CN114032086A - Application of long-afterglow nano probe combination in detection of bacterial biofilm - Google Patents
Application of long-afterglow nano probe combination in detection of bacterial biofilm Download PDFInfo
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Abstract
The invention discloses an application of a long afterglow nanoprobe combination in detecting bacterial biofilm, belonging to the field of analysis and detection. The long-afterglow nano probe combination comprises at least three probes formed by modifying organic ligands with different charges and hydrophilicity on rare earth element doped lanthanum oxide long-afterglow nano materials with different luminescent colors, wherein the rare earth element doped lanthanum oxide long-afterglow nano materials are obtained by a high-temperature thermal decomposition method. The probe combination can be prepared into a detection kit and combined with machine learning for identifying the bacterial biofilm. The invention can not only realize the accurate identification of different bacterial biofilms and the accurate identification of different subspecies bacterial biofilms, but also realize the detection of bacterial biofilms on biomedical implants such as cardiac pacemakers. The probe combination based on the invention is used for detecting the bacterial biofilm, has simple and convenient operation, high sensitivity and good accuracy, and is expected to be used for clinical diagnosis of bacterial biofilm infection.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to application of a long-afterglow nanoprobe combination in detection of a bacterial biofilm.
Technical Field
Biomedical implants, such as vascular stents, cardiac pacemakers, and the like, have been revolutionizing modern medicine by restoring the function of damaged tissues and organs of the human body. However, the use of medical implants also increases the risk of bacterial infection. In fact, implant infection is one of the most common and serious complications of the use of biological materials. Infection often leads to prosthesis failure, requires implant replacement, and often results in chronic or recurrent disease, placing an economic and physical burden on the patient. At present, 60 percent of implant infections are caused by bacterial biofilms, and the extracellular matrix of the bacterial biofilms enables bacteria to have strong drug resistance and tolerance, so that the infection of the implants becomes difficult to treat. While the most effective treatment methods currently need to be developed based on an understanding of the species of bacteria known to cause infection, timely, reliable identification of bacterial biofilms attached to implants is critical to providing an effective treatment regimen.
At present, the most common detection method in clinic is a gold-labeled culture method, but the method has long detection period, is not suitable for detecting bacteria which cannot be cultured, and is easy to generate false positive phenomena and the like. Among molecular-based diagnostic methods, methods such as PCR and fluorescence in situ hybridization are detection methods based on gene and genome analysis, but these methods have difficulty in achieving rapid detection. Thus, rapid detection of bacterial biofilms remains a serious challenge today.
A bacterial biofilm is a population of bacteria that is tightly packed with extracellular matrix attached to a solid surface. The extracellular matrix is mainly composed of polysaccharide, protein, nucleic acid, lipid and other substances. The chemical composition of the extracellular matrix of different types of bacterial biofilms can vary, resulting in bacterial biofilms with different physicochemical properties, such as charge, hydrophilicity and hydrophobicity, and the like. Thus, it is hoped that the identification of the biofilm is carried out by the physicochemical properties of the biofilm. The long afterglow is a phenomenon that the light can still continuously emit after the excitation light stops, and has good application prospect in the field of biological analysis. Due to different physicochemical properties of the surfaces of different bacterial biofilms, the long-afterglow nano combined probe and the bacterial biofilms can present different optical signals after the action, and the identification of the bacterial biofilms is realized by further combining machine learning to perform cluster analysis on the optical signals. Therefore, the long-afterglow nanoprobe kit based on machine learning assistance can realize accurate detection of the bacterial biofilm.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a long-afterglow nanoprobe combination and application thereof in bacterial biofilm detection.
The purpose of the invention is realized by the following technical scheme:
a long-afterglow nanoprobe combination comprises at least three rare earth element doped lanthanum oxysulfide (La) with different luminescent colors2O2S) modifying the long-afterglow nano-material with a probe consisting of macromolecular polymer ligands with different charges and hydrophilicity and hydrophobicity.
Further, the rare earth elements include terbium (Tb), dysprosium (Dy), europium (Eu), and the like.
