AIE reagent and method for rapid detection of antimicrobial drug sensitivity

Abstract

The present invention discloses an AIE reagent and method for rapid detection of antimicrobial drug sensitivity. The AIE molecules of the present invention include AIE-1 molecules, preferably also AIE-2 molecules, for in vitro antimicrobial drug sensitivity detection. The AIE reagent has high sensitivity, low fluorescence background and good photostability, and the reagent can be directly added to the drug sensitivity test diluent, and long-term monitoring of bacterial growth can be performed in real time. Through the bacterial growth curve, the drug sensitivity results are quickly obtained and the requirements of MIC value determination are met. The method for rapid detection of antimicrobial drug sensitivity of the present invention can obtain the results of drug sensitivity test within 8 hours, and accurately judge the MIC value of antimicrobial drugs, is applicable to different culture media, and has low requirements for detection equipment, greatly improves the efficiency of in vitro antimicrobial drug sensitivity test, and can provide a solution for antimicrobial treatment quickly.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial sensitivity detection, and in particular to an AIE reagent and method for rapidly detecting the sensitivity of antimicrobial drugs. Background Art

[0002] With the widespread use of antibiotics, the speed at which bacteria develop resistance is gradually accelerating, making drug selection difficult. Rapid detection technology for bacterial resistance is an important means to address global public health challenges. At present, the antimicrobial sensitivity tests used in clinical microbiology laboratories mainly include paper diffusion method, dilution method, E-test method, etc. These methods all observe the growth of bacteria with the naked eye, and require bacteria to grow to a certain number, which takes at least 12 hours or even longer. The automated bacterial drug sensitivity detection system has improved the detection speed to a certain extent, but these instruments generally use turbidimetry to observe the dynamic changes in the number of bacteria. Limited by the low sensitivity of turbidimetry, it also takes about 8 hours to give drug sensitivity results. In addition, these instruments are expensive and are limited in use in primary hospitals. Therefore, it is necessary to develop a rapid detection method that has low equipment requirements, can give drug sensitivity test results in a shorter time, and meets the requirements of MIC (minimum inhibitory concentration), which can quickly provide solutions for antimicrobial treatment. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an AIE reagent and method for rapidly detecting the sensitivity of antimicrobial drugs.

[0004] The present invention first provides an application of an AIE molecule (including an AIE-1 molecule and may also include an AIE-2 molecule) in in vitro antimicrobial drug sensitivity detection, which is used to determine the sensitivity of antimicrobial drugs and to determine the minimum inhibitory concentration (MIC value) of antimicrobial drugs.

[0005] Secondly, the present invention provides an AIE reagent for rapidly detecting the sensitivity of antibiotics, which is used for in vitro antibiotic sensitivity tests. It has high sensitivity, low fluorescence background and good photostability, and can obtain drug sensitivity test results in a shorter time, rapidly judge the sensitivity of antibiotics, and accurately determine the MIC value.

[0006] Finally, the present invention provides a method for rapidly detecting the sensitivity of antimicrobial drugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides an application of an AIE molecule in in vitro antimicrobial drug sensitivity detection, wherein the AIE molecule comprises an AIE-1 molecule, and the AIE-1 molecule is selected from at least one of Formula 1;

[0009]

[0010] Formula 1

[0011] In Formula 1, R is independently selected from hydrogen, amino, alkyl having 1 to 20 carbon atoms, unsaturated hydrocarbon having 2 to 20 carbon atoms, heteroalkyl having 1 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, heterocycloalkyl having 2 to 20 carbon atoms, aryl having 6 to 30 carbon atoms, and heteroaryl having 5 to 30 carbon atoms;

[0012] R 1 Select one of the following structures:

[0013]

[0014]

[0015] Z is an anion;

[0016] R 2 Select one of the following structures:

[0017]

[0018] n 1 An integer from 1 to 5 (1, 2, 3, 4, 5).

[0019] Preferably, in Formula 1, the heteroalkyl group includes an alkoxy group or an alkylamino group;

[0020] Preferably, in Formula 1, the anion is a halide ion (fluoride ion, chloride ion, bromide ion, iodide ion, etc.), NO 2 - , BF 4 - , BF 6 - 、NO 3 - ,PO 4 3- 、SO 3 2- At least one of .

[0021] Preferably, the AIE-1 molecule is selected from , At least one of .

[0022] Preferably, the AIE molecule further comprises an AIE-2 molecule; the AIE-2 molecule is selected from at least one of Formula 2;

[0023]

[0024] Formula 2

[0025] In formula 2, R 1 , R 2 , R 3 , R 4 , R 5 Each independently selected from a substituted or unsubstituted straight-chain alkyl group having 1 to 20 carbon atoms, a substituted or unsubstituted branched-chain alkyl group having 3 to 20 carbon atoms, and a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms;

[0026] n 2 An integer from 2 to 10 (2, 3, 4, 5, 6, 7, 8, 9, 10);

[0027] X - It is an anion.

