Fluorescent probe, preparation method and application thereof, and method for simultaneously detecting bacterial quantity and bacterial drug resistance

Abstract

The invention relates to the technical field of bacterial detection, in particular to a fluorescent probe, a preparation method and application thereof, and a method for simultaneously detecting bacterial quantity and bacterial drug resistance. The invention provides a fluorescent probe. The fluorescent probe is prepared from the following components: blue carbon quantum dots (BCDs) and a lanthanide coordination compound sensitized luminescent material (Tb-GMP), the invention also provides a method for simultaneously detecting the number of bacteria and the drug resistance of bacteria, and the method is simple to operate, low in cost, less in time consumption and easy to implement, does not need large-scale experimental equipment, and only needs the assistance of an ultraviolet lamp; the visual detection of the bacterial quantity and bacterial drug resistance can be realized through the change of the system color caused by the change of the fluorescence intensity; and a data analysis system and a linear equation can be further utilized for calculation, so that a detection result can be obtained in a short time, the detection efficiency is improved, and the accuracy is high.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteria detection, and in particular to a fluorescent probe and a preparation method and application thereof, and a method for simultaneously detecting the number of bacteria and bacterial drug resistance. Background Art

[0002] Antibiotics have been used to treat bacterial infections for more than 70 years, but the abuse of antibiotics in the treatment of human diseases and the addition of antibiotics to animal feed have exacerbated bacterial resistance. The increase in morbidity and mortality caused by resistant bacterial infections has greatly threatened global public health. Therefore, it is of great significance to rapidly detect bacteria and accurately assess bacterial resistance, strictly control the spread of resistant bacteria, study the mechanism of resistance, and improve the diagnosis and treatment of clinical infectious diseases.

[0003] Commonly used bacterial resistance tests include drug sensitivity tests and drug resistance genotype tests. Drug sensitivity tests include agar diffusion method and dilution method, which can determine whether the pathogenic microorganisms in a certain infectious disease are sensitive to specific drugs, thereby screening effective antibacterial drugs. The test results of this method are intuitive, but it depends on the growth of microorganisms, is time-consuming, and the operation is relatively cumbersome. Drug resistance genotype detection refers to the use of PCR, DNA sequencing, gene probes and other methods to detect drug-resistant bacteria. Although the detection of bacteria is fast and accurate, the technical cost is high and requires professional equipment and personnel to operate. In order to overcome the problems of the above methods, electrochemical detection methods, microfluidic chip-based methods, nanomaterial-based methods, etc. have also been reported. However, there are also problems such as complex operation, low sensitivity, long time, high cost, poor repeatability, and less intuitive results.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorescent probe and its preparation method and application, a method for simultaneously detecting the number of bacteria and bacterial drug resistance. The detection method of the present invention is not only simple to operate, low in cost, less time-consuming, easy to implement, does not require large-scale experimental equipment, but also has high sensitivity, visualization and high accuracy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a fluorescent probe, which is composed of the following components in parts by volume: 7 to 9 parts of blue carbon quantum dots BCDs and 180 to 220 parts of lanthanide coordination compound sensitized luminescent material Tb-GMP.

[0008] The present invention also provides a method for preparing the fluorescent probe, comprising the following steps: mixing blue carbon quantum dots BCDs and lanthanide coordination compound sensitized luminescent material Tb-GMP to obtain the fluorescent probe.

[0009] The present invention also provides application of the fluorescent probe in detecting bacterial quantity and bacterial drug resistance.

[0010] The present invention also provides a method for simultaneously detecting the number of bacteria and bacterial drug resistance, comprising the following steps:

[0011] (1) diluting the bacterial flora to be tested with physiological saline to obtain a bacterial suspension; mixing the bacterial suspension with a copper ion solution to obtain a copper ion bacterial suspension mixed solution;

[0012] (2) mixing the copper ion bacterial suspension mixture and the fluorescent probe to obtain a bacterial probe mixture, detecting the fluorescence intensity of the fluorescent probe and the bacterial probe mixture, and calculating the number of bacteria;

[0013] (3) mixing the bacterial probe mixture and the antibacterial drug solution for 5 to 15 minutes to obtain a mixed solution, and detecting the fluorescence intensity of the mixed solution;

[0014] (4) Centrifuging the mixed solution, taking the supernatant, detecting the fluorescence intensity of the supernatant, and calculating the bacterial resistance;

[0015] The calculation formula of the bacterial quantity is: fluorescence intensity of the bacterial probe mixture / fluorescence intensity of the fluorescent probe;

[0016] The calculation formula of the bacterial resistance is: fluorescence intensity of the supernatant / fluorescence intensity of the mixed solution-number of bacteria;

[0017] In step (2), the fluorescent probe is a fluorescent probe.

