A high-sensitivity rapid fluorescence probe without metal toxicity, a preparation method and application in food pathogenic bacteria detection

Abstract

This invention discloses a metal-free, highly sensitive, rapid fluorescent probe, its preparation method, and its application in the detection of pathogenic bacteria in food, belonging to the field of food detection technology. The probe consists of an organic fluorescent molecular graphene composite matrix, an organic-inorganic hybrid dopant, and a specific recognition molecule. The dopant is carboxylated cellulose and silica nanoparticles, with a mass ratio of 1:6~10 to the composite matrix. The specific recognition molecule is a pathogenic bacteria aptamer, grafted onto the probe surface via spray coating. The probe triggers a fluorescence response by the specific binding of the aptamer to the target pathogenic bacteria, completing detection within 5 minutes with a sensitivity ≥90% and a specificity ≥98%. The probe contains no metal components, posing no toxicity or environmental risk, and is suitable for rapid on-site food detection and live animal detection. Simultaneously, the preparation cost is reduced by 90%, and the cost per sample is reduced by 40%, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a non-metallic, highly sensitive, rapid fluorescent probe, its preparation method, and its application in the detection of pathogenic bacteria in food. Background Technology

[0002] Food safety is fundamental to people's livelihood, and pathogenic bacteria contamination is a core hidden danger in food safety production and distribution. Zoonotic pathogens such as Brucella and Salmonella can be transmitted through the food chain, seriously threatening human and animal health. Currently, the fluorescent probe detection technology widely used in the field of food pathogen detection has several technical drawbacks: First, detection is time-consuming and sample processing is complex, requiring 3-5 hours per test, which cannot meet the needs of rapid on-site screening in scenarios such as farms and food processing plants; second, detection costs are high, with core probes and equipment relying on imports, making it difficult for small and medium-sized farmers and food enterprises to afford; third, traditional fluorescent probes contain metal components, posing a toxicity risk, and nano-solid particles injected into livestock are difficult to metabolize, easily accumulating in the liver and kidneys and causing tissue damage; improper disposal of waste probes can also cause secondary environmental pollution; fourth, detection sensitivity and specificity are insufficient, with traditional probe sensitivity mostly between 50% and 75%, easily affected by interference in complex food systems, resulting in low quantitative accuracy.

[0003] Therefore, there is an urgent need in the field of food testing to develop a rapid, low-cost, non-toxic, and highly sensitive fluorescent probe detection technology to detect pathogenic bacteria in food. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a metal-free, highly sensitive, rapid fluorescent probe, its preparation method, and its application in the detection of pathogenic bacteria in food. It achieves technical effects such as completing detection within 5 minutes, sensitivity ≥90%, specificity ≥98%, a 90% reduction in preparation cost, and a 40% reduction in single-sample detection cost. At the same time, the probe contains no metal components, making it suitable for rapid on-site food detection and live animal detection scenarios, with no toxicity or environmental pollution risks. This invention is achieved through the following technical solution: In a first aspect, the present invention provides a highly sensitive and rapid fluorescent probe without metal toxicity, comprising an organic fluorescent molecule-graphene composite host, an organic-inorganic hybrid dopant, and a specific recognition molecule; The organic-inorganic hybrid dopant is carboxylated cellulose and silica nanoparticles; The mass ratio of the organic-inorganic hybrid dopant to the organic fluorescent molecule-graphene composite host is 1:6~10.

[0005] Furthermore, the mass ratio of organic fluorescent molecules to graphene in the organic fluorescent molecule-graphene composite matrix is ​​5:8.1~9.9; the mass ratio of carboxylated cellulose to silica nanoparticles is 2:2.7~3.3; and the amount of the specific recognition molecule added is 0.5‰~2‰ of the total mass of the organic fluorescent molecule-graphene composite matrix and the organic-inorganic hybrid dopant.

[0006] Furthermore, the organic fluorescent molecule is a TADF organic fluorescent molecule (an organic fluorescent molecule that combines aggregation-induced emission and thermally activated delayed fluorescence properties); the graphene is biocompatible graphene with a purity ≥99.5%; the silica nanoparticles have a particle size of 10~30 nm; the carboxylated cellulose has a molecular weight of 8000~12000; and the specific recognition molecule is a food pathogen-specific aptamer or monoclonal antibody.

