A ratio fluorescent sensing material conjugated with rare earth doped peptide nanospheres and a preparation method and application thereof

Abstract

The present application belongs to the field of nanomaterial preparation and food detection, and provides a kind of conjugated rare earth doped peptide nanosphere ratio fluorescent sensing material and its preparation method and application.Soluble europium salt, dipeptide and photosensitizer are mixed in solvent, and self-assembled into rare earth doped peptide nanosphere under mechanical force, then mixed with fluorescent molecules to react to obtain the fluorescent sensing material.The fluorescent sensing material has double signal output, anti-interference and high sensitivity, and has the advantage of low cost compared with noble metal nanoprobes, and is suitable for constructing rapid detection system to meet the needs of food safety, environmental monitoring and clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and food detection, specifically relating to the preparation of a rare earth doped material and its application in the detection of biogenic amines. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] It is reported that due to inefficiencies in the management of food processing, transportation, and consumption, approximately 1.3 billion metric tons of edible food waste are generated globally each year. In the European Union alone, more than 88 million tons of food are wasted annually, costing around €143 billion. The share of food produced and ultimately wasted accounts for 8% of global greenhouse gas emissions, 20% of freshwater consumption, and 30% of global farmland use. In addition to economic and environmental costs, consuming spoiled food also leads to an increase in the incidence of foodborne illnesses. Therefore, sustainable food production management has become a key strategy for preventing food waste and foodborne illnesses, and for helping to optimize natural resources. This means that the need to monitor food spoilage throughout the entire farm-to-table process is urgently needed.

[0004] Food spoilage is often accompanied by the production and release of biogenic amines, which are formed by the decarboxylation of amide groups promoted by external microorganisms. Therefore, biogenic amines have become important biomarkers for monitoring food quality. Based on this, simple, low-cost, and rapid in-situ monitoring techniques for biogenic amines should be developed for on-demand spoilage analysis. Considering the recent attention given to fluorescence sensors, such as their excellent signal resolution, cost-effectiveness, and adaptability to portable devices, these sensors are considered capable of identifying and quantifying spoilage in packaged foods, assisting in resource management of the supply chain, and helping consumers make informed purchasing decisions. However, the measurement accuracy of traditional fluorescence extinguishing / on response sensors is easily affected by other factors, such as luminescent concentration and external environment (including temperature and humidity). Furthermore, the human eye has relatively limited sensitivity to changes in fluorescence brightness, hindering the development of visual detection modes. In contrast, ratiometric fluorescence sensing systems based on two luminescent elements not only possess excellent self-calibration capabilities but also meet the needs of visual visualization, as the human eye more easily detects changes in fluorescence color. Therefore, exploring novel sensing materials may be one of the keys to achieving a revolution in biogenic amine ratiometric fluorescence detection technology.

[0005] As a classic sensor material, rare-earth-doped nanostructures possess tunable luminescence properties, excellent photostability, and large Stokes / anti-Stokes shifts, making them highly promising for sensing applications. Some studies have employed strategies such as antenna sensitization to enhance and modulate the luminescence intensity of corresponding ions. However, most sensitization processes implemented to date involve irritating chemicals, which is undesirable for ideal optical sensors; furthermore, the energy mismatch between organic ligands and the emission levels of rare-earth ions is not conducive to strong luminescence from the corresponding metal ions. Moreover, for ratiometric fluorescence sensors based on rare-earth-doped nanostructures, breaking free from the limitations of ratiometric methods reliant on fixed reference signals can improve the detection sensitivity and visualization of sensor materials within the desired detection range, which is more conducive to integration with portable sensing terminals. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a ratiometric fluorescence sensing material composed of conjugated rare earth-doped peptide nanospheres. This ratiometric fluorescence sensing material is flexibly modulated, safe, and environmentally friendly, and simultaneously possesses dual dynamic detection signals and reference signals for the target analyte.

[0007] Another objective of this invention is to provide a method for preparing the above-mentioned material, which has mild reaction conditions, avoids the use of harsh chemicals, requires no 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 the detection of 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 solution.

