CN116465870A - Detection system of antibiotic molecule and antibacterial application thereof - Google Patents
Detection system of antibiotic molecule and antibacterial application thereof Download PDFInfo
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- CN116465870A CN116465870A CN202310453721.7A CN202310453721A CN116465870A CN 116465870 A CN116465870 A CN 116465870A CN 202310453721 A CN202310453721 A CN 202310453721A CN 116465870 A CN116465870 A CN 116465870A
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- 230000003115 biocidal effect Effects 0.000 title claims abstract description 36
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 12
- 239000002091 nanocage Substances 0.000 claims abstract description 60
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 29
- 230000004044 response Effects 0.000 claims abstract description 27
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 26
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 10
- 239000003782 beta lactam antibiotic agent Substances 0.000 claims abstract description 10
- 239000002132 β-lactam antibiotic Substances 0.000 claims abstract description 10
- 229940124586 β-lactam antibiotics Drugs 0.000 claims abstract description 10
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims abstract description 3
- 229960003085 meticillin Drugs 0.000 claims abstract description 3
- -1 tetraphenyl ethylene (TPE) cation Chemical class 0.000 claims abstract description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 16
- 239000012498 ultrapure water Substances 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 239000010453 quartz Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 206010041925 Staphylococcal infections Diseases 0.000 claims 1
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 claims 1
- 238000002983 circular dichroism Methods 0.000 abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 230000009977 dual effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- JLZUZNKTTIRERF-UHFFFAOYSA-N tetraphenylethylene Chemical group C1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 JLZUZNKTTIRERF-UHFFFAOYSA-N 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract description 2
- 230000001954 sterilising effect Effects 0.000 abstract description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 23
- 241000894006 Bacteria Species 0.000 description 15
- 239000002609 medium Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 229960002793 amoxicillin sodium Drugs 0.000 description 6
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C([O-])=O)(C)C)=CC=CC=C1 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 6
- 229960001931 ampicillin sodium Drugs 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 229960003669 carbenicillin Drugs 0.000 description 6
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 6
- MGFZNWDWOKASQZ-UMLIZJHQSA-M methicillin sodium Chemical compound [Na+].COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C([O-])=O)C(C)(C)S[C@@H]21 MGFZNWDWOKASQZ-UMLIZJHQSA-M 0.000 description 6
- 229940019826 methicillin sodium Drugs 0.000 description 6
- ILVPFTMKCHREDJ-UHFFFAOYSA-N methyl 5-amino-2-fluorobenzoate Chemical compound COC(=O)C1=CC(N)=CC=C1F ILVPFTMKCHREDJ-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000001985 kidney epithelial cell Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000032770 biofilm formation Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000010065 bacterial adhesion Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 150000002678 macrocyclic compounds Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/19—Dichroism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a detection system of antibiotic molecules and an antibacterial application thereof, which utilize a tetraphenyl ethylene (TPE) cation molecular cage as a recognition response host of antibiotics. Wherein, the TPE unit endows the molecular cage with excellent fluorescence performance and self-adaptive chiral conversion capability, and realizes dual response to fluorescence and Circular Dichroism (CD) spectrum of the beta-lactam antibiotic; the octacation characteristic not only ensures good water solubility of the molecular cage, but also is more beneficial to the action of the molecular cage and the negatively charged biological film to break down the biological film, thereby realizing the effects of sterilization and bacteriostasis on Staphylococcus Aureus (SA) and methicillin-resistant staphylococcus aureus (MRSA).
Description
Technical Field
The invention relates to the field of medical detection, in particular to a detection system of antibiotic molecules and an antibacterial application thereof.
Background
Antibiotics are widely used for preventing and treating diseases caused by bacterial infection due to their bactericidal or bacteriostatic effects. However, with the massive use of various antibiotics, the number of cases in which bacteria develop resistance to the antibiotics has increased exponentially, greatly reducing the effectiveness of the antibiotics, and posing a serious threat to human health and the ecological environment, such as impairing the function of human organs or having potential oncogenic mutagenesis. Therefore, it is necessary and urgent to develop a material having a sensitive responsiveness to antibiotics and having an antibacterial effect.
Supermolecular materials, particularly those based on macrocyclic supermolecular materials, have unprecedented advantages in antibiotic detection due to their encapsulatable cavities, multifunctional structures, and their dynamic adjustability of host-guest complexation. However, most of the macrocycles used for antibiotic identification have limited optical response due to lack of luminophores, and detection of antibiotics still needs to be achieved by means of conventional liquid chromatography, mass spectrometry, capillary electrophoresis, electrochemistry and other technologies, which are complicated, time-consuming, and not highly selective and sensitive. Thus, a macrocyclic ring with an optical response is a very attractive class of molecular tools for identifying detection antibiotics.
In addition, bacterial biofilm formation isolates the bacteria from the host immune system and antibiotics, making the bacteria more susceptible to drug resistance. However, because its surface consists of an anionic matrix, its electrostatic interaction with the cationic host is believed to be one of the key forces affecting bacterial adhesion to the surface and inhibiting early biofilm formation. Therefore, the macrocyclic host with rich cations is expected to disrupt the biofilm by acting with the negatively charged biofilm, thereby achieving bacteriostatic effects.
Disclosure of Invention
In view of the above technical problems, the present invention utilizes a tetraphenyl ethylene (TPE) cationic molecular cage as a recognition response host for antibiotics. Wherein, the TPE unit endows the molecular cage with excellent fluorescence performance and self-adaptive chiral conversion capability, and realizes dual response to fluorescence and Circular Dichroism (CD) spectrum of the beta-lactam antibiotic; the octacation characteristic not only ensures good water solubility of the molecular cage, but also is more beneficial to the action of the molecular cage and the negatively charged biological film to break down the biological film, thereby realizing the effects of sterilization and bacteriostasis. The invention provides the following technical scheme:
according to the invention, the tetra-styrene TPE cation molecular cage is used as an identification response host of antibiotics, the beta-lactam antibiotics are dripped into the aqueous solution of the TPE molecular cage, and after being stirred and mixed uniformly, the dual response detection of fluorescence and CD can be obtained through a fluorescence and circular dichroism meter.
The fluorescence response detection of the molecular cage to the beta-lactam antibiotics comprises the steps of weighing 4.75mg of molecular cage solid sample, dissolving the molecular cage solid sample in 0.5mL of ultrapure water, carrying out ultrasonic dissolution, preparing the molecular cage solid sample into 4.8mM of concentrated solution for standby, simultaneously respectively weighing a certain amount of antibiotic sample PQ, AMP, CAR, AMX and MET in a sample bottle, respectively adding a certain amount of ultrapure water, carrying out ultrasonic dissolution, respectively preparing the antibiotic sample PQ, AMP, CAR, AMX and MET into 4.8mM of concentrated solution of the antibiotics, sucking 6.25 mu L of concentrated solution of the molecular cage by a pipetting gun, adding 2993.75 mu L of ultrapure water to dilute the concentration to 10 mu M, then sucking 2.4mL of diluted solution of the molecular cage of 10 mu M into a quartz cuvette, sequentially adding 1.0 mu L (0.2 equivalent) of concentrated solution of the antibiotics, and testing the fluorescence response behavior of the molecular cage to the antibiotics by a fluorescence spectrometer.
The CD response detection of the molecular cage to the beta-lactam antibiotics comprises the steps of weighing 4.75mg of molecular cage solid sample, dissolving the molecular cage solid sample in 0.5mL of ultrapure water, carrying out ultrasonic dissolution, preparing the molecular cage solid sample into 4.8mM of concentrated solution for standby, simultaneously respectively weighing a certain amount of antibiotic sample PQ, AMP, CAR, AMX and MET in a sample bottle, respectively adding a certain amount of ultrapure water, carrying out ultrasonic dissolution, respectively preparing the molecular cage solid sample into 4.8mM of concentrated solution of the antibiotic, sucking 12.5 mu L of concentrated solution of the molecular cage by a pipetting gun, adding 2987.5 mu L of ultrapure water to dilute the concentration to 20 mu M, then sucking 2.4mL of diluted solution of the molecular cage of 20 mu M into a quartz cuvette, sequentially adding 2.0 mu L (0.2 equivalent) of concentrated solution of the antibiotic, and testing the CD response behavior of the molecular cage to the antibiotics by a circular dichroscope.
The tetraphenyl ethylene TPE cation molecular cage provided by the invention has low cytotoxicity to human kidney epithelial cells 293T, and compared with single antibiotics, the molecular cage and the compound of the molecular cage and the antibiotics show good antibacterial activity to Staphylococcus Aureus (SA) and methicillin-resistant staphylococcus aureus (MRSA). Human kidney epithelial cells 293T were cultured in fresh medium containing different concentrations of molecular cages in a sterile environment usingCell viability was measured by the method and fluorescence was read using a microplate reader (Molecular Devices), each set of experimentsRepeat five times. Adding material solution (molecular cage, molecular cage and host-guest complex of antibiotics, antibiotics) into a liquid culture medium, diluting the culture solution of a 96-well plate row by row after uniformly mixing, adding bacterial solution into each well, placing the mixture in a shaking table at 37 ℃ for overnight culture, and visually observing whether each well liquid becomes turbid, if so, indicating that bacteria grow, if not, indicating that the bacteria are killed, and the minimum killing concentration is the obtained MIC.
