CN114032086B - 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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- C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
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Abstract
The invention discloses an application of a long afterglow nano probe combination in detecting a bacterial biofilm, belonging to the field of analysis and detection. The long afterglow nanometer probe combination comprises at least three probes formed by modifying different charges and hydrophobic organic ligands on rare earth element doped lanthanum oxysulfide long afterglow nanometer materials with different luminous colors, wherein the rare earth element doped lanthanum oxysulfide long afterglow nanometer materials are obtained through a high temperature thermal decomposition method. The probe combination can be prepared into a detection kit and used for identifying a bacterial biofilm in combination with machine learning. The invention can not only realize the accurate identification of different bacterial biofilms and the accurate identification of different subspecies of bacterial biofilms, but also realize the detection of the bacterial biofilms on biomedical implants such as cardiac pacemakers. The detection of the bacterial biofilm based on the probe combination has the advantages of 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 nano probe combination in detection of bacterial biofilm.
Technical Field
Biomedical implants such as vascular stents, cardiac pacemakers, etc., which are capable of restoring the function of damaged tissues and organs of the human body, have thoroughly revolutionized modern medicine. 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 using biological materials. Infections often lead to failure of the prosthesis, require implant replacement, and often lead to chronic or recurrent disease, adding to the economic and physical burden of the patient. At present, 60% of implant infections are caused by bacterial biofilms, the extracellular matrix of which makes the bacteria more resistant and tolerant, which also makes the implant infections difficult to treat. The most effective treatment methods at present need to be developed based on knowledge of the bacterial species known to cause the infection, so timely and reliable identification of bacterial biofilms adhering to implants is critical to providing effective treatment regimens.
The most commonly used detection method in clinic at present 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 cause false positive phenomenon and the like. Among molecular-based diagnostic methods, methods such as PCR and fluorescence in situ hybridization are detection methods based on genetic and genomic analysis, but these methods are difficult to achieve rapid detection. Thus, it is still a serious challenge to rapidly detect bacterial biofilms.
Bacterial biofilms are a population of bacteria that adhere to solid surfaces and are tightly surrounded by extracellular matrix. The extracellular matrix mainly comprises polysaccharide, protein, nucleic acid, lipid and other substances. The chemical composition of the extracellular matrix of different bacterial biofilms may vary, resulting in bacterial biofilms having different physicochemical properties, such as charge, hydrophilicity, etc. Therefore, it is promising to identify a biological membrane by its physicochemical properties. The long afterglow is a phenomenon that the light can still continue to emit after the excitation light stops, and has good application prospect in the field of biological analysis. Because the physicochemical properties of the surfaces of different bacterial biofilms are different, the long afterglow nano combined probe and the bacterial biofilms can show different optical signals after acting, and the optical signals are further combined with machine learning to realize the identification of the bacterial biofilms. Therefore, the long persistence nanometer probe kit based on the machine learning assistance can realize the accurate detection of the bacterial biofilm.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provides a long afterglow nano probe combination and application thereof in bacterial biofilm detection.
The aim of the invention is achieved by the following technical scheme:
a long afterglow nano-probe combination contains at least three rare earth element doped lanthanum oxysulfide (La) 2 O 2 S) modifying probes formed by high molecular polymer ligands with different charges and hydrophilcity on the long afterglow nano material.
Further, the rare earth elements include terbium (Tb), dysprosium (Dy), europium (Eu), etc.
Further, the preparation method of the rare earth element doped lanthanum oxysulfide long afterglow nanomaterial is a high temperature pyrolysis 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 nanomaterial is synthesized under the conditions of no water and no oxygen and the temperature of 300-320 ℃. Wherein, the mol ratio of lanthanum acetate to sodium acetate to sulfur powder is preferably 1:1:1, the mol ratio of oleic acid to oleylamine to 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 rare earth elements (the ratio of the atomic number of the rare earth elements to the total atomic number 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-La 2 O 2 S long afterglow nano material and Dy-La 2 O 2 S long afterglow nano material and Eu-La 2 O 2 S long afterglow nanometer 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) the long afterglow nano material and the polyetherimide are subjected to vigorous stirring reaction for 3-5 hours under the conditions that the solvent is DMSO and the temperature is 80-100 ℃ to obtain the polyetherimide modified long afterglow nano probe. Wherein the dosage ratio of the long afterglow nanomaterial, polyetherimide and DMSO is preferably 20-50 mg:60-150 mg:5-20 mL; further, the mass ratio of the long afterglow nanometer material to the polyetherimide is 1:3.