Further, the preparation method of the rare earth element doped lanthanum oxysulfide long-afterglow nano material is a high-temperature thermal decomposition method, and specifically comprises the following steps: lanthanum acetate, sodium acetate, rare earth element acetate and sulfur powder are used as precursors, oleic acid, oleylamine and octadecene are used as solvents, and the rare earth element doped lanthanum oxysulfide long-afterglow nano material is synthesized under the conditions of no water, no oxygen and the temperature of 300-320 ℃. The molar ratio of lanthanum acetate, sodium acetate and sulfur powder is preferably 1:1:1, the molar ratio of oleic acid, oleylamine and octadecene is 5:34:40, and the reaction time of the mixed system at 300-320 ℃ is preferably 20-80 min. The doping amount of the rare earth elements (the ratio of the number of the rare earth elements to the total number of atoms of the material) in the long-afterglow nano material is preferably 1-6%. The rare earth element acetate comprises terbium acetate, dysprosium acetate, europium acetate and the like, and the corresponding long afterglow nano material is Tb-La2O2S long afterglow nano material, Dy-La2O2S long afterglow nano material, Eu-La2O2S long afterglow nano material.
Further, the high molecular polymer ligand comprises polyetherimide, polyethylene glycol, dextran and the like.
When the high molecular polymer ligand is polyetherimide, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: and (3) violently stirring and reacting the long-afterglow nano material and the polyetherimide for 3-5 hours at the temperature of 80-100 ℃ in the presence of DMSO as a solvent to obtain the polyetherimide modified long-afterglow nano probe. Wherein the dosage ratio of the long afterglow nano material, the polyetherimide and the DMSO is preferably 20-50 mg: 60-150 mg: 5-20 mL; furthermore, the mass ratio of the long afterglow nano material to the polyetherimide is 1: 3.
When the high molecular polymer ligand is polyethylene glycol, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: dissolving the long-afterglow nano material and polyethylene glycol in acetone, dropwise adding tween-80 into the solution under stirring, and violently stirring the mixed solution for reaction for 12-24 hours to obtain the polyethylene glycol modified long-afterglow nano probe. The temperature of the reaction is preferably room temperature. Wherein the dosage ratio of the long-afterglow nano material, the polyethylene glycol, the acetone and the tween-80 is preferably 20-50 mg, 2-5 mg, 8-15 mL and 8-15 mL; furthermore, the mass ratio of the long afterglow nano material to the polyethylene glycol is 10:1, and the volume ratio of the acetone to the tween-80 is 1: 1.
When the macromolecular polymer ligand is glucan, the preparation method of the corresponding long-afterglow nanoprobe comprises the following steps: and mixing the long-afterglow nano material and the glucan in water, and stirring the mixed solution to react for 12-24 hours to obtain the glucan modified long-afterglow nano probe. The temperature of the reaction is preferably room temperature. Wherein the dosage ratio of the long-afterglow nano material to the glucan to the water is preferably 20-50 mg to 200-500 mg to 5-10 mL; furthermore, the mass ratio of the long afterglow nano material to the glucan is 1: 5.
Further, the long afterglow nano probe combination comprises polyether imide modified Eu-La2O2S long afterglow nanoprobe and polyethylene glycol modified Tb-La2O2S long afterglow nanoprobe and glucoseGlycan-modified Dy-La2O2S long afterglow nano probe made of Tb-La2O2S、Dy-La2O2S、Eu-La2O2S obtaining the corresponding long-afterglow nanoprobe according to the preparation method of the long-afterglow nanoprobe.
The long-afterglow nano probe combination is applied to the detection of bacterial biofilms.
The long-afterglow nano probe combination is applied to the preparation of a bacterial biofilm detection kit.
A bacterial biofilm detection kit, which comprises the long afterglow nanoprobe combination.
The use method of the long afterglow nano probe combination comprises the following steps: after incubation and cultivation of the long afterglow nano probe combination and the bacterial biofilm for 10-60 min, washing by PBS, detecting and collecting fluorescence signals and afterglow signals of different colors of a sample under ultraviolet excitation by an instrument, and performing data processing on the signals by a machine learning algorithm to realize the distinguishing of different bacterial biofilms.
Further, the bacterial biofilm may be a bacterial biofilm attached to a biomedical implant. The biomedical implant comprises a blood vessel stent, a cardiac pacemaker and the like.
Further, the bacterial biofilm may be a biofilm of 3 to 6 subspecies of staphylococcus aureus. The invention has the following advantages and effects:
(1) the raw materials used in the invention have rich sources and low price.
(2) The bacterial biofilm detection method constructed in the invention is based on the comprehensive action of various probes and the comprehensive analysis of various fluorescence and afterglow data, and has high and rapid detection accuracy.
(3) The bacterial biofilm detection method constructed by the invention can realize the differentiation of different subspecies biofilms of the same kind of bacteria.