Based on the technical scheme, the invention has the following beneficial effects:
(1) According to the invention, no extra fluorescent dye is required to be introduced, and double-response detection of fluorescence and CD of the beta-lactam antibiotic can be realized by utilizing a single molecular cage, so that the method is simple and convenient to operate, high in sensitivity and low in cost.
(2) The molecular cage used in the invention shows low cytotoxicity and biocompatibility to human kidney epithelial cells 293T in vitro experiments. And compared with the single antibiotics, the molecular cage and the host-guest complex of the molecular cage and the antibiotics have better antibacterial effect on SA and MRSA bacteria.
Compared with the prior art, the invention can be combined with a molecular cage for researching drug delivery, realizes response detection and drug delivery to antibiotic molecules by utilizing a single molecular cage platform, can achieve the effects of resisting and inhibiting bacteria, and opens up a new way for reasonable use of antibiotics.
Drawings
FIG. 1 shows a molecular cage and antibiotics according to the present invention: penicillin sodium (PQ), carbenicillin sodium (CAR), ampicillin sodium (AMP), amoxicillin sodium (AMX), methicillin sodium (MET)).
FIG. 2 is a graph showing the fluorescence response of molecular cages (10. Mu.M) according to example 1 of the present invention to sodium Penicillin (PQ) in water.
FIG. 3 is a bar graph of the fluorescence response of the molecular cage (10. Mu.M) of example 1 of the present invention to five antibiotics in water.
FIG. 4 is a graph showing the CD response of molecular cages (20. Mu.M) according to example 2 of the present invention to sodium Penicillin (PQ) in water.
FIG. 5 is a bar graph of CD response of molecular cages (20. Mu.M) according to example 2 of the invention to five antibiotics in water.
FIG. 6 is a chart showing a molecular cage cytotoxicity test according to example 3 of the present invention.
FIG. 7 is a graph showing the antibacterial activity of the molecular cages, the molecular cage-antibiotic complex and the antibiotic according to example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Fluorescence response detection of molecular cages to β -lactam antibiotics:
a4.75 mg molecular cage solid sample was weighed and dissolved in 0.5mL of ultrapure water, and sonicated to prepare a 4.8mM concentrated solution for use. Simultaneously, a certain amount of antibiotic sample PQ, AMP, CAR, AMX and MET are respectively weighed in sample bottles, a certain amount of ultrapure water is respectively added, and the mixture is ultrasonically dissolved to prepare 4.8mM antibiotic concentrated solutions respectively. 6.25. Mu.L of the concentrated solution of the molecular cage was pipetted, 2993.75. Mu.L of ultrapure water was added to dilute the concentration to 10. Mu.M, then 2.4mL of the 10. Mu.M diluted solution of the molecular cage was pipetted into a quartz cuvette, and 1.0. Mu.L (0.2 eq.) of the concentrated solution of the antibiotic was successively added, and the fluorescence response behavior of the molecular cage to the antibiotic was tested by a fluorescence spectrometer.
Example 2
Detection of CD response of molecular cages to β -lactam antibiotics:
in the same manner as in example 1, 12.5. Mu.L of the concentrated solution of the molecular cage was pipetted with a pipette, the concentration thereof was diluted to 20. Mu.M by adding 2987.5. Mu.L of ultrapure water, then 2.4mL of the diluted solution of the molecular cage of 20. Mu.M was pipetted into a quartz cuvette, and 2.0. Mu.L (0.2 eq.) of the concentrated solution of the antibiotic was successively added, and CD response behavior of the molecular cage to the antibiotic was tested by a circular dichroism spectrometer.
Example 3
Cytotoxicity test of molecular cages:
DMEM (Gibco) medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco), 1.0X105U/L penicillin (Hyclone) and 100mg/L streptavidin (Hyclone) was used as complete growth medium. 293T cells were seeded in 96-well plates at a density of 10000 cells per well and after 24h of culture, the medium was replaced with fresh medium containing molecular cages of different concentrations. Cells were inoculated on TCP without extract as positive control group. After further culturing for 24 hours, adoptingCell viability was measured by the method. The medium was replaced with a medium containing 10. Mu.LAlam +>100. Mu.L of complete growth medium of the reagent. After 4h incubation, 90. Mu.L of medium per well was transferred to a 96 well blackboard (Costar). Fluorescence was read using a microplate reader (molecular devices) using 560nm as excitation wavelength and 600nm as emission wavelength, and each set of experiments was repeated 5 times.
