Ratio fluorescence sensing material conjugated with rare earth doped peptide nanospheres as well as preparation method and application of ratio fluorescence sensing material

Abstract

The invention belongs to the field of nano material preparation and food detection, and provides a ratio fluorescence sensing material conjugated with rare earth doped peptide nanospheres as well as a preparation method and application of the ratio fluorescence sensing material. The preparation method comprises the following steps: mixing soluble europium salt, dipeptide and a photosensitizer in a solvent, self-assembling into rare earth doped peptide nanospheres under mechanical force, and then mixing and reacting with fluorescent molecules to obtain the rare earth doped peptide nanospheres. The fluorescent sensing material disclosed by the invention has double-signal output, interference resistance and high sensitivity; compared with a noble metal nano probe, the cost is low; the method is suitable for constructing a rapid detection system, and meets the requirements of food safety, environmental monitoring and clinical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of nano material preparation and food detection, and particularly relates to the preparation of a rare earth doped material and the application of the same in detecting biogenic amines. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] It is reported that due to inefficient management during food processing, transportation, and consumption, approximately 1.3 billion metric tons of edible food waste is generated globally each year. In the EU alone, over 88 million tons of food are wasted annually, at a cost of approximately €143 billion. The share of food produced that is ultimately wasted accounts for 8% of global greenhouse gas emissions, 20% of freshwater consumption, and 30% of global farmland use. In addition to the economic and environmental costs, consuming spoiled food also leads to increased incidence of foodborne illness. Therefore, sustainable food production management has become a key strategy for preventing food waste and foodborne illness, and helping to optimize natural resources. This means that there is an urgent need to monitor food spoilage throughout the entire process chain, from farm to table.

[0004] Food spoilage is often accompanied by the production and release of biogenic amines. These amines are formed by the decarboxylation of amide groups promoted by external microorganisms, making them important biomarkers for monitoring food quality. Therefore, simple, low-cost, and rapid in situ monitoring technologies for biogenic amines are needed to enable on-demand spoilage analysis. Given the recent interest in fluorescent sensors, due to their excellent signal resolution, cost-effectiveness, and compatibility with portable devices, these sensors are believed to be capable of identifying and quantifying spoilage in packaged foods, assisting resource management in supply chain processes and aiding consumer pre-purchase decision-making. However, the accuracy of conventional fluorescence off / on response sensors is easily affected by other factors, such as luminophore concentration and the external environment (including temperature and humidity). Furthermore, the human eye's relatively limited sensitivity to changes in fluorescence brightness hinders the development of visual detection methods. Ratiometric fluorescence sensing systems based on two luminescent elements not only offer excellent self-calibration capabilities but also meet the needs of visual visualization, as the human eye more readily detects changes in fluorescence color. Therefore, exploring novel sensing materials may be a key to revolutionizing ratiometric fluorescence detection of biogenic amines.

[0005] As a classic sensor material, rare earth element-doped nanostructures have tunable luminescence properties, excellent photostability, and large Stokes / anti-Stokes shifts, and therefore have great potential in sensing. Some studies have adopted strategies such as antenna sensitization to enhance and regulate the luminescence intensity of the corresponding ions. However, most of the sensitization processes implemented to date involve irritating chemicals, which is not desirable for ideal optical sensors; and the energy mismatch between the organic ligands and the emission energy levels of rare earth ions is not conducive to the strong luminescence of the corresponding metal ions. In addition, for ratiometric fluorescence sensors based on rare earth-doped nanostructures, breaking the limitations of the ratiometric measurement method that relies on a fixed reference signal can improve the detection sensitivity and visualization of the sensor material within the required detection range, which is more conducive to integration with portable sensing terminals. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres. The ratiometric fluorescent sensing material can be flexibly modulated and is safe and environmentally friendly. It also has a dual-dynamic detection signal and a reference signal for the target object.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned material, which has mild reaction conditions, avoids the use of harsh chemicals, does not require complex purification steps, and has a short synthesis time.

[0008] Another object of the present invention is to provide an application of the above-mentioned material in detecting biogenic amines, which can respond to the total content of volatile biogenic amines in spoiled food with short response time and high sensitivity.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] A method for preparing a ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres comprises the following steps: (1) Soluble europium salt, dipeptide and photosensitizer are mixed in a solvent and self-assembled into a suspension of rare earth-doped peptide nanospheres under mechanical force; (2) The suspension of rare earth-doped peptide nanospheres is mixed with fluorescent molecules to react and obtain a ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres.