[0018] Preferably, in step (1), the initial concentration of the physiological saline is 0.70-0.90%; the concentration of the bacterial suspension is 1×10 5 ~1×10 8 CFU / mL.

[0019] Preferably, in step (1), the initial concentration of the copper ion solution is 20-30 mg / L; and the volume ratio of the bacterial suspension and the copper ion solution is 10:4-6.

[0020] Preferably, in step (2), the volume ratio of the copper ion bacterial suspension mixture and the fluorescent probe is 4 to 6:10.

[0021] Preferably, in step (3), the volume ratio of the bacterial probe mixture to the antibacterial drug solution is 3:0.5-1.5.

[0022] Preferably, in step (4), the centrifugal speed is 7500-8500 rpm; and the centrifugal time is 5-15 min.

[0023] Beneficial effects of the present invention:

[0024] The invention provides a method for simultaneously detecting the number of bacteria and bacterial drug resistance. Since the surface of bacteria carries negative charge, the negative charge on the surface of bacteria is the result of the joint action of multiple factors such as isoelectric point and cell wall components. The isoelectric point of Gram-positive bacteria is pH2-3, and the isoelectric point of Gram-negative bacteria is pH4-5. In general culture fluid, bacteria carry negative charge. At the same time, the teichoic acid of the bacterial cell wall contains a large number of phosphate groups with strong acidity. After the acidic groups are dissociated, the surface of bacteria carries negative charge. The copper ion surface carries positive charge. Therefore, after the copper ion is mixed with a solution of a bacterial flora to be tested, the copper ion can be adsorbed on the surface of the bacteria. Then, a certain amount of the solution of the bacterial flora to be tested with the copper ion adsorbed on the surface is absorbed and added to a fluorescent probe solution. The copper ion coordinates with the fluorescent probe, and metal-coordinated charge transfer occurs, so that the charge distribution of the probe has an obvious shift, resulting in quenching of the fluorescence of Tb-GMP. Therefore, the number of the bacterial flora to be tested can be reflected by the degree of fluorescence reduction of Tb-GMP.

[0025] After adding the antimicrobial solution, the oxygen atoms on the carboxyl and ketone groups on the surface of the antimicrobial further sensitize Tb 3+ The fluorescence is enhanced, and then centrifugation is performed to remove the bottom bacterial sediment and absorb the supernatant, which is composed of the fluorescent probe solution and the copper ions shed from the surface of the destroyed bacteria. As the copper ions in the system decrease, the peak value of Tb-GMP rises. Therefore, the rise in the peak value can reflect the drug resistance of bacteria. The stronger the drug resistance of bacteria, the fewer the destroyed bacterial colonies, and the fewer the number of free copper ions in the system after centrifugation, the more the peak value of Tb-GMP rises.

[0026] The detection method of the present invention is simple to operate, low in cost, time-consuming, and easy to implement. It does not require large-scale experimental equipment. It only needs to use ultraviolet light to assist. The change in the color of the system caused by the change in fluorescence intensity can realize the visual detection of the number of bacteria and bacterial resistance, and the result is more intuitive; it can also further use the data analysis system and linear equations for calculation, and the test results can be obtained in a short time, which improves the detection efficiency and has high accuracy. Therefore, the detection method of the present invention has high sensitivity and accuracy, and is efficient, visualized and versatile.

[0027] Sensitivity: The fluorescent probe in the present invention has a sensitive fluorescence response to copper ions and is indirectly sensitive to detecting bacteria, thereby being able to detect trace bacterial populations;

[0028] High efficiency: Compared with traditional bacterial resistance detection, the present invention utilizes the quenching effect of copper ions attached to the surface of bacteria on the fluorescence of fluorescent probes, which greatly shortens the detection time and improves the detection efficiency;

[0029] Visualization: The present invention utilizes the on and off of the TB-GMP fluorescence in the fluorescent probe to achieve the mutual conversion between blue and green, which can intuitively judge the number and drug resistance status of the tested bacterial flora, facilitating real-time analysis and monitoring;

[0030] Accuracy: The fluorescence of the fluorescent probe decreases or increases when the copper ions in the system increase or decrease, which can accurately reflect the number and drug resistance of bacteria.