Example 4
Molecular cages, molecular cage and antibiotic complex, antibiotic antibacterial activity test:
(1) Bacterial culture: a quantity of SA and MRSA bacteria was removed from the dishes with an inoculating loop in 6-8 mM HB broth and incubated overnight at 37℃on a shaker, which is the first generation bacteria. Then, 50. Mu.L of the bacterial liquid was taken from the first-generation bacteria and cultured in 5 mM HB medium at 37℃for 9 hours, which is the second-generation bacteria, and the concentration of the obtained second-generation bacteria was considered to be 10 8 . Then the second generation bacteria are selected as the experimental object, and are diluted to 10 in gradient 5 。
(2) Antibacterial activity test: 100. Mu.L of 2 XMHB liquid medium was added to the first row of the 96-well plate, and 100. Mu.L of 1 XMHB medium was added to the next 7 rows. Preparing 4 times target material (molecular cage, host-guest complex of molecular cage and antibiotic, antibiotic) concentration, adding 100 μl of material solution into the first row, mixing, and sucking 100 μl of liquid to the next row of holesIn this step, the dilution was 2-fold, and the reaction was repeated until the final row of wells was aspirated and 100. Mu.L of the wells were discarded. If the concentration starts from 1200. Mu.M, the well plate concentration is 1200. Mu.M, 600. Mu.M, 300. Mu.M, 150. Mu.M, 75. Mu.M, 37.5. Mu.M, 18.75. Mu.M, 9.375. Mu.M in this order. After dilution was completed, 100. Mu.L of 10 was added to each well 5 Bacterial liquid, three groups were repeated, and one group was blank (no bacterial liquid was added and only 1×mhb was added).
Shaking overnight at 37deg.C, and observing the results. And (5) visually checking whether each hole of liquid becomes turbid, if so, indicating that bacteria grow, if not, indicating that bacteria are killed, and if not, obtaining the minimum killing concentration as the MIC.
Claims (4)
1. A detection system of antibiotic molecules is characterized in that a tetraphenyl ethylene cation molecular cage is used as a recognition response host of the antibiotic.
2. The detection system of an antibiotic molecule according to claim 1, wherein the fluorescent response of the molecular cage to the β -lactam antibiotic is detected as follows:
weighing 4.75mg of molecular cage solid sample, dissolving in 0.5mL of ultrapure water, carrying out ultrasonic dissolution, preparing a 4.8mM concentrated solution for later use, simultaneously respectively weighing a certain amount of antibiotic sample PQ, AMP, CAR, AMX and MET in a sample bottle, respectively adding a certain amount of ultrapure water, carrying out ultrasonic dissolution, respectively preparing 4.8mM concentrated solution of the antibiotic, sucking 6.25 mu L of concentrated solution of the molecular cage by a pipette, adding 2993.75 mu L of ultrapure water to dilute the concentration to 10 mu M, then sucking 2.4mL of 10 mu M dilute solution of the molecular cage into a quartz cuvette, sequentially adding 1.0 mu L of 0.2 equivalent concentrated solution of the antibiotic, and testing the fluorescent response behavior of the molecular cage to the antibiotic by a fluorescence spectrometer.
3. The detection system of an antibiotic molecule according to claim 1, wherein the CD response of said molecular cage to a β -lactam antibiotic is detected as follows:
weighing 4.75mg of molecular cage solid sample, dissolving in 0.5mL of ultrapure water, carrying out ultrasonic dissolution, preparing a 4.8mM concentrated solution for later use, simultaneously respectively weighing a certain amount of antibiotic sample PQ, AMP, CAR, AMX and MET in a sample bottle, respectively adding a certain amount of ultrapure water, carrying out ultrasonic dissolution, respectively preparing 4.8mM concentrated solution of the antibiotic, sucking 12.5 mu L of concentrated solution of the molecular cage by a pipette, adding 2987.5 mu L of ultrapure water to dilute the concentration to 20 mu M, then sucking 2.4mL of 20 mu M diluted solution of the molecular cage into a quartz cuvette, sequentially adding 2.0 mu L of 0.2 equivalent concentrated solution of the antibiotic, and testing the CD response behavior of the molecular cage to the antibiotic by a circular dichroscope.
4. The detection system of an antibiotic molecule according to claim 1, wherein said molecular cages and complexes of molecular cages with antibiotics are used for the antibacterial action against staphylococcus aureus SA and methicillin resistant staphylococcus aureus MRSA.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310453721.7A CN116465870A (en) | 2023-04-25 | 2023-04-25 | Detection system of antibiotic molecule and antibacterial application thereof |
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| CN202310453721.7A CN116465870A (en) | 2023-04-25 | 2023-04-25 | Detection system of antibiotic molecule and antibacterial application thereof |
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| CN116465870A true CN116465870A (en) | 2023-07-21 |
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