A bacterial biofilm detection probe, detection method and application

Abstract

The application discloses a bacterial biofilm detection probe, a detection method and application, relates to the technical field of microorganism detection and fluorescent probe application, and the fluorescent probe is of formula Ia structure. The bacterial biofilm detection probe detection method comprises the following steps: bacterial culture to form a biofilm, PBS removal of planktonic bacteria, addition of the probe incubation for 5 min, and detection of 520 nm fluorescence intensity by using a fluorescence enzyme label instrument. The application applies the fluorescent probe to biofilm detection, compared with traditional classic biofilm detection methods such as crystal violet, has the advantages of no need of cleaning, short detection period, simple operation, high accuracy, strong linear correlation, adaptation to gram-positive / negative bacteria and fungal biofilm, and provides an effective tool for detection of bacterial biofilm. The application can be used for high-throughput screening of active drugs with inhibition of biofilm formation or destruction of bacterial biofilm structure, and solves the technical problems of complicated steps, long time consumption and poor reproducibility in the prior art.

Description

Technical Field

[0001] This invention relates to the field of microbial detection and fluorescent probe application technology, specifically to a bacterial biofilm detection probe, detection method, and application. Background Technology

[0002] Biofilms are three-dimensional microbial communities formed by bacteria themselves, consisting of an extracellular polymer matrix. They are composed of fibrin, extracellular DNA, nucleic acids, and extracellular polysaccharides. These components intertwine to construct a complex three-dimensional network structure that encapsulates the bacteria. Compared to planktonic bacteria, bacteria within biofilms can exhibit 10-1000 times greater resistance to antimicrobial drugs. Drug-resistant bacterial infections have become a major global public health threat, with over 80% of chronic human infections directly related to biofilms. Approximately 700,000 people die globally each year from drug-resistant bacterial infections, a number projected to reach 10 million by 2050. Therefore, preventing bacterial biofilm formation and disrupting mature bacterial biofilms are of significant research importance for reducing the risk of drug-resistant infections and improving human safety.

[0003] However, anti-biofilm drugs remain scarce. One of the core bottlenecks is that existing biofilm detection methods are unsuitable for large-scale, high-throughput drug screening, making it difficult to quickly identify potentially active candidate molecules. For example, while scanning electron microscopy and confocal microscopy can characterize bacterial biofilm structures at the microscopic level, it is difficult to quickly assess the destructive or inhibitory effects of drug molecules on biofilms at the macroscopic level, and the detection equipment is expensive. Plate counts can accurately determine the number of viable bacteria, but they cannot assess the direct impact of drugs on biofilms. Crystal violet staining is currently the classic method for in vitro biofilm detection. Using this method, biofilm determination requires multiple steps, including biofilm culture, washing to remove airborne bacteria, fixation, crystal violet staining, washing to remove excess dye, dissolving and binding dye, and quantitative detection. This process is not only cumbersome, but the multiple washing steps are also likely to cause biofilm detachment, resulting in inaccurate staining results and poor reproducibility. Furthermore, electron microscopy and confocal microscopy equipment are expensive and have low throughput, making them unsuitable for large-scale drug screening. Plate counts only measure the number of viable bacteria and cannot directly evaluate the effect of drugs on biofilms.

[0004] Therefore, in order to accelerate the research and development of anti-biofilm drugs, it is urgent to develop a method suitable for high-throughput drug screening that can conveniently and accurately detect bacterial biofilms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bacterial biofilm detection probe, detection method, and application, which solves the problems of long detection time, poor reproducibility, and cumbersome operation steps in the existing detection methods mentioned in the background.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a bacterial biofilm detection probe, comprising a fluorescent detection probe, wherein the fluorescent detection probe has the following structure:

[0007] Among them, anion X - Anion X is anion with one or more charges. - For F - Cl - ,Br - I - AcO - PF6 - BF4 - CF3COO - SO4 2- CO3 2- SO3 2- ClO4 - PO4 3- One of them.

[0008] Preferably, the anion X - For Br - .