[0028] Further preferably, in Formula 2, the anion is a halide ion (fluoride ion, chloride ion, bromide ion, iodide ion, etc.), NO 2 - , BF 4 - , BF 6 - 、NO 3 - ,PO 4 3- 、SO 3 2- At least one of;

[0029] Further preferably, in Formula 2, the substituents in the substituted or unsubstituted straight-chain alkyl group having 1 to 20 carbon atoms, the substituted or unsubstituted branched-chain alkyl group having 3 to 20 carbon atoms, and the substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms are -COOH, -OH, -NH 2 , -CHO, -CN or -NO 2 .

[0030] Further preferably, the AIE-2 molecule is selected from , At least one of .

[0031] More preferably, the molar ratio of the AIE-1 molecules to the AIE-2 molecules is 1:(5-10).

[0032] Preferably, the AIE molecules of the present invention are used to determine the minimum inhibitory concentration of antibiotics and judge the sensitivity of antibiotics in in vitro antibiotic sensitivity testing.

[0033] The present invention provides an AIE reagent for rapidly detecting the sensitivity of antimicrobial drugs, comprising AIE molecules; the AIE molecules include the above-mentioned AIE-1 molecules.

[0034] Preferably, the AIE molecule of the AIE reagent for rapid detection of antimicrobial drug sensitivity also includes the above-mentioned AIE-2 molecule.

[0035] More preferably, the molar ratio of the AIE-1 molecules to the AIE-2 molecules is 1:(5-10).

[0036] Preferably, the AIE reagent for rapid detection of antimicrobial drug sensitivity further comprises a solvent, and the solvent is preferably any one of water, dimethyl sulfoxide, and N,N-dimethylformamide.

[0037] The present invention provides an application of the above-mentioned AIE reagent for rapid detection of antibiotic sensitivity in in vitro antibiotic sensitivity detection.

[0038] The present invention provides a method for rapidly detecting the sensitivity of antimicrobial drugs, comprising the following steps:

[0039] (1) preparing a bacterial solution, adding the above-mentioned AIE reagent, and mixing evenly to obtain a mixed bacterial solution containing the AIE reagent;

[0040] (2) preparing different gradient concentrations of the antimicrobial drug solution to be tested by gradient dilution, adding the mixed bacterial solution of step (1) to obtain the test solution, culturing the bacteria, and monitoring the fluorescence value at different time points;

[0041] Calculate the relative fluorescence intensity F at different antimicrobial drug concentrations and culture time n hours respectively n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0042] (3) Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 Make comparisons to determine the minimum inhibitory concentration of antimicrobial drugs and judge the sensitivity of antimicrobial drugs;

[0043] As the concentration of antibiotics increases, the first slope k a When ≤0.01, the antimicrobial drug concentration a corresponding to the slope is determined as the minimum inhibitory concentration of the antimicrobial drug.

[0044] Preferably, in step (2), in the test solution, the concentration of bacteria is any concentration between 0.5 McFarland turbidity and 0.5 McFarland turbidity diluted 1000 times, and the concentration of AIE molecules is 5-12 μM;

[0045] Further preferably, when the AIE molecule is an AIE-1 molecule, the concentration of the AIE-1 molecule in the detection solution in step (2) is 5-10 μM;

[0046] Further preferably, when the AIE molecules are AIE-1 molecules and AIE-2 molecules, the concentration of AIE molecules-1 in the detection solution in step (2) is 1-2 μM, and the concentration of AIE molecules-2 is 5-10 μM.

[0047] Preferably, the conditions for culturing the bacteria in step (2) are conditions suitable for bacterial growth.

[0048] Preferably, the fluorescence value can be monitored by any instrument with a luminescence detection function. Preferably, an ELISA instrument is used to directly perform real-time culture and monitoring.

[0049] Preferably, the gradient dilution concentration of the antimicrobial drug solution to be tested refers to the CLSI (Clinical and Laboratory Standards Institute) standard.

[0050] As a preferred embodiment, the method for rapid detection of antimicrobial drug sensitivity comprises the following steps:

[0051] (1) Cultivate the bacteria on a plate at 30-37°C for 15-20 h, pick a single colony from the plate, use the culture medium to prepare a 0.5 McFarland turbidity bacterial solution, and then dilute it 0-500 times for later use;

[0052] The AIE molecule is dissolved in ultrapure water to prepare an AIE reagent with an AIE molecule concentration of 5-12 mM;

[0053] The AIE reagent was added to the bacterial solution and mixed evenly to prepare a mixed bacterial solution containing AIE molecules at a final concentration of 10-24 μM;

[0054] (2) The antimicrobial drug stock solution is diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations, and each gradient is dispensed into a 96-well microplate at 100 μL / well; then the mixed bacterial solution of step (1) is dispensed into the antimicrobial drug solution of the 96-well microplate at 100 μL / well to obtain a number of antimicrobial drug test solutions of different gradient concentrations; after the 96-well plate is prepared, it is placed in a fluorescence reader for regular monitoring, and the excitation wavelength is set to 460 nm and the emission wavelength is set to 560 nm;

