CN110003888B - Fluorescent nano probe and preparation method thereof - Google Patents
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- CN110003888B CN110003888B CN201910291426.XA CN201910291426A CN110003888B CN 110003888 B CN110003888 B CN 110003888B CN 201910291426 A CN201910291426 A CN 201910291426A CN 110003888 B CN110003888 B CN 110003888B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/14—Macromolecular compounds
- C09K2211/1441—Heterocyclic
- C09K2211/145—Heterocyclic containing oxygen as the only heteroatom
Abstract
The invention discloses a fluorescent nano probe and a preparation method thereof, wherein the fluorescent nano probe is composed of a polysaccharide ligand, a photosensitizer and a fluorescent nano material, the fluorescent nano material is mixed with an aqueous solution of the polysaccharide ligand and then added with a sodium borohydride solution for oscillation reaction, the unreacted polysaccharide ligand is removed by ultrafiltration and centrifugation, the photosensitizer solution is added for oscillation reaction, and the unreacted photosensitizer molecules are removed by ultrafiltration and centrifugation, so that the final fluorescent nano probe is prepared. The fluorescent nano probe prepared by the method has high sensitivity and can detect 10 in vivo5CFU number of bacteria, and photodynamic bacterial infection treatment can be realized through photosensitizer molecules loaded on the bacteria; in addition, the fluorescent nano probe not only has good fluorescence stability, but also has excellent biological safety, and is expected to be used for clinical bacterial infection detection and treatment.
Description
Technical Field
The invention belongs to the technical field of fluorescent nanoprobes, and relates to a fluorescent nanoprobe with broad-spectrum bacteria detection and photodynamic therapy functions and a preparation method thereof.
Background
The World Health Organization (WHO) issued the first basic diagnostic list (EDL) in 2018, emphasizing that a rapid, sensitive, specific and reasonably priced diagnosis is crucial for the treatment of infectious diseases caused by viruses, parasites and bacteria (see: nat. microbiol.3, 847 (2018)). More than 3 million people die annually from sepsis, endocarditis and other related diseases caused by bacteria (see: nat. med.17, 1142-1146 (2011); nat. med.10, S122-S129 (2004); nat. commun.9, 917 (2018)). Therefore, there is an urgent need to develop a method for diagnosing pathogenic bacteria in vivo with high specificity and sensitivity and eliminating them at the early stage of infection. Although various methods for bacterial detection have been developed so far, such as bacterial culture, biochemical identification, immunoassay, Polymerase Chain Reaction (PCR), sequencing and the like, most of these methods are long-lasting and complicated in procedure (see: Science 314, 1464-1467 (2006); nat. Commun.5, 5427 (2014)). Thus, even if a pathogenic infection can be successfully diagnosed by these methods, the gold phase of infection treatment may have been missed, resulting in an increased bacterial infection.
Fluorescence imaging is a simple, rapid and sensitive method of detecting bacteria. Although a large number of fluorescent nanoprobes for bacterial detection diagnosis and treatment have been developed, some of them have poor specificity and cannot completely distinguish bacterial infection inflammation from other inflammatory diseases (see: j. am. chem. soc.132, 12349-. Most of the reported fluorescent nanoprobes can only detect one specific type of bacteria, gram-positive or gram-negative bacteria (see: nat. Commun.9, 1969(2018)), but both types of bacteria can cause clinical bacterial infection (such as septicemia, skin burn infection, non-sterile instrument infection and the like), so that the realization of broad-spectrum detection of bacteria is very important. In addition, the fluorescence of the currently reported fluorescent nanoprobes is generally single excitation and single emission, and the single emission signals of the fluorescent nanoprobes are easily affected by the local probe concentration fluctuation, so that the accuracy of bacterial imaging detection is influenced (see: ACS Nano 11, 4428-4438 (2017)).
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing a fluorescent nanoprobe with both broad-spectrum bacteria detection and photodynamic therapy functions, aiming at the problems that the existing fluorescent nanoprobe is not high in specificity and sensitivity, has a limited range of detected bacteria, and cannot realize broad-spectrum bacteria detection.
In order to achieve the purpose, the polysaccharide ligand is connected with the fluorescent nano material, and the polysaccharide ligand can enter the interior of the bacteria through a sugar-specific transport channel on a bacterial membrane but cannot enter cells which do not express the channel on the surface of the membrane, so that the polysaccharide ligand can achieve specific bacterial targeted detection. The photosensitizer is adsorbed to the surface of the nano material, and the photosensitizer not only has strong fluorescence, but also can generate singlet oxygen under the illumination of visible light to kill bacteria.
