Multifunctional nano-material fluorescence-colorimetric dual-mode food-borne pathogenic bacterium detection and killing method and system

Abstract

The invention discloses a multifunctional nano material fluorescence-colorimetric dual-mode food-borne pathogenic bacterium detection and killing method and a system thereof. According to the method, a metal organic framework material (MIL-53 (Fe)) with nano-enzyme activity is used as a signal switch and a generator, carbon nanodots (CDs) of a fluorescence indication and photo-thermal sterilization unit are combined to form a basic platform, and masking of an aptamer on a nano-enzyme active site and competitive release under triggering of target bacteria are used as a core mechanism. A colorimetric and ratiometric fluorescence double-signal channel is activated to realize self-verification high-precision detection; cDs in the system are used as a photo-thermal element, and in-situ photo-thermal sterilization is realized after detection. The platform design that detection objects can be switched by replacing the aptamers shows good universality and application prospects, and a sensitive and efficient new strategy is provided for detection and elimination of food-borne pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of microbial detection and sterilization technology, specifically to a method and system for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode configuration, achieving dual-mode detection and in-situ sterilization of foodborne pathogens. Background Technology

[0002] The explosive growth of foodborne pathogens urgently necessitates the development of integrated detection and sterilization technologies that combine rapid response capabilities with cost-effectiveness. Currently, numerous patents exist regarding integrated detection-sterilization design methods. For example, patent application 202010708452.0 proposes a nanocomposite material with enzyme-responsive detection and dual antibacterial functions. This nanocomposite material achieves responsive fluorescence detection of bacteria and possesses both photodynamic and photothermal dual-mode sterilization properties. Authorized patent 201980040725.4, through the synthesis of antibiotic compounds with both antibiotic and aggregation-induced emission (AIE) properties, achieves synergistic sterilization by combining drugs and reactive oxygen species. Furthermore, the inherent imaging capabilities of this compound enable its use as an imaging tool for bacterial monitoring. Invention patent 202310083222.3 synthesizes a boric acid-modified AIE nanoparticle that can detect and kill Staphylococcus aureus within 60 min; authorized invention patent 202410481149.X utilizes a photoelectric detection system that combines photoelectrochemical synergistic antibacterial and in-situ bacterial detection to achieve integrated antibacterial and in-situ detection; authorized patent 202311105877.2 synthesizes a near-infrared luminescent cationic free radical bacterial fluorescence imaging antibacterial agent, which can also be used as a fluorescent probe for detecting the interaction between bacteria and immune cells.

[0003] While the aforementioned patents demonstrate good performance in integrated detection and sterilization, it is evident that these inventions employ single-mode signal responses, lacking mutual calibration between signal molecules. In practical applications, they are easily drowned out by background noise and susceptible to non-specific interference from various components in the food matrix, leading to false positive or false negative results. Therefore, developing a detection method capable of providing multiple signal outputs and possessing self-calibration capabilities to resist interference is a pressing technical problem in this field.

[0004] Furthermore, in terms of material selection, MIL-53(Fe), as an environmentally friendly and low-cost metal-organic framework, not only possesses good water stability but is also easily functionalized, thus showing broad application potential in multiplex detection, biosensing, and catalysis. Meanwhile, carbon nanoparticles (CDs) are a class of zero-dimensional carbon nanomaterials with excellent biocompatibility and low toxicity. Based on their superior photoluminescence properties, they exhibit excellent photothermal conversion efficiency through elemental doping of carbon dots. How to effectively integrate the characteristics of these materials to overcome the shortcomings of existing single-mode detection technologies is currently an important research direction. Summary of the Invention

[0005] Based on the above background, this invention utilizes single-stranded DNA aptamers modified on the surface of MIL-53(Fe). Specific base sequences and conformations can specifically recognize foodborne pathogens, thus designing a "switch" for enzyme activity triggered by the target bacteria. This invention fully explores and utilizes the photoluminescent and photothermal properties of CDs, using the different properties of the same material to continuously complete two tasks in the same system, avoiding material complexity while achieving more accurate dual-mode detection and efficient sterilization. MIL-53(Fe), as a signal switch and generator, together with the CDs of the fluorescence indicator and photothermal sterilization unit, constitutes a stable and powerful basic platform. The aptamer, as a flexibly replaceable recognition element, can target and bind to specific bacteria with high affinity. By simply replacing the aptamer, specific detection and killing of the vast majority of foodborne pathogens can be achieved.