When the high molecular polymer ligand is polyethylene glycol, the preparation method of the corresponding long afterglow nano probe 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 vigorously stirring the mixed solution to react for 12-24 h 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 nanometer material, the polyethylene glycol, the acetone and the tween-80 is preferably 20-50 mg:2-5 mg:8-15 mL:8-15 mL; further, the mass ratio of the long afterglow nanometer material to the polyethylene glycol is 10:1, and the volume ratio of the acetone to the tween-80 is 1:1.
When the high molecular polymer ligand is glucan, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: 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 nanometer material, the glucan and the water is preferably 20-50 mg:200-500 mg:5-10 mL; further, the mass ratio of the long afterglow nanometer material to the dextran is 1:5.
Further, the long persistence nano-probe combination comprises polyetherimide modified Eu-La 2 O 2 S long afterglow nano probe and polyethylene glycol modified Tb-La 2 O 2 S long afterglow nano probe and dextran modified Dy-La 2 O 2 S long afterglow nanometer probe, tb-La long afterglow nanometer material 2 O 2 S、Dy-La 2 O 2 S、Eu-La 2 O 2 S, the corresponding long afterglow nano probe can be obtained according to the preparation method of the long afterglow nano probe.
The long afterglow nano probe combination is applied to detection of bacterial biofilm.
The long afterglow nanometer probe combination is applied to the preparation of a bacterial biofilm detection kit.
A bacterial biofilm detection kit, comprising the long afterglow nano probe combination.
The application method of the long afterglow nano probe combination comprises the following steps: after incubating and culturing the long afterglow nano probe combination and the bacterial biofilm for 10-60 min, washing by PBS, detecting and collecting fluorescent signals and afterglow signals of different colors of a sample under the excitation of ultraviolet light by an instrument, and carrying out data processing on the signals by a machine learning algorithm to realize the differentiation of different bacterial biofilms.
Further, the bacterial biofilm may be a bacterial biofilm attached to a biomedical implant. The biomedical implant comprises a vascular stent, a cardiac pacemaker and the like.
Further, the bacterial biofilm may be a biofilm of 3 to 6 staphylococcus aureus subspecies. 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 effect of various probes and the comprehensive analysis of various fluorescence and afterglow data, and has high detection accuracy and rapidness.
(3) The bacterial biofilm detection method constructed in the invention can realize the distinction of different subspecies of biofilms of the same bacteria.
(4) The bacterial biofilm detection method constructed in the invention has the advantages of simple and convenient operation, high sensitivity and good accuracy, can realize the detection of bacterial biofilms on implants such as cardiac pacemakers and the like, and is expected to be applied to the diagnosis of clinical bacterial biofilm infection.
Drawings
FIG. 1 shows a long afterglow nanomaterial Tb-La obtained in embodiment 1 of the invention 2 O 2 S transmission electron microscope image.
FIG. 2 is a graph showing the emission spectra of the long persistence nanoprobe prepared in examples 2 to 4 of the present invention.
FIG. 3 is a graph showing the results of identifying biofilms of different bacteria 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 embodiments of the present invention are not limited thereto
Example 1: preparation of long afterglow nano material
La having three color luminescence of green light, red light and yellow light was used in the present invention 2 O 2 The long afterglow nanometer material of S has different luminescence according to the different doped rare earth elements. The terbium (Tb) doped emits green light, the europium (Eu) doped emits red light, and the dysprosium (Dy) doped emits yellow light.
Tb-La with green light 2 O 2 The preparation of the S long afterglow nanometer material is as follows: lanthanum acetate (0.5 mmol,0.158 g), sodium acetate (0.5 mmol,0.041 g), terbium acetate (0.0046 g) and sulfur powder (0.5 mmol,0.016 g) were weighed into a 100mL three-neck round bottom flask, 2.5mmol oleic acid, 20mmol octadecene and 17mmol oleylamine were added to the flask, and a high temperature resistant magnet was added, an air condenser tube and a three-way valve cock were inserted into the middle bottleneck of the three-neck flask, and a balloon filled with high purity argon gas was connected to the cock. The other bottle mouth of the three-mouth flask is provided with a temperature probe, the remaining bottle mouth is plugged by a sealing plug, and all the interfaces of the whole device are sealed by raw rubber belts. After the apparatus was set up, the vacuum was pulled at room temperature for half an hour, and then the solution was heated to 120 ℃ and maintained at that temperature for half an hour while continuing to pull the vacuum to remove water and oxygen from the apparatus. And after the vacuumizing process is finished, the cock is rotated to enable the device to be under the argon protection condition, the device is heated, the temperature is increased to 310 ℃, and the device is kept for 40 minutes. After the reaction is finished, the device is naturally cooled to room temperature. Centrifuging the reacted solution at 10000rpm for 5min, collecting the product, washing with cyclohexane and ethanol (3:1, v/v) for three times, and oven drying the nanomaterial at 60deg.C to obtain Tb-La capable of generating green fluorescence emission 2 O 2 S long afterglow nanometer material.