[0009] A method for detecting bacterial biofilms includes the following steps: S1: Static culture of bacteria at 37℃ for 24–72 hours to form a bacterial biofilm. S2: Remove the upper culture medium and wash with PBS to remove airborne bacteria to obtain a biofilm suspension; S3: Add the solution of the fluorescent detection probe described in claim 1 or 2 to the biofilm suspension and mix it to make the final probe concentration 5–1000 μg / mL, and incubate at 25°C in the dark for 1–120 min; S4: Detection was performed using a fluorescent microplate reader with an excitation wavelength of 300–400 nm and an emission wavelength of 500–600 nm. The concentration of bacterial biofilm was quantified based on the fluorescence intensity.

[0010] Preferably, the final concentration of the probe in S3 is 50. μ g / mL, incubation time is 5 min.

[0011] Preferably, the excitation wavelength in S4 is 365nm and the emission wavelength is 520nm.

[0012] The bacterial biofilm includes biofilms formed by Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Shewanella, or Trichoderma reesei. The application of bacterial biofilm detection probes in detecting bacterial biofilms: The fluorescent probes can be used for high-throughput screening of active drugs that inhibit bacterial biofilm formation or destroy mature bacterial biofilms.

[0013] This invention provides a bacterial biofilm detection probe, detection method, and application, which have the following beneficial effects: 1. This bacterial biofilm detection probe, detection method, and application utilizes a fluorescent detection probe for detection. Compared to the traditional crystal violet staining method, which requires multiple steps such as fixation, staining, decolorization, and dissolution, and typically takes more than 2 hours, this method only requires 5 minutes of light-protected incubation to complete the labeling, significantly shortening the detection cycle and improving detection efficiency. It is particularly suitable for high-throughput screening scenarios and can significantly improve the screening efficiency of anti-biofilm active drugs.

[0014] 2. This bacterial biofilm detection probe, detection method, and application, compared with existing biofilm detection methods, generally require multiple PBS washing, methanol fixation, decolorization and dissolution steps. The washing process is very likely to cause biofilm detachment and structural damage, resulting in large deviations and poor repeatability. In contrast, this method requires no washing, fixation, or decolorization during the entire detection process. The probe directly interacts with the biofilm and generates a fluorescent signal, avoiding biofilm detachment and loss. It has high reproducibility, and the stability and reproducibility of the detection results are significantly better than traditional methods.

[0015] 3. The bacterial biofilm detection probe, detection method, and application: The fluorescent probe exhibits excellent specific binding ability to the biofilm, and the fluorescence signal intensity is highly linearly correlated with the biofilm concentration (R0). 2 >0.95), and can be accurately quantified in a wide concentration range of 1%–100%. Compared with the crystal violet method, the present invention has low background interference, high signal-to-noise ratio and lower detection limit, and is especially suitable for the accurate detection of low-content biofilms.

