CN1896738A - Fluorescent nano-particle with surface biological function, its production and use - Google Patents

Abstract

The invention relates to a surface biofunctionalized core-shell fluorescent nanoparticle, a preparation method and application thereof. The particles have a core-shell structure, the inner core layer is wrapped with fluorescent substances; the outer shell layer is composed of fluorescent transparent substances; and the surface of the outer shell layer is decorated with organic functional groups. The nanoparticles are synthesized by inverse microemulsion method, and then the surface is chemically modified to become biofunctional nanoparticles. The nanoparticle has important application prospects in the fields of cell biology, ultramicrochemistry, biomacromolecule detection, and medical in vivo diagnosis.

Description

Fluorescent nanoparticles with surface biofunctionalization and their preparation and application

Background technique

In recent years, biological detection technology has developed rapidly, among which the development of ultra-sensitive fluorescence detection technology provides conditions for the study of biomolecules in complex environments. The establishment of ultra-sensitive fluorescence detection technology depends on the successful development of non-toxic and biocompatible luminescent materials.

Organic fluorescent substances are classic fluorescent substances. Although organic fluorescent substances have their shortcomings due to their inherent properties, such as fluorescence efficiency problems, etc., compared with current inorganic fluorescent substances, organic fluorescent substances still have a wide range of applications. Such as applied to flow cytometry, applied to antibody labeling and so on.

Biofunctional materials have always been a hot field in the field of materials research. In biofunctional materials, nanomaterials have attracted more and more attention from scientists due to their special physical and chemical properties since the 1980s. Technology research and solving major problems in the field of biology have also become one of the important frontier research fields.

However, the combination of organic fluorescent materials and nanomaterials as functional materials in the biological field has not been studied in depth. There have been studies abroad that this type of fluorescent materials have been chemically modified on the surface of nanomaterials. However, there is no relevant report on encapsulating fluorescent substances in nanomaterials, and there is no activation of the surface of nanoparticles, or "surface biofunctionalization", even though the surface can be connected to nucleic acids, proteins, nucleotides, amino acids, antibodies, Peptides, animal and plant cells, subcellular structures, viruses, bacteria and other biological macromolecules or the ability of the organism itself.

Contents of the invention

technical problem to be solved

The technical problem to be solved in the present invention is to provide a core-shell type fluorescent nanoparticle with surface biofunctionalization and its preparation method and application, so as to overcome the fluorescent substance in the prior art that is only modified on the surface of nanomaterials and cannot make fluorescent nanomaterials At the same time, it has the defect of biomolecular binding function.

Technical solutions

One of the contents of the present invention is to provide a core-shell fluorescent nanoparticle with surface biofunctionalization, which has a core-shell structure with a core and an outer shell: the inner core layer contains fluorescent substances; the outer shell layer is composed of fluorescent transparent substances; the outer shell The surface of the layer is a modified layer of organic functional groups.

A preferred solution of the above biologically functionalized fluorescent nanoparticles is that the organic functional group is an amino group, a carboxyl group or a mercapto group, or a combination thereof.

Another preferred version of the above-mentioned biofunctionalized fluorescent nanoparticles is that the fluorescent substance is fluorescein isothiocyanate, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, phycoerythrin, or Its combination is preferably fluorescein isothiocyanate FITC.

Another preferred solution of the above-mentioned biologically functionalized fluorescent nanoparticles is that the shell layer component is silicon dioxide, agarose, olefin polymer, polyacrylonitrile or epoxy compound, or a combination thereof.

Those skilled in the art can understand without special experiments that the components for preparing the shell layer can be inorganic coatings such as aminosilane and mercaptosilane, or organic coatings such as glycosides and proteins. The inorganic coating layer is preferably aminosilane, and the organic coating layer is preferably glycoside. Preferably, its composition is selected from silica, agarose, olefin polymer, polyacrylonitrile, epoxy compound, or a combination thereof; most preferably, the shell composition of the inner core layer is silica.

The second content of the present invention is to provide a method for preparing said surface biofunctionalized core-shell fluorescent nanoparticles, comprising the following steps:

(1) provide the aqueous solution of isopropanol and fluorescent substance;

(2) adding ammonia water and tetraethyