A method for quickly evaluating ultraviolet disinfection efficiency and a detection assembly

Abstract

This invention discloses a rapid evaluation method and detection component for ultraviolet (UV) disinfection efficiency, relating to the field of food microbial disinfection and detection. The method includes: fluorescently labeling a bacterial sample to be tested using a membrane potential difference fluorescent dye to obtain a labeled sample; subjecting the labeled sample to UV treatment, followed by fluorescence detection; and calculating the UV disinfection efficiency of the bacterial sample to be tested based on the obtained fluorescence signal and a preset mathematical model of UV disinfection efficiency. This invention is the first to demonstrate that membrane potential is a core active parameter for evaluating UV disinfection effectiveness, overcoming the shortcomings of traditional membrane permeability or enzyme-active dyes that cannot accurately distinguish between live and dead bacteria within a short time after UV irradiation; and clearly defining an R / G value ≤ 0.74 as the critical threshold for determining that the target pathogen has been inactivated (disinfection is qualified). This invention has significant advantages such as accurate characterization, short measurement time, and the ability to achieve point-of-care testing (POCT) without the need for large instruments.

Description

Technical Field

[0001] This invention relates to the field of food microbial disinfection technology, specifically to a method and detection component for rapid evaluation of ultraviolet disinfection efficiency. Background Technology

[0002] Foodborne illnesses have become a major cause of global health and food safety problems. To reduce the incidence of such incidents, microbial decontamination treatment must be implemented during food production and before consumption. Ultraviolet (UV) technology, due to its lack of toxic byproducts, minimal impact on food sensory characteristics, and low energy consumption, has been widely used in food substrates, food contact surface treatment, and water and air disinfection. However, UV radiation is limited to surface disinfection of solid substrates, and the irradiation power of UV equipment decreases with prolonged use, leading to reduced sterilization efficiency. Therefore, accurate and rapid evaluation of UV disinfection efficiency is crucial.

[0003] Existing methods for evaluating the efficiency of ultraviolet (UV) disinfection mainly include plate culture, ATP assay, and molecular biology methods. Among these, plate culture, considered the "gold standard," requires a culture time of 24-72 hours, a cumbersome process that cannot meet the needs of rapid detection. In recent years, flow cytometry combined with fluorescent active dyes has provided a rapid platform for bacterial detection. However, the inventors discovered in their research that fluorescent dyes commonly used to evaluate bacterial viability (such as the membrane permeability dye SYTO 9 / PI, the intracellular esterase activity dye cFDA, and the transcriptional activity dye Pyronin Y) have serious limitations in evaluating the effectiveness of UV disinfection: although short-term UV irradiation can effectively kill bacteria, the dead cells do not immediately cause significant changes in bacterial membrane permeability, esterase activity, or transcriptional activity within a relatively short period (e.g., within 30 minutes), making these parameters unreliable for assessing the effectiveness of UV disinfection.

[0004] Therefore, there is an urgent need to develop a rapid evaluation method for UV disinfection efficiency that can accurately characterize the bacterial state after UV irradiation, has a short detection time, and is applicable to point-of-care testing (POCT). Summary of the Invention

[0005] To address these issues, this invention provides a rapid evaluation method and detection component for ultraviolet disinfection efficiency based on membrane potential, thereby solving problems such as long detection time, inability to accurately evaluate ultraviolet disinfection efficiency in real time, and complexity of existing detection methods.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides a method for rapid evaluation of ultraviolet disinfection efficiency, the method comprising:

[0008] (1) The bacterial samples to be tested were fluorescently labeled using membrane potential difference fluorescent dyes to obtain labeled samples;

[0009] (2) The labeled sample is subjected to ultraviolet treatment, followed by fluorescence detection. The ultraviolet disinfection efficiency of the bacterial sample to be tested is calculated based on the obtained fluorescence signal and the preset mathematical model of ultraviolet disinfection efficiency.

[0010] The membrane potential difference fluorescent dye can distinguish between dead and surviving bacteria that have been inactivated by ultraviolet light based on changes in bacterial membrane potential.

[0011] Furthermore, the bacterial sample to be tested contains at least one of Staphylococcus lentus, Staphylococcus aureus, Listeria monocytogenes, or Escherichia coli.