[0006] This invention represents a breakthrough in developing a closed-loop probe technology based on aptamer-modified MOF combined with CDs. The aptamer specifically recognizes target bacteria and triggers changes in MOF enzyme activity, simultaneously driving the TMB colorimetric response and the CDs / OPD ratio fluorescence response, greatly simplifying the operation and enabling dual-mode detection. A 655 nm laser excites carbon dots to generate a high temperature of 70 °C and •OH / ¹O2 reactive oxygen species, achieving a 99.9% sterilization rate in 15 minutes. In single-signal detection mode, the introduction of colorimetric-fluorescence dual-mode signal detection endows this invention with powerful practicality and anti-interference capabilities. The colorimetric and fluorescence signals originate from two distinct chemical mechanisms, independent yet mutually reinforcing. When the trends of the two signals are consistent, the results are extremely reliable, fundamentally eliminating false positives or false negatives—a significant advantage that single-mode sensors cannot match. Furthermore, the changes in the colorimetric and fluorescence signals originate from a shared reaction system, eliminating the need for repeated operations or the addition of two different reagents to obtain both signals. In summary, this design completes the entire process of "identification-dual-mode response-sterilization" within a single system, and eliminates the risk of drug resistance through a physical sterilization mechanism with zero chemical addition, achieving green sterilization and providing an innovative solution for food safety.

[0007] This invention provides a multifunctional nanomaterial-based fluorescence-colorimetric dual-mode detection and eradication method and system for foodborne pathogens. It achieves a colorimetric / ratio fluorescence dual-signal response by triggering the MIL-53(Fe) enzyme activity recovery mechanism through target bacteria, enabling signal self-verification and eliminating false positives or false negatives. Utilizing the specificity of foodborne pathogen aptamers, selectivity is enhanced, and the detection range is broadened by modifying different foodborne pathogen aptamers. This system combines dual-signal response, environmental stability, biocompatibility, and anti-interference capabilities, enabling rapid and accurate detection in complex food matrices. It also utilizes the photothermal effect of carbon dots to achieve in-situ green sterilization, and features simple preparation, high sensitivity, strong selectivity, and wide applicability.

[0008] The present invention adopts the following technical solution: A method for detecting and eliminating foodborne pathogens using a dual-mode fluorescence-colorimetric assay using multifunctional nanomaterials, characterized by comprising the following steps: (1) After preparing aptamer-modified MIL-53(Fe) and carbon nanodots, an apt-MIL-53(Fe) / CDs probe was constructed; (2) Mix the sample to be tested with the apt-MIL-53(Fe) / CDs probe solution, incubate, centrifuge and collect the precipitate to obtain the reactant; (3) Add HEPES buffer, TMB solution, H2O2 solution and OPD substrate to the reactants and react. After the reaction is completed, measure the absorbance and fluorescence intensity ratio of the solution, and then compare it with the standard curve of foodborne pathogens to obtain the concentration of foodborne pathogens in the solution. (4) The tested sample is vertically irradiated with a semiconductor laser to achieve in-situ sterilization.