[0055] Calculate the relative fluorescence intensity F at different antimicrobial drug concentrations and culture time n hours n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0056] (3) Fitting F at different antimicrobial drug concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 Make comparisons to determine the minimum inhibitory concentration of antimicrobial drugs and judge the sensitivity of antimicrobial drugs;

[0057] As the concentration of antibiotics increases, the first slope k a When ≤0.01, the antimicrobial drug concentration a corresponding to the slope is determined as the minimum inhibitory concentration of the antimicrobial drug.

[0058] The present invention has the following beneficial effects:

[0059] 1. The present invention provides the use of AIE molecules (including AIE-1 molecules and AIE-2 molecules) in in vitro antimicrobial drug sensitivity tests. The present invention creatively discovered that AIE-1 molecules can be used for in vitro antimicrobial drug sensitivity tests, with high sensitivity, low fluorescence background and good photostability, and can obtain drug sensitivity test results in a shorter time, quickly determine the sensitivity of antimicrobial drugs, and accurately determine the minimum inhibitory concentration (MIC value) of antimicrobial drugs.

[0060] 2. Compared with the prior art, the AIE reagent for rapid detection of antimicrobial drug sensitivity of the present invention has lower fluorescence background and better photostability (no fluorescence quenching occurs during long-term monitoring). The reagent can be directly added to the drug sensitivity test diluent to perform long-term monitoring of bacterial growth in real time. The detection sensitivity is high, and the drug sensitivity results can be quickly obtained through the bacterial growth curve and meet the requirements of MIC value determination.

[0061] 3. The method for rapid detection of antibiotic sensitivity of the present invention can obtain the results of drug sensitivity test within 8 hours and accurately determine the MIC value of antibiotics. It is applicable to different culture media and has low requirements for detection equipment (monitoring can be achieved using an ordinary multifunctional plate reader with luminescence detection function). It greatly improves the efficiency of in vitro antibiotic sensitivity tests and can quickly provide solutions for antibacterial treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1a and Figure 1b This is a graph showing the change in fluorescence intensity when AIE reagent I in Example 1 tests different bacterial concentrations.

[0063] Figure 2a It is a straight line diagram of the fitting relationship between relative fluorescence intensity and time when AIE reagent I tests Pseudomonas aeruginosa under the action of different concentrations of ceftazidime in Example 7.

[0064] Figure 2b It is a straight line diagram of the fitting relationship between relative fluorescence intensity and time when AIE reagent II tests Pseudomonas aeruginosa under the action of different concentrations of ceftazidime in Example 8.

[0065] Figure 3 This is a straight line diagram of the fitting relationship between relative fluorescence intensity and time when the AIE reagent IV in Example 1 was used to test Staphylococcus aureus under the action of different concentrations of levofloxacin.

[0066] Figure 4a This is a curve of OD variation over time when AIE reagent V was used to test Staphylococcus aureus under the action of different concentrations of ceftazidime in Comparative Example 2.

[0067] Figure 4b This is a curve chart showing the change in fluorescence intensity over time when AIE reagent V in comparative example 2 was used to test Staphylococcus aureus under the action of different concentrations of ceftazidime.

[0068] Figure 5 This is a straight line graph of the fitting relationship between relative fluorescence intensity and time when the AIE reagent VI in Example 3 was used to test Pseudomonas aeruginosa under the action of different concentrations of ceftazidime.

[0069] Figure 6a This is a straight line graph of the fitting relationship between relative fluorescence intensity and time when the AIE reagent VII in Example 4 was used to test Pseudomonas aeruginosa under the action of different concentrations of levofloxacin.

[0070] Figure 6b They are respectively the straight line graphs of the fitting relationship between relative fluorescence intensity and time when the AIE reagent VII in Comparative Example 4 tested Staphylococcus aureus under the action of different concentrations of levofloxacin. DETAILED DESCRIPTION

[0071] The present invention is described in detail below in conjunction with examples, but the implementation manner and protection scope of the present invention are not limited to the following examples.

[0072] The AIE-1 molecule used in the embodiments of the present invention is selected from the following:

[0073]

[0074] The AIE-2 molecule is selected from one of the following:

[0075] .

[0076] The culture medium of the embodiment of the present invention adopts Mueller-Hinton broth medium (MH broth medium).

[0077] The fluorescence detection instrument used in the embodiment of the present invention is a Bioteck multifunctional microplate reader.