[0010] A method for preparing a ratiometric fluorescence sensing material with conjugated rare-earth-doped peptide nanospheres includes the following steps:

[0011] (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;

[0012] (2) The suspension of rare earth-doped peptide nanospheres was mixed with fluorescent molecules to obtain a ratio fluorescence sensing material with conjugated rare earth-doped peptide nanospheres.

[0013] The amino acids that make up the dipeptide are independently selected from histidine, tryptophan, phenylalanine, or tyrosine.

[0014] The photosensitizer is selected from one of benzyl ketone, benzophenone, o-phenanthroline and salicylic acid.

[0015] The solvent is selected from at least one of methanol, ethanol, acetonitrile, and dimethyl sulfoxide.

[0016] The molar ratio of the dipeptide, photosensitizer, and europium is 16:(0~50):(0.5~50), and the photosensitizer content is not 0; preferably, it is 16:(0.5~50):(0.5~50).

[0017] 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.

[0018] The molar ratio of the fluorescent molecule to europium is (1~5)×10⁻⁶. -7 : 1; preferably 4×10 -7 : 1.

[0019] A ratiometric fluorescence sensing material of conjugated rare earth-doped peptide nanospheres obtained by the above preparation method.

[0020] The ratiometric fluorescence sensing material of the conjugated rare earth-doped peptide nanospheres has a spherical morphology with an average particle size of 10-100 nm.

[0021] Application of the above-mentioned ratiometric fluorescence sensing material of conjugated rare earth-doped peptide nanospheres in the detection of biogenic amines.

[0022] The biogenic amine is histamine, cadaverine, putrescine, tyramine, or tryptamine.

[0023] A test strip prepared from the ratio fluorescence sensing material of the above-mentioned conjugated rare earth-doped peptide nanospheres.

[0024] The test strip is obtained by coating the ratio fluorescence sensing material of the above-mentioned conjugated rare earth-doped peptide nanospheres onto an inert carrier, such as a glass fiber membrane.

[0025] A method for detecting the content of biogenic amines in food includes the following steps:

[0026] The suspension of the above-mentioned ratiometric fluorescence sensing material was brought into contact with food or placed in the same sealed space, and then the intensity of the fluorescence emission peaks at 510 nm and 618 nm was measured, and the ratio was calculated; or,

[0027] The test strips were sealed in the same space as the food, and the fluorescence color of the test strips was observed under ultraviolet light.

[0028] When the ratio (I) 510 / I 618 An increase in the concentration of biogenic amines or a green color on the test strip indicates an increase in the biogenic amine content in the food.

[0029] The food is selected from high-protein foods such as meat or dairy products, including aquatic products such as shellfish, crustaceans, and fish, livestock and poultry meat, milk, milk powder, or cheese.

[0030] The present invention has the following advantages:

[0031] (1) The preparation process of rare earth doped peptide nanospheres in this invention is in-situ coordination doping of rare earth ions and peptide chains, without the need for complex purification steps, and the synthesis time is shortened to within 2 hours (the synthesis of traditional rare earth complexes requires 6-12 hours).

[0032] (2) The reaction conditions of the ratio fluorescence sensing material of rare earth doped peptide nanospheres in this invention are mild (room temperature, normal pressure), avoiding the use of harsh chemicals and conforming to the principles of green chemistry.

[0033] (3) The ratiometric fluorescence sensing material of rare earth-doped peptide nanospheres in this invention has good fluorescence characteristics. The characteristic emission peak of the rare earth-doped peptide nanospheres and the autofluorescence peak of the fluorescein molecule constitute a dual signal output. The ratio of emission intensity (I0.05) is used to measure the fluorescence intensity. 510 / I 618 ) It quantifies the concentration of target substances, effectively eliminating interference from light source fluctuations and uneven probe concentrations in single-wavelength detection, and improves sensitivity by several times compared to traditional single fluorescent probes;

[0034] (4) The linear dipeptide sequence in this invention can be synthesized on a large scale in solid phase, with rare earth doping amount <1 wt% and fluorescein molecule addition amount <1 wt%, and the overall cost is reduced by 60%-70% compared with noble metal nanoprobes (such as gold nanoparticles);