[0016] 4. This bacterial biofilm detection probe, detection method, and application have the advantages of strong versatility, covering biofilms of Gram-positive bacteria, Gram-negative bacteria, and fungi. Existing methods are mostly targeted at single bacterial species and have limited applicability. The probe of this scheme can effectively identify and label biofilms formed by a variety of bacteria and fungi, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Shewanella, and Trichoderma reesei. It has a wide range of applicable bacterial species and strong versatility, which greatly reduces the technical threshold for detecting different types of biofilms. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the fluorescence emission spectrum scan in the range of 400-700 nm after the biofilm detection probe Ia of the present invention was co-incubated with biofilms of different concentrations of Escherichia coli for 5 min, with 365 nm as the excitation light source. Figure 2This is a schematic diagram of fluorescence after co-incubation of the biofilm detection probe Ia of the present invention with biofilms of different concentrations (1%-100%) of Escherichia coli; Figure 3 This is a schematic diagram of the linear fitting curve between the fluorescence emission intensity at 520 nm in each test well and the concentration of Escherichia coli-derived biofilm, as measured by a fluorescence microplate reader in this invention. Figure 4 This is a schematic diagram illustrating biofilms derived from different concentrations of Escherichia coli stained using the crystal violet staining method according to the present invention. Figure 5 For the present invention Figure 4 Linear fitting curves of absorbance values ​​at 595 nm and concentration of E. coli-derived biofilm in each test well; Figure 6 The fluorescence emission spectrum scan of the biofilm detection probe Ia of this invention in the range of 400-700 nm after co-incubating with biofilms of different concentrations of Staphylococcus aureus for 5 min, with 365 nm as the excitation light source; Figure 7 This is a schematic diagram of the fluorescence after co-incubation of the biofilm detection probe Ia of the present invention with biofilms of different concentrations (1%-100%) of Staphylococcus aureus; Figure 8 This is a linear fitting curve of the fluorescence emission intensity at 520 nm in each test well and the concentration of Staphylococcus aureus-derived biofilm, measured using a fluorescence microplate reader in this invention. Figure 9 This is a schematic diagram of different concentrations of Staphylococcus aureus-derived biofilms stained using the crystal violet staining method in this invention; Figure 10 For the present invention Figure 9 Linear fitting curves of absorbance values ​​at 595 nm and the concentration of Staphylococcus aureus-derived biofilm in each test well. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0019] This invention provides a technical solution: a bacterial biofilm detection probe, comprising... The synthetic structure is as follows: A fluorescent probe in which the anion X - =Br - The fluorescent probe is denoted as probe Ia.

[0020] Example 2 This invention provides a technical solution: Based on Embodiment 1, this embodiment discloses a method for detecting Escherichia coli biofilm using probe Ia, comprising the following steps: S1: Cultivation and Acquisition of Escherichia coli Biofilm: Escherichia coli cultured to the logarithmic growth phase was adjusted to 0.5 MCF with physiological saline, and then diluted 1:100 with LB broth to prepare a bacterial suspension; 200 μL of the suspension was prepared per well. μ L inoculated the bacterial suspension into 96-well plates and incubated at 37°C for 48 hours. The supernatant suspension was then carefully aspirated, and the plates were slowly washed three times with PBS. The lower layer was the biofilm formed by *E. coli*, and the relative concentration of biofilms was recorded as 100%. The biofilm was then peeled from the well walls using a cell scraper and serially diluted with PBS to obtain bacterial biofilm suspensions with relative concentrations of 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, and 1%. S2: Determination of the characteristic wavelength of probe Ia in detecting E. coli biofilm: 100 μL of bacterial biofilms of three different concentrations (low (relative biofilm concentration of 1%), medium (relative biofilm concentration of 5%), and high (relative biofilm concentration of 10%) were transferred and incubated with 100 μL of PBS solution containing 100 μg / mL probe Ia at 25°C in the dark for 5 min (referred to as BF(Low)+Probe, BF(Medium)+Probe, and BF(High)+Probe, respectively). Subsequently, the fluorescence emission spectrum of the incubation solution in the range of 400-700 nm was scanned using a fluorescence spectrophotometer with 365 nm as the excitation wavelength; probe Ia solution (denoted as Probe) and bacterial biofilm suspension (denoted as BF) were used as controls; The results are as follows Figure 1 As shown, the probe itself or the biofilm itself has very weak fluorescence emission in the 500-600nm wavelength range. However, after co-incubating the probe with the biofilm, the fluorescence emission of the probe in the 500-600nm range gradually increases with the increase of the biofilm concentration. Therefore, this fluorescence emission band is expected to be selected as the characteristic band for detecting biofilm concentration, and 520nm is selected as the characteristic emission wavelength as the maximum emission wavelength.