[0011] The amino acids constituting the dipeptide are independently selected from histidine, tryptophan, phenylalanine or tyrosine.

[0012] The photosensitizer is selected from one of acetophenone, benzophenone, o-phenanthroline and salicylic acid.

[0013] The solvent is selected from at least one of methanol, ethanol, acetonitrile and dimethyl sulfoxide.

[0014] The molar ratio of the dipeptide, photosensitizer and europium is 16: (0-50): (0.5-50), and the content of the photosensitizer is not 0; preferably 16: (0.5-50): (0.5-50).

[0015] The fluorescent molecule is selected from one of AF488 NHS ester, rhodamine 110, 6-carboxyfluorescein and fluorescein isothiocyanate. Preferably, the fluorescent molecule is selected from fluorescein isothiocyanate.

[0016] The molar ratio of the fluorescent molecule to europium is (1-5)×10 -7 : 1; preferably 4×10 -7 : 1.

[0017] A ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres obtained by the above preparation method.

[0018] The ratio fluorescence sensing material conjugated with rare earth doped peptide nanospheres has a spherical shape and an average particle size of 10-100 nm.

[0019] Application of the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres in detecting biogenic amines.

[0020] The biogenic amine is histamine, cadaverine, putrescine, tyramine or tryptamine.

[0021] A test paper prepared from the ratio fluorescence sensing material conjugated with rare earth-doped peptide nanospheres.

[0022] The test paper is obtained by coating the ratiometric fluorescent sensing material conjugated with the rare earth-doped peptide nanospheres on an inert carrier, such as a glass fiber membrane.

[0023] A method for detecting the content of biogenic amines in food comprises the following steps: The suspension of the ratiometric fluorescent sensor material is placed in contact with the food or in the same closed space, and then the intensity of the fluorescence emission peaks at 510 nm and 618 nm is detected and the ratio is calculated; or, Seal the above-mentioned test paper and food in the same space and observe the fluorescent color of the test paper under ultraviolet light.

[0024] When the ratio (I 510 / I 618 ) increases or the test paper turns green, indicating that the content of biogenic amines in the food is increased.

[0025] The food is selected from foods with high protein content such as meat or dairy products, such as shellfish, crustaceans, fish and other aquatic products, livestock and poultry meat, milk, milk powder or cheese, etc.

[0026] The present invention has the following advantages: (1) The preparation process of the rare earth-doped peptide nanospheres in the present invention is the in-situ coordination doping of rare earth ions with peptide chains, which does not require complex purification steps and shortens the synthesis time to within 2 hours (traditional rare earth complex synthesis requires 6-12 hours); (2) The reaction conditions of the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres in the present invention are mild (room temperature, normal pressure), avoiding the use of harsh chemicals and complying with the principles of green chemistry; (3) The ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres in the present invention has good fluorescence characteristics. The characteristic emission peak of the rare earth doped peptide nanospheres and the autofluorescence peak of the fluorescein molecule form a dual signal output. The emission intensity ratio (I 510 / I 618 ) Quantify the concentration of the target, effectively eliminating interference from light source fluctuations and uneven probe concentrations in single-wavelength detection, and its sensitivity is several times higher than that of traditional single fluorescent probes; (4) The linear dipeptide sequence of the present invention can be synthesized on a large scale in solid phase, with the rare earth doping amount less than 1 wt% and the fluorescein molecule addition amount less than 1 wt%. The overall cost is reduced by 60%-70% compared with precious metal nanoprobes (such as gold nanoparticles); (5) The ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres in the present invention can be loaded on a test strip and combined with smartphone RGB analysis to achieve rapid on-site detection (response time < 2 minutes), meeting the needs of food safety, environmental monitoring and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Fluorescence emission spectra (A) and fluorescence intensity values ​​at 616 nm (B) of rare earth-doped peptide nanospheres prepared by self-assembly of dipeptide phenylalanine, o-phenanthroline, and europium chloride at different molar ratios; Figure 2 Fluorescence emission spectra (A) and fluorescence intensity values ​​at 616 nm (B) of rare earth-doped peptide nanospheres prepared by self-assembly of dipeptide phenylalanine, o-phenanthroline, and europium chloride at different molar ratios; Figure 3 The fluorescence emission spectra and color diagrams of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared by adding different amounts of fluorescein isothiocyanate; Figure 4 1. Transmission electron microscopy image (A) and particle size distribution diagram (B) of rare earth-doped peptide nanospheres prepared in Example 4; Figure 5 1. Transmission electron microscopy (A) and particle size distribution (B) of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 5; Figure 6The fluorescence intensity changes and linear relationships of the ratiometric fluorescent sensing material (A) and the rare earth-doped peptide nanospheres (B) prepared in Example 4 to different concentrations of histamine, tyramine, and tryptamine are shown; Figure 7 The fluorescence intensity changes and linear relationships of the ratiometric fluorescent sensing material (A) and the rare earth-doped peptide nanospheres (B) prepared in Example 5 to different concentrations of histamine are shown; Figure 8 The fluorescence response of the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres prepared in Examples 4 and 5 to the target at different adsorption times; Figure 9 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 6 to different concentrations of histamine; Figure 10 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 7 to different concentrations of histamine; Figure 11 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 8 to different concentrations of histamine; Figure 12 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 9 to different concentrations of histamine; Figure 13 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 10 to different concentrations of histamine; Figure 14 The fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 11 to different concentrations of histamine; Figure 15 The present invention relates to the application of a test strip loaded with the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres prepared in Example 4 in in situ monitoring of the spoilage of raw shrimp samples (A) and raw fish samples (B). DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0029] Example 1 Preparation of rare earth-doped peptide nanospheres The dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: (0, 1, 3, 6, 10, 15, 30, 50) mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing.