[0009] Preferably, step (1) specifically includes: Preparation of S1 and MIL-53(Fe): Terephthalic acid and ferric chloride hexahydrate were dissolved in N,N-dimethylformamide to obtain mixed solution I; mixed solution I was stirred and heated, cooled and filtered to collect the precipitate, washed and dried under vacuum to obtain MIL-53(Fe); S2. Identification and modification of foodborne pathogens: The aptamer of the foodborne pathogen was dissolved in Tris-EDTA buffer to obtain mixed solution II; after heating and cooling mixed solution II, MIL-53(Fe) solution was added to obtain mixed solution III; mixed solution III was stirred in the dark to obtain aptamer-modified MIL-53(Fe), i.e., apt-MIL-53(Fe); Preparation of S3 and CDs: Citric acid and urea were dissolved in dimethyl sulfoxide to obtain mixed solution IV; mixed solution IV was stirred and heated, mixed with anhydrous ethanol, centrifuged to collect the precipitate, and freeze-dried to obtain CDs; S4. Construction of apt-MIL-53(Fe) / CDs: Mix apt-MIL-53(Fe) and CDs in proportion to construct the apt-MIL-53(Fe) / CDs probe.

[0010] Preferably, in S1, The mass-to-volume ratio of terephthalic acid, ferric chloride hexahydrate, and N,N-dimethylformamide in the mixed solution I is 1.66 g: 2.7 g: 100 mL; the stirring is carried out in a fume hood at a speed of 500 r / min to 600 r / min for 1 to 5 h; the heating temperature is 100 to 200 °C for 10 to 30 h; the washing is performed by washing three times each with N,N-dimethylformamide and ethanol; the vacuum drying temperature is 50 to 100 °C for 10 to 15 h.

[0011] Preferably, in S2, The foodborne pathogen aptamer is an aptamer of at least one of Staphylococcus aureus, Escherichia coli O157:H7, Salmonella, Listeria, or Pseudomonas aeruginosa; the ratio of the foodborne pathogen aptamer to Tris-EDTA buffer is 1OD: 15~30μL. The heating temperature of the mixed solution II is 60 ~ 95 ℃, and the time is 5 ~ 10 min; In the mixed solution III, the volume ratio of MIL-53(Fe) solution to mixed solution II is 1000:1, and the concentration of MIL-53(Fe) solution is 2~5 mg / mL; the stirring time of mixed solution III is 2 h, and it is repeated 3 times.

[0012] Preferably, in S3, The mass-to-volume ratio of citric acid, urea and dimethyl sulfoxide in the mixed solution IV is 2 g: 6 g: 30 mL; The heating temperature is 140~200 ℃, and the heating time is 5~10 h; the centrifugation conditions are 5000~10000 r / min, 5~10 min, repeated 3 times; the freeze-drying conditions are -50~-80 ℃, 24~72 h. In S4, a 3 mg / mL apt-MIL-53(Fe) solution and a 0.4 mg / mL CDs solution were mixed at a volume ratio of 1:1 and stirred for 2–5 h.

[0013] Preferably, the incubation conditions are 30~45℃ for 15~25 min; the centrifugation conditions are 5000~10000 rpm for 10~15 min; and the volume ratio of the sample to be tested to the apt-MIL-53(Fe) / CDs probe solution is 1:1.

[0014] Preferably, step (3) specifically involves: adding 0.1 M HEPES buffer, 110 mM TMB solution, 100 mM H2O2 solution and 2.5 mM OPD substrate sequentially to the reactants and reacting at room temperature for 5-20 min; the volume ratio of HEPES buffer, TMB solution, H2O2 solution and OPD substrate is 75-80:10:2:5. After the reaction, the absorbance of the solution at 652 nm was measured, and the fluorescence signal was measured at 556 nm and 720 nm to obtain the fluorescence intensity ratio (F). 556 / F 720 ).

[0015] Preferably, in step (3), the standard curve of the foodborne pathogen is obtained by the following method: foodborne pathogens with concentration gradients are mixed with apt-MIL-53(Fe) / CDs probe solution, the incubation and centrifugation in step (2) are repeated, and then the reaction is carried out under the same conditions as in step (3). The absorbance at 652 nm is measured, and the fluorescence signal is measured at 556 nm and 720 nm to obtain the fluorescence intensity ratio (F). 556 / F 720 ), to obtain the absorbance standard curve and the fluorescence intensity ratio standard curve of foodborne pathogens.