The terbium acetate (Tb) is changed into dysprosium acetate (Dy) or europium acetate (Eu) with equal mass, and Dy-La with yellow fluorescence emission is prepared under the condition that other raw materials and reaction conditions are the same 2 O 2 S and Eu-La with red fluorescence emission 2 O 2 S long afterglow nanometer material.
Tb-La to be obtained 2 O 2 S long afterglow nanomaterial is dissolved in cyclohexane and dripped into a copper mesh for sample preparation, and observed by a transmission microscope, wherein the sample is shown as Tb-La in figure 1 2 O 2 Transmission electron microscope image of S long afterglow nano material, dy-La prepared by using same 2 O 2 S and Eu-La 2 O 2 The transmission electron microscope of the S-shaped long afterglow nanomaterial is also very similar to that of FIG. 1, and is in the shape of a pentagonal nanosheet with a size of about 20nm.
Example 2: PEI modified Eu-La 2 O 2 Preparation of S long afterglow nano material probe
Eu-La having Red light emission prepared in example 1 2 O 2 S long persistence nanomaterial was weighed 25mg dispersed into 8mL of DMSO (dimethylsulfoxide) solution and sonicated for 30min. 150mg PEI (polyetherimide, MW=25000) was further weighed out and dispersed in another 8mL DMSO. The two solutions were mixed and reacted for 4-5 h with vigorous stirring (with magnetic stirring at 1000-1500 rpm) in an oil bath at 95 ℃. Centrifuging the solution at 6000rpm for 5min after the reaction is finished, centrifugally washing the precipitate with ultrapure water for 3 times, and dispersing the precipitate in water to obtain PEI modified Eu-La 2 O 2 S nano probe.
Example 3: PEG modified Tb-La 2 O 2 Preparation of S long afterglow nano material probe
25mg of Tb-La having green light emission prepared in example 1 was weighed 2 O 2 S long persistence nanomaterial and 2.5mg PEG (polyethylene glycol, mw=2000) were dissolved in 8mL acetone and the solution was further sonicated for 30min to make it further soluble. 8mL of Tween-80 was added dropwise to the mixed solution under vigorous stirring, and the mixed solution was stirred vigorously at room temperature (using magnetonsStirring and reacting for 12h at the magnetic rotation speed of 1000-1500 rpm). Centrifuging at 6000rpm for 5min after the reaction, centrifuging with ultrapure water for 3 times, and dispersing into water to obtain PEG modified Tb-La 2 O 2 S nano probe.
Example 4: dextran (Dextran) -modified Dy-La 2 O 2 Preparation 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 solution 2 O 2 S long afterglow nanometer material, and stirring the mixed solution at room temperature for reaction for 12h. Centrifuging at 6000rpm for 5min after the reaction, washing with ultrapure water for 3 times, and dispersing into water to obtain dextran modified Dy-La 2 O 2 S nano probe.
Example 5: detection kit for combining probes
The probes of the long afterglow nanomaterials prepared in examples 2 to 4 were combined, and the specific steps were: 100mg/L PEI-Eu-La of the same volume is taken respectively 2 O 2 S、100mg/L PEG-Tb-La 2 O 2 S and 100mg/L Dextran-Dy-La 2 O 2 And mixing the S probes in an equal volume to obtain the detection kit.
Application example 1: detection kit for identifying different bacterial biofilms
The biofilms of 5 different types of bacteria (escherichia coli, baumannii, pseudomonas aeruginosa, pseudomonas maltophilia and staphylococcus aureus) were cultured in 96-well plates, the biofilms were grown in an adhesive LB (1L of water containing 0.1% glucose, 1mM magnesium sulfate, 0.15M ammonium sulfate and 34mM citric acid, and the solution was mixed and sterilized after adjusting the ph=7.0 with HCl or NaOH) for 24 hours, the bacterial biofilms were grown to form a culture solution (the bacterial biofilms were washed with PBS, 200 μl per well and washed 2-3 times), 200 μl of a detection kit solution was added to the same bacterial biofilms, the probe and the bacterial biofilms were incubated at room temperature for 20 minutes, the supernatant was gently removed after incubation was washed 2 times with 200 μl PBS, and 100 μl PBS was added to each well after washing. And irradiating the sample after the probe acts with the bacterial biofilm for 5min by using a portable ultraviolet lamp, and then placing the sample into a living animal imaging instrument to read the afterglow luminous intensity value of the marked bacterial biofilm. And continuously measuring the fluorescence intensity of the labeled bacterial biofilm by using an enzyme-labeled instrument. Samples were excited at 260nm and fluorescence intensity values were collected for three values 550nm, 586nm and 620 nm. The random forest algorithm is trained by using the afterglow luminous intensity value and the fluorescence intensity value, and the trained random forest algorithm is used for identifying unknown bacterial biofilms.