[0078] Embodiment 1:

[0079] Weigh the AIE-1 (B1) molecule with a balance, add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent I (10 mM), which is stored at 4 °C;

[0080] Escherichia coli 25922 and Staphylococcus aureus 29213 were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate and diluted into a series of concentrations using MH broth medium. AIE reagent I was added to each concentration (the final concentration of AIE-1 molecule was 10 μM). 200uL was taken into a 96-well plate, and the OD value and fluorescence intensity at each concentration were detected. The results are shown in Figure 2. Figure 1a and Figure 1b As shown. Figure 1a and Figure 1b It can be seen that as the bacterial concentration increases, the fluorescence intensity changes and has a good linear relationship with the OD value.

[0081] Embodiment 2:

[0082] (1) Culture Escherichia coli 25922 on a plate culture medium at 37°C for 18 h, pick a single colony from the plate, prepare a 0.5 McFarland turbidity bacterial solution with MH broth, and then dilute it 50 times for later use;

[0083] Weigh the AIE-1 molecule (B1) with a balance, add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent I (10 mM), which is stored at 4°C;

[0084] (2) Use MH broth medium to dilute the antimicrobial drug stock solution (ampicillin, ceftazidime, levofloxacin, imipenem) in a gradient dilution to prepare different gradient concentrations of the antimicrobial drug solutions to be tested (see Table 1 for the concentration gradient). The prepared antimicrobial drug solutions are dispensed into a 96-well microplate at 100 μL / well for each gradient concentration;

[0085] Table 1:

[0086]

[0087] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent I and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 2, and the final concentration of AIE-1 molecules is 10 μM);

[0088] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 4 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0089] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0090] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0091] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0092] The results are shown in Table 2 and Table 3. It can be seen that Escherichia coli is resistant to ampicillin, but sensitive to ceftazidime, levofloxacin and imipenem, and the measured MIC value is consistent with the traditional broth dilution method. This result also shows that the AIE reagent of the present invention can monitor the growth of bacteria in real time, and can give drug sensitivity results and MIC values ​​in about 4 hours, greatly shortening the time of drug sensitivity test.

[0093] Table 2:

[0094]

[0095] Table 3: MIC values ​​of each drug against Escherichia coli

[0096]

[0097] Embodiment 3:

[0098] (1) The following eight bacterial species (Klebsiella pneumoniae, Salmonella 1, Salmonella 19, Escherichia coli 25922, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213, MRSA, Enterococcus faecium) were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate. MH broth was used to prepare 0.5 McFarland turbidity bacterial liquid, which was then diluted 50 times for later use;

[0099] Weigh the AIE-1 molecule (B1) with a balance, add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent I (10 mM), which is stored at 4°C;

[0100] (2) Using MH broth medium, the antibiotic stock solution of levofloxacin was diluted in a gradient manner to prepare different gradient concentrations of the antibiotic solution to be tested. The prepared antibiotic solution was dispensed into a 96-well microplate at 100 μL / well for each gradient concentration;

[0101] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent I and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 4, and the final concentration of AIE-1 molecules is 10 μM);

[0102] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 6 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0103] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0104] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0105] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0106] The results are shown in Tables 4 and 5. It can be seen that MRSA is resistant to levofloxacin, while Klebsiella pneumoniae, Salmonella 1, Salmonella 19, Escherichia coli 25922, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213, and Enterococcus faecium are all sensitive to levofloxacin, and the measured MIC values ​​are consistent with the traditional broth dilution method. This result also shows that the AIE reagent of the present invention can monitor the growth of bacteria in real time, and can give drug sensitivity results and MIC values ​​in about 6 hours, greatly shortening the time of drug sensitivity test.

[0107] Table 4:

[0108]

[0109] Table 5: MIC values ​​of levofloxacin against various bacteria

[0110]

[0111] Embodiment 4:

[0112] (1) The following 9 bacterial species (Klebsiella pneumoniae, Salmonella 1, Salmonella 19, Escherichia coli 25922, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213, Enterococcus faecium, Staphylococcus squirrel 29061, Streptococcus agalactiae) were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate. MH broth was used to prepare 0.5 McFarland turbidity bacterial liquid, which was then diluted 50 times for later use;

[0113] Weigh AIE-1 molecules (B1) and AIE-2 molecules (A1) with a balance, add ultrapure water to dissolve, filter and sterilize with a 0.22 μm filter membrane to prepare AIE reagent II (the molar ratio of AIE-1 molecules (B1) and AIE-2 molecules (A1) is 1:5, and the concentrations are 2 mM and 10 mM, respectively), and store at 4°C;

[0114] (2) Using MH broth medium, the antibiotic stock solution of levofloxacin was diluted in a gradient manner to prepare different gradient concentrations of the antibiotic solution to be tested. The prepared antibiotic solution was dispensed into a 96-well microplate at 100 μL / well for each gradient concentration;

[0115] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent II and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 6, the final concentration of AIE-1 molecules is 2 μM, and the final concentration of AIE-2 molecules is 10 μM);

[0116] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 6 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0117] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0118] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0119] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0120] The results are shown in Tables 6 and 7. It can be seen that Klebsiella pneumoniae, Salmonella 1, Salmonella 19, Escherichia coli 25922, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213, Enterococcus faecium, Staphylococcus squirrel 29061, and Streptococcus agalactiae are all sensitive to levofloxacin, and the measured MIC values ​​are consistent with the traditional broth dilution method. This result also shows that the AIE reagent of the present invention can monitor the growth of bacteria in real time, and can give drug sensitivity results and MIC values ​​in about 6 hours, greatly shortening the time of drug sensitivity test.