(4) The bacterial biofilm detection method constructed by the invention is simple and convenient to operate, high in sensitivity and good in accuracy, can realize detection of bacterial biofilms on implants such as cardiac pacemakers and the like, and is expected to be applied to diagnosis of clinical bacterial biofilm infection.
Drawings
FIG. 1 shows a long afterglow nanomaterial Tb-La prepared in example 1 of the present invention2O2Transmission electron micrograph of S.
FIG. 2 is an emission spectrum of the long-afterglow nanoprobe prepared in example 2-4 of the present invention.
FIG. 3 is a graph showing the results of the identification of biofilms of different species in application example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto
Example 1: preparation of long afterglow nano material
La having three colors of green, red and yellow light emission is used in the present invention2O2The long afterglow nano material of S has different luminescence according to different doped rare earth elements. Terbium (Tb) doped emits green light, europium (Eu) doped emits red light, and dysprosium (Dy) doped emits yellow light.
With green light-emitting Tb-La2O2The preparation of the S long afterglow nano material is as an example: lanthanum acetate (0.5mmol, 0.158g), sodium acetate (0.5mmol, 0.041g), terbium acetate (0.0046g) and sulfur powder (0.5mmol, 0.016g) were weighed and added into a 100mL three-necked round bottom flask, then 2.5mmol oleic acid, 20mmol octadecene and 17mmol oleylamine were added into the flask, respectively, and a high temperature resistant magneton was added, an air condenser and a three-way valve were inserted into the middle neck of the three-necked flask, and a balloon filled with high purity argon gas was connected to the cock. The other bottle of the three-mouth flask is provided with a temperature probe, the rest bottle is plugged by a sealing plug, and all the joints of the whole device are sealed by raw adhesive tapes. After the device was set up, it was evacuated to room temperature for half an hour, then the solution was heated to 120 ℃ and held at that temperature for half an hour while vacuum was continued to remove water and oxygen from the device. After the vacuum-pumping process is finished, the rotating cock makes the device be inThe apparatus was heated and the temperature was raised to 310 ℃ for 40min under argon. After the reaction, the apparatus was naturally cooled to room temperature. Centrifuging the solution after reaction at 10000rpm for 5min, collecting the product, washing with cyclohexane and ethanol (3:1, v/v), drying the nanometer material in a 60 ℃ oven after washing for three times to obtain Tb-La generating green light fluorescence emission2O2S long afterglow nano material.
The terbium (Tb) acetate is replaced by dysprosium (Dy) acetate or europium (Eu) acetate with the same mass, and Dy-La with yellow fluorescence emission is prepared under the condition that other raw materials and reaction conditions are the same2O2S and Eu-La with red fluorescence emission2O2S long afterglow nano material.
Tb-La to be obtained2O2S long afterglow nano material is dissolved in cyclohexane and dropped into copper net for sample preparation, and observed by transmission microscope, wherein the figure 1 is Tb-La2O2Transmission electron microscope picture of S long afterglow nano material, Dy-La prepared by using the same2O2S and Eu-La2O2The transmission electron microscope of the S long afterglow nano material is also very similar to the transmission electron microscope shown in the figure 1, and the S long afterglow nano material is in a pentagonal nanosheet shape and is about 20nm in size.
Example 2: PEI modified Eu-La2O2Preparation of S long afterglow nano material probe
Eu-La having Red light emission prepared in example 12O2S long afterglow nano material 25mg is weighed and dispersed into 8mL DMSO (dimethyl sulfoxide) solution, and ultrasonic treatment is carried out for 30 min. Further 150mg PEI (polyetherimide, MW 25000) was weighed out and dispersed in another 8mL DMSO. Mixing the two solutions, and reacting for 4-5 h under vigorous stirring (using magneton stirring, the magneton rotation speed is 1000-. Centrifuging the solution at 6000rpm for 5min after the reaction is finished, centrifuging and washing the precipitate for 3 times by ultrapure water, and dispersing the precipitate in water to obtain PEI modified Eu-La2O2And (4) an S nano probe.
Example 3: PEG-modified Tb-La2O2Preparation of S long afterglow nano material probe
25mg of Tb-La having green light emission prepared in example 1 were weighed out2O2S long persistence nanomaterial and 2.5mg PEG (polyethylene glycol, MW 2000) were dissolved in 8mL acetone, and to further dissolve it, the solution required further sonication for 30 min. 8mL of Tween-80 was added dropwise to the mixed solution under vigorous stirring, and the mixed solution was reacted for 12 hours under vigorous stirring at room temperature (using magneton stirring at a magneton rotation speed of 1000-. Centrifuging at 6000rpm for 5min after reaction, centrifuging with ultrapure water for 3 times, and dispersing in water to obtain PEG-modified Tb-La2O2And (4) an S nano probe.