[0012] Further, in step (1), the membrane potential difference fluorescent dye includes DiBAC4 (3), DiOC2 (3), DiIC1 (5), FluoVolt, Di-4-ANEPPS, Di-8-ANEPPS, Di-4-ANEPPDHQ, Di-2-ANEPEQ, Di-3-ANEPPDHQ, RH414, RH421, and RH795;

[0013] The conditions for fluorescent labeling are: bacterial concentration 1×10⁻⁶. 5 -1×10 8 CFU / mL, final concentration of membrane potential difference fluorescent dye 1-100μM, incubate at 10-40℃ in the dark for 1-60 minutes.

[0014] The preferred membrane potential difference fluorescent dye is DiOC2(3). This dye emits red fluorescence when it aggregates at high concentrations in live bacteria, while it exists as a monomer in dead bacteria that have lost membrane potential due to ultraviolet inactivation and emits green fluorescence. This is beneficial for quickly and accurately evaluating the efficiency of ultraviolet disinfection based on the relative intensity and ratio changes of red and green signals.

[0015] Further, in step (2), the conditions for ultraviolet treatment are: a disinfection wavelength of 254 nm or 222 nm, and an ultraviolet irradiation dose of 1-100 mJ / cm². 2 .

[0016] Furthermore, the evaluation method is a laboratory quantitative detection method, and step (2) includes:

[0017] The UV-treated sample was placed in a multi-functional microplate reader for fluorescence detection to obtain the peak fluorescence intensity Y (in au). The UV disinfection efficiency X (in %) was then calculated by substituting the values ​​into the following exponential fitting function:

[0018] Y = 95755.49243×e (X / 16.16984) +15909500

[0019] The detection parameters of the multifunctional microplate reader are: excitation wavelength 488 nm and emission wavelength 500-650 nm.

[0020] Furthermore, the evaluation method is an on-site real-time detection method, and step (2) includes:

[0021] Before the ultraviolet treatment, the labeled sample was loaded onto a porous biomaterial to remove free liquid. After the ultraviolet treatment, the sample was irradiated with an external fluorescence excitation device and the fluorescence image was acquired by an image acquisition device.

[0022] Colorimetric analysis was performed on the fluorescence image to obtain the dimensionless ratio of the red channel value to the green channel value, R / G. This value was then substituted into the following fitting function to calculate the ultraviolet disinfection efficiency X, in % (%).

[0023] Y = -1809.54955+1810.48031 / [1+e (X-189.27173) / 11.76 ],

[0024] The excitation wavelength of the external fluorescence excitation device is 488 nm.

[0025] Furthermore, the porous biomaterial is a mixed cellulose ester membrane with a pore size of 0.22 or 0.45 μm;

[0026] The image acquisition device is a smartphone camera, used in conjunction with a 500 nm long-pass filter.

[0027] Furthermore, the method also includes step (3): when the R / G value of the measured fluorescence image is ≤0.74, it is determined that the ultraviolet disinfection efficiency of the bacterial sample to be tested has reached the sterilization standard or is qualified for disinfection.

[0028] According to a second aspect of the present invention, the present invention provides a detection component for rapid evaluation of ultraviolet disinfection efficiency, characterized in that it comprises:

[0029] Porous biomaterial substrates;

[0030] And a membrane potential difference fluorescent dye preloaded or to be loaded onto the substrate;

[0031] The detection component is used to perform on-site real-time detection and disinfection qualification determination as described in any of the preceding items.

[0032] The embodiments of the present invention have the following advantages:

[0033] (1) High accuracy: This invention is the first to confirm and utilize the characteristic that "ultraviolet irradiation will immediately destroy the bacterial membrane potential", and screen out the membrane potential as the core active parameter for evaluating the ultraviolet disinfection effect, overcoming the technical bias that traditional membrane permeability or enzyme-active dyes cannot accurately distinguish between live and dead bacteria in a short time after ultraviolet irradiation.

[0034] (2) The present invention has a fast detection speed, and the entire evaluation process can be completed within 30 minutes, which greatly shortens the detection cycle.