[0121] Table 6:

[0122]

[0123] Table 7: MIC values ​​of levofloxacin against various bacteria

[0124]

[0125] Embodiment 5:

[0126] (1) Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29213 were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate. MH broth was used to prepare 0.5 McFarland turbidity bacterial liquid, which was then diluted 50 times for later use;

[0127] AIE-1 molecules (B2) and AIE-2 molecules (A2) were weighed on a balance, dissolved in ultrapure water, and sterilized by filtration with a 0.22 μm filter membrane to prepare AIE reagent III (the molar ratio of AIE-1 molecules (B2) and AIE-2 molecules (A2) was 1:7.5, and the concentrations were 1 mM and 7.5 mM, respectively), and stored at 4°C;

[0128] (2) Using MH broth medium, the antibiotic stock solution of levofloxacin was diluted in a gradient manner to prepare different gradient concentrations of the antibiotic solution to be tested. The prepared antibiotic solution was dispensed into a 96-well microplate at 100 μL / well for each gradient concentration;

[0129] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent III and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 8, the final concentration of AIE-1 molecules is 1 μM, and the final concentration of AIE-2 molecules is 7.5 μM);

[0130] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 6 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0131] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0132] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0133] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0134] The results are shown in Tables 8 and 9. It can be seen that Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29213 are sensitive to levofloxacin, and the measured MIC values ​​are consistent with the traditional broth dilution method. This result also shows that the AIE reagent of the present invention can monitor the growth of bacteria in real time, and can give drug sensitivity results and MIC values ​​in about 6 hours, greatly shortening the time of drug sensitivity test.

[0135] Table 8:

[0136]

[0137] Table 9: MIC values ​​of levofloxacin against various bacteria

[0138]

[0139] Embodiment 6:

[0140] (1) Pseudomonas aeruginosa 27853 was cultured on a plate culture medium at 37°C for 18 h, and a single colony was picked from the plate. A 0.5 McFarland turbidity bacterial solution was prepared using MH broth medium. The working bacterial solutions of undiluted, 5-fold, 50-fold, and 500-fold were prepared for use;

[0141] Weigh AIE-1 molecules (B1) and AIE-2 molecules (A1) with a balance, add ultrapure water to dissolve, filter and sterilize with a 0.22 μm filter membrane to prepare AIE reagent II (the molar ratio of AIE-1 molecules (B1) and AIE-2 molecules (A1) is 1:5, and the concentrations are 2 mM and 10 mM, respectively), and store at 4°C;

[0142] (2) Using MH broth medium, the antibiotic stock solution of levofloxacin was diluted in a gradient manner to prepare different gradient concentrations of the antibiotic solution to be tested. The prepared antibiotic solution was dispensed into a 96-well microplate at 100 μL / well for each gradient concentration;

[0143] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL AIE reagent II, and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 10, the final concentration of AIE-1 molecules is 2 μM, and the final concentration of AIE-2 molecules is 10 μM);

[0144] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 6 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0145] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0146] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0147] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0148] The results are shown in Table 10. It can be seen that the levofloxacin susceptibility test was carried out using different McLaurel turbidity bacterial solutions, and the measured MIC value was 2 μg / mL, which is consistent with the traditional broth dilution method. This result shows that the AIE reagent of the present invention is suitable for different McLaurel turbidity bacterial solutions, and the susceptibility test results can be obtained quickly and accurately.

[0149] Table 10:

[0150]

[0151] Embodiment 7:

[0152] (1) Pseudomonas aeruginosa 27853 was cultured on a plate culture medium at 37°C for 18 h, and a single colony was picked from the plate. A 0.5 McFarland turbidity bacterial solution was prepared using MH broth medium, and then diluted 50 times for later use;

[0153] Weigh the AIE-1 molecule (B1) with a balance, add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent I (10 mM), which is stored at 4°C;

[0154] (2) Using MH broth medium, the antimicrobial drug stock solution ceftazidime was diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL). The prepared antimicrobial drug solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0155] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent I and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 11, and the final concentration of the AIE-1 molecule is 10 μM);

[0156] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 8 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0157] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0158] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0159] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0160] The results are shown in Tables 11, 12 and Figure 2a As shown, it can be seen that when AIE reagent I is used in the ceftazidime anti-Pseudomonas aeruginosa susceptibility test, it takes 7-8 hours to accurately interpret the results.