[0007] Furthermore, the metal-free, highly sensitive, and rapid fluorescent probe also includes a hydrophilic polyester substrate material, an organic fluorescent molecule-graphene composite host, and an organic-inorganic hybrid dopant loaded on the hydrophilic polyester substrate material.

[0008] In a second aspect, the present invention provides a method for preparing the aforementioned metal-free, highly sensitive, and rapid fluorescent probe, comprising the following steps: (1) Hybrid dispersion: Organic fluorescent molecules, graphene, carboxylated cellulose, and silica nanoparticles were added to deionized water and mixed and dispersed to obtain a uniform and stable mixture; (2) Substrate loading: The mixture obtained in step (1) is coated onto the surface of a hydrophilic polyester substrate using a slot coating process. The wet film thickness after coating is controlled to be 0.2~0.5 mm, and then dried. (3) Microwave-assisted bonding: The coated substrate is placed in a microwave reactor for microwave-assisted bonding to enhance the interfacial bonding stability between organic fluorescent molecules and graphene. (4) Recognition molecule modification: The specific recognition molecule is dissolved in anhydrous ethanol to prepare a solution with a mass concentration of 0.1%~0.5%. The solution is coated onto the microwave-treated substrate surface using a spray coating process. The spray pressure is 0.1~0.3MPa and the coating amount is 0.5~2mL / m². (5) Drying and shaping: The substrate modified with specific recognition molecules in step (4) is dried under constant temperature and normal pressure to obtain a high-sensitivity and fast fluorescent probe without metal toxicity.

[0009] Furthermore, before the hybrid is dispersed, QM / MM technology is used for pre-optimization to obtain the optimal ratio of raw materials: the molecular configuration and energy level structure of TADF organic fluorescent molecules are optimized by QM / MM multi-scale simulation method, and the optimal composite ratio of organic fluorescent molecules and graphene, the optimal ratio of organic-inorganic hybrid dopants and dispersion mode are determined.

[0010] Furthermore, the specific method for QM / MM technology pre-optimization is as follows: The quantum mechanics (QM) part employs density functional theory to perform quantum chemical calculations on the active center (pyridine ring + triphenylamine group) of the TADF organic fluorescent molecule at the B3LYP / 6-31G(d) basis set level. This optimizes the energy level difference between the singlet state S1 and the triplet state T1 from the conventional 0.3–0.4 eV to 0.1–0.2 eV, improving intersystem crossing efficiency and delayed fluorescence quantum yield. The molecular mechanics (MM) part uses molecular dynamics simulations based on GROMACS software to optimize the spatial arrangement of the graphene-organic fluorescent molecule-organic-inorganic hybrid dopant composite system, aligning the luminescent groups of the organic fluorescent molecule towards the graphene surface and reducing nonradiative transition losses of fluorescence energy. Combining the simulation results from both parts, the optimal ratio and dispersion parameters of each component are determined.

[0011] Furthermore, in step (1), the amount of deionized water added is 1 to 10 times the total mass of the organic fluorescent molecules, graphene, carboxylated cellulose and silica nanoparticles.

[0012] Furthermore, the process conditions for microwave-assisted bonding in step (3) are power of 300~800W and processing time of 1~5min; the process conditions for drying in step (5) are temperature of 25~45℃ and drying time of 5~20min, and the relative humidity of the environment is maintained at 30%~50% during the drying process.

[0013] In a third aspect, the present invention provides the application of the aforementioned metal-free, highly sensitive, rapid fluorescent probe in the detection of pathogenic bacteria in food.

[0014] Furthermore, the foodborne pathogens mentioned are one or more of Brucella, Salmonella, Vibrio parahaemolyticus, and Listeria monocytogenes.