[0035] (5) The ratio fluorescence sensing material of rare earth doped peptide nanospheres in this invention can be loaded onto test strips and combined with RGB analysis of smartphones to achieve rapid on-site detection (response time < 2 minutes), meeting the needs of food safety, environmental monitoring and clinical diagnosis. Attached Figure Description

[0036] Figure 1 The fluorescence emission spectra (A) and fluorescence intensity at 616 nm (B) of rare earth-doped peptide nanospheres prepared by self-assembly of dipeptide phenylalanine, o-phenanthroline and europium chloride in different molar ratios are shown.

[0037] Figure 2 The fluorescence emission spectra (A) and fluorescence intensity at 616 nm (B) of rare earth-doped peptide nanospheres prepared by self-assembly of dipeptide phenylalanine, o-phenanthroline and europium chloride in different molar ratios are shown.

[0038] Figure 3 The fluorescence emission spectrum and color spectrum of ratio fluorescence sensing materials of conjugated rare earth doped peptide nanospheres prepared by adding different amounts of fluorescein isothiocyanate are shown.

[0039] Figure 4These are transmission electron microscopy (TEM) images (A) and particle size distribution diagrams (B) of the rare earth-doped peptide nanospheres prepared in Example 4.

[0040] Figure 5 The images show transmission electron microscopy (TEM) images (A) and particle size distribution diagrams (B) of the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres prepared in Example 5.

[0041] Figure 6 The ratio fluorescence sensing material (A) and rare earth doped peptide nanospheres (B) prepared in Example 4 show the fluorescence intensity changes and linear relationships of different concentrations of histamine, tyramine and tryptamine.

[0042] Figure 7 The ratio fluorescence sensing material (A) and rare earth-doped peptide nanospheres (B) prepared in Example 5 show the fluorescence intensity changes and linear relationships of different concentrations of histamine.

[0043] Figure 8 The ratio fluorescence sensing materials of the conjugated rare earth doped peptide nanospheres prepared in Examples 4 and 5 are the fluorescence responses of the target analytes to different adsorption times.

[0044] Figure 9 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of the conjugated rare earth doped peptide nanospheres prepared in Example 6 to different concentrations of histamine.

[0045] Figure 10 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of conjugated rare earth doped peptide nanospheres prepared in Example 7 to different concentrations of histamine;

[0046] Figure 11 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of the conjugated rare earth doped peptide nanospheres prepared in Example 8 to different concentrations of histamine.

[0047] Figure 12 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of conjugated rare earth doped peptide nanospheres prepared in Example 9 to different concentrations of histamine;

[0048] Figure 13 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of conjugated rare earth doped peptide nanospheres prepared in Example 10 to different concentrations of histamine.

[0049] Figure 14 This is the fluorescence intensity change and linear relationship of the ratiometric fluorescence sensing material of conjugated rare earth doped peptide nanospheres prepared in Example 11 to different concentrations of histamine.

[0050] Figure 15This describes the application of a test strip loaded with ratioluminescent sensing material of conjugated rare-earth-doped peptide nanospheres prepared in Example 4 in in-situ monitoring of spoilage in raw shrimp sample (A) and raw fish sample (B). Detailed Implementation

[0051] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0052] Example 1: Preparation of rare earth-doped peptide nanospheres

[0053] 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 mixture was vortexed vigorously to obtain a suspension of rare earth-doped peptide nanospheres.

[0054] The fluorescence emission spectra and intensities of the obtained series of rare earth-doped peptide nanospheres are as follows: Figure 1 As shown, the fluorescence emission peak at 618 nm in rare earth-doped peptide nanospheres, attributed to europium ions, increases with the addition ratio of o-phenanthroline, reaching a maximum at 10 mM.

[0055] Example 2: Preparation of rare earth-doped peptide nanospheres

[0056] Dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed 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 mixture was vortexed vigorously to obtain a suspension of rare earth-doped peptide nanospheres.