[0161] Table 11:

[0162]

[0163] Table 12: MIC values ​​of ceftazidime against Pseudomonas aeruginosa

[0164]

[0165] Embodiment 8:

[0166] (1) Pseudomonas aeruginosa 27853 was cultured on a plate culture medium at 37°C for 18 h, and a single colony was picked from the plate. A 0.5 McFarland turbidity bacterial solution was prepared using MH broth medium, and then diluted 50 times for later use;

[0167] Weigh AIE-1 molecules (B1) and AIE-2 molecules (A1) with a balance, add ultrapure water to dissolve, filter and sterilize with a 0.22 μm filter membrane to prepare AIE reagent II (the molar ratio of AIE-1 molecules (B1) and AIE-2 molecules (A1) is 1:5, and the concentrations are 2 mM and 10 mM, respectively), and store at 4°C;

[0168] (2) Using MH broth medium, the antimicrobial drug stock solution ceftazidime was diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL, respectively). The prepared antimicrobial drug solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0169] Each 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent II and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final drug concentration gradient is shown in Table 13, the final concentration of AIE-1 molecules is 2 μM, and the final concentration of AIE-2 molecules is 10 μM);

[0170] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 8 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0171] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0172] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0173] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0174] The results are shown in Tables 13, 14 and Figure 2b As shown, it can be seen that AIE reagent II can accurately interpret the results within 4-6 hours when conducting ceftazidime anti-Pseudomonas aeruginosa susceptibility test.

[0175] Table 13:

[0176]

[0177] Table 14: MIC values ​​of ceftazidime against Pseudomonas aeruginosa

[0178]

[0179] By comparing Examples 3 / 4 / 7 / 8, it can be seen that the detection sensitivity of bimolecular AIE reagents is higher than that of monomolecular AIE reagents. Bimolecular AIE reagents can accurately interpret the results of drug sensitivity tests for most bacteria within 4-6 hours, and have wider applicability; monomolecular AIE reagents require 7-8 hours to accurately interpret the results of drug sensitivity tests for a certain part of bacteria, and the required time is relatively long. The present invention preferably contains AIE reagents containing AIE-1 molecules and AIE-2 molecules.

[0180] Comparative Example 1:

[0181] (1) Cultivate Staphylococcus aureus 29213 on a plate culture medium at 37°C for 18 h, pick a single colony from the plate, prepare a 0.5 McFarland turbidity bacterial solution with MH broth, and then dilute it 50 times for later use;

[0182] Weigh the AIE molecule (structure: ), add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent IV (10 mM), which is stored at 4 °C;

[0183] (2) Using MH broth medium, the antimicrobial drug stock solution levofloxacin was diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL, respectively). The prepared antimicrobial drug solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0184] Each 1 mL of the bacterial solution from step (1) was mixed evenly with 2 μL of AIE reagent IV and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions (AIE molecules) with different gradient concentrations. The final concentration was 10 μM);

[0185] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 5 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0186] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0187] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 Make a comparison.

[0188] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0189] The results are as follows Figure 3 As shown in the figure, the entire bacteria did not undergo fluorescence changes even in the blank group, while the traditional broth dilution method of the control group proved that the blank group grew well. Subsequent proof showed that the AIE reagent IV in Example 1 would affect bacterial growth, resulting in false sensitivity.

[0190] Comparative Example 2:

[0191] (1) Cultivate Staphylococcus aureus 29213 on a plate culture medium at 37°C for 18 h, pick a single colony from the plate, prepare a 0.5 McFarland turbidity bacterial solution with MH broth, and then dilute it 50 times for later use;

[0192] Weigh the AIE molecule (structure: ), add ultrapure water to dissolve, filter with a 0.22 μm filter membrane to sterilize, and prepare AIE reagent V (10 mM), which is stored at 4 °C;

[0193] (2) Using MH broth medium, the antimicrobial drug stock solution ceftazidime was diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL). The prepared antimicrobial drug solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0194] Each 1 mL of the bacterial solution from step (1) was mixed evenly with 2 μL of AIE reagent V and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions (AIE molecules) with different gradient concentrations. The final concentration was 10 μM);

[0195] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 25 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0196] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0197] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0198] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0199] The results are as follows Figure 4a and Figure 4b As shown in the figure, it can be seen that the AIE reagent V of comparative example 2 has a good staining effect on bacteria, and the addition of AIE reagent V does not affect bacterial growth. After 24 hours, the MIC value of the control group monitored by the traditional broth dilution method was 4 μg / mL, while the MIC value of comparative example 2 was 16 μg / mL. The sensitivity of the AIE reagent V of comparative example 2 is low and the MIC value cannot be accurately judged.