[0015] Furthermore, after contacting the sample to be tested with a highly sensitive and rapid fluorescent probe free of metal toxicity, incubating it at room temperature for 2-5 minutes, the pathogenic bacteria are qualitatively or quantitatively detected by color change or fluorescence signal intensity. Furthermore, the specific method for detecting food pathogens using the metal-free, highly sensitive, rapid fluorescent probe prepared according to this invention is as follows: (1) Sample preparation: Liquid food samples (such as raw milk and drinking water) are taken directly without pretreatment; solid / semi-solid food samples (such as meat and meat products) are homogenized and the supernatant is used as the test sample; live animal test samples are blood or mucosal swab extracts. (2) Probe reaction: Immerse the metal-free, high-sensitivity, rapid fluorescent probe prepared in this invention into the test sample, or drop 50 μL of the test sample onto the detection area of ​​the probe test paper and incubate at room temperature for 2-5 min; (3) Detection results: The intensity of the probe fluorescence signal is read by a portable fluorescence detector or the fluorescence color is observed by the naked eye (positive results show obvious fluorescence color, negative results show no fluorescence color); the fluorescence signal intensity is linearly positively correlated with the concentration of pathogenic bacteria, and the detection limit can reach 1 CFU / mL (liquid sample) / 1 CFU / g (solid sample).

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The high-sensitivity rapid fluorescent probe prepared by the present invention, which is free of metal toxicity, can be used to detect pathogens in food. The detection efficiency is greatly improved. The entire detection process only takes 2-5 minutes, which is more than 100 times more efficient than the detection time of 3-5 hours of traditional fluorescent probes. It is suitable for rapid screening scenarios such as farms, food processing plants, and market supervision sites. It can respond quickly to epidemics and avoid economic losses caused by the spread of pathogens. (2) This invention relies on the original QM / MM technology to optimize the luminescence mechanism, reducing the cost of fluorescent probe preparation by 90% and the cost of single sample detection by 40%, eliminating import dependence and meeting the needs of small and medium-sized farmers and small and medium-sized food enterprises. (3) The high-sensitivity rapid fluorescent probe prepared by the present invention is a composite system of organic fluorescent molecules and graphene, without any metal components, which solves the toxicity problems of traditional metal probes such as liver and kidney accumulation and tissue damage. At the same time, the waste probe can be naturally degraded, without the risk of secondary environmental pollution. (4) The high-sensitivity rapid fluorescent probe prepared by the present invention has a sensitivity of ≥90%, a specificity of ≥98%, a good linear relationship, no obvious interference in complex food systems, and a detection limit as low as 1 CFU / mL, which is far below the national standard limit. It can accurately identify pathogenic bacteria contamination in the early stage and avoid batch food contamination recall. (5) The high-sensitivity rapid fluorescent probe prepared by the present invention is easy to operate when detecting pathogenic bacteria in food. The detection process only requires three steps: "sample addition - room temperature incubation - result interpretation". No professional operation training is required. The portable fluorescence detector is suitable for field / on-site use and can also be quickly interpreted by the naked eye, reducing the operation threshold. Attached Figure Description

[0017] Figure 1The graph shows the linear relationship between the fluorescence signal intensity of a highly sensitive and rapid fluorescent probe without metal toxicity and the concentration of Brucella bacteria.

[0018] Figure 2 The graph shows the linear relationship between the fluorescence signal intensity of a highly sensitive and rapid fluorescent probe without metal toxicity and the concentration of Salmonella.

[0019] Figure 3 This is a linear relationship curve between the fluorescence signal intensity of a highly sensitive and rapid fluorescent probe without metal toxicity and the concentration of Vibrio parahaemolyticus.

[0020] Figure 4 The graph shows the linear relationship between the fluorescence signal intensity of a highly sensitive and rapid fluorescent probe without metal toxicity and the concentrations of two pathogenic bacteria. Detailed Implementation