Example 4: dextran (Dextran) -modified Dy-La2O2Preparation of S long afterglow nano material probe
250mg of dextran (MW 70000) was weighed out and dissolved in 8mL of water, and 25mg of Dy-La having yellow light emission prepared in example 1 was added to the solution2O2And (3) stirring the mixed solution of the S long-afterglow nano material for reaction for 12 hours at room temperature. Centrifuging at 6000rpm for 5min after reaction, washing with ultrapure water for 3 times, and dispersing in water to obtain dextran-modified Dy-La2O2And (4) an S nano probe.
Example 5: combining probes to construct a detection kit
The combination of the probes of the long afterglow nano materials prepared in the embodiments 2 to 4 comprises the following specific steps: respectively taking 100mg/L PEI-Eu-La of the same volume2O2S、100mg/L PEG-Tb-La2O2S and 100mg/L Dextran-Dy-La2O2And (3) mixing the S probe with an equal volume to obtain the detection kit.
Application example 1: detection kit for identifying different bacterial biofilms
5 different types of bacteria (escherichia coli, acinetobacter baumannii, pseudomonas aeruginosa, pseudomonas maltophilia, staphylococcus aureus) biofilm were cultured by using a 96-well plate, the biofilms were grown for 24 hours in adherent LB (1L of water containing 0.1% glucose, 1mM magnesium sulfate, 0.15M ammonium sulfate and 34mM citric acid, the solutions were mixed and then sterilized after adjusting pH to 7.0 with HCl or NaOH), the surface of the cultured solution was washed after the biofilm growth (the bacterial biofilm was washed with PBS, 200. mu.L per well and 2-3 times), 200. mu.L of a detection kit solution was added to the same bacterial biofilm, a probe and the bacterial biofilm were incubated at room temperature for 20 minutes, the supernatant was gently removed after incubation, the bacterial biofilm was washed with 200. mu.L of PBS for 2 times, and 100. mu.L of PBS was added to each well after washing. And irradiating the sample with the probe and the bacterial biofilm for 5min by using a portable ultraviolet lamp, and then putting the sample into a small animal living body imaging instrument to read the afterglow luminous intensity value of the marked bacterial biofilm. And continuously measuring the fluorescence intensity of the marked bacterial biofilm by using a microplate reader. The sample was excited at 260nm and fluorescence intensity values were collected for three values, 550nm, 586nm and 620 nm. And training a random forest algorithm by using the afterglow luminous intensity numerical value and the fluorescence intensity numerical value, wherein the trained random forest algorithm is used for identifying unknown bacterial biofilms.
FIG. 3 shows the results of the identification of different types of bacterial biofilms, the accuracy of the Escherichia coli biofilm identification is 84.6%, the Acinetobacter baumannii biofilm identification is 69.0%, the Pseudomonas aeruginosa biofilm identification is 88.2%, the Pseudomonas maltophilia biofilm identification is 100%, and the Staphylococcus aureus biofilm identification is 78.1%. The method has high accuracy in identifying different types of bacterial biofilms.
Application example 2: detection kit for identifying different subspecies bacterial biofilms
Further, a detection kit is used for distinguishing different staphylococcus aureus subspecies (ATCC: 6568, 12600, 29213, RN4220 and 43300) biofilms, a 96-well plate is adopted for culturing 5 staphylococcus aureus subspecies, and all culture mediums are adherent LB. After adding 200. mu.L of detection kit solution into 5 staphylococcus aureus subspecies bacterial biofilms, incubating the probe and the bacterial biofilms for 20min, gently removing supernatant, washing twice with PBS, and adding 100. mu.L of PBS for data measurement. The sample is subjected to afterglow data determination by a small animal living body imager, and fluorescence intensities of three values of 550nm, 586nm and 620nm under the excitation of 260nm are determined by an enzyme-labeling instrument. And identifying the staphylococcus aureus subspecies biofilm by using the trained random forest algorithm. The identification accuracy of the biofilm of staphylococcus aureus (ATCC: 6568) is 100%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 12600) is 75.0%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 29213) is 85.7%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: RN4220) is 90.9%, and the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 43300) is 100%.