[0057] The fluorescence emission spectra and intensities of the obtained series of rare earth-doped peptide nanospheres are as follows: Figure 2 As shown, the fluorescence emission peak intensity at 618 nm in rare earth-doped peptide nanospheres, attributed to europium ions, increases with the increase of europium chloride addition ratio, reaching a maximum at 6 mM.

[0058] Example 3: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0059] (1) Dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and then vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0060] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 0.6, 1.5, 2.4, and 3.0 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged at 10,000 rpm, washed with ethanol:water (8:2 v / v), and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0061] The fluorescence emission spectra and fluorescence colors of the obtained series of ratioluminescent sensing materials composed of conjugated rare-earth-doped peptide nanospheres are as follows: Figure 3 As shown, by simply adjusting the amount of fluorescein isothiocyanate added, ratiometric fluorescence sensing materials of conjugated rare earth-doped peptide nanospheres with different initial fluorescence intensity ratios and colors were obtained. Considering maximizing fluorescence response and visual detection (red to green fluorescence), a final concentration of 2.4 nM of fluorescein isothiocyanate was selected for subsequent research.

[0062] Example 4: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0063] 1. Material Preparation

[0064] (1) Dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and then vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0065] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged at 10,000 rpm, washed with ethanol:water (8:2 v / v), and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0066] Transmission electron microscopy (TEM) images of the rare-earth-doped peptide nanospheres and fluorescent sensing materials obtained in steps (1) and (2) are shown below. Figure 4 and 5 As shown, the rare earth-doped peptide nanospheres have an average particle size of 49.8 nm, are dispersed as single particles, and have a spherical appearance. The obtained fluorescent sensing material still maintains a spherical microstructure, with a particle size slightly larger than the rare earth-doped peptide nanospheres, at 64.3 nm.

[0067] 2. Response to biogenic amines

[0068] Accurately measure 1.5 mL of ratiometric fluorescence sensing material or a suspension of rare-earth-doped peptide nanospheres, and thoroughly mix with 1.5 mL of standard working solutions of histamine (final concentration 0-1200 ppm), tyramine (final concentration 0-1000 ppm), or tryptophan (final concentration 0-900 ppm). Incubate for 1 min before fluorescence measurement. The parameters of the fluorescence spectrophotometer are set as follows: excitation wavelength 360 nm, emission wavelength range 400-800 nm. A linear relationship is established between the concentration of the standard working solution (x-axis) and the fluorescence intensity ratio (y-axis).

[0069] The results are as follows Figure 6 As shown, the fluorescence emission peak intensities at 510 nm and 618 nm in the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres tend to amplify and quench as the concentration of histamine, tyramine, or tryptamine solution increases, respectively. However, the fluorescence emission peak intensity at 618 nm in the rare-earth-doped peptide nanospheres only tends to quench as the concentration of these biogenic amine solutions increases. Both methods use ratiometric fluorescence and single-signal fluorescence to indicate the biogenic amine content, respectively.

[0070] According to the corresponding linear regression curve, the ratiometric fluorescence sensing material with conjugated rare-earth-doped peptide nanospheres, compared to rare-earth-doped peptide nanospheres, exhibits dual-signal emission (…). I 510 / I 618 The peak exhibits a larger slope K value due to the synergistic quantification of the target analyte concentration, indicating higher sensitivity. Furthermore, this ratiometric detection can largely eliminate interference from light source fluctuations and probe concentration inconsistencies in single-wavelength detection, thus improving the accuracy of fluorescent probe detection.

[0071] Example 5: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0072] 1. Material Preparation

[0073] (1) Dipeptide histidine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0074] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratiometric fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres. The microstructure of the suspension was spherical with an average particle size of 65.7 nm.

[0075] 2. Response to biogenic amines

[0076] The linear relationship between the ratiometric fluorescence sensing material of conjugated 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 the rare-earth-doped peptide nanospheres, which means higher sensitivity.