[0016] Preferably, in step (4), a 655 nm semiconductor laser is used to vertically irradiate the sample for 15 to 25 minutes, with a laser power density of 1 W / cm². 2 The irradiation distance is 8 to 10 cm.

[0017] A multifunctional nanomaterial fluorescence-colorimetric dual-mode foodborne pathogen detection and eradication system based on the method described in this invention, characterized in that it comprises: Probe construction module: used to prepare apt-MIL-53(Fe) / CDs probes; Reaction control module: Performs sample incubation, centrifugation, and substrate addition operations; Signal detection module: Acquires colorimetric and fluorescence signals; Photothermal sterilization module: Provides 655 nm laser irradiation.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) In the prior art, single-mode detection is easily affected by environmental interference, which may lead to false positive or false negative results. In this invention, the dual-channel signals of colorimetry and ratio fluorescence not only verify each other independently, greatly improving the reliability of the detection results, but also the introduction of ratio fluorescence significantly enhances the sensitivity and quantitative accuracy of the detection, overcoming the inherent limitations of single-mode detection.

[0019] (2) After the target bacteria are detected and confirmed, this invention only requires the application of a laser of a specific wavelength. These carbon dots can quickly and efficiently convert light energy into heat energy, rapidly raising the temperature to about 70°C in the local area. This photothermal sterilization method is a physical sterilization mechanism, so it is not easy to induce bacterial drug resistance, and it works quickly, avoiding the secondary pollution or residual toxicity problems that may be caused by traditional chemical sterilization.

[0020] (3) This invention proposes a multifunctional integrated platform, which greatly simplifies the operation process, shortens the total time from detection to sterilization, significantly improves the processing efficiency, and minimizes the risk of contamination and operational errors caused by sample transfer. It provides new strategies and ideas for developing a multifunctional sensing system with targeting, detection and inactivation of bacteria. Attached Figure Description

[0021] Figure 1 The image shows the X-ray diffraction (XRD) pattern of MIL-53 (Fe) synthesized in Example 1 of this invention.

[0022] Figure 2 The UV-Vis absorption spectra of apt-MIL-53(Fe) and MIL-53(Fe) synthesized in Example 1 of this invention are shown.

[0023] Figure 3 The image shows the XRD pattern of CDs synthesized in Example 1 of this invention.

[0024] Figure 4 This is a linear fitting standard curve for the colorimetric signal detected in Embodiment 2 of the present invention.

[0025] Figure 5 F detected in Example 2 of the present invention 556 / F 720 The standard curve for linear fitting of ratio fluorescence signal.

[0026] Figure 6 This is a specificity verification diagram for Example 2 of the present invention. Figure 6 (a) The absorbance at 652 nm after incubation with different strains at the same concentration. Figure 6 (b) F in the detection system after incubation with different strains at the same concentration 556 / F 720 Ratio fluorescence diagram.

[0027] Figure 7 This is a plate diagram showing the colony growth of Staphylococcus aureus under different irradiation times in Example 3 of the present invention.

[0028] Figure 8 This is a plate image of Staphylococcus aureus colony growth on an actual sample from Example 4 of the present invention after 15 minutes of irradiation. Figure 8 (a) is a diagram showing the sterilization effect of simulated polluted tap water. Figure 8 (b) is a diagram showing the sterilization effect of simulated contaminated milk. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative effort are within the scope of protection of the present application.

[0030] The beneficial effects of the present invention are verified using the following embodiments: Example 1 This embodiment provides a method for preparing aptamer-modified MIL-53(Fe) (apt-MIL-53(Fe)) and carbon nanodots (CDs) and constructing apt-MIL-53(Fe) / CDs probes, including the following steps: 1. Preparation of MIL-53(Fe) Weigh 1.66 g of terephthalic acid and 2.7 g of ferric chloride hexahydrate, dissolve them in 100 mL of N,N-dimethylformamide to form a mixed solution. Stir at 550 r / min for 3 h in a fume hood. Transfer the mixed solution to a polytetrafluoroethylene reactor and heat in an oven at 150 ℃ for 24 h. After cooling to room temperature, filter the solution. Wash the precipitate three times each with N,N-dimethylformamide and ethanol. Dry the solid product in a vacuum drying oven at 60 ℃ for 12 h to obtain a yellowish-brown MIL-53(Fe) powder, which is stored at 4 ℃. Figure 1 The XRD pattern of MIL-53(Fe) shows that the 2θ peak positions are 9.18°, 12.59°, 17.56°, 18.15°, and 25.36°, indicating successful synthesis.