[0021] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0023] In the following examples, the TADF organic fluorescent molecules selected were TADF organic fluorescent molecules with aggregation-induced emission properties (specifically, 2,4,6-triphenylpyridine derivative TPY-TPA, CAS No. 1422211-99-0, purity 99%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product model: T199286), biocompatible graphene (purity 99.5%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product model: G119517), carboxylated cellulose (number average molecular weight 10000, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product model: C110234), silica nanoparticles (particle size 20nm, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product model: S104379), and deionized water; Example 1 Preparation of high-sensitivity, rapid fluorescent probes without metal toxicity: (1) Hybrid dispersion: 5g of TADF organic fluorescent molecules, 9g of biocompatible graphene, 0.8g of carboxylated cellulose, 1.2g of silica nanoparticles and 100mL of deionized water were mixed in a stirring tank, and a laboratory vibratory disperser (model: Shanghai Huxi HX-FS200) was turned on. The vibration frequency was adjusted to 200Hz and dispersed for 5 minutes to obtain a uniform mixture. (2) Substrate loading: The mixture in step (1) was coated onto the surface of a hydrophilic polyester substrate using a laboratory small multi-roller coating machine (model: Suzhou Keying KYT-150) at a roller speed of 5m / min. The wet film thickness after coating was controlled to be 0.3mm, and the thickness of the probe functional coating formed after drying was 0.1mm. (3) Microwave-assisted bonding: The coated substrate was placed in a 500W microwave reactor and microwave bonded for 2 min; (4) Recognition molecule modification: Brucella specific recognition aptamer (Brucella specific recognition aptamer is a commercially available product, purchased from Aochuang Biotechnology (Shandong) Co., Ltd., product number AC-BRU-001, batch number 20240601, purity ≥95%, the product has been verified by affinity and has no cross-reactivity with Brucella) was selected as the specific recognition molecule. 0.02g of the aptamer was dissolved in 20mL of anhydrous ethanol to prepare a 0.1% solution. The solution was uniformly coated on the microwave-treated substrate surface using a small spray coating instrument (pressure 0.2MPa), with a coating amount of 1mL / m². (5) Drying and shaping: The modified substrate in step (4) is dried at 35°C for 10 min and cut into 1cm×3cm test paper, which is a high-sensitivity rapid fluorescent probe without metal toxicity. It is then sealed and stored in a desiccator.

[0024] Example 2 Unlike Example 1, the specific recognition molecule in Example 2 is a Salmonella-specific recognition aptamer (the Salmonella-specific recognition aptamer is a commercially available product, purchased from Shandong Weizhen Biotechnology Co., Ltd., product code WZ-SAL-002, batch number 20240602, purity ≥95%, HPLC purified grade finished product, the sequence has been specifically verified, and there is no cross-reactivity with other bacteria). The remaining steps are the same as in Example 1.

[0025] Example 3 Unlike Example 1, the specific recognition molecule in Example 3 is the Vibrio parahaemolyticus specific recognition aptamer (the Vibrio parahaemolyticus specific recognition aptamer is a commercially available product, purchased from Shandong Shuoboyuan Intelligent Technology Co., Ltd., product code SBY-VP-003, batch number 20240603, purity ≥95%, sequence specificity verified, no cross-reactivity with other bacteria), and the remaining steps are the same as in Example 1.

[0026] Example 4 Unlike Example 1, the specific recognition molecules in Example 4 are a Salmonella-specific recognition aptamer (a commercially available product purchased from Shandong Weizhen Biotechnology Co., Ltd., product code WZ-SAL-002, batch number 20240602, purity ≥95%, HPLC purified grade, sequence verified for specificity, with no cross-reactivity with other bacteria) and a Listeria monocytogenes-specific recognition aptamer (a commercially available product purchased from Qingdao Zhongchuang Huike Biotechnology Co., Ltd., product code ZCHK-LM-004, batch number 20240604, purity ≥95%, a product verified by the National Pathogenic Bacteria Identification Network). These molecules are loaded onto two independent regions of a hydrophilic polyester substrate (the amounts of Salmonella-specific recognition aptamer and Listeria monocytogenes added are the same as those for Brucella-specific recognition aptamer). The remaining steps are the same as in Example 1.

[0027] Application Example 1 Application of the metal-free, highly sensitive, and rapid fluorescent probe prepared in Example 1 in the detection of Brucella in raw milk: (1) Samples: Raw milk samples were selected and divided into negative samples (no Brucella) and positive samples (Brucella concentration 1 CFU / mL, 10 CFU / mL, 100 CFU / mL). (2) Detection: Take 50 μL of each raw milk sample and drop it onto the metal-free, high-sensitivity, rapid fluorescent probe modified with Brucella aptamer prepared by the method in Example 1. Incubate at room temperature for 3 min. Brucella in the sample binds to the aptamer specifically recognized on the probe surface, triggering a fluorescence signal response. The fluorescence signal intensity is detected using a portable fluorescence detector. (3) Detection results: Negative samples showed no fluorescence signal, while positive samples all showed obvious fluorescence signals. The linear relationship curve between the fluorescence signal intensity of the high-sensitivity rapid fluorescent probe without metal toxicity and the concentration of Brucella is shown in the figure below. Figure 1 As shown, the fluorescence signal intensity increases linearly with increasing Brucella concentration, the detection limit is 1 CFU / mL, the concordance rate with the gold standard detection result is ≥99%, and the detection time is 3 min.