[0077] Application Example 1: Response time of ratiometric fluorescence sensing material composed of rare-earth-doped peptide nanospheres to biogenic amines

[0078] 1.5 mL of the ratiometric fluorescence sensing material of the conjugated rare-earth-doped peptide nanospheres from Examples 4 and 5 were accurately weighed and thoroughly mixed with 1.5 mL of histamine standard working solution (concentration of 1200 ppm). Fluorescence measurements were performed after 0–3 min, and the degree of dynamic adsorption equilibrium was determined by the change in the fluorescence intensity ratio. The parameters of the fluorescence spectrophotometer were set as follows: excitation wavelength of 360 nm and emission wavelength range of 400–800 nm.

[0079] The results are as follows Figure 8 As shown, the ratio of fluorescence intensity of the conjugated rare-earth-doped peptide nanospheres in Examples 4 and 5 after 1 minute is ( I 510 / I 618 The values ​​of the ratio fluorescence sensing material with conjugated rare earth doped peptide nanospheres have almost stopped changing, indicating that the dynamic response time of the ratio fluorescence sensing material to the target has reached equilibrium. The shorter equilibrium time helps to quickly identify biogenic amines in the system, thereby efficiently meeting the needs of in-situ food safety monitoring.

[0080] Example 6: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0081] 1. Material Preparation

[0082] (1) Dipeptide tryptophan, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and then vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0083] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0084] 2. Response to biogenic amines

[0085] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and the results are as follows: Figure 9 As shown, the ratiometric fluorescence sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0086] Example 7: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0087] 1. Material Preparation

[0088] (1) Dipeptide tyrosine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0089] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0090] 2. Response to biogenic amines

[0091] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and the results are as follows: Figure 10 As shown, the ratiometric fluorescence sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0092] Example 8: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0093] 1. Material Preparation

[0094] (1) Dipeptide phenylalanine histidine, o-phenanthroline and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and then vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0095] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0096] 2. Response to biogenic amines

[0097] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and the results are as follows: Figure 11 As shown, the ratiometric fluorescence sensing material here can also exhibit a dual dynamic fluorescence response to histamine, that is, the intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0098] Example 9: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0099] 1. Material Preparation

[0100] (1) Dipeptide phenylalanine, salicylic acid and europium chloride were mixed and dissolved in 10 mL of methanol in a ratio of 16 mM: 10 mM: 6 mM, and the mixture was vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0101] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0102] 2. Response to biogenic amines

[0103] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and 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 intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0104] Example 10: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0105] 1. Material Preparation

[0106] (1) Dipeptide phenylalanine, benzophenone and europium chloride were mixed in 10 mL of methanol at a ratio of 16 mM: 10 mM: 6 mM and vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0107] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0108] 2. Response to biogenic amines

[0109] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and 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 intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0110] Example 11: Preparation of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres

[0111] 1. Material Preparation

[0112] (1) Dipeptide phenylalanine, o-phenanthroline and europium chloride were mixed in 10 mL of dimethyl sulfoxide in a ratio of 16 mM: 10 mM: 6 mM and then vortexed vigorously to obtain a suspension of rare earth doped peptide nanospheres.

[0113] (2) All the prepared rare earth-doped peptide nanosphere suspensions were placed in a 25 mL round bottom flask, and fluorescein isothiocyanate was added to a final concentration of 2.4 nM. The mixture was then magnetically stirred at room temperature for 24 h to carry out an electrophilic addition reaction. After the reaction was completed, the reaction system was centrifuged and washed at 10,000 rpm, and then resuspended in 10 mL methanol to obtain a ratio fluorescence sensing material suspension of conjugated rare earth-doped peptide nanospheres.

[0114] 2. Response to biogenic amines

[0115] The linear relationship between the ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres and histamine solutions of different concentrations was established according to the method in Example 4, and the results are as follows: Figure 14As shown, the ratiometric fluorescence sensing material can also exhibit a dual dynamic fluorescence response to histamine, that is, the intensity of the fluorescence emission peak at 510 nm gradually increases with the increase of histamine concentration, while the intensity of the fluorescence emission peak at 618 nm gradually quenches with the increase of histamine concentration.