[0035] (3) Wide range of applicable scenarios, supports on-site POCT and clear judgment criteria: The innovative design of test strips based on MCE membrane and image colorimetric (R / G value) analysis method has freed us from the dependence on large flow cytometers; In particular, R / G≤0.74 has been clearly defined as a conservative and reliable critical threshold for complete sterilization, providing a clear quantitative standard for non-professionals to make immediate and intuitive judgments on disinfection qualification on-site (such as canteens, hospitals, markets, laboratories), which has extremely high engineering application value. Attached Figure Description

[0036] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0037] Figure 1 The following is a flow cytometry analysis result of four fluorescent dyes used in Example 1 to distinguish between live and dead bacteria after heat treatment: a is SYTO9 / PI; b is Pyronin Y; c is DiOC2(3); d is cFDA.

[0038] Figure 2 This is a flow cytometry analysis result of four fluorescent dyes used in Example 2 to distinguish bacteria before and after ultraviolet irradiation.

[0039] Figure 3 For the comparison of counting results of flow cytometry (FCM) and plate counting (PCM) in Example 3, A is a bar chart; B is a linear fitting curve.

[0040] Figure 4The flow cytometry results of DiOC2(3) in Example 4 distinguishing different bacteria under heat treatment and UV treatment are as follows: a is Staphylococcus albus; b is Staphylococcus aureus; c is Listeria monocytogenes; d is Escherichia coli.

[0041] Figure 5 The results of the analysis on the correlation between fluorescence intensity and disinfection efficiency in Example 5 are shown in Figure a. Fluorescence emission spectra of bacterial solutions with different activity levels are shown in Figure b. Fluorescence intensity-disinfection efficiency fitting curve is shown in Figure b.

[0042] Figure 6 The fitting result between the R / G value of the MCE filter membrane and the disinfection efficiency calculated by the plate counting method in Example 6;

[0043] Figure 7 To verify the ability of paper-based sensors to evaluate the UV disinfection effect in Example 7, a shows the color change of the sensor at different UV treatment times; b is a comparison chart of the disinfection effect measured using the plate method and the measurement using paper-based sensors. Detailed Implementation

[0044] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1

[0046] To investigate whether membrane integrity, intracellular enzyme activity, membrane potential, and transcriptional activity can be used to accurately characterize cell activity, this embodiment selected membrane integrity dye SYTO9 / PI, intracellular enzyme activity dye cFDA, membrane potential difference dye DiOC2(3), and transcriptional activity dye Pyronin Y, respectively, and used a flow cytometer to study their ability to distinguish between live and dead bacteria.

[0047] I. Materials and Methods

[0048] 1. Staphylococcus lentus, CICC 10897, purchased from the China Industrial Microbial Culture Collection Center, is a disinfection indicator bacterium in the "Disinfection Technical Specifications 2002 Edition".

[0049] 2. The green specific nucleic acid dye SYTO9 was purchased from Thermol Fisher.

[0050] Propidium iodide (PI), a bacterium-specific dye that can penetrate damaged cell membranes, was purchased from Thermol Fisher.

[0051] cFDA, an intracellular enzyme activity-specific dye, was purchased from Thermol Fisher.

[0052] The cell membrane potential difference labeling dye DiOC2(3) was purchased from Thermol Fisher.

[0053] The RNA-specific dye Pyronin Y was used to label transcriptional activity and was purchased from Thermol Fisher.

[0054] 3. Flow cytometry

[0055] Staphylococcus albus was activated in nutrient broth (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride) for 5 h to reach mid-log phase. The bacterial suspension was collected by centrifugation at 8000 g for 5 min and washed twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8 (cells / mL). Simultaneously, a portion of the live bacterial suspension was treated at 80℃ for 10 min to prepare a dead bacterial suspension (approximately 10 cells / mL). 8 (cells / mL).

[0056] The bacterial suspensions were incubated with 5 μM SYTO9 and 2 μg / mL PI at 37°C in the dark for 15 min, respectively; with 200 μM cFDA at 37°C in the dark for 30 min; with 30 μM DiOC2(3) at room temperature in the dark for 30 min; and with Pyronin Y at 4°C in the dark for 20 min. After incubation, the suspensions were centrifuged at 8000 g for 5 min, washed once with PBS, and analyzed using an Apogee A50 flow cytometer.