[0031] Versatility: The system used in the present invention can detect copper ion concentration, the number of bacterial colonies to be tested, the concentration of antibiotics and the drug resistance of bacteria in a certain aspect, thus realizing the versatility of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flow chart of the method for simultaneously detecting bacterial count and bacterial resistance;

[0033] Figure 2 is the fluorescence spectrum of the bacterial probe mixture in Example 1;

[0034] Figure 3 In Example 1, under ultraviolet light, different concentrations of antibacterial drug solutions and bacterial probe mixtures (the concentration of bacterial suspension is 1×10 8 CFU / mL) fluorescence changes after mixing;

[0035] Figure 4 The fluorescence changes of the copper ion bacterial suspension mixture prepared from bacterial suspensions of different concentrations and the fluorescent probe after mixing under ultraviolet light in Example 2. DETAILED DESCRIPTION

[0036] The present invention provides a fluorescent probe, which is composed of the following components in parts by volume:

[0037] Blue carbon quantum dots BCDs (Blue Carbon Quantum Dots) 7-9 parts, preferably 7.3-7.7 parts, more preferably 8 parts;

[0038] The lanthanide complex sensitized light-emitting material Tb-GMP is 180 to 220 parts, preferably 190 to 210 parts, and more preferably 200 parts.

[0039] In the present invention, the intrinsic fluorescence of the fluorescent probe is green;

[0040] The particle size of the blue carbon quantum dots BCDs is 1 to 4 nm, preferably 2 to 3 nm, and more preferably 2.5 nm;

[0041] The potential of the blue carbon quantum dots BCDs is -17.2 mV; the potential of the lanthanide coordination compound sensitized luminescent material Tb-GMP is 19.3 mV; and the potential of the fluorescent probe is -13.3 mV.

[0042] The present invention also provides a method for preparing the fluorescent probe, comprising the following steps: mixing blue carbon quantum dots BCDs and lanthanide coordination compound sensitized luminescent material Tb-GMP to obtain the fluorescent probe.

[0043] The present invention also provides application of the fluorescent probe in detecting bacterial quantity and bacterial drug resistance.

[0044] The present invention also provides a method for simultaneously detecting the number of bacteria and bacterial drug resistance, comprising the following steps:

[0045] (1) diluting the bacterial flora to be tested with physiological saline to obtain a bacterial suspension; mixing the bacterial suspension with a copper ion solution to obtain a copper ion bacterial suspension mixed solution;

[0046] (2) mixing the copper ion bacterial suspension mixture and the fluorescent probe to obtain a bacterial probe mixture, detecting the fluorescence intensity of the fluorescent probe and the bacterial probe mixture, and calculating the number of bacteria;

[0047] (3) mixing the bacterial probe mixture and the antibacterial drug solution for 5 to 15 minutes to obtain a mixed solution, and detecting the fluorescence intensity of the mixed solution;

[0048] (4) Centrifuging the mixed solution, taking the supernatant, detecting the fluorescence intensity of the supernatant, and calculating the bacterial resistance;

[0049] The calculation formula of the bacterial quantity is: fluorescence intensity of the bacterial probe mixture / fluorescence intensity of the fluorescent probe;

[0050] The calculation formula of the bacterial resistance is: fluorescence intensity of the supernatant / fluorescence intensity of the mixed solution-number of bacteria;

[0051] In step (2), the fluorescent probe is a fluorescent probe.

[0052] In the present invention, in step (1), the initial concentration of the physiological saline is 0.70-0.90%, preferably 0.75-0.85%, and more preferably 0.80%; the concentration of the bacterial suspension is 1×10 5 ~1×10 8 CFU / mL, preferably 1×10 6 ~1×10 7 CFU / mL, more preferably 1×10 7 CFU / mL.

[0053] In the present invention, in step (1), the initial concentration of the copper ion solution is 20-30 mg / L, preferably 23-27 mg / L, and more preferably 25 mg / L; the volume ratio of the bacterial suspension and the copper ion solution is 10:4-6, preferably 10:5;

[0054] The method of mixing the bacterial suspension and the copper ion solution is stirring and mixing;

[0055] After the bacterial suspension and the copper ion solution are mixed, as the number of bacteria in the bacterial suspension increases, the number of adsorbed copper ions will also increase.