[0021] S3: Linear detection of E. coli biofilm by probe Ia: The classic biofilm detection method—crystal violet staining—was used as a control for the following experiments: S3.1: 100 μ L concentration is 100 μ g / mL detection probe PBS solution and 100 μThe aforementioned suspensions containing different concentrations of biofilm (100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1%) were incubated for 5 min. Subsequently, the fluorescence emission intensity at 520 nm in each test well was measured using a fluorescence microplate reader with 365 nm as the excitation wavelength. A linear fitting analysis was performed on the relationship between biofilm concentration and fluorescence intensity. The results are as follows: Figure 2 As shown, the fluorescence intensity emitted by probe Ia is positively correlated with the biofilm concentration; See Figure 3 The fluorescence emission intensity of probe Ia at 520 nm showed a strong linear correlation with the biofilm concentration, with the regression equation being: y = 0.4742x + 10.212, R0 2 =0.9525, where y represents the fluorescence intensity at 520 nm and x represents the relative concentration of the biofilm.

[0022] The above test results show that the biofilm detection method provided by the present invention has high detection accuracy for bacterial biofilms derived from Escherichia coli over a wide concentration range. S3.2: Crystal Violet Staining Method: After centrifugation, discard the supernatant liquid from each test well. Then, add 200 ml of the supernatant to the test well containing a concentration gradient biofilm suspension (100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1%). μ After fixing with anhydrous methanol at room temperature for 15 minutes, the solution was inverted and allowed to air dry naturally. Then, it was stained with 0.1% crystal violet solution at room temperature in the dark for 20 minutes. Excess crystal violet solution was discarded, and the solution was gently rinsed with deionized water until colorless. 200 μL of the solution was then added to each well. μ Incubate with 33% glacial acetic acid at room temperature with shaking for 30 min. Detect the absorbance of each test well at 595 nm using a microplate reader. Perform linear regression analysis between the absorbance values ​​of each test well and the biofilm concentration gradient. Results are as follows: Figure 4 As shown, the crystal violet staining intensity of each test well increased in a dependent manner with increasing biofilm concentration. The absorbance value at 595 nm was linearly correlated with the biofilm concentration, with a regression equation of y = 0.0326x - 0.2797, R0. 2 =0.9582, y represents the absorbance value at 595 nm, and x represents the relative concentration of the biofilm (see...). Figure 5 ).

[0023] The above results demonstrate that the biofilm detection method provided by this invention, along with the crystal violet staining method, exhibits comparable detection accuracy within a high concentration range of biofilms. However, the crystal violet staining method is more cumbersome, involving the fixation, washing, and dissolution of various reagents. During this process, it is difficult to completely avoid biofilm loss due to washing, leading to reduced detection accuracy and a risk of decreased repeatability. This further illustrates the convenience and reliability of the biofilm detection method of this invention, which uses this wavelength as the characteristic wavelength for biofilm detection.

[0024] Example 3 This invention provides a technical solution: Based on Embodiment 1 and Embodiment 2, this embodiment discloses a method for detecting Staphylococcus aureus biofilm using probe Ia. The probe Ia molecule from Embodiment 1 is selected as the biofilm detection probe for detecting Staphylococcus aureus biofilm. The biofilm was derived from Staphylococcus aureus, which was used as a representative of Gram-positive bacteria to verify whether the biofilm detection method in this protocol could detect biofilms produced by Gram-positive bacteria rapidly, accurately, and efficiently.

[0025] The preparation, detection, and crystal violet staining methods for the biofilm are the same as in Example 2, except that the biofilm is derived from Staphylococcus aureus and includes the following steps: S1: Cultivation and acquisition of Staphylococcus aureus biofilm: Biofilms cultured to the logarithmic growth phase were collected. S.aureus Adjust the concentration to 0.5 MCF with physiological saline, then dilute Staphylococcus aureus with LB broth at a ratio of 1:100 to prepare a bacterial suspension; prepare a suspension at 200 μL per well. μ L inoculated the bacterial suspension into 96-well plates and incubated at 37°C for 48 hours. The supernatant was then carefully aspirated, and the plates were slowly washed three times with PBS. The lower layer was the Staphylococcus aureus biofilm, with the relative concentration of biofilms recorded as 100%. The biofilm was then peeled from the well walls using a cell scraper and serially diluted with PBS to obtain bacterial biofilm suspensions with relative concentrations of 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, and 1%.