[0030] The fluorescence emission spectra and intensities of the obtained series of rare earth doped peptide nanospheres are shown in Figure 2. Figure 1 As shown: The fluorescence emission peak intensity at 618 nm attributed to europium ions in rare earth-doped peptide nanospheres increases with the increase of the addition ratio of o-phenanthroline and reaches a maximum value at 10 mM.

[0031] Example 2 Preparation of rare earth doped peptide nanospheres The dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed and dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: (0.5, 1, 3, 6, 12, 20, 30, 50) mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing.

[0032] The fluorescence emission spectra and intensities of the obtained series of rare earth doped peptide nanospheres are shown in Figure 2. Figure 2 As shown: The fluorescence emission peak intensity at 618 nm attributed to europium ions in rare earth-doped peptide nanospheres increases with the increase of the addition ratio of europium chloride and reaches a maximum value at 6 mM.

[0033] Example 3 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres (1) The dipeptide phenylalanine, o-phenanthroline, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to the final concentrations of 0.6, 1.5, 2.4, and 3.0 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged at 10,000 rpm, washed with ethanol:water (8:2 v / v), and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0034] The fluorescence emission spectra and fluorescence colors of the ratiometric fluorescence sensing materials of the series conjugated rare earth doped peptide nanospheres were obtained as shown in Figure 3 As shown in the figure, by simply adjusting the amount of fluorescein isothiocyanate added, a ratiometric fluorescent sensing material of conjugated rare earth-doped peptide nanospheres with different initial fluorescence intensity ratios and colors was obtained. Considering the maximization of fluorescence response and naked eye visualization detection (red to green fluorescence), a final concentration of 2.4 nM fluorescein isothiocyanate was selected for subsequent studies.

[0035] Example 4 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide phenylalanine, o-phenanthroline, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged at 10,000 rpm, washed with ethanol:water (8:2 v / v), and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0036] Transmission electron microscopy of rare earth doped peptide nanospheres and fluorescent sensing materials obtained in steps (1) and (2) is shown in FIG. Figure 4 and 5 As shown: the average particle size of the rare earth doped peptide nanospheres is 49.8 nm, they are single particle dispersed, and have a spherical appearance; the obtained fluorescent sensing material still maintains a spherical microstructure, and its particle size is slightly larger than that of the rare earth doped peptide nanospheres, at 64.3 nm.

[0037] 2. Response to biogenic amines Accurately measure 1.5 mL of a suspension of ratiometric fluorescent sensing material conjugated with rare-earth-doped peptide nanospheres or rare-earth-doped peptide nanospheres and thoroughly mix with 1.5 mL of a standard working solution of histamine (final concentration 0-1200 ppm), tyramine (final concentration 0-1000 ppm), or tryptamine (final concentration 0-900 ppm). Incubate for 1 minute before measuring fluorescence. The fluorescence spectrophotometer was set to an excitation wavelength of 360 nm and an emission wavelength range of 400-800 nm. A linear relationship was established, with the standard working solution concentration as the horizontal axis and the fluorescence intensity ratio as the vertical axis.