[0057] The flow cytometer parameters are as follows: excitation wavelength 488 nm, analysis flow rate 5-20 μL / min, detection time 10-100 s, and analysis of different live-dead bacterial solutions and corresponding measured live-dead Staphylococcus aureus areas through red-green dual fluorescence channels.

[0058] II. Experimental Results

[0059] See results Figure 1 By comparing the volume ratio added to the bacterial solution with the volume ratio detected by the corresponding flow cytometer, it was found that all four cell activity dyes could clearly distinguish between live and dead bacteria. This result indicates that flow cytometry combined with cell activity characterization dyes can quickly and accurately analyze the number of live bacteria in the sample, verifying the ability of these four activity dyes to distinguish between live and dead bacterial homogenates after conventional heat treatment.

[0060] Example 2

[0061] To further explore whether membrane integrity, intracellular enzyme activity, membrane potential and transcriptional activity can be used to accurately characterize cell viability after UV treatment, this example also selected membrane integrity dye SYTO9 / PI, intracellular enzyme activity dye cFDA, membrane potential difference dye DiOC2(3) and transcriptional activity dye Pyronin Y, respectively, and carried out a study on their ability to distinguish between live and dead bacteria after UV treatment using a flow cytometer.

[0062] I. Materials and Methods

[0063] 1. Staphylococcus lentus, CICC 10897, purchased from the China Industrial Microbial Culture Collection Center, is a disinfection indicator bacterium in the "Disinfection Technical Specifications 2002 Edition".

[0064] 2. Flow cytometry

[0065] Staphylococcus albus was activated in nutrient broth medium for 5 h to the mid-log phase, centrifuged at 8000 g for 5 min to collect the bacterial suspension, and washed twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8 1 mL of bacterial suspension was incubated with 5 μM SYTO9 and 2 μg / mL PI in the dark for 15 min, then with 200 μM cFDA at 37°C in the dark for 30 min, then with 30 μM DiOC2(3) at room temperature in the dark for 30 min, and finally with Pyronin Y at 4°C in the dark for 20 min. After incubation, the suspension was centrifuged at 8000 g for 5 min and washed once with PBS. Subsequently, a portion of the stained bacterial suspension was irradiated with ultraviolet light to prepare a dead bacterial suspension (approximately 10 cells / mL). 8 (cells / mL). Measurements were performed using an Apogee A50 flow cytometer.

[0066] Among them, the dead bacteria in the bacterial solution were obtained using an irradiance of 0.155 W / m². 2 Irradiation with 222 nm ultraviolet light for 30 min (27.9 mJ / cm²) 2 The flow cytometer parameters were: excitation wavelength 488 nm, analysis flow rate 5-20 μL / min, detection time 10-100 s, acquisition of red and green dual fluorescence channel signals, and analysis of bacterial solutions before and after ultraviolet irradiation and the corresponding measured live and dead areas of Staphylococcus aureus.

[0067] II. Experimental Results

[0068] The results are as follows Figure 2As shown, apart from the membrane potential dye DiOC2(3), the other three fluorescent dyes failed to clearly distinguish between UV-irradiated and unirradiated bacterial populations. These results indicate that although 30 min of UV irradiation effectively kills bacteria, dead cells retain certain survival-related characteristics. Specifically, death caused by abnormal DNA replication does not immediately cause changes in bacterial membrane permeability, esterase activity, or transcriptional activity, making these parameters unreliable for assessing the effectiveness of UV disinfection over a relatively short period. The results of this example validate the ability of membrane potential dyes to evaluate the effectiveness of UV disinfection.

[0069] Example 3

[0070] Because flow cytometry relies on automated analysis of bacterial suspensions, while plate counting relies on manual plating and culturing followed by counting, the two methods differ in their detection principles and operational procedures. To verify the consistency of the counting results of the two methods on bacterial suspensions after UV irradiation and to further ensure the reliability of the experimental data, a control experiment comparing plate counting and flow cytometry was added.

[0071] I. Materials and Methods

[0072] 1. Staphylococcus lentus, CICC 10897, purchased from the China Industrial Microbial Culture Collection Center, is a disinfection indicator bacterium in the "Disinfection Technical Specifications 2002 Edition".