[0026] S2: Determination of the characteristic wavelength for detecting Staphylococcus aureus biofilm using probe Ia: To illustrate the detection principle of the biofilm detection method in this invention, 100 μL of bacterial biofilms at three different concentrations (low (relative biofilm concentration of 1%), medium (relative biofilm concentration of 5%), and high (relative biofilm concentration of 10%) were respectively incubated with 100 μL of PBS solution containing 100 μg / mL probe Ia at 25°C in the dark for 5 min (denoted as BF(Low)+Probe, BF(Medium)+Probe, and BF(High)+Probe, respectively). Then, using 365 nm as the excitation wavelength, the fluorescence emission spectrum of the incubation solution in the range of 400-700 nm was scanned using a fluorescence spectrophotometer. Probe Ia solution (denoted as Probe) and bacterial biofilm suspension (denoted as BF) were used as controls. The results are as follows: Figure 6 As shown, the probe itself or the biofilm itself has very weak fluorescence emission in the 500-600nm wavelength range. However, after co-incubating the probe with the biofilm, the fluorescence emission of the probe in the 500-600nm range gradually increases with the increase of the biofilm concentration. Similar to Gram-negative bacteria, this fluorescence emission band is expected to be selected as the characteristic band for detecting the biofilm concentration of Gram-positive bacteria, and 520nm as the maximum emission wavelength can be selected as the characteristic emission peak.

[0027] S3: Linear detection of Staphylococcus aureus biofilm by probe Ia: To further illustrate the convenience and reliability of the biofilm detection method of this invention, which uses this band as the characteristic band for Staphylococcus aureus biofilm detection, the inventors selected the classic biofilm detection method—crystal violet staining—as a control and conducted the following experiments: a) 100 μ L concentration is 100 μ g / mL detection probe PBS solution and 100 μ The aforementioned suspensions containing different concentrations of biofilm (100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1%) were incubated for 5 min. Subsequently, the fluorescence emission intensity at 520 nm in each test well was measured using a fluorescence microplate reader, and a linear fitting analysis was performed on the relationship between biofilm concentration and fluorescence intensity. See the results below. Figure 7 The fluorescence intensity emitted by probe Ia is positively correlated with the biofilm concentration; See Figure 8 The fluorescence emission intensity of probe Ia at 520 nm showed a strong linear correlation with the biofilm concentration, with the regression equation being: y = 0.7945x + 14.9306, R0. 2 =0.9658, where y represents the fluorescence intensity at 520 nm and x represents the relative concentration of the biofilm.

[0028] The above test results show that the biofilm detection method of the present invention has high detection accuracy for biofilms of Gram-positive bacteria, represented by Staphylococcus aureus, over a wide concentration range.

[0029] b) Crystal violet staining method: Add 200 μL of crystal violet solution to the test wells containing a concentration gradient of Staphylococcus aureus biofilm suspension (100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1%). μ After fixing with anhydrous methanol at room temperature for 15 minutes, the solution was inverted and allowed to air dry naturally. Then, it was stained with 0.1% crystal violet solution at room temperature in the dark for 20 minutes. Excess crystal violet solution was discarded, and the solution was gently rinsed with deionized water until colorless. 200 μL of the solution was then added to each well. μ Incubate with 33% glacial acetic acid at room temperature with shaking for 30 min. Detect the absorbance of each test well at 595 nm using a microplate reader. Perform linear regression analysis between the absorbance values ​​of each test well and the biofilm concentration gradient. Results are as follows: Figure 9 As shown, the crystal violet staining intensity of each test well increased in a dependent manner with increasing biofilm concentration. The absorbance value at 595 nm was linearly correlated with the biofilm concentration, with a regression equation of y = 0.0534x - 0.0902, R0. 2 =0.9570, where y represents the absorbance value at 595 nm and x represents the relative concentration of the biofilm. See [reference needed]. Figure 10 .

[0030] As can be seen from the above results, the biofilm detection method provided by this invention and the crystal violet staining method both have comparable detection accuracy in the high concentration range of biofilms. However, the biofilm detection method provided by this invention has significantly simpler operation steps than the crystal violet staining method, and has greater advantages in improving detection accuracy and high-throughput screening efficiency.