Specifically, the invention provides the following technical scheme:
the preparation method of the fluorescent nano probe comprises the following steps:
the fluorescent nano material comprises fluorescent silicon nano particles, composite fluorescent silicon dioxide nano particles, II-IV group quantum dots, fluorescent nano microspheres, carbon dots and the like.
The polysaccharide ligand is glucose polymer, including maltodextrin, amylose and the like.
Step 2, removing unreacted polysaccharide ligand in the step 1 through ultrafiltration and centrifugation to purify the complex of the polysaccharide ligand coupled fluorescent nano material;
preferably, the ultrafiltration centrifugation condition is 7500rpm/min for 15 min.
the photosensitizer comprises chlorin e6(Ce6), methylene blue, methylene benzyl, hematoporphyrin derivative and the like.
Step 4, removing unreacted photosensitizer molecules from the mixture prepared in the step 3 through ultrafiltration and centrifugation to obtain a target product;
preferably, the ultrafiltration centrifugation condition is 7000rpm/min for centrifugation treatment for 10 min.
Theoretically, on one hand, after a polysaccharide ligand of a targeted bacterium is connected to a small-sized fluorescent nano material, the polysaccharide ligand can guide the whole nano probe system to enter the inside of the bacterium through a specific transfer channel on a bacterial cell membrane, and the specific bacterial fluorescence imaging function of the whole nano probe system is realized; on the other hand, the photosensitizer loaded by the nanoprobe generates singlet oxygen under the irradiation of visible light, and has the function of killing bacteria.
The fluorescent nano probe constructed by the invention mainly comprises three components: polysaccharide ligand micromolecules of targeted bacteria, photosensitizer micromolecules with photoresponse therapeutic action and fluorescent nano-material particles with drug loading and imaging functions. Wherein polysaccharide ligand micromolecules are connected to the surface of the fluorescent nano-particles in a covalent bond mode, and on the other hand, photosensitizer micromolecules are adsorbed to the surface of the fluorescent nano-material through electrostatic interaction.
The invention selects polysaccharide molecules as bacteria targeting molecules, overcomes the limitation that most of the existing bacteria detection fluorescent nanoprobes can only detect one type of bacteria (gram-positive bacteria or gram-negative bacteria), and realizes broad-spectrum bacteria detection. Meanwhile, polysaccharide molecules can lead the whole fluorescent nano probe to enter the interior of bacteria through a sugar specificity transport channel on a bacterial membrane, and the channel cannot be expressed on a mammalian cell membrane, so that the high specificity of the probe for detecting the bacteria is created. The fluorescent nano probe prepared by the invention can detect 10 in vivo5The CFU number of bacteria represents the high sensitivity of the in vivo bacterial detection of the probe. The prepared fluorescent nano probe can not only detect bacteria, but also realize photodynamic bacterial infection treatment through photosensitizer molecules loaded on the fluorescent nano probe. In addition, the fluorescent nano probe not only has good fluorescence stability, but also has excellent biological safety, and is expected to be used for clinical bacterial infection detection and treatment.
Drawings
FIG. 1 is a diagram of the preparation method and action mechanism of the fluorescent nanoprobe of the invention;
FIG. 2 is a laser confocal fluorescence imaging diagram of four different types of bacteria (EC: Escherichia coli, SA: Staphylococcus aureus, ML: Micrococcus luteus, PA: Pseudomonas aeruginosa) respectively incubated with GP-Ce6-SiNPs after the fluorescent nanoprobe of the invention is used for in vitro broad-spectrum bacteria imaging detection;
FIG. 3 shows that the fluorescent nanoprobe of the invention is used for in vivo broad-spectrum bacterial infection imaging detection, wherein a is to infect PA (Pseudomonas aeruginosa 1X 10)7CFU, left) and SA (Staphylococcus aureus 1X 10)7CFU, right) and imaging and corresponding fluorescence intensity bar chart after GP-Ce6-SiNPs are injected into the tail vein of the old rat; b is the bacterium mixed with PA and SA infected (1X 10)7CFU) intravenous injection of GP-Imaging Ce6-SiNPs and corresponding fluorescence intensity histogram, and using PBS as an experimental control group; c is for infection PA or SA (1X 10)5CFU) and imaging after GP-Ce6-SiNPs are injected into the tail vein of the old rat and corresponding fluorescence intensity histograms, and PBS is used as an experimental control group;
FIG. 4 is the analysis data of the fluorescent nanoprobe of the invention used for in vitro broad-spectrum bacterial photodynamic therapy, wherein a is the illumination (660nm, 12 mW/cm) after the incubation of EC (Escherichia coli), SA (Staphylococcus aureus) and GP-Ce6-SiNPs2) Scanning Electron Microscope (SEM) images before and after and live and dead bacteria staining fluorescence imaging images; b is the result of quantification of viable bacteria in the staining pattern of viable and dead bacteria in panel a; respectively irradiating untreated EC (Escherichia coli) and SA (Staphylococcus aureus) and EC (Escherichia coli) and SA (Staphylococcus aureus) incubated with GP-Ce6-SiNPs under 660nm illumination for 0, 5, 10 and 15 minutes to obtain a bacterium plate culture result (c), a bacterium liquid turbidity (d) and a bacterium CFU counting result (e);
FIG. 5 is the analysis data of the fluorescent nanoprobe of the present invention for photodynamic therapy of broad spectrum bacterial infection in vivo, wherein a is the tail vein injection of GP-Ce6-SiNPs and PBS respectively to SA (Staphylococcus aureus) and PA (Pseudomonas aeruginosa) infected mice, and one group of them is selected for illumination (660nm, 12 mW/cm)2) Taking a photograph of the wound every day for 40 minutes; b is the relative infected area (S/S) of the mouse corresponding to the graph a0) (ii) a Pathological section analysis (c) and bacterial count quantification (d) were performed on wound skin of mice treated with day 7 SA infection and day 9 PA infection, respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments of the present invention.
FIG. 1 is a diagram of the preparation method and action mechanism of the fluorescent nanoprobe of the invention. Wherein a is the synthesis step of GP-Ce6-SiNPs, NaBH4Is sodium borohydride; b is a schematic diagram of GP-Ce6-SiNPs used for broad-spectrum bacteria detection and treatment. GP-Ce6-SiNPs can enter the interior of the bacteria through sugar-specific ABC transport channels on the bacterial membrane. Under the excitation light of 405nm, GP-Ce6-SiNPs can respectively presentAnd the fluorescent material shows green fluorescence and red fluorescence and is used for bacterial imaging. Under 660nm laser irradiation, GP-Ce6-SiNPs can generate singlet oxygen to kill bacteria, and ROS is a reactive oxygen species.
Example 1
300. mu.L of photochemically-derived green fluorescent silicon nanoparticles (SiNPs) (having amino groups on the surface) (25mg/mL) were taken, 200. mu.L of Glucose Polymer (GP) solution (10mg/mL) was mixed therein, and the mixture was put on a shaker at 70 ℃ for shaking reaction for 6 hours, followed by addition of sodium borohydride solution (10. mu.g/mL) and shaking reaction at room temperature overnight. After the reaction, in order to remove excessive unreacted GP molecules, the solution was centrifuged by a 3K ultrafiltration tube 7500rprn/min for 15 minutes each time until the lower layer solution was centrifuged and almost no GP molecules were contained, thus obtaining a solution of GP-SiNPs on the upper layer. 200. mu.L of the solution of GP-SiNPs obtained by the reaction was added with 50. mu.L of chlorin e6(Ce6) solution (200. mu.M), and the mixture was placed on a shaker at room temperature and reacted for 12 hours with shaking. After the reaction is finished, centrifuging for 10 minutes at 7000rpm/min of a 3K ultrafiltration tube for removing redundant unadsorbed Ce6 molecules until the lower layer solution is centrifuged and almost does not contain Ce6 molecules, and finally preparing the GP-Ce6-SiNPs fluorescent nano probe. Gram-positive bacteria (staphylococcus aureus and micrococcus luteus) and gram-negative bacteria (escherichia coli and pseudomonas aeruginosa) are selected and cultured. The above-mentioned species of bacteria were inoculated into LB (lysis broth medium) and cultured on a shaker at a rotation speed of 250rpm/min at a temperature of 37 ℃ for 12 hours. The bacterial suspension obtained from the culture was washed 2 to 3 times by centrifugation in a PBS solution (phosphate buffered saline) and resuspended in the PBS solution for use. A certain amount of GP-Ce6-SiNPs (200 mu L and 10mg/mL) is added into the bacterial suspension, and the bacterial suspension is washed and sliced after being shaken and incubated in a shaking table (250rpm/min and 37 ℃) for 2 hours, and the bacteria are imaged and detected by a laser confocal microscope. As shown in FIG. 2, the GP-Ce6-SiNPs probe of the invention can successfully perform imaging detection on the four different types of selected bacteria.