[0073] 2. Analytical Methods

[0074] Staphylococcus albus was activated in nutrient broth medium (to mid-log phase 5 h), centrifuged at 8000 g for 5 min to collect the bacterial suspension, and washed twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8 First, 15 mL of live bacterial suspension was incubated with 30 μM DiOC2(3) at room temperature in the dark for 30 min. After incubation, the suspension was centrifuged at 8000 g for 5 min and washed once with PBS. 15 mL of the bacterial suspension was then transferred to a sterile plastic petri dish and subjected to the aforementioned standard conditions for ultraviolet irradiation (irradiance of 0.155 W / m²). 2 The samples were irradiated with 222 nm ultraviolet light for 0, 1, 3, 5, 7, and 10 min, with three parallel samples at each time point. Unstained viable bacterial suspensions were simultaneously subjected to the same ultraviolet irradiation treatment. After irradiation, sample preparation and detection using two counting methods were performed simultaneously. Measurements and analyses were conducted using an Apogee A50 flow cytometer, and the plate count method was used to analyze the viable bacterial count of unstained viable bacteria after ultraviolet irradiation.

[0075] Live and dead bacterial suspensions were serially diluted 10-fold. 100 μL of the appropriately diluted bacterial suspension was evenly spread on nutrient agar plates (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 20 g / L agar). The plates were incubated at 37°C for 24 h. Single colonies that grew on the plates were counted manually and multiplied by the dilution factor to calculate the number of live bacteria in the sample.

[0076] II. Experimental Results

[0077] The example demonstrates how adjusting the UV irradiation time modulates the degree of bacterial inactivation to obtain bacterial culture samples with gradient activities. Linear regression analysis was performed on the detection results of the two counting methods to... Figure 3 The fitted curves clearly show a good linear relationship between flow cytometry cell counting results and plate counting results, with a correlation coefficient R0. 2 Greater than 0.99.

[0078] The results clearly demonstrate that instrumental flow cytometry and manual plate counting methods exhibit a significant and good linear correlation for bacterial suspensions with different activities. Both methods can accurately reflect the number of viable bacteria in the bacterial suspension and can serve as effective quantitative methods for subsequent evaluation of ultraviolet disinfection effects, providing methodological support for the reliability of data in subsequent disinfection experiments.

[0079] Example 4

[0080] To verify that membrane potential dyes can also be used as core activity parameters for evaluating the disinfection efficiency of ultraviolet light against foodborne pathogens, this embodiment uses membrane potential dye DiOC2(3) to evaluate the disinfection of three foodborne pathogens and verify the universality of the method.

[0081] I. Materials and Methods

[0082] 1. *Staphylococcus aureus*, ATCC 29213, purchased from the China General Microbiological Culture Collection Center. 2. *Listeria monocytogenes*, ATCC 19111, purchased from the China General Microbiological Culture Collection Center. 3. *Escherichia coli*, ATCC 10798, a representative strain of foodborne pathogens, purchased from the China General Microbiological Culture Collection Center.

[0083] 2. Flow cytometry

[0084] Staphylococcus albus, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were activated in nutrient broth for 5 h to mid-log phase. The bacterial suspension was collected by centrifugation at 8000 g for 5 min and washed twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8 Simultaneously, a portion of the live bacterial suspension was subjected to UV treatment at 222 nm for 30 min and heat treatment at 80℃ for 10 min, respectively, to prepare a dead bacterial suspension (approximately 10 cells / mL). 8 (cells / mL) to facilitate comparison between the two.

[0085] Each bacterial culture was incubated with 30 μM DiOC2(3) at room temperature in the dark for 30 min. After incubation, the culture was centrifuged at 8000 g for 5 min, washed once with PBS, and analyzed using an Apogee A50 flow cytometer.

[0086] The flow cytometer parameters are: excitation wavelength 488 nm, analysis flow rate 5-20 μL / min, detection time 10-100 s, and analysis of bacterial solutions in different live and dead states and the corresponding measured live and dead bacterial regions through red and green dual fluorescence channels.

