analysis system, which comprises three stages: calibration, RGB signal processing and data output, and specifically comprises a calibration module for minimizing errors caused by external influences, for calibration and linear equation calculation using two known concentrations within a linear concentration range; an RGB signal processing module for identifying and analyzing RGB values, digitizing images after the user uploads a photo of a film of two known analyte concentrations; a data output module for the user to select a calculation linear equation (R, G, B, R / B, B / R, R / G, G / R, B / G, G / B) and an output type (color ratio or log color ratio), input the corresponding concentration in the specified standard part, and identify the calculable linear equation; and an identification analysis module for identifying and calculating the concentration of the analyzed substance after inputting a film photo of an unknown concentration. The system ensures that colors can be digitized and enables portable sensor devices to perform quantitative analysis on site.

[0040] A batch of probe suspension was prepared by adding 200 mg of curcumin ratio-type fluorescent sensor fluorescent probe material crystal into 270 mL of deionized water and ultrasonicating for 30 minutes. 2.7 mL of the suspension was taken and placed in a cuvette, 300 µL of different concentrations of CUR standard solution (concentration range 5×10 -5 ~ 5×10 -3 M) was added. The cuvette was inserted into the slot of the 3D printing device, the UV light source was turned on to irradiate the sample, and the front of the cuvette was photographed using a smartphone camera; at this time, it was observed that the fluorescence color of the solution in the cuvette after excitation gradually changed from blue to green with the increase of CUR concentration; the automatic identification real-time quantitative analysis system installed in the application program on the mobile phone analyzed the photographed fluorescence photos, extracted the blue (B) and green (G) channel intensity values of the images, and calculated the B / G ratio; experiments showed that within the CUR concentration range of 5×10 -5 ~ 5×10 -3 M, B / G and CUR concentration had a linear relationship. Using the pre-established calibration curve, the real-time quantitative analysis system could automatically convert the CUR concentration in the sample and display the results; compared with the traditional large instrument analysis method, the system is low in cost and simple in operation, and can be used for on-site rapid screening of curcumin content in food or environmental samples, and has important practical value.

Claims

1. A method for detecting curcumin and its derivatives, characterized in that, A curcumin-based ratiometric fluorescent sensor probe was used, the probe material being a zinc-organic metal complex [Zn2(btec)(H2O)2]. n Wherein, btec is a 1,2,4,5-benzenetetracarboxylate ligand; the zinc organometallic complex [Zn2(btec)(H2O)2] n The crystal structure is a three-dimensional coordination polymer with binuclear Zn. 2+ A three-dimensional porous network composed of clusters and rigid polycarboxylic acid ligands; The zinc-organic metal complex [Zn2(btec)(H2O)2] n The product is obtained by dissolving Zn(NO3)2 and H4btec in a mixed solvent of water and acetonitrile, stirring at room temperature, heating and reacting, and then crystallizing by temperature-programmed cooling. The molar ratio of Zn(NO3)2 to H4btec is 2:1, the reaction temperature is 100-120℃, and the reaction time is 60-80 hours.

2. The method for detecting curcumin and its derivatives according to claim 1, characterized in that, The fluorescent probe material of the curcumin ratiometric fluorescent sensor is dispersed in the liquid sample to be tested and excited by a near-ultraviolet light source. The change in the ratio of fluorescence quenching of the fluorescent probe material to fluorescence enhancement of curcumin enables the detection of curcumin.

3. The method for detecting curcumin and its derivatives according to claim 2, characterized in that, Based on the intrinsic emission of the fluorescent probe material at 426 nm and the emission of the analyte curcumin at 538 nm, a fluorescence ratio signal is formed. When detecting curcumin, the emission wavelengths are set to 426 nm and 538 nm. Quantitative analysis is performed by observing the linear relationship between the fluorescence intensity ratio I538 / I426 and the curcumin concentration, with a linear range of 10. -9 M to 2×10 -7 M.

4. The method for detecting curcumin and its derivatives according to claim 3, characterized in that, The excitation wavelength of the near-ultraviolet light source is 328 nm.

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