Example 2
300. mu.L of photochemically-derived green fluorescent silicon nanoparticles (SiNPs) (having amino groups on the surface) (25mg/mL) were taken, 200. mu.L of Glucose Polymer (GP) solution (10mg/mL) was mixed therein, and the mixture was put on a shaker at 70 ℃ for shaking reaction for 6 hours, followed by addition of sodium borohydride solution (10. mu.g/mL) and shaking reaction at room temperature overnight. After the reaction is finished, in order to remove excessive unreacted GP molecules, the solution is centrifuged for 15 minutes at 7500rpm/min by a 3K ultrafiltration tube until the solution at the lower layer is centrifuged and almost no GP molecules are contained, and the solution of GP-SiNPs at the upper layer is obtained. 200. mu.L of the solution of GP-SiNPs obtained by the reaction was added with 50. mu.L of chlorin e6(Ce6) solution (200. mu.M), and the mixture was placed on a shaker at room temperature and reacted for 12 hours with shaking. After the reaction is finished, centrifuging for 10 minutes at 7000rpm/min of a 3K ultrafiltration tube for removing redundant unadsorbed Ce6 molecules until the lower layer solution is centrifuged and almost does not contain Ce6 molecules, and finally preparing the GP-Ce6-SiNPs fluorescent nano probe.
Gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (pseudomonas aeruginosa) are selected and cultured. The above two kinds of bacteria were inoculated into LB (lysis broth medium) and cultured on a shaker at a rotation speed of 250rpm/min at a temperature of 37 ℃ for 12 hours. The bacterial suspension obtained from the culture was washed 2 to 3 times by centrifugation in a PBS (phosphate buffered saline) solution and resuspended in a PBS solution for use.
And establishing different groups of mouse bacterial infection inflammation models. One group of: a suspension of Staphylococcus aureus (50. mu.L, 0.9X 10) was obtained7CFU) and Pseudomonas aeruginosa suspension (50. mu.L, 0.7X 10)7CFU) were injected subcutaneously into the right and left thighs of mice, respectively. Two groups are as follows: mixing PBS solution (50 μ L) and mixed suspension of Staphylococcus aureus and Pseudomonas aeruginosa (50 μ L, 0.8 × 10)7CFU) were injected subcutaneously into the left and right thighs of the mice, respectively. Three groups: PBS solution (50. mu.L) and Staphylococcus aureus suspension (50. mu.L, 1.4X 10)5CFU) or Pseudomonas aeruginosa suspension (50. mu.L, 1.2X 10)5CFU) were injected subcutaneously into the left and right thighs of the mice, respectively. After mice were infected with bacteria for 24 hours, GP-Ce6-SiNPs (100. mu.L, 10mg/mL) were injected separately from the tail vein. After 24 hours of injection of the material, the mice were placed in a small animal imager for fluorescence imaging detection of the infected site. And cutting the skin of the infected part of the mouse, and separating the fine parts of the infected skin part by tissue pulverization and centrifugationAnd (4) bacteria, and the number of bacteria at the infected part is determined by a plate counting method. As shown in FIG. 3, the GP-Ce6-SiNPs probe of the invention can successfully target and detect different types of bacterial infection sites in a living body, and can sensitively detect in vivo 105CFU number of bacteria.