[0028] Application Example 2 Application of the metal-free, highly sensitive, and rapid fluorescent probe prepared in Example 2 for detecting Salmonella in fresh meat: (1) Samples: Fresh pork samples were selected, and the supernatant was taken as the test samples after homogenization. They were divided into negative samples and positive samples (Salmonella concentration 1 CFU / mL, 20 CFU / mL, 100 CFU / mL). (2) Detection steps: Take 50 μL of the supernatant of each fresh pork sample and drop it onto the metal-free, high-sensitivity, rapid fluorescent probe modified with Salmonella aptamer prepared by the method in Example 2. Incubate at room temperature for 5 min. The Salmonella in the sample binds to the specific recognition aptamer on the probe surface, triggering a fluorescence signal response. Observe the color development with the naked eye and quantify it with a fluorescence detector. (3) Detection results: Negative samples showed no fluorescence, while positive samples all showed bright fluorescence. The linear relationship curve between the fluorescence signal intensity of the high-sensitivity rapid fluorescent probe without metal toxicity and the concentration of Salmonella is shown in the figure below. Figure 2 As shown, by Figure 2 It was found that the fluorescence signal was linearly correlated with the concentration of Salmonella, with a detection limit of 1 CFU / mL, sensitivity of 95%, specificity of 99%, and detection time of 5 min.

[0029] Application Example 3 Application of the metal-free, highly sensitive, and rapid fluorescent probe prepared in Example 3 in the detection of Vibrio parahaemolyticus in aquatic products: (1) Samples: Fresh shrimp samples were selected, and the supernatant was collected after homogenization. The samples were divided into negative and positive samples (Vibrio parahaemolyticus concentrations of 1 CFU / mL, 10 CFU / mL, and 100 CFU / mL). (2) Detection: Take 50 μL of the supernatant of each shrimp sample and drop it onto the metal-free, high-sensitivity, rapid fluorescent probe modified with Vibrio parahaemolyticus aptamer prepared in Example 3. Incubate at room temperature for 4 min. Vibrio parahaemolyticus in the sample binds to the specific recognition aptamer on the probe surface, triggering a fluorescence signal response. Read the fluorescence signal. (3) Detection results: The linear relationship curve between the fluorescence signal intensity of the high-sensitivity rapid fluorescent probe without metal toxicity and the concentration of Vibrio parahaemolyticus is shown in the figure below. Figure 3 As shown, negative samples showed no signal, while positive samples showed a linear increase in fluorescence signal. The detection limit was 1 CFU / mL, which meets the national standard limit requirements. The detection time was 4 min.

[0030] Application Example 4 Application of high-sensitivity, rapid fluorescent probes without metal toxicity for the simultaneous detection of multiple pathogens in fresh beef: (1) Samples: Fresh beef samples were selected, and the supernatant was taken as the test samples after homogenization. They were divided into negative samples and positive samples (positive samples contained both Salmonella and Listeria monocytogenes, with concentration gradients of 1 CFU / mL, 10 CFU / mL, and 100 CFU / mL). (2) Detection: Take 50 μL of the supernatant of each beef sample and drop it onto the metal-free, high-sensitivity, rapid fluorescent probe array prepared in Example 4 (this step uses the aptamer fluorescent probe array method, which fixes the specific functionalized fluorescent probes for different pathogens into independent array detection sites. The specific binding of the target and the corresponding site aptamer triggers the site fluorescence response, realizing the simultaneous and rapid detection of multiple pathogens in a single sample). Incubate at room temperature for 5 min. The corresponding pathogens in the sample bind to the specific recognition aptamers on the probe surface, triggering a specific fluorescence signal response. Read the fluorescence signal. (3) Detection results: The linear relationship curve between the fluorescence signal intensity of the high-sensitivity rapid fluorescent probe without metal toxicity and the concentration of the two pathogenic bacteria is shown in the figure below. Figure 4 As shown, there was no signal in the negative sample, while the fluorescence signals of the two pathogens in the positive sample increased linearly with the corresponding bacterial concentration. The detection limit was 1 CFU / mL for both samples, with no cross-reactivity, meeting the national standard limit requirements. The detection time was 5 minutes.