[0031] 2. Preparation of apt-MIL-53(Fe) 1 OD Staphylococcus aureus aptamer (sequence: 5'-GCA ATG GTA CGG TAC TTC CTC GGCACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACTTTG CTAA-3') was dissolved in 30 μL Tris-EDTA buffer, and the solution was then heated at 95 °C for 10 min, followed by cooling to room temperature. 10 μL of the aptamer solution was added to 10 mL of ultrapure water to prepare a 3 mg / mL MIL-53(Fe) solution. The mixture was stirred in the dark for 2 h, and this process was repeated three times to obtain the apt-MIL-53(Fe) solution. Figure 2 The image shows a comparison of the UV spectra of apt-MIL-53(Fe) and MIL-53(Fe). Compared with MIL-53(Fe) (i.e., MOF in the attached figure), apt-MIL-53(Fe) shows many absorption peaks between 230 nm and 300 nm, indicating that the aptamer modification was successful.

[0032] 3. Preparation of CDs 2 g of citric acid and 6 g of urea were dissolved in 30 mL of dimethyl sulfoxide, stirred thoroughly, and then transferred to a reaction vessel. The mixture was heated in an oven at 160 °C for 6 h. The dark-colored reaction solution was collected, mixed with 60 mL of anhydrous ethanol, centrifuged at 8000 r / min for 5 min, the supernatant was discarded, and the mixture was washed three times. The precipitate was freeze-dried at -50 °C for 48 h to obtain black CDs powder, which was stored at 4 °C. Figure 3 The XRD pattern of CDs showed two amorphous peaks, indicating successful synthesis.

[0033] 4. Preparation of apt-MIL-53(Fe) / CDs probe Mix 1 mL of 3 mg / mL apt-MIL-53(Fe) solution with 1 mL of 0.4 mg / mL CDs solution. Stir for 2 h to obtain the apt-MIL-53(Fe) / CDs probe solution.

[0034] Example 2 This embodiment provides a procedure for dual-mode detection of Staphylococcus aureus, as detailed below: 1. Preparation of bacterial suspension First, Staphylococcus aureus was streaked onto LB agar and cultured for 24 h to activate the culture. Then, a single colony was picked and inoculated into 5 mL of LB broth and cultured overnight with shaking (37 ℃, 180 r / min). The resulting bacterial suspension was washed with physiological saline, centrifuged at 3200×g, and the OD of the bacterial suspension was adjusted.598 = 0.2, resulting in a bacterial concentration of approximately 10. 9 CFU / mL. Serial dilutions yielded a concentration of 10. 0 ~10 7 Staphylococcus aureus suspension at CFU / mL.

[0035] All experiments were conducted under sterile conditions.

[0036] 2. Sample processing Take 100 μL of probe solution and 100 μL of bacterial culture to be tested (10 0 ~10 7 Mix (CFU / mL), incubate at 37 °C for 25 min, centrifuge at 8000 rpm for 15 min, discard the supernatant, and retain the precipitate as the reactant.

[0037] 3. Dual signal response: 780 μL of 0.1 M HEPES, 100 μL of 10 mM TMB, 20 μL of 100 mM H2O2, and 50 μL of 2.5 mM OPD substrate were added sequentially to the reactants, and the mixture was reacted at room temperature for 10 min.