[0056] In the present invention, in step (2), the volume ratio of the copper ion bacterial suspension mixture and the fluorescent probe is 4 to 6:10, preferably 5:10;

[0057] The copper ion bacterial suspension mixture and the fluorescent probe are mixed by oscillating, and the mixing time is 5 to 15 minutes, more preferably 8 to 12 minutes, and more preferably 10 minutes;

[0058] After the copper ion bacterial suspension mixture and the fluorescent probe are mixed, the fluorescent probe, whose intrinsic fluorescence is green, is quenched, and the color can be observed to change from green to blue under ultraviolet irradiation.

[0059] In the present invention, in step (3), the volume ratio of the bacterial probe mixture and the antibacterial drug solution is 3:0.5-1.5, preferably 3:0.8-1.2, and more preferably 3:1;

[0060] When the bacterial probe mixture and the antibacterial drug solution are mixed, a HEPES buffer solution needs to be added. The volume ratio of the HEPES buffer solution to the antibacterial drug solution is 1:0.5-1.5, preferably 1:0.8-1.2, and more preferably 1:1.

[0061] In the present invention, in step (3), the method of mixing the bacterial probe mixture and the antibacterial drug solution is oscillating mixing;

[0062] The mixing time of the bacterial probe mixture and the antibacterial drug solution is preferably 8 to 12 minutes, more preferably 10 minutes;

[0063] After the bacterial probe mixture and the antimicrobial drug solution are mixed, the antimicrobial drugs cause some bacteria to die or change their surface structure, and the copper ions adsorbed on their surface diffuse into the solution. When irradiated with ultraviolet light, as the concentration of the antimicrobial drugs increases, the fluorescence gradually changes from blue to green. In this process, the degree of decrease in the fluorescence intensity of the fluorescent probe can reflect the number of bacterial colonies to be tested.

[0064] In the present invention, in step (4), the centrifugal speed is 7500-8500 rpm, preferably 7800-8200 rpm, and more preferably 8000 rpm; the centrifugal time is 5-15 min, preferably 8-12 min, and more preferably 10 min;

[0065] The bacterial colonies are removed by centrifugation, along with the copper ions adsorbed on the surface of live bacteria. The fluorescent probe and the copper ions detached from the dead bacteria remain in the supernatant.

[0066] Since the copper ion concentration in the supernatant is lower than that in the mixed solution, the fluorescence of the supernatant is enhanced compared with that of the mixed solution. By recording the fluorescence intensity of the supernatant, the drug resistance of bacteria can be reflected by the increase in the peak value of the characteristic peak of the fluorescent probe.

[0067] In the present invention, the detection conditions for measuring the fluorescence intensity of the bacterial probe mixture, the fluorescence intensity of the fluorescent probe, the fluorescence intensity of the supernatant and the fluorescence intensity of the mixed solution are: the excitation light is 345nm, the voltage is 450V, the slit is 10nm, and the fluorescence intensity of 365-670nm is detected and recorded.

[0068] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0069] Example 1 A fluorescent probe

[0070] 8 μL of blue carbon quantum dots BCDs (particle size 2 nm, potential -17.2 mV) and 200 μL of lanthanide coordination compound sensitized luminescent material Tb-GMP (potential 19.3 mV) were mixed to obtain a fluorescent probe (BCDs@Tb-GMP) with a potential of -13.3 mV.

[0071] Example 2 A fluorescent probe

[0072] 7 μL of blue carbon quantum dots BCDs (particle size 3 nm, potential -17.2 mV) and 220 μL of lanthanide coordination compound sensitized luminescent material Tb-GMP (potential 19.3 mV) were mixed to obtain a fluorescent probe with a potential of -13.3 mV.

[0073] Example 3

[0074] 9 μL of blue carbon quantum dots BCDs (particle size 4 nm, potential -17.2 mV) and 180 μL of lanthanide coordination compound sensitized luminescent material Tb-GMP (potential 19.3 mV) were mixed to obtain a fluorescent probe with a potential of -13.3 mV.