[0087] II. Experimental Results

[0088] See results Figure 4 After DiOC2(3) staining, live cells and dead cells (treated by heat and UV) were able to achieve good separation. Furthermore, the fluorescence populations of cells treated by heat and UV largely overlapped, indicating that the membrane potential of bacteria treated by these two sterilization methods underwent significant changes. In conclusion, the ability of membrane potential dyes to evaluate the effectiveness of UV disinfection has been further validated and is applicable to a variety of foodborne pathogens.

[0089] Example 5

[0090] To determine the correlation between fluorescence intensity and disinfection efficiency, we considered the characteristic that increased UV disinfection efficiency leads to an increase in the number of inactivated bacteria. During UV disinfection, the green fluorescence intensity in the DiOC2(3) detection system increased. The fluorescence intensity was measured using an ELISA reader to obtain the fluorescence spectra corresponding to different concentrations and ratios of live and dead bacteria. The UV disinfection efficiency was then calculated using a functional relationship.

[0091] I. Materials and Methods

[0092] 1. Activate *S. lentus* in nutrient broth for 5 h to mid-log phase, centrifuge at 8000 g for 5 min to collect the bacterial suspension, and wash twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8Cells / mL). A portion of the live bacterial suspension was treated at 80°C for 10 min to prepare a dead bacterial suspension (approximately 10 cells / mL). 8 S. lentus was prepared with live-to-dead cell ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 (corresponding to disinfection efficiencies of 0%–100%). Each group of bacteria was dispersed in 1 mL of PBS to simulate the decrease in live bacteria and increase in dead bacteria during actual disinfection.

[0093] Add 30 μM DiOC2(3) to the above solution, mix thoroughly, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 8000 g for 5 min, wash once with PBS, and resuspend to ensure uniform bacterial concentration before instrument analysis.

[0094] 2. Fluorescence analysis

[0095] Add 200 μL of bacterial suspension to an opaque 96-well plate for fluorescence detection and analysis.

[0096] The excitation wavelength of the microplate reader was set to 488 nm. Fluorescence emission spectra were recorded in the wavelength range of 500-650 nm, and the fluorescence intensity of the peak at 512 nm was measured.

[0097] II. Experimental Results

[0098] Fluorescence spectra of bacterial solutions under different disinfection efficiencies are shown in the figure. Figure 5 (a) As the disinfection efficiency (%) gradually increases, the peak value of its fluorescence spectrum at around 510 nm gradually increases. A fitting curve was constructed with disinfection efficiency as the x-axis and fluorescence intensity at 510 nm as the y-axis; the fitting curve is shown in [Figure number missing]. Figure 5 (b) The results showed that there was a good exponential correlation between fluorescence intensity and disinfection efficiency, with a correlation coefficient R0. 2 Greater than 0.98.

[0099] Based on the functional relationship, the corresponding disinfection efficiency against S. lentus can be obtained by measuring the fluorescence intensity. Then, in accordance with the guidance of the "Disinfection Technical Specifications 2002 Edition", the disinfection effect of ultraviolet light is evaluated using S. lentus as an indicator strain.

[0100] Example 6

[0101] To enable convenient point-of-care testing (POCT) of UV disinfection effectiveness without relying on large-scale laboratory analysis, this embodiment develops a bacterial strip for evaluating UV disinfection effectiveness. By analyzing the RGB values ​​of the captured fluorescence images, a semi-quantitative evaluation of UV disinfection efficiency can be achieved.

[0102] I. Materials and Methods

[0103] 1. Staphylococcus lentus, CICC 10897, purchased from the China Industrial Microbial Culture Collection Center, is a disinfection indicator bacterium in the "Disinfection Technical Specifications 2002 Edition".

[0104] 2. Preparation of fungal tablets

[0105] S. lentus was activated in nutrient broth for 5 h to mid-log phase, centrifuged at 8000 g for 5 min to collect the bacterial suspension, and washed twice with sterile phosphate-buffered saline (PBS) to prepare a live bacterial suspension (approximately 10 μL). 8 (cells / mL). A portion of the live bacterial suspension was treated at 80℃ for 10 min to prepare a dead bacterial suspension. *S. lentus* was prepared according to a live-to-dead cell ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, and each group of bacteria was evenly dispersed in 1 mL of PBS. 500 μL of each bacterial suspension stained with 30 μM DiOC2(3) was slowly added to an MCE filter membrane lined with filter paper, ensuring even distribution of the bacterial suspension. The filter paper was used to absorb the liquid, leaving fluorescently labeled bacteria on the MCE filter membrane. The disinfection efficiency was calculated by recording the RGB values ​​on the membrane and the plate count results, establishing a correlation between the two.