[0038] The results are as follows Figure 6 As shown in the figure, the fluorescence emission peak intensities at 510 nm and 618 nm in the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres show amplification and quenching trends, respectively, with the increase of the concentration of histamine, tyramine or tryptamine solution, while the fluorescence emission peak intensity at 618 nm in the rare earth-doped peptide nanospheres only shows a quenching trend with the increase of the concentration of these biogenic amine solutions. Both indicate the biogenic amine content with ratiometric fluorescence and single signal fluorescence, respectively.

[0039] According to the corresponding linear regression curve, the ratio fluorescence sensing material conjugated with rare earth doped peptide nanospheres is relatively better than that of rare earth doped peptide nanospheres due to the dual signal emission ( I 510 / I 618) peaks, resulting in a larger slope K value due to the coordinated quantification of target concentration, indicating higher sensitivity. Furthermore, this ratiometric detection largely eliminates interference from single-wavelength detection, such as light source fluctuations and uneven probe concentrations, thereby improving the accuracy of fluorescent probe detection.

[0040] Example 5 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide histidylhistidine, o-phenanthroline, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres. The microstructure of the suspension was spherical, and the average particle size was 65.7 nm.

[0041] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres or rare earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 7 As shown, the ratiometric fluorescence sensing material has a larger slope than that of the rare earth-doped peptide nanospheres, which means higher sensitivity.

[0042] Application Example 1 Response time of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres to biogenic amines 1.5 mL of the ratiometric fluorescent sensing material conjugated with the rare earth-doped peptide nanospheres described in Examples 4 and 5 was accurately weighed and thoroughly mixed with 1.5 mL of a standard histamine working solution (1200 ppm). Fluorescence measurements were performed after 0-3 minutes, and the dynamic adsorption equilibrium was determined by changes in the fluorescence intensity ratio. The fluorescence spectrophotometer parameters were set as follows: an excitation wavelength of 360 nm and an emission wavelength range of 400-800 nm.

[0043] The results are as follows Figure 8 As shown, the fluorescence intensity ratio of the ratio fluorescence sensing material conjugated with rare earth doped peptide nanospheres in Examples 4 and 5 after 1 minute ( I 510 / I 618) almost no longer changes, indicating that the dynamic response time of the ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres to the target has reached equilibrium. The shorter equilibrium time helps to quickly realize the identification of biogenic amines in the system, thereby efficiently meeting the needs of in situ monitoring of food safety.

[0044] Example 6 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide tryptophanyltryptophan, o-phenanthroline, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0045] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 9 As shown, the ratiometric fluorescent sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0046] Example 7 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) Dissolve dipeptide tyrosyltyrosine, o-phenanthroline, and europium chloride in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and vortex vigorously to obtain a suspension of rare earth-doped peptide nanospheres. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0047] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 10 As shown, the ratiometric fluorescent sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0048] Example 8 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide phenylalanylhistidine, o-phenanthroline, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0049] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 11 As shown, the ratiometric fluorescent sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0050] Example 9 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide phenylalanine, salicylic acid, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0051] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 12 As shown, the ratiometric fluorescence sensing material can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0052] Example 10 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide phenylalanine, benzophenone, and europium chloride were dissolved in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0053] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 13 As shown, the ratiometric fluorescence sensing material can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0054] Example 11 Preparation of ratiometric fluorescence sensing material conjugated with rare earth-doped peptide nanospheres 1. Material Preparation (1) The dipeptide phenylalanine, o-phenanthroline, and europium chloride were dissolved in 10 mL of dimethyl sulfoxide at a ratio of 16 mM: 10 mM: 6 mM, and the suspension of rare earth-doped peptide nanospheres was obtained after vigorous vortexing. (2) The entire prepared rare earth-doped peptide nanosphere suspension was placed in a 25 mL round-bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The suspension was placed under magnetic stirring at room temperature for 24 h for electrophilic addition reaction. After the reaction, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL of methanol to obtain a ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres.

[0055] 2. Response to biogenic amines The linear relationship between the ratiometric fluorescence sensing material conjugated with rare earth doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4. The results are as follows: Figure 14 As shown, the ratiometric fluorescence sensing material can also exhibit a dual dynamic fluorescence response to histamine, that is, the fluorescence emission peak intensity at 510 nm gradually increases with the increase of histamine concentration, and the fluorescence emission peak intensity at 618 nm gradually quenches with the increase of histamine concentration.