[0038] 4. Signal Detection and Results: Colorimetric detection: Measure the absorbance at 652 nm and compare it with the standard curve for Staphylococcus aureus (absorbance versus Staphylococcus aureus concentration (10⁻¹⁰)). 0 10 1 10 2 10 3 10 4 10 5 10 6 and 10 7 The logarithm of CFU / mL was compared. Figure 4 The results showed that the higher the absorbance, the higher the concentration of Staphylococcus aureus. Ratio fluorescence mode detection: Fluorescence intensity at 556 nm and 720 nm was measured using a fluorescence spectrophotometer, and F was calculated. 556 / F 720 The ratio, then compared with the standard curve of Staphylococcus aureus (F) 556 / F 720 Ratio and Staphylococcus aureus concentration (10) 0 10 1 10 2 10 3 10 4 10 5 10 6 and 107 The logarithm of (CFU / mL) was compared. Figure 5 The results show that F 556 / F 720 The higher the ratio, the higher the concentration of Staphylococcus aureus; Specificity validation: To evaluate the selectivity of this sensor for Staphylococcus aureus, 100 μL of 10 7 The effectiveness of the sensor was tested in other samples containing Gram-positive strains such as Bacillus subtilis and Gram-negative strains such as Escherichia coli, as well as mixtures, at CFU / mL concentrations. Figure 6 The results in (a) and (b) indicate that the sensor is effective only against Staphylococcus aureus.

[0039] The detection results of Staphylococcus aureus in the colorimetric detection mode in Example 2 are shown in Table 1; Table 1

[0040] The detection results of Staphylococcus aureus under ratiometric fluorescence mode in Example 2 are shown in Table 2; Table 2

[0041] Figure 4 This is the linear fitting standard curve of the colorimetric signal detected in Embodiment 2 of the present invention; Figure 5 F detected in Example 2 of the present invention 556 / F 720 Standard curve for linear fitting of ratio fluorescence signal; Figure 6 This is a specificity verification graph for Example 2. In the graph, a represents the absorbance at 652 nm after incubation with different strains at the same concentration, and b represents the absorbance of F in the detection system after incubation with different strains at the same concentration. 556 / F 720 Ratio fluorescence diagram.

[0042] Example 3 This embodiment provides a process and method for in-situ sterilization operation and effect verification, as follows: All experiments were conducted under sterile conditions; The sample after detection was placed under a 655 nm semiconductor laser (power density 1 W / cm²) at a distance of 10 cm and irradiated vertically for 0 min, 5 min, 10 min, or 15 min. Finally, a concentration of 10 was selected. 7 The bactericidal effect of the bacterial suspension with CFU / mL was verified. The concentration was 10... 7The bacterial suspension at CFU / mL was serially diluted 10-fold, and 100 μL of the suspension was spread onto nutrient agar plates at each irradiation time. After incubation at 37 ℃ for 24 h, the surviving bacteria were counted using standard plate counting. Each group was repeated in triplicate. A blank control was performed using the same method. The killing effect on Staphylococcus aureus is shown in Table 3. Table 3

[0043] Figure 7 This is a plate graph showing the colony growth of Staphylococcus aureus under different irradiation times. From... Figure 7 It can be seen that the irradiation for 15 minutes in Example 3 can kill Staphylococcus aureus by >99.9%.

[0044] Example 4 This embodiment provides a method for detecting and killing Staphylococcus aureus in milk and tap water, as detailed below: Take commercial milk, dilute the milk sample 1000 times, and add Staphylococcus aureus to it until the final concentration is 10. 3 CFU / mL and 10 7 CFU / mL was used as a simulated contaminated milk sample; Staphylococcus aureus was added to laboratory tap water to a final concentration of 10. 3 CFU / mL and 10 7 CFU / mL, used as simulated contaminated tap water.