[0106] Take a color photo of the paper-based sensor with your mobile phone, and then use the built-in Color Picker APP or ImageJ on your phone to convert the colors to RGB values. The focus is on extracting the red (R) value and the green (G) value, and calculating the R / G relative value.

[0107] II. Experimental Results

[0108] See results Figure 6 The R / G value was calculated by analyzing the RGB values ​​of the fluorescence image. A fitted curve with disinfection efficiency as the x-axis and R / G value as the y-axis was obtained: y = -1809.54955 + 1810.48031 / [1 + e] (x-189.27173) / 11.76 Correlation coefficient R 2 Greater than 0.99. Therefore, this sensor can be used to evaluate the UV disinfection effect of actual samples: the bacterial solution stained with DiOC2(3) was dropped onto the MCE filter membrane to prepare the sensor. After the sensor and the real sample to be disinfected were exposed to UV irradiation at the same time, the RGB value and R / G value of the bacterial sheet were recorded by passing the filter through the mobile phone camera. Combined with the above fitting curve, the UV irradiation under this condition can be evaluated in real time.

[0109] Example 7

[0110] To verify the ability of the developed paper sensor based on mycelium sheets to evaluate the effectiveness of ultraviolet disinfection in real food samples, this embodiment uses artificially contaminated lettuce and compares the disinfection efficiency measured by mycelium sheets with the disinfection efficiency calculated by plate counting.

[0111] The disinfection efficiency is calculated as follows: Disinfection efficiency = (Number of bacteria before UV disinfection - Number of bacteria after UV disinfection) / Number of bacteria before UV disinfection × 100%.

[0112] I. Materials and Methods

[0113] 1. Lettuce was used as the actual food sample in this example (purchased from a local supermarket in Beijing). The lettuce was refrigerated at 4°C. After being treated at 121°C for 15 minutes, the sample was cut into uniform sizes suitable for artificial contamination. 500 μL of 10... 8 A solution of Staphylococcus aureus, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes at a concentration of [cells / mL] was applied to the surface of a food matrix and dried at 37°C for 2 hours. The solution was then subjected to an irradiation of 0.155 W / m². 2 After 20 minutes of irradiation with 222 nm ultraviolet light, the food samples were recovered with PBS and plate counts were performed. The disinfection efficiency was then calculated.

[0114] 2. By establishing a functional relationship between R / G value and disinfection efficiency, the fluorescence signal of the captured bacterial flakes is converted into disinfection efficiency.

[0115] Plate counting method:

[0116] The recovered bacterial suspension was serially diluted 10-fold. 100 μL of the appropriately diluted bacterial suspension was evenly spread on nutrient agar plates and incubated at 37°C for 24 h. The number of single colonies that grew on the plates was counted manually and multiplied by the dilution factor to calculate the number of viable bacteria in the sample.

[0117] The disinfection efficiency obtained from plate counting is calculated as follows: Disinfection efficiency = (number of bacteria before UV disinfection - number of bacteria after UV disinfection) / number of bacteria before UV disinfection × 100%.

[0118] II. Experimental Results

[0119] Figure 7The results showed that during the 20-minute UV exposure, the disinfection efficiency detected by the paper-based sensor gradually increased, while the number of bacteria in the exposed lettuce samples gradually decreased, which translates to a gradual increase in disinfection efficiency. Notably, during the 20-minute detection period of the paper-based sensor, the UV disinfection efficiency against bacteria reached saturation (log kill value greater than or equal to 3), corresponding to an R / G ratio of 0.74 for the paper-based bacterial strip. Figure 7 (b) Arrow markings. The UV efficiency evaluation method constructed in this embodiment can efficiently detect bacterial activity after UV disinfection. In the UV disinfection efficiency detection method of this invention, the final R / G value of the detected bacterial strip is determined to be below 0.74 as qualified.