Application example 3: probe for detecting bacterial biofilm on cardiac pacemaker
The detection kit is further used to distinguish bacterial biofilms originating from pacemakers. And (3) putting the cardiac pacemaker attached with the biofilm into a beaker containing PBS for ultrasonic treatment for 1-3min, and taking the PBS after ultrasonic treatment for bacterial culture to obtain bacterial colonies. The resulting bacteria were cultured in a 96-well plate to form a bacterial biofilm, 200. mu.L of the detection kit solution was added to the bacterial biofilm, the probe was incubated with the bacterial biofilm for 20min, the supernatant was gently removed, washed twice with PBS, and 100. mu.L of PBS was added for data measurement. The sample is subjected to afterglow data determination by a small animal living body imager, and fluorescence intensities of three values of 550nm, 586nm and 620nm under the excitation of 260nm are determined by an enzyme-labeling instrument. And identifying the biofilm by using a trained random forest algorithm. The identification accuracy of the Escherichia coli biofilm is 65.4%, the identification accuracy of the Acinetobacter baumannii biofilm is 88.5%, the identification accuracy of the Pseudomonas aeruginosa biofilm is 96.2%, the identification accuracy of the Pseudomonas maltophilia biofilm is 100%, and the identification accuracy of the Staphylococcus aureus biofilm is 88.5%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A long afterglow nanoprobe combination is characterized in that: the probe comprises at least three high molecular polymer ligands which are used for modifying different charges and hydrophilicity and hydrophobicity on rare earth element doped lanthanum oxysulfide long-afterglow nano materials with different luminescent colors.
2. The long persistence nanoprobe assembly of claim 1, wherein: the preparation method of the rare earth element doped lanthanum oxysulfide long-afterglow nano material comprises the following steps: lanthanum acetate, sodium acetate, rare earth element acetate and sulfur powder are used as precursors, oleic acid, oleylamine and octadecene are used as solvents, and the rare earth element doped lanthanum oxysulfide long-afterglow nano material is synthesized under the conditions of no water, no oxygen and the temperature of 300-320 ℃.
3. The long persistence nanoprobe assembly of claim 1, wherein: the high molecular polymer ligand comprises polyetherimide, polyethylene glycol and glucan.
4. The long persistence nanoprobe assembly of claim 3, wherein:
when the high molecular polymer ligand is polyetherimide, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: reacting the long-afterglow nano material with polyetherimide for 3-5 h at the temperature of 80-100 ℃ in the presence of DMSO as a solvent to obtain a polyetherimide modified long-afterglow nano probe;
when the high molecular polymer ligand is polyethylene glycol, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: dissolving the long-afterglow nano material and polyethylene glycol in acetone, dropwise adding tween-80 into the solution under stirring, and reacting the mixed solution for 12-24 hours to obtain the polyethylene glycol modified long-afterglow nano probe;
when the macromolecular polymer ligand is glucan, the preparation method of the corresponding long-afterglow nanoprobe comprises the following steps: and mixing the long-afterglow nano material and the glucan in water, and reacting the mixed solution for 12-24 hours to obtain the glucan modified long-afterglow nano probe.
5. The long persistence nanoprobe assembly of claim 1, wherein: Eu-La containing polyetherimide modification2O2S long afterglow nanoprobe and polyethylene glycol modified Tb-La2O2S long afterglow nanoprobe and dextran-modified Dy-La2O2S long afterglow nanoprobe.
6. Use of the long persistence nanoprobe combination of any one of claims 1 to 5 in the detection of bacterial biofilms.
7. Use of the long persistence nanoprobe assembly of any one of claims 1 to 5 in the preparation of a kit for detecting a bacterial biofilm.
8. A bacterial biofilm detection kit, comprising: comprising the long persistence nanoprobe assembly of any one of claims 1 to 5.
9. The method of using the long persistence nanoprobe assembly of any of claims 1 to 5, wherein: the method comprises the following steps: after incubation and cultivation of the long afterglow nano probe combination and the bacterial biofilm for 10-60 min, washing by PBS, detecting and collecting fluorescence signals and afterglow signals of different colors of a sample under ultraviolet excitation by an instrument, and performing data processing on the signals by a machine learning algorithm to realize the distinguishing of different bacterial biofilms.
10. The use according to claim 6 or 7, the test kit according to claim 8 or the method of use according to claim 9, characterized in that: the bacterial biofilm can be a bacterial biofilm attached to a biomedical implant; the bacterial biofilm can be a biofilm of 3-6 staphylococcus aureus subspecies.
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