[0031] Example 4 This invention provides a technical solution: based on Example 1, the probe Ia molecule from Example 1 is selected as a biofilm detection probe, and Escherichia coli is used as a representative of Gram-negative bacteria to illustrate the application of the biofilm detection method proposed in this invention in high-throughput screening of drug molecules that inhibit the formation of biofilms in Gram-negative bacteria.

[0032] Overnight cultured *E. coli* was adjusted to 0.5 MCF with physiological saline. The *E. coli* was then diluted 1:100 with LB broth to prepare a bacterial suspension. 100 μL of the suspension was added to a 96-well plate, followed by an equal volume of physiological saline solution containing the test compound. An equal volume of physiological saline was used as a control. The plates were incubated at 37°C for 24 h. The bacterial suspension in each well was slowly aspirated, and the wells were rinsed three times with PBS. 100 μL of a 50 μg / mL probe Ia solution was added, and staining was performed for 5 min. The fluorescence intensity at 520 nm was measured using a fluorescence microplate reader with an excitation wavelength of 365 nm. Groups with decreased fluorescence intensity compared to the physiological saline group indicated that the compound inhibited *E. coli* biofilm formation; the lower the fluorescence intensity, the stronger the inhibitory effect on *E. coli* biofilm formation.

[0033] Example 5 This invention provides a technical solution: Based on Example 1, the probe Ia molecule from Example 1 is selected as a biofilm detection probe in this example, and Staphylococcus aureus is used as a representative of Gram-positive bacteria to illustrate the application of the biofilm detection method proposed in this invention in high-throughput screening of drug molecules that have the ability to inhibit the formation of biofilms by Gram-positive bacteria.

[0034] Overnight cultured Staphylococcus aureus was adjusted to 0.5 MCF with physiological saline. The bacteria were then diluted 1:100 with TSB broth to prepare a bacterial suspension. 100 μL of the suspension was added to a 96-well plate, followed by an equal volume of physiological saline solution containing the test compound. An equal volume of physiological saline was used as a control. The plates were incubated at 37°C for 24 h. The bacterial suspension in each well was slowly aspirated, and the wells were rinsed three times with PBS. 100 μL of a 50 μg / mL probe Ia solution was added, and staining was performed for 5 min. The fluorescence intensity at 520 nm was measured using a fluorescence microplate reader with an excitation wavelength of 365 nm. Groups with decreased fluorescence intensity compared to the physiological saline group indicated that the compound inhibited Staphylococcus aureus biofilm formation; the lower the fluorescence intensity, the stronger the inhibitory effect on Staphylococcus aureus biofilm formation.

[0035] Example 6 This invention provides a technical solution: Based on Example 1, the probe Ia molecule from Example 1 is selected as a biofilm detection probe in this example, and Escherichia coli is used as a representative of Gram-negative bacteria to illustrate the application of the biofilm detection method proposed in this invention in high-throughput screening of drug molecules that have the effect of destroying mature biofilms.

[0036] Overnight cultured *E. coli* was adjusted to 0.5 MCF with physiological saline. The *E. coli* was then diluted 1:100 with LB broth to prepare a bacterial suspension. 200 μL of the suspension was added to a 96-well plate and incubated at 37°C for 48 h to form a mature biofilm. The suspension was slowly aspirated from each well, and the plate was washed three times with PBS. Then, 200 μL of physiological saline solution containing the test compound was added, and the plate was incubated at 37°C for 24 h. An equal volume of physiological saline was used as a control group. After incubation, the suspension was slowly aspirated from each well, and the wells were slowly rinsed three times with PBS. Then, 100 μL of a 50 μg / mL probe Ia solution was added, and staining was performed for 5 min. The fluorescence intensity at 520 nm was measured using a fluorescence microplate reader with an excitation wavelength of 365 nm. Groups with decreased fluorescence intensity compared to the physiological saline group indicated that the compound had the ability to disrupt the formation of the mature *E. coli* biofilm; the lower the fluorescence intensity, the stronger the ability of the compound to disrupt the *E. coli* biofilm.