[0120] This embodiment demonstrates that although the paper-based sensor developed and designed in this invention does not provide a precise value for disinfection efficiency, its detection principle and performance trend make it a promising candidate for a wide range of methods for evaluating ultraviolet disinfection efficiency. Applying this technical feature to the rapid ultraviolet disinfection efficiency method of this invention enables real-time detection of ultraviolet disinfection efficiency, significantly improving the detection efficiency and expanding the application scenarios for ultraviolet disinfection efficiency detection.

[0121] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A rapid evaluation method for ultraviolet disinfection efficiency, characterized in that, The method includes: (1) The bacterial samples to be tested were fluorescently labeled using membrane potential difference fluorescent dyes to obtain labeled samples; (2) The labeled sample is subjected to ultraviolet treatment, followed by fluorescence detection. The ultraviolet disinfection efficiency of the bacterial sample to be tested is calculated based on the obtained fluorescence signal and the preset mathematical model of ultraviolet disinfection efficiency. The membrane potential difference fluorescent dye can distinguish between dead and surviving bacteria that have been inactivated by ultraviolet light based on changes in bacterial membrane potential.

2. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 1, characterized in that, The bacterial sample to be tested contains at least one of Staphylococcus lentus, Staphylococcus aureus, Listeria monocytogenes, or Escherichia coli.

3. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 1, characterized in that, In step (1), The membrane potential difference fluorescent dyes include DiBAC4(3), DiOC2(3), DiIC1(5), FluoVolt, Di-4-ANEPPS, Di-8-ANEPPS, Di-4-ANEPPDHQ, Di-2-ANEPEQ, Di-3-ANEPPDHQ, RH414, RH421, and RH795. The conditions for fluorescent labeling are: bacterial concentration 1×10⁻⁶. 5 -1×10 8 CFU / mL, final concentration of membrane potential difference fluorescent dye 1-100μM, incubate at 10-40℃ in the dark for 1-60 minutes.

4. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 1, characterized in that, In step (2), the conditions for ultraviolet treatment are: a disinfection wavelength of 254 nm or 222 nm, and an ultraviolet irradiation dose of 1-100 mJ / cm². 2 .

5. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 1, characterized in that, The evaluation method is a laboratory quantitative detection method, and step (2) includes: The UV-treated sample was placed in a multi-functional microplate reader for fluorescence detection to obtain the peak fluorescence intensity Y (in au). The UV disinfection efficiency X (in %) was then calculated by substituting the values ​​into the following exponential fitting function: Y = 95755.49243×e (X / 16.16984) +15909500, The detection parameters of the multifunctional microplate reader are: excitation wavelength 488 nm and emission wavelength 500-650 nm.

6. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 1, characterized in that, The evaluation method is an on-site real-time detection method, and step (2) includes: Before the ultraviolet treatment, the labeled sample was loaded onto a porous biomaterial to remove free liquid. After the ultraviolet treatment, the sample was irradiated with an external fluorescence excitation device and the fluorescence image was acquired by an image acquisition device. Colorimetric analysis was performed on the fluorescence image to obtain the dimensionless ratio (R / G) of the red channel values ​​to the green channel values. This value was then substituted into the following fitting function to calculate the ultraviolet disinfection efficiency (X), in percentage (%). Y = -1809.54955+1810.48031 / [1+e (X-189.27173) / 11.76 ], The excitation wavelength of the external fluorescence excitation device is 488 nm.

7. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 6, characterized in that, The porous biomaterial is a mixed cellulose ester membrane with a pore size of 0.22 or 0.45 μm; The image acquisition device is a smartphone camera, used in conjunction with a 500 nm long-pass filter.

8. The rapid evaluation method for ultraviolet disinfection efficiency according to claim 6, characterized in that, The method further includes step (3): When the R / G value of the measured fluorescence image is ≤0.74, the ultraviolet disinfection efficiency of the bacterial sample to be tested is determined to meet the sterilization standard or be qualified for disinfection.

9. A detection component for rapid evaluation of ultraviolet disinfection efficiency, characterized in that, include: Porous biomaterial substrates; And a membrane potential difference fluorescent dye preloaded or to be loaded onto the substrate; The detection component is used to perform on-site real-time detection and disinfection qualification determination as described in any one of claims 6-8.

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