[0031] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions I described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A highly sensitive and rapid fluorescent probe without metal toxicity, characterized in that, It includes organic fluorescent molecules-graphene composite hosts, organic-inorganic hybrid dopants, and specific recognition molecules; The organic-inorganic hybrid dopant is carboxylated cellulose and silica nanoparticles; The mass ratio of the organic-inorganic hybrid dopant to the organic fluorescent molecule-graphene composite host is 1:6~10.

2. The metal-free, highly sensitive, rapid fluorescent probe according to claim 1, characterized in that, The mass ratio of organic fluorescent molecules to graphene in the organic fluorescent molecule-graphene composite matrix is ​​5:8.1~9.9; the mass ratio of carboxylated cellulose to silica nanoparticles is 2:2.7~3.3; and the amount of the specific recognition molecule added is 0.5‰~2‰ of the total mass of the organic fluorescent molecule-graphene composite matrix and the organic-inorganic hybrid dopant.

3. The metal-free, highly sensitive, rapid fluorescent probe according to claim 2, characterized in that, The organic fluorescent molecule is a TADF organic fluorescent molecule; the graphene is biocompatible graphene with a purity ≥99.5%; the silica nanoparticles have a particle size of 10~30nm; the carboxylated cellulose has a molecular weight of 8000~12000; and the specific recognition molecule is a food pathogen-specific aptamer or monoclonal antibody.

4. The metal-free, highly sensitive, rapid fluorescent probe according to claim 2, characterized in that, The aforementioned metal-free, highly sensitive, and rapid fluorescent probe also includes a hydrophilic polyester substrate material, an organic fluorescent molecule-graphene composite host, and an organic-inorganic hybrid dopant loaded on the hydrophilic polyester substrate material.

5. A method for preparing a metal-free, highly sensitive, rapid fluorescent probe according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Hybrid dispersion: Organic fluorescent molecules, graphene, carboxylated cellulose, and silica nanoparticles were added to deionized water and mixed and dispersed to obtain a uniform and stable mixture; (2) Substrate loading: The mixture obtained in step (1) is coated onto the surface of a hydrophilic polyester substrate using a slot coating process, and the wet film thickness after coating is controlled to be 0.2~0.5 mm, and then dried; (3) Microwave-assisted bonding: The coated substrate is placed in a microwave reactor for microwave-assisted bonding to enhance the interfacial bonding stability between organic fluorescent molecules and graphene. (4) Recognition molecule modification: The specific recognition molecule is dissolved in anhydrous ethanol to prepare a solution with a mass concentration of 0.1%~0.5%. The solution is coated onto the microwave-treated substrate surface using a spray coating process. The spray pressure is 0.1~0.3MPa and the coating amount is 0.5~2mL / m². (5) Drying and shaping: The substrate modified with specific recognition molecules in step (4) is dried to obtain a high-sensitivity and fast fluorescent probe without metal toxicity.

6. The method for preparing a metal-free, highly sensitive, and rapid fluorescent probe according to claim 5, characterized in that, In step (1), the amount of deionized water added is 1 to 10 times the total mass of the organic fluorescent molecules, graphene, carboxylated cellulose and silica nanoparticles.

7. The preparation method according to claim 5, characterized in that, The process conditions for microwave-assisted bonding in step (3) are power 300~800W and processing time 1~5min; the process conditions for drying in step (5) are temperature 25~45℃ and drying time 5~20min, and the relative humidity of the environment is maintained at 30%~50% during the drying process.

8. The application of a metal-free, highly sensitive, rapid fluorescent probe according to any one of claims 1 to 4, or a metal-free, highly sensitive, rapid fluorescent probe prepared by the method according to claims 5 to 7, in the detection of foodborne pathogens.

9. The application according to claim 8, characterized in that, The foodborne pathogens mentioned are one or more of Brucella, Salmonella, Vibrio parahaemolyticus, and Listeria monocytogenes.

10. The application according to claim 8, characterized in that, After the sample to be tested is in contact with a highly sensitive and rapid fluorescent probe that is free of metal toxicity, it is incubated at room temperature for 2-5 minutes. The qualitative or quantitative detection of pathogenic bacteria is achieved by observing color changes or fluorescence signal intensity.

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