[0200] Comparative Example 3:

[0201] (1) Pseudomonas aeruginosa 27853 was cultured on a plate culture medium at 37°C for 18 h, and a single colony was picked from the plate. A 0.5 McFarland turbidity bacterial solution was prepared using MH broth medium, and then diluted 50 times for later use;

[0202] Weigh the AIE molecules using a balance ( ) and AIE-2 molecules (A1), dissolved in ultrapure water, and sterilized by filtration with a 0.22 μm filter membrane to obtain AIE reagent VI (AIE molecules ( ) and AIE-2 molecules (A1) at a molar ratio of 1:5, with concentrations of 2 mM and 10 mM, respectively, and stored at 4 °C;

[0203] (2) Using MH broth medium, the antimicrobial drug stock solution ceftazidime was diluted in a gradient manner to prepare antimicrobial drug solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL). The prepared antimicrobial drug solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0204] Each 1 mL of the bacterial solution from step (1) was mixed evenly with 2 μL of AIE reagent VI and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions (AIE molecules) with different gradient concentrations. The final concentration was 2 μM, and the final concentration of AIE-2 molecules was 10 μM);

[0205] (3) After the 96-well plate is prepared, place it in an ELISA reader (96-well plate, etc.) and monitor it regularly for 8 hours (measure the fluorescence value every hour), set the excitation wavelength to 460 nm, and the emission wavelength to 560 nm;

[0206] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0207] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0208] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0209] The results are as follows Figure 5As shown in Table 15, it can be seen that the AIE reagent VI of Comparative Example 3 has a good staining effect on bacteria, and the addition of AIE reagent VI does not affect bacterial growth. After 24 hours, the MIC value of the control group monitored by the traditional broth dilution method was 2 μg / mL, while the MIC of Comparative Example 3 was greater than 32 μg / mL. The AIE reagent VI of Comparative Example 3 cannot accurately judge the MIC value.

[0210] Table 15:

[0211]

[0212] Comparative Example 4:

[0213] (1) Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29213 were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate. MH broth was used to prepare 0.5 McFarland turbidity bacterial liquid, which was then diluted 50 times for later use;

[0214] AIE-1 molecules (B1) and AIE-2 molecules (A1) were weighed on a balance, dissolved in ultrapure water, and sterilized by filtration with a 0.22 μm filter membrane to prepare AIE reagent VII (the molar ratio of AIE-1 molecules (B1) and AIE-2 molecules (A1) was 1:2, and the concentrations were 4 mM and 8 mM, respectively), and stored at 4°C;

[0215] (2) Gradual dilution of the antimicrobial stock solution of levofloxacin was performed to prepare antimicrobial solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL). The prepared antimicrobial solutions were dispensed into a 96-well microplate at a rate of 100 μL / well for each gradient concentration;

[0216] Every 1 mL of the bacterial solution in step (1) was mixed evenly with 2 μL of AIE reagent VII, and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final concentration of AIE-1 molecule (B1) was 4 μM, and the final concentration of AIE-2 molecule (A1) was 8 μM);

[0217] (3) After the 96-well plate is prepared, place it in a fluorescence reader (96-well plate, etc.) and monitor it regularly for 8 hours (measure the fluorescence value every hour), set the excitation wavelength to 460nm and the emission wavelength to 560nm;

[0218] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0219] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0220] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0221] The results are as follows Figure 6a , Figure 6b As shown in Table 16, it can be seen that the addition of AIE reagent VII in Comparative Example 4 does not affect bacterial growth. After 24 hours, the corresponding MIC values ​​of Pseudomonas aeruginosa and Staphylococcus aureus monitored and judged by the traditional broth dilution method in the control group were 2 μg / mL and 0.5 μg / mL, respectively, while the MIC values ​​of Comparative Example 4 were greater than 32 μg / mL and 32 μg / mL, respectively. The sensitivity of AIE reagent VII in Comparative Example 4 was low and the MIC value could not be accurately judged.

[0222] Table 16:

[0223]

[0224] Comparative Example 5:

[0225] (1) Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29213 were cultured on plate culture medium at 37°C for 18 h, and single colonies were picked from the plate. MH broth was used to prepare 0.5 McFarland turbidity bacterial liquid, which was then diluted 50 times for later use;

[0226] Weigh the AIE-2 molecule (A1) with a balance, add ultrapure water to dissolve, filter and sterilize with a 0.22 μm filter membrane to prepare AIE reagent VIII (the concentration of AIE-2 molecule (A1) is 10 mM), and store at 4°C;

[0227] (2) Gradual dilution of the antimicrobial stock solution of levofloxacin was performed to prepare antimicrobial solutions of different gradient concentrations (the concentrations were 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL), and each gradient concentration of the prepared antimicrobial solution was dispensed into a 96-well microplate at 100 μL / well;

[0228] Each 1 mL of the bacterial solution from step (1) was mixed evenly with 2 μL of AIE reagent VIII and dispensed into the antimicrobial drug solution to be tested in a 96-well microplate at a rate of 100 μL / well to obtain several antimicrobial drug test solutions with different gradient concentrations (the final concentration of the AIE-2 molecule (A1) was 10 μM);