[0037] Example 7 This invention provides a technical solution: Based on Example 1, the probe Ia molecule from Example 1 is selected as a biofilm detection probe in this example, and Staphylococcus aureus is used as a representative of Gram-positive bacteria to illustrate the application of the biofilm detection method proposed in this invention in high-throughput screening of drug molecules that have the effect of destroying mature biofilms.

[0038] Overnight cultured Staphylococcus aureus was adjusted to 0.5 MCF with physiological saline, and a bacterial suspension was prepared by diluting the Staphylococcus aureus 1:100 with TSB broth. 200 μL of the bacterial suspension was added to a 96-well plate and incubated at 37°C for 48 h to form a mature biofilm. The bacterial suspension in each well was slowly aspirated, and the plate was washed three times with PBS. 200 μL of PBS was then added to the plate. μ L of physiological saline solution containing the test compound was incubated at 37°C for 24 h, with an equal volume of physiological saline as a control. After incubation, the bacterial suspension in each test well was slowly aspirated and discarded. The airborne bacteria in the wells were then slowly rinsed three times with PBS, and 100 L of PBS was added. μ After staining with a 50 μg / mL aqueous solution of probe Ia for 5 min, the fluorescence intensity at 520 nm was measured using a fluorescence microplate reader with 365 nm as the excitation wavelength. The groups with a decrease in fluorescence intensity compared to the saline group indicated that the compound had the ability to disrupt the formation of mature Staphylococcus aureus biofilm. The lower the fluorescence intensity, the stronger the ability of the compound to disrupt Staphylococcus aureus biofilm.

[0039] In summary, this invention proposes a biofilm detection probe, its application in biofilm detection, and its detection method. It overcomes the technical bottlenecks of traditional biofilm detection methods, such as cumbersome operation, long time consumption, poor reproducibility, and low throughput. It provides a rapid, simple, sensitive, universal, high-throughput, safe, and environmentally friendly bacterial biofilm detection technology, which has significant scientific research value and clinical application prospects.

[0040] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bacterial biofilm detection probe, characterized in that: Includes a fluorescent detection probe, wherein the fluorescent detection probe has a structure of formula Ia: ， Among them, anion X - Anion X is anion with one or more charges. - For F - Cl - ,Br - I - AcO - PF6 - BF4 - CF3COO - SO4 2- CO3 2- SO3 2- ClO4 - PO4 3- One of them.

2. The bacterial biofilm detection probe according to claim 1, characterized in that: The anion X - For Br - .

3. A method for detecting bacterial biofilms, characterized in that, Includes the following steps: S1: Static culture of bacteria at 37℃ for 24–72 h to form a bacterial biofilm; S2: Remove the upper culture medium and wash with PBS to remove airborne bacteria to obtain a biofilm suspension; S3: Add the solution of the fluorescent detection probe described in claim 1 or 2 to the biofilm suspension and mix it to make the final probe concentration 5–1000 μg / mL, and incubate at 25°C in the dark for 1–120 min; S4: Detection was performed using a fluorescent microplate reader with an excitation wavelength of 300–400 nm and an emission wavelength of 500–600 nm. The concentration of bacterial biofilm was quantified based on the fluorescence intensity.

4. The method for detecting bacterial biofilms according to claim 3, characterized in that: The final concentration of the probe in S3 is 50. μ g / mL, incubation time is 5 min.

5. The method for detecting bacterial biofilms according to claim 3, characterized in that: The excitation wavelength in S4 is 365nm, and the emission wavelength is 520nm.

6. The method for detecting bacterial biofilms according to claim 3, characterized in that: The bacterial biofilm includes biofilms formed by Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Shewanella, or Trichoderma reesei.

7. The application of a bacterial biofilm detection probe according to claim 1 or 2 in the detection of bacterial biofilms.

8. The application of the bacterial biofilm detection probe according to claim 1 or 2 in high-throughput screening of drugs with activity that inhibits biofilm formation or disrupts bacterial biofilm structure.

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