[0116] Application Example 2: Application of ratiometric fluorescence sensing material composed of rare earth-doped peptide nanospheres in in-situ monitoring of meat spoilage.

[0117] An untreated glass fiber membrane was immersed in a ratiometric fluorescence sensing material suspension of conjugated rare-earth-doped peptide nanospheres prepared in Example 4. After incubation for 6 hours, the membrane was removed and dried in a 60°C oven for 5 hours to obtain a test strip based on this ratiometric fluorescence sensing material. This test strip can be used as a smart tag for the spoilage monitoring of raw shrimp and raw fish samples, where red fluorescence represents freshness and green fluorescence represents spoilage. Specifically, the obtained test strip was placed in a sealed petri dish, and the raw shrimp or raw fish sample was also stored in the petri dish. At two different storage temperatures, 25°C and -20°C, the fluorescence color images of the test strip in the corresponding petri dish were captured using a smartphone after 0, 4, 8, 16, and 24 hours for spoilage monitoring.

[0118] The results are as follows Figure 15 As shown, when stored at 25 ℃, the fluorescence color of the test strips fixed to the top of the shrimp and fish packaging boxes gradually changed from red to green as the storage time increased, indicating that the seafood had gradually spoiled. The released biogenic amines came into contact with the sensing material of the conjugated rare earth-doped peptide nanospheres loaded on the test strips, causing them to fluoresce. However, the fluorescence color of the test strips near the raw shrimp and raw fish samples stored at -20 ℃ for 24 hours did not change significantly, indicating that they remained fresh. This is because the low temperature environment can inhibit the growth of microorganisms in food to a certain extent, thereby delaying the spoilage of the food.

[0119] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing a ratiometric fluorescence sensing material composed of conjugated rare-earth-doped peptide nanospheres, characterized in that, Includes 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 was mixed with fluorescent molecules to obtain a ratio fluorescence sensing material with conjugated rare earth-doped peptide nanospheres. The amino acids that make up the dipeptide are independently selected from histidine, tryptophan, phenylalanine or tyrosine. The photosensitizer is selected from benzyl ketone, benzophenone, o-phenanthroline or salicylic acid; The fluorescent molecule is selected from AF488 NHS ester, Rhodamine 110, 6-carboxyfluorescein, or fluorescein isothiocyanate. The molar ratio of the dipeptide, photosensitizer, and europium is 16:(0.5~50):(0.5~50); The solvent is selected from at least one of methanol, ethanol, acetonitrile, and dimethyl sulfoxide.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the fluorescent molecule to europium is (1~5)×10⁻⁶. -7 :

1.

3. The preparation method according to claim 1, characterized in that, The fluorescent molecule is selected from fluorescein isothiocyanate.

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

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

6. The application of a ratiometric fluorescence sensing material of conjugated rare earth-doped peptide nanospheres as described in claim 4 or 5 in the detection of biogenic amines.

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

8. A test strip prepared using a ratiometric fluorescence sensing material of conjugated rare-earth-doped peptide nanospheres as described in claim 4 or 5.

9. A method for preparing a test strip as described in claim 8, characterized in that, The test strip is obtained by coating a ratio fluorescence sensing material composed of conjugated rare-earth-doped peptide nanospheres onto an inert carrier.

10. A method for detecting the content of biogenic amines in food, characterized in that, Includes the following steps: A suspension of the ratiometric fluorescence sensing material of the conjugated rare-earth-doped peptide nanospheres as described in claim 4 or 5 is brought into contact with food or placed in the same sealed space, and then the intensity of the fluorescence emission peaks at 510 nm and 618 nm is detected, and the ratio is calculated; or, The test strip as described in claim 8 is sealed in the same space as the food, and the fluorescence color of the test strip is observed under ultraviolet light.

11. The method according to claim 10, characterized in that, The food products are selected from meat or dairy products with high protein content.

12. The method according to claim 11, characterized in that, The meat is selected from shellfish, crustaceans, fish, or poultry; the dairy products are selected from milk, milk powder, or cheese.

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