[0056] Application Example 2: Application of ratiometric fluorescence sensing materials conjugated with rare earth-doped peptide nanospheres in in situ monitoring of meat spoilage A glass fiber membrane without any pretreatment was immersed in the ratiometric fluorescent sensing material suspension conjugated with rare earth-doped peptide nanospheres prepared in Example 4, incubated for 6 hours, and then removed and dried in a 60°C oven for 5 hours to obtain a test strip based on the ratiometric fluorescent sensing material. The test strip can be used as a smart label for spoilage monitoring of raw shrimp and raw fish samples, where red fluorescence indicates freshness and green fluorescence indicates spoilage. Specifically, the obtained test strip was placed in a sealed culture dish, and the raw shrimp or raw fish samples were stored in the culture dish together. At two different storage temperatures of 25°C and -20°C, a smartphone was used to take photos and record the fluorescent color images of the test strip in the corresponding culture dish after 0, 4, 8, 16, and 24 hours for spoilage monitoring.

[0057] The results are as follows Figure 15 As shown, when stored at 25°C, the fluorescence color of the test strips affixed to the top of the shrimp and fish packaging gradually changed from red to green over time, indicating that the seafood had gradually deteriorated. The released biogenic amines contacted the sensing material conjugated with rare-earth-doped peptide nanospheres loaded on the test strips, causing them to fluorescently respond. However, the fluorescence color of the test strips near the raw shrimp and fish samples stored at -20°C for 24 hours did not change significantly, indicating that they remained fresh. This is because low temperatures can inhibit the growth of microorganisms in food to a certain extent, thereby delaying food spoilage.

[0058] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for preparing a ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres, characterized in that: The following steps are involved: (1) Soluble europium salt, dipeptide and photosensitizer are mixed in a solvent and self-assembled into a suspension of rare earth-doped peptide nanospheres under mechanical force; (2) mixing a suspension of rare earth-doped peptide nanospheres with fluorescent molecules to obtain a ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres; The constituent amino acids of the dipeptide are independently selected from histidine, tryptophan, phenylalanine or tyrosine; The photosensitizer is selected from acetophenone, benzophenone, o-phenanthroline or salicylic acid; The fluorescent molecule is selected from AF488 NHS ester, rhodamine 110, 6-carboxyfluorescein or fluorescein isothiocyanate.

2. The preparation method according to claim 1, characterized in that The molar ratio of the dipeptide, photosensitizer and europium is 16:(0-50):(0.5-50), and the content of the photosensitizer is not 0; The molar ratio of the fluorescent molecule to europium is (1-5)×10 -7 :

1.

3. The preparation method according to claim 1, characterized in that The solvent is selected from at least one of methanol, ethanol, acetonitrile and dimethyl sulfoxide; The molar ratio of the dipeptide, photosensitizer and europium is 16: (0.5-50): (0.5-50); The fluorescent molecule is selected from fluorescein isothiocyanate.

4. A ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres obtained by the preparation method according to any one of claims 1 to 3.

5. The ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres according to claim 4, characterized in that: The morphology is spherical and the average particle size is 10-100 nm.

6. Use of the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres as claimed in claim 4 or 5 in detecting biogenic amines and a test paper prepared therefrom.

7. The use according to claim 6, characterized in that The biogenic amine is histamine, cadaverine, putrescine, tyramine or tryptamine.

8. A method for preparing the test paper according to claim 6, characterized in that: The test paper is obtained by coating the ratio fluorescence sensing material conjugated with the rare earth-doped peptide nanospheres on an inert carrier.

9. A method for detecting the content of biogenic amines in food, characterized in that: The following steps are involved: The method comprises placing a suspension of the ratiometric fluorescent sensing material conjugated with rare earth-doped peptide nanospheres as claimed in claim 4 or 5 in contact with food or placing the suspension in the same closed space, and then detecting the intensity of the fluorescence emission peaks at 510 nm and 618 nm to calculate the ratio; or The test paper as claimed in claim 6 and food are sealed in the same space, and the fluorescent color of the test paper is observed under ultraviolet light.

10. The method according to claim 9, characterized in that The food is selected from foods with high protein content such as meat or dairy products, such as shellfish, crustaceans, fish and other aquatic products, livestock and poultry meat, milk, milk powder or cheese, etc.

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