Example 3
300. mu.L of photochemically-derived green fluorescent silicon nanoparticles (SiNPs) (having amino groups on the surface) (25mg/mL) was mixed with 200. mu.L of Glucose Polymer (GP) solution (10mg/mL), and the mixture was placed on a shaker at 70 ℃ for shaking reaction for 6 hours, followed by addition of sodium borohydride solution (10. mu.g/mL) and shaking reaction at room temperature overnight. After the reaction, in order to remove excessive unreacted GP molecules, the solution was centrifuged by a 3K ultrafiltration tube 7500rprn/min for 15 minutes each time until the lower layer solution was centrifuged and almost no GP molecules were contained, thus obtaining a solution of GP-SiNPs on the upper layer. 200. mu.L of the solution of GP-SiNPs obtained by the reaction was added with 50. mu.L of chlorin e6(Ce6) solution (200. mu.M), and the mixture was placed on a shaker at room temperature and reacted for 12 hours with shaking. After the reaction is finished, centrifuging for 10 minutes at 7000rpm/min of a 3K ultrafiltration tube for removing redundant unadsorbed Ce6 molecules until the lower layer solution is centrifuged and almost does not contain Ce6 molecules, and finally preparing the GP-Ce6-SiNPs fluorescent nano probe.
Gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli) are selected and cultured. The above two kinds of bacteria were inoculated into LB (lysis broth) medium and cultured on a shaker at a rotation speed of 250rpm/min at a temperature of 37 ℃ for 12 hours. The bacterial suspension obtained from the culture was washed 2 to 3 times by centrifugation in a PBS solution (phosphate buffered saline) and resuspended in the PBS solution for use.
A certain amount of GP-Ce6-SiNPs (200. mu.L, 10mg/mL) was added to the bacterial suspension, and after incubation for 2 hours with shaking on a shaker (250rpm/min, 37 ℃), resuspended in PBS by centrifugation. The bacterial suspension after co-incubation with the material was placed under light (660nm, 12 mW/cm)2) Irradiation was carried out for 0, 5, 10 and 15 minutes, respectively. The light-irradiated bacteria were subjected to slide-making, and the morphological changes of the bacteria were observed by a scanning microscope while staining with live and dead bacterial dyes (propidium iodide (PI) and SYT)O9) was stained at room temperature and filmed, the bacteria were examined by imaging with a confocal laser microscope to determine the live-dead state of the bacteria, and the number of the bacteria after the light irradiation was measured by plate counting. As shown in FIG. 4, after the bacteria and GP-Ce6-SiNPs probe are incubated, the bacteria can be obviously killed by illumination, and the number of the bacteria is sharply reduced. The probe has good in vitro antibacterial ability.
Example 4
300. mu.L of green fluorescent silicon nanoparticles (SiNPs) (having amino groups on the surface) (25mg/mL) obtained by photochemical method was taken, 200. mu.L of Glucose Polymer (GP) solution (10mg/mL) was mixed therein, and the mixture was put on a shaker at 70 ℃ for shaking reaction for 6 hours, and then sodium borohydride solution (10. mu.g/mL) was added thereto for shaking reaction at room temperature overnight. After the reaction is finished, in order to remove excessive unreacted GP molecules, the solution is centrifuged for 15 minutes at 7500rpm/min by a 3K ultrafiltration tube until the solution at the lower layer is centrifuged and almost no GP molecules are contained, and the solution of GP-SiNPs at the upper layer is obtained. 200. mu.L of the solution of GP-SiNPs obtained by the reaction was added with 50. mu.L of chlorin e6(Ce6) solution (200. mu.M), and the mixture was placed on a shaker at room temperature and reacted for 12 hours with shaking. After the reaction is finished, centrifuging for 10 minutes at 7000rpm/min of a 3K ultrafiltration tube for removing redundant unadsorbed Ce6 molecules until the lower layer solution is centrifuged and almost does not contain Ce6 molecules, and finally preparing the GP-Ce6-SiNPs fluorescent nano probe.
Gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (pseudomonas aeruginosa) are selected and cultured. The above two kinds of bacteria were inoculated into LB (lysis broth medium) and cultured on a shaker at a rotation speed of 250rpm/min at a temperature of 37 ℃ for 12 hours. The bacterial suspension obtained from the culture was washed 2 to 3 times by centrifugation in a PBS solution (phosphate buffered saline) and resuspended in the PBS solution for use.