FIG. 3 shows the results of the identification of various bacterial biofilms, such as 84.6% for E.coli biofilms, 69.0% for Bowman's Acinetobacter, 88.2% for Pseudomonas aeruginosa biofilms, 100% for Pseudomonas maltophilia biofilms, and 78.1% for Staphylococcus aureus biofilms. It is demonstrated that the method has very high accuracy in identifying different kinds of bacterial biofilms.
Application example 2: detection kit for identifying bacterial biofilms of different subspecies
Further, the detection kit is used for distinguishing biological films of different staphylococcus aureus subspecies (ATCC: 6568, 12600, 29213, RN4220 and 43300), 5 staphylococcus aureus subspecies are cultured by using a 96-well plate, and all culture mediums are attached LB. 200. Mu.L of the detection kit solution was added to 5 staphylococcus aureus subspecies of bacterial biofilms, after incubation of the probes with the bacterial biofilms for 20min, the supernatant was gently removed, washed twice with PBS, and data were measured by adding 100. Mu.L of PBS. The afterglow data of the sample is measured by a living animal imager, and the fluorescence intensities of three values of 550nm, 586nm and 620nm under the excitation of 260nm are measured by an enzyme-labeled instrument. And identifying the staphylococcus aureus subspecies biofilm by using a trained random forest algorithm. The identification accuracy of the biofilm of staphylococcus aureus (ATCC: 6568) was 100%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 12600) was 75.0%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 29213) was 85.7%, the identification accuracy of the biofilm of staphylococcus aureus (ATCC: RN 4220) was 90.9%, and the identification accuracy of the biofilm of staphylococcus aureus (ATCC: 43300) was 100%.
Application example 3: detection of bacterial biofilm on cardiac pacemaker by using probe
The detection kit is further utilized to distinguish bacterial biofilms originating on cardiac pacemakers. Placing the cardiac pacemaker with the biological film in 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 96-well plates to form a bacterial biofilm, 200. Mu.L of a detection kit solution was added to the bacterial biofilm, and after incubation of the probe 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 afterglow data of the sample is measured by a living animal imager, and the fluorescence intensities of three values of 550nm, 586nm and 620nm under the excitation of 260nm are measured by an enzyme-labeled instrument. And identifying the biofilm by using a trained random forest algorithm. The identification accuracy of the escherichia coli biological film is 65.4%, the identification accuracy of the Acinetobacter baumannii biological film is 88.5%, the identification accuracy of the pseudomonas aeruginosa biological film is 96.2%, the identification accuracy of the pseudomonas maltophilia biological film is 100%, and the identification accuracy of the staphylococcus aureus biological film is 88.5%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. A long afterglow nanometer probe combination is characterized in that: the fluorescent probe consists of three probes formed by modifying high polymer ligands with different charges and hydrophobicity on rare earth element doped lanthanum oxysulfide long afterglow nano materials with different luminescent colors;
the preparation method of the rare earth element doped lanthanum oxysulfide long afterglow nanomaterial 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 nanomaterial is synthesized under the conditions of no water and no oxygen and the temperature of 300-320 ℃;
the three rare earth element acetates are terbium acetate, dysprosium acetate and europium acetate;
the three high polymer ligands are polyetherimide, polyethylene glycol and dextran;
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 hours under the conditions that the solvent is DMSO and the temperature is 80-100 ℃ 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 nano probe 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 a polyethylene glycol modified long-afterglow nano probe;
when the high molecular polymer ligand is glucan, the preparation method of the corresponding long afterglow nanoprobe comprises the following steps: mixing the long afterglow nanometer material and the glucan in water, and reacting the mixed solution for 12-24 hours to obtain the glucan modified long afterglow nanometer probe.
2. The long persistence nanoprobe combination of claim 1, wherein: eu-La modified by polyetherimide 2 O 2 S long afterglow nano probe and polyethylene glycol modified Tb-La 2 O 2 S long afterglow nano probe and dextran modified Dy-La 2 O 2 S long afterglow nanometer probe.
3. Use of a long persistence nanoprobe combination according to claim 1 or 2 in the preparation of a bacterial biofilm detection kit.
4. A bacterial biofilm detection kit is characterized in that: a long persistence nanoprobe combination comprising the composition of claim 1 or 2.
5. The use according to claim 3 or the detection kit according to claim 4, characterized in that: the bacterial biofilm is attached to the biomedical implant; or the bacterial biofilm is a biofilm of 3-6 staphylococcus aureus subspecies.
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