[0045] 100 μL of simulated contaminated milk and 100 μL of simulated contaminated tap water were respectively mixed with 100 μL of probe solution, incubated at 37 ℃ for 25 min, and then centrifuged at 8000 rpm for 15 min. The supernatant was discarded, and the precipitate was retained as the reactant. 780 μL of 0.1 M HEPES, 100 μL of 10 mM TMB, 20 μL of 100 mM H2O2, and 50 μL of 2.5 mM OPD substrate were added sequentially to the reactant, and the reaction was carried out at room temperature for 10 min. Subsequently, the absorbance at 652 nm was measured, and then... Figure 4 The linear fitting standard curves of the colorimetric signals were compared. Furthermore, the fluorescence intensity at 556 nm and 720 nm was measured using a fluorescence spectrophotometer, and Fo was calculated. 556 / F 720 The ratio, and then with Figure 5 F 556 / F 720The linear fitting standard curves of the ratio fluorescence signals were compared. As shown in Table 4, the recoveries of colorimetric and ratio fluorescence detection for simulated contaminated milk were 94.7% to 108.2% and 97.8% to 105.6%, respectively, while the recoveries of colorimetric and ratio fluorescence detection for simulated contaminated tap water were 98.4% to 107.3% and 96.9% to 101.4%, respectively.

[0046] Table 4

[0047] The amount of Staphylococcus aureus added after testing was 10. 7 Samples with CFU / mL were irradiated with a 655 nm semiconductor laser (power density 1 W / cm²) at a distance of 10 cm for 15 min vertically. Then, 100 μL of the sample was spread onto nutrient agar plates and incubated at 37 ℃ for 24 h. Afterward, the surviving bacteria were counted using standard plate counting. Figure 8 As shown, compared with the unirradiated control group, no bacterial colonies grew on the plates of the irradiated group, indicating that complete sterilization was achieved in tap water and milk. Figure 8 Image 'a' shows the sterilization effect of simulated contaminated tap water. Figure 8 b is a diagram showing the sterilization effect of simulated contaminated milk.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting and killing foodborne pathogens using a dual-mode fluorescence-colorimetric method with multifunctional nanomaterials, characterized in that, Includes the following steps: (1) After preparing aptamer-modified MIL-53(Fe) and carbon nanodots, an apt-MIL-53(Fe) / CDs probe was constructed; (2) Mix the sample to be tested with the apt-MIL-53(Fe) / CDs probe solution, incubate, centrifuge and collect the precipitate to obtain the reactant; (3) Add HEPES buffer, TMB solution, H2O2 solution and OPD substrate to the reactants and react. After the reaction is completed, measure the absorbance and fluorescence intensity ratio of the solution, and then compare it with the standard curve of foodborne pathogens to obtain the concentration of foodborne pathogens in the solution. (4) The tested sample is vertically irradiated with a semiconductor laser to achieve in-situ sterilization.

2. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 1, characterized in that, Step (1) is as follows: Preparation of S1 and MIL-53(Fe): Terephthalic acid and ferric chloride hexahydrate were dissolved in N,N-dimethylformamide to obtain mixed solution I; mixed solution I was stirred and heated, cooled and filtered to collect the precipitate, washed and dried under vacuum to obtain MIL-53(Fe); S2. Identification and modification of foodborne pathogens: The aptamer of the foodborne pathogen was dissolved in Tris-EDTA buffer to obtain mixed solution II; after heating and cooling mixed solution II, MIL-53(Fe) solution was added to obtain mixed solution III; mixed solution III was stirred in the dark to obtain aptamer-modified MIL-53(Fe), i.e., apt-MIL-53(Fe); Preparation of S3 and CDs: Citric acid and urea were dissolved in dimethyl sulfoxide to obtain mixed solution IV; mixed solution IV was stirred and heated, mixed with anhydrous ethanol, centrifuged to collect the precipitate, and freeze-dried to obtain CDs; S4. Construction of apt-MIL-53(Fe) / CDs: Mix apt-MIL-53(Fe) and CDs in proportion to construct the apt-MIL-53(Fe) / CDs probe.

3. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 2, characterized in that, In S1, The mass-to-volume ratio of terephthalic acid, ferric chloride hexahydrate, and N,N-dimethylformamide in the mixed solution I is 1.66 g: 2.7 g: 100 mL; the stirring is carried out in a fume hood at a speed of 500 r / min to 600 r / min for 1 to 5 h; the heating temperature is 100 to 200 °C for 10 to 30 h; the washing is performed by washing three times each with N,N-dimethylformamide and ethanol; the vacuum drying temperature is 50 to 100 °C for 10 to 15 h.

4. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 3, characterized in that, In S2, The foodborne pathogen aptamer is an aptamer of at least one of Staphylococcus aureus, Escherichia coli O157:H7, Salmonella, Listeria, or Pseudomonas aeruginosa; the ratio of the foodborne pathogen aptamer to Tris-EDTA buffer is 1OD: 15~30μL. The heating temperature of the mixed solution II is 60 ~ 95 ℃, and the time is 5 ~ 10 min; In the mixed solution III, the volume ratio of MIL-53(Fe) solution to mixed solution II is 1000:1, and the concentration of MIL-53(Fe) solution is 2~5 mg / mL; the stirring time of mixed solution III is 2 h, and it is repeated 3 times.

5. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 4, characterized in that, In S3, The mass-to-volume ratio of citric acid, urea and dimethyl sulfoxide in the mixed solution IV is 2 g: 6 g: 30 mL; The heating temperature is 140~200 ℃, and the heating time is 5~10 h; the centrifugation conditions are 5000~10000 r / min, 5~10 min, repeated 3 times; the freeze-drying conditions are -50~-80 ℃, 24~72 h. In S4, a 3 mg / mL apt-MIL-53(Fe) solution and a 0.4 mg / mL CDs solution were mixed at a volume ratio of 1:1 and stirred for 2–5 h.

6. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 1, characterized in that, In step (2), the incubation conditions are 30~45℃ for 15~25min; the centrifugation conditions are 5000~10000rpm for 10~15min; and the volume ratio of the sample to be tested to the apt-MIL-53(Fe) / CDs probe solution is 1:

1.

7. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 1, characterized in that, Step (3) specifically involves adding 0.1 M HEPES buffer, 110 mM TMB solution, 100 mM H2O2 solution, and 2.5 mM OPD substrate sequentially to the reactants and reacting at room temperature for 5-20 min; the volume ratio of HEPES buffer, TMB solution, H2O2 solution, and OPD substrate is 75-80:10:2:

5. After the reaction, the absorbance of the solution at 652 nm was measured, and the fluorescence signal was measured at 556 nm and 720 nm to obtain the fluorescence intensity ratio (F). 556 / F 720 ).

8. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode method according to claim 7, characterized in that, In step (3), the standard curve of the foodborne pathogens is obtained by the following method: foodborne pathogens with concentration gradients are mixed with apt-MIL-53(Fe) / CDs probe solution, the incubation and centrifugation in step (2) are repeated, and then the reaction is carried out under the same conditions as in step (3). The absorbance at 652 nm is measured, and the fluorescence signal is measured at 556 nm and 720 nm to obtain the fluorescence intensity ratio (F). 556 / F 720 ), to obtain the absorbance standard curve and the fluorescence intensity ratio standard curve of foodborne pathogens.

9. The method for detecting and killing foodborne pathogens using multifunctional nanomaterials in a fluorescence-colorimetric dual-mode approach according to claim 1, characterized in that, In step (4), a 655 nm semiconductor laser is used to vertically irradiate the sample for 15 to 25 minutes, with a laser power density of 1 W / cm². 2 The irradiation distance is 8 to 10 cm.

10. A multifunctional nanomaterial fluorescence-colorimetric dual-mode foodborne pathogen detection and eradication system based on the method of any one of claims 1 to 9, characterized in that, include: Probe construction module: used to prepare apt-MIL-53(Fe) / CDs probes; Reaction control module: Performs sample incubation, centrifugation, and substrate addition operations; Signal detection module: Acquires colorimetric and fluorescence signals; Photothermal sterilization module: Provides 655 nm laser irradiation.

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