Respectively establishing mouse inflammation models infected by gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (pseudomonas aeruginosa). Collecting Staphylococcus aureus suspension (50 μ L, 1.0X 10)7CFU) or Pseudomonas aeruginosa suspension (50. mu.L, 1.0X 10)7CFU) were injected subcutaneously into the right thigh of the mice, respectively. The mice are infected with the bacteria for 24 hoursAfter that, GP-Ce6-SiNPs (100. mu.L, 10mg/mL) or PBS (100. mu.L) were injected from the tail vein, respectively. After 24 hours of injection of the material or PBS, the infected mice were placed under light (660nm, 12 mW/cm)2) Irradiation was carried out for 40 minutes. All mice infected wounds were then recorded by photographing daily and wound area size was measured. After a period of treatment, mice were excised for infected skin tissue, histological sections were analyzed, and the number of bacteria at the infected site was determined by plate counting. As shown in FIG. 5, the mice injected with the GP-Ce6-SiNPs probe of the present invention and irradiated with light had the fastest healing rate of infected wounds and the least number of bacteria at the infected sites after treatment, indicating that the probe had good in vivo antibacterial ability.
Example 5
Mixing the carbon dots and maltodextrin in a concentration ratio of 4: 1-2: 1, carrying out oscillation reaction at 70 ℃ for 4-6 hours, adding a sodium borohydride solution (1-10 mu g/mL), and carrying out oscillation reaction at room temperature overnight to prepare the stable maltodextrin modified carbon dot nano material. The complex of maltodextrin coupled to the carbon dot nanomaterial was purified by ultrafiltration centrifugation (7500rpm/min, 15min) to remove unreacted maltodextrin. Adding 200 mu M methylene blue into the solution of the compound, and carrying out oscillation reaction for 12-16 hours at room temperature; the resulting mixture was centrifuged by ultrafiltration (7000rpm/min, 10min) to remove unreacted methylene blue molecules; finally obtaining the maltodextrin-methylene blue-carbon point fluorescent nano probe. Gram-positive bacteria (staphylococcus aureus and micrococcus luteus) and gram-negative bacteria (escherichia coli and pseudomonas aeruginosa) are selected and cultured. The above-mentioned species of bacteria were inoculated into LB (lysis broth medium) and cultured on a shaker at a rotation speed of 250rpm/min at a temperature of 37 ℃ for 12 hours. The bacterial suspension obtained from the culture was washed 2 to 3 times by centrifugation in a PBS solution (phosphate buffered saline) and resuspended in the PBS solution for use. Adding a certain amount of fluorescent nano probe (200 mu L, 10mg/mL) into the bacterial suspension, carrying out shaking incubation on a shaking table (250rpm/min, 37 ℃) for 2 hours, then cleaning and flaking the bacterial suspension, and carrying out imaging detection on the bacteria through a laser confocal microscope.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. The preparation method of the fluorescent nano probe is characterized by comprising the following steps;
s1, mixing the fluorescent nano-material and the polysaccharide ligand solution according to the concentration ratio of 4: 1-2: 1, carrying out oscillation reaction at 70 ℃ for 4-6 hours, adding a sodium borohydride solution, carrying out oscillation reaction at room temperature overnight, and preparing to obtain a complex of the polysaccharide ligand coupled fluorescent nano-material;
s2, removing unreacted polysaccharide ligand in the S1 through ultrafiltration and centrifugation to obtain a purified polysaccharide ligand coupled fluorescent nano material compound;
s3, adding a photosensitizer solution into the complex solution of the polysaccharide ligand coupled fluorescent nano material purified by S2, and carrying out oscillation reaction for 12-16 hours at room temperature;
s4, removing unreacted photosensitizer molecules by ultrafiltration and centrifugation of the mixture prepared in the step S3 to obtain a target product;
the polysaccharide ligand is glucose polymer.
2. The method of claim 1, wherein the fluorescent nanomaterial is at least one of a fluorescent silicon-based nanoparticle, a composite fluorescent silica nanoparticle, a group II-IV quantum dot, a fluorescent nanosphere, and a carbon dot.
3. The method of claim 1, wherein the polysaccharide ligand is at least one of maltodextrin and amylose.
4. The method of claim 1, wherein the photosensitizer is at least one of chlorin e6, methylene blue, hematoporphyrin derivative.
5. The method of claim 1, wherein in S2, the ultrafiltration centrifugation is performed at 7500rpm/min for 15 min.
6. The method of claim 1, wherein in S4, the ultrafiltration centrifugation is performed at 7000rpm/min for 10 min.
7. A fluorescent nanoprobe prepared by the method of any one of claims 1 to 6.
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| 载光敏剂磁性纳米粉的制备及其光敏活性;李东红 等;《应用化学》;20080930;第25卷;1065-1068 * |
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