[0229] (3) After the 96-well plate is prepared, place it in a fluorescence reader (96-well plate, etc.) and monitor it regularly for 8 hours (measure the fluorescence value every hour), set the excitation wavelength to 460nm and the emission wavelength to 560nm;

[0230] (4) Calculate the relative fluorescence intensity F after incubation for n hours under different concentrations of antimicrobial solutions n =(I n -I 0 ) / I 0 , where I n is the fluorescence value after the culture time n hours, I 0 is the initial fluorescence value at 0 h of culture time;

[0231] Fitting F at different antibiotic concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a The slope k of the straight line when the antibiotic concentration is 0 0 By comparison, as the concentration of antibiotics increases, the first slope k a When ≤0.01, the antibiotic concentration a corresponding to the slope is determined as the MIC value of the antibiotic.

[0232] (5) Conduct the traditional broth dilution method antibiotic sensitivity test simultaneously, and determine the MIC value by visually observing the turbidity after 24 h of incubation.

[0233] The results are shown in Table 17. It can be seen that when AIE reagent VIII was added to Comparative Example 5, the MIC values ​​of Pseudomonas aeruginosa and Staphylococcus aureus detected by the traditional broth dilution method in the control group after 24 hours were 2 μg / mL and 0.5 μg / mL, respectively, while the MIC values ​​of Comparative Example 5 were greater than 32 μg / mL and less than 0 μg / mL, respectively. The sensitivity of AIE reagent VIII in Comparative Example 5 was low, the effect was poor, and the MIC value could not be accurately determined.

[0234] Table 17:

[0235]

[0236] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. Application of AIE molecules in in vitro antimicrobial drug sensitivity detection, characterized in that: The AIE molecule includes an AIE-1 molecule, and the AIE-1 molecule is selected from at least one of Formula 1; Formula 1 In Formula 1, R is each independently selected from hydrogen or methoxy; R1 is selected from , Z is a halide ion; R2 is selected from or , n1 is 1 or 2.

2. The use according to claim 1, characterized in that: The AIE-1 molecule is selected from , At least one of .

3. The use according to claim 1, characterized in that: The AIE molecules further include AIE-2 molecules; the AIE-2 molecules are selected from at least one of Formula 2; the molar ratio of the AIE-1 molecules to the AIE-2 molecules is 1:(5-10); Formula 2 In Formula 2, R1 and R2 are each independently selected from methyl or ethyl; R3, R4, and R5 are selected from methyl; n2 is 2 or 3; X - It is a halide ion.

4. The use according to claim 3, characterized in that: The AIE-2 molecule is selected from , At least one of .

5. The use according to claim 1, characterized in that: Used to determine the minimum inhibitory concentration of antimicrobial drugs.

6. An AIE reagent for rapid detection of antimicrobial drug sensitivity, characterized in that: Including AIE molecules; the AIE molecules include the AIE-1 molecules in the application according to any one of claims 1-2.

7. The AIE reagent for rapid detection of antimicrobial drug sensitivity according to claim 6, characterized in that: The AIE molecules also include the AIE-2 molecules in the application according to any one of claims 3 to 4; the molar ratio of the AIE-1 molecules to the AIE-2 molecules is 1:(5-10).

8. The AIE reagent for rapid detection of antimicrobial drug sensitivity according to claim 6, characterized in that: Also included are solvents.

9. A method for rapid detection of antimicrobial drug sensitivity, characterized in that: The following steps are involved: (1) preparing a bacterial solution, adding the AIE reagent according to any one of claims 6 to 8, and mixing them evenly to obtain a mixed bacterial solution containing the AIE reagent; (2) preparing different gradient concentrations of the antimicrobial drug solution to be tested by gradient dilution, adding the mixed bacterial solution of step (1) to obtain the test solution, culturing the bacteria, and monitoring the fluorescence value at different time points; Calculate the relative fluorescence intensity F at different antimicrobial drug concentrations and culture time n hours respectively n =(I n -I0) / I0, where I n is the fluorescence value at the incubation time of n hours, and I0 is the initial fluorescence value at the incubation time of 0 hours; (3) Fitting F at different antimicrobial drug concentrations n The linear relationship with time is determined by dividing the slope of the straight line by the slope k when the antibiotic concentration is a. a Compare with the slope k0 of the straight line when the antibiotic concentration is 0 to determine the minimum inhibitory concentration of the antibiotic and judge the sensitivity of the antibiotic; As the concentration of antibiotics increases, the first slope k a When ≤0.01, the antimicrobial drug concentration a corresponding to the slope is determined as the minimum inhibitory concentration of the antimicrobial drug.

10. The method for rapid detection of antimicrobial drug sensitivity according to claim 9, characterized in that: In step (2), in the test solution, the concentration of bacteria is any concentration between 0.5 McFarland turbidity and 0.5 McFarland turbidity diluted 1000 times, and the concentration of AIE molecules is 5-12 μM.

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