[0075] Example 4

[0076] A method for simultaneously detecting bacterial counts and bacterial resistance (e.g. Figure 1 (shown)

[0077] (1) Escherichia coli strains (purchased from Guangzhou Huankai Microbiology Technology Co., Ltd.) were selected and placed in LB broth liquid culture medium and incubated at 37°C for 24 hours to obtain Escherichia coli flora. The Escherichia coli flora was prepared with 0.85% saline to concentrations of 0 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL bacterial suspension; take 1mL bacterial suspension and 500μL copper ion solution (concentration: 25mg / L) and stir to mix, so that the Escherichia coli flora adsorbs copper ions to obtain a copper ion bacterial suspension mixture;

[0078] (2) Add 100 μL of the fluorescent probe described in Example 1 to a disposable 96-well plate, and then add 50 μL of the copper ion bacterial suspension mixture, shake and mix for 10 minutes to obtain a bacterial probe mixture, and use a fluorescence detection device F7000 to detect the fluorescence intensity of the bacterial probe mixture and the fluorescent probe. The specific steps are: set the detection temperature to a constant temperature of 30°C, the excitation wavelength to 345 nm, detect and record the fluorescence intensity value in the band of 365 to 670 nm, and the fluorescence spectrum of the bacterial probe mixture is as follows: Figure 2 As shown, the number of bacteria was calculated using the formula: fluorescence intensity of the bacterial probe mixture / fluorescence intensity of the fluorescent probe;

[0079] (3) using 0.03 mmol / L sodium hydroxide solution to dilute enrofloxacin to a concentration of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 70 mg / L to obtain an antibacterial drug solution; adding 50 μL of HEPES buffer solution to the bacterial probe mixture in step (2), and then adding different concentrations of antibacterial drug solutions respectively, shaking the reaction for 10 minutes to obtain a mixed solution, and irradiating the mixed solution with an ultraviolet lamp (such as Figure 3 As shown), it was found that the fluorescence gradually changed from blue to green, and the fluorescence intensity of the mixed solution was detected by referring to the method described in step (2);

[0080] (4) The mixed solution was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min. The precipitate was removed and the supernatant was collected. The fluorescence intensity of the supernatant was detected according to the method described in step (2) and the bacterial resistance was calculated using the following formula: fluorescence intensity of the supernatant / fluorescence intensity of the mixed solution-number of bacteria.

[0081] according to Figure 2It can be seen that at 545nm, when the concentration of bacterial suspension is 0CFU / mL, the fluorescence intensity of the bacterial probe mixture is 374.6; when the concentration of bacterial suspension is 1×10 5 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 344.5; the concentration of the bacterial suspension was 1×10 6 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 308.3; the concentration of the bacterial suspension was 1×10 7 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 276.7; the concentration of the bacterial suspension was 1×10 8 CFU / mL, the fluorescence intensity of the bacterial probe mixture is 154.9; according to the fluorescence intensity (y) and the bacterial concentration (x), a curve equation is drawn, and the number of bacteria (concentration) in the sample can be calculated by substituting the fluorescence intensity after the sample measurement into the equation;

[0082] When the concentration of the bacterial suspension is 0 CFU / mL, that is, only physiological saline is absorbed for reaction, at this time, it is the intrinsic fluorescence of the fluorescent probe. Therefore, when the concentration of the bacterial suspension is 0 CFU / mL, the fluorescence intensity of the fluorescent probe is 374.6;

[0083] from Figure 3 It can be seen that in the bacterial system without adding the antimicrobial solution, the bacteria grow normally. As the number of E. coli increases, more copper ions are adsorbed on its surface, and the fluorescence of the fluorescent probe is quenched more. The peak value of Tb-GMP will indirectly decrease with the increase of the number of E. coli. After adding the antimicrobial solution, as the concentration of the antimicrobial solution increases, the fluorescence of Tb-GMP increases, and some E. coli are killed and the number decreases. The destroyed E. coli and the resistant E. coli are removed by centrifugation, the copper ions in the system decrease, and the fluorescence of Tb-GMP increases again.

[0084] After adding enrofloxacin, the fluorescence of TB-GMP was sensitized. Based on TB-GMP, at 545nm, when the concentration of bacterial suspension was 0 CFU / mL, when the concentration of the antimicrobial solution was 20 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 2069; when the concentration of the antimicrobial solution was 30 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 3085; when the concentration of the antimicrobial solution was 40 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 4070; when the concentration of the antimicrobial solution was 50 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 5035; when the concentration of the antimicrobial solution was 60 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 6088; when the concentration of the antimicrobial solution was 70 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 6770;

[0085] Based on TB-GMP, at 545 nm, the concentration of bacterial suspension was 1×10 8 CFU / mL, when the concentration of the antimicrobial solution is 20 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 1976; when the concentration of the antimicrobial solution is 30 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 2880; when the concentration of the antimicrobial solution is 40 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 3997; when the concentration of the antimicrobial solution is 50 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 4924; when the concentration of the antimicrobial solution is 60 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 6003; when the concentration of the antimicrobial solution is 70 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 6887;

[0086] When the concentration was >60mg / L, the fluorescence intensity recovered. The minimum bactericidal concentration of enrofloxacin for the Escherichia coli in this experiment was 60-70mg / L.

[0087] Example 5

[0088] (1) A Staphylococcus aureus strain (purchased from Guangzhou Huankai Microbiology Technology Co., Ltd.) was selected and placed in LB broth liquid culture medium, and incubated at 37°C for 24 hours to obtain Staphylococcus aureus flora. The Staphylococcus aureus flora was prepared with 0.85% saline to concentrations of 0 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL bacterial suspension; 1mL bacterial suspension and 500μL copper ion solution (concentration: 25mg / L) were stirred and mixed to allow the large Staphylococcus aureus flora to adsorb copper ions to obtain a copper ion bacterial suspension mixture;

[0089] (2) Add 100 μL of the fluorescent probe described in Example 1 to a disposable 96-well plate, and then add 50 μL of the copper ion bacterial suspension mixture, shake and mix for 10 minutes to obtain a bacterial probe mixture, and irradiate the bacterial probe mixture with an ultraviolet lamp (such as Figure 4 As shown), it was found that fluorescence quenching occurred after the fluorescent probe and the copper ion bacterial suspension were mixed, so that the fluorescence changed from green to blue; the fluorescence intensity of the bacterial probe mixture and the fluorescent probe was detected using the fluorescence detection equipment F7000, and the specific steps were: setting the detection temperature to a constant temperature of 30°C, the excitation wavelength to 345nm, detecting and recording the fluorescence intensity value in the band of 365-670nm, and calculating the number of bacteria. The calculation formula was: fluorescence intensity of the bacterial probe mixture / fluorescence intensity of the fluorescent probe;

[0090] (3) diluting ciprofloxacin with deionized water to a concentration of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 70 mg / L to obtain an antibacterial drug solution; adding 50 μL of HEPES buffer solution to the bacterial probe mixture in step (2), and then adding different concentrations of antibacterial drug solutions respectively, shaking the reaction for 10 minutes to obtain a mixed solution, irradiating the mixed solution with an ultraviolet lamp, and finding that the fluorescence gradually changes from blue to green, and detecting the fluorescence intensity of the mixed solution by referring to the method described in step (2);

[0091] (4) The mixed solution was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min. The precipitate was removed and the supernatant was collected. The fluorescence intensity of the supernatant was detected according to the method described in step (2) and the bacterial resistance was calculated using the following formula: fluorescence intensity of the supernatant / fluorescence intensity of the mixed solution-number of bacteria.

[0092] from Figure 4 It can be seen that in the bacterial system without adding antimicrobial solution, the bacteria grow normally. As the number of Staphylococcus aureus increases, more copper ions are adsorbed on the bacterial surface in the system, and the fluorescence of the fluorescent probe is quenched more. The peak value of Tb-GMP will indirectly decrease with the increase in the number of Staphylococcus aureus.

[0093] At 545 nm, when the concentration of bacterial suspension was 0 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 385.7; when the concentration of bacterial suspension was 1×10 5 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 347.1; the concentration of the bacterial suspension was 1×10 6 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 330.6; the concentration of the bacterial suspension was 1×10 7 CFU / mL, the fluorescence intensity of the bacterial probe mixture was 223.4; the concentration of the bacterial suspension was 1×10 8 CFU / mL, the fluorescence intensity of the bacterial probe mixture is 145.7; based on the fluorescence intensity (y) and the bacterial concentration (x), a curve equation is drawn, and the number of bacteria (concentration) in the sample can be calculated by substituting the fluorescence intensity after the sample measurement into the equation;

[0094] When the concentration of the bacterial suspension is 0 CFU / mL, that is, only physiological saline is absorbed for reaction, at this time, it is the intrinsic fluorescence of the fluorescent probe. Therefore, when the concentration of the bacterial suspension is 0 CFU / mL, the fluorescence intensity of the fluorescent probe is 385.7;

[0095] After adding the antimicrobial solution, as the concentration of the antimicrobial solution increases, the fluorescence of Tb-GMP increases, and some Staphylococcus aureus are killed, and the number decreases; then the destroyed Staphylococcus aureus and drug-resistant Staphylococcus aureus are removed by centrifugation, the copper ions in the system decrease, and the fluorescence of Tb-GMP increases again;

[0096] After adding ciprofloxacin, the fluorescence of TB-GMP was sensitized. Based on TB-GMP, at 545nm, when the concentration of bacterial suspension was 0 CFU / mL, when the concentration of the antimicrobial solution was 20 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 2001; when the concentration of the antimicrobial solution was 30 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 2432; when the concentration of the antimicrobial solution was 40 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 3171; when the concentration of the antimicrobial solution was 50 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 4226; when the concentration of the antimicrobial solution was 60 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 5007; when the concentration of the antimicrobial solution was 70 mg / L, the fluorescence intensity of the copper ion bacterial community mixture was 6308;

[0097] Based on TB-GMP, at 545 nm, the concentration of bacterial suspension was 1×10 8 CFU / mL, when the concentration of the antimicrobial solution is 20 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 1950; when the concentration of the antimicrobial solution is 30 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 2451; when the concentration of the antimicrobial solution is 40 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 3215; when the concentration of the antimicrobial solution is 50 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 4358; when the concentration of the antimicrobial solution is 60 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 5121; when the concentration of the antimicrobial solution is 70 mg / L, the fluorescence intensity of the copper ion-bacteria mixture is 6341;

[0098] The minimum bactericidal concentration of enrofloxacin for the experimental bacteria Staphylococcus aureus is 10-20 mg / L.

[0099] From the above examples, it can be seen that the present invention provides a fluorescent probe and its preparation method and application, and a method for simultaneously detecting the number of bacteria and bacterial resistance. The detection method of the present invention is not only simple to operate, low in cost, less time-consuming, easy to implement, does not require large-scale experimental equipment, but also has high sensitivity, visualization, and high accuracy.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that The fluorescent probe is composed of the following components in volume fractions: 7 to 9 parts of blue carbon quantum dots BCDs and 180 to 220 parts of lanthanide coordination compound sensitized luminescent material Tb-GMP.

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The method comprises the following steps: mixing blue carbon quantum dots BCDs and lanthanide coordination compound sensitized luminescent material Tb-GMP to obtain a fluorescent probe.

3. Use of the fluorescent probe according to claim 1 in detecting bacterial count and bacterial drug resistance.

4. A method for simultaneously detecting bacterial count and bacterial drug resistance, characterized in that: The steps include: (1) diluting the bacterial flora to be tested with physiological saline to obtain a bacterial suspension; mixing the bacterial suspension with a copper ion solution to obtain a copper ion bacterial suspension mixed solution; (2) mixing the copper ion bacterial suspension mixture and the fluorescent probe to obtain a bacterial probe mixture, detecting the fluorescence intensity of the fluorescent probe and the bacterial probe mixture, and calculating the number of bacteria; (3) mixing the bacterial probe mixture and the antibacterial drug solution for 5 to 15 minutes to obtain a mixed solution, and detecting the fluorescence intensity of the mixed solution; (4) Centrifuging the mixed solution, taking the supernatant, detecting the fluorescence intensity of the supernatant, and calculating the bacterial resistance; The calculation formula of the bacterial quantity is: fluorescence intensity of the bacterial probe mixture / fluorescence intensity of the fluorescent probe; The calculation formula of the bacterial resistance is: fluorescence intensity of the supernatant / fluorescence intensity of the mixed solution-number of bacteria; In step (2), the fluorescent probe is the fluorescent probe described in claim 1.

5. The method according to claim 4, characterized in that In step (1), the initial concentration of the physiological saline is 0.70-0.90%; the concentration of the bacterial suspension is 1×10 5 ~1×10 8 CFU / mL.

6. The method according to claim 5, characterized in that In step (1), the initial concentration of the copper ion solution is 20-30 mg / L; the volume ratio of the bacterial suspension and the copper ion solution is 10:4-6.

7. The method according to claim 6, characterized in that In step (2), the volume ratio of the copper ion bacterial suspension mixture and the fluorescent probe is 4 to 6:

10.

8. The method according to claim 7, characterized in that In step (3), the volume ratio of the bacterial probe mixture and the antibacterial drug solution is 3:0.5-1.

5.

9. The method according to claim 8, characterized in that In step (4), the centrifugal speed is 7500-8500 rpm; the centrifugal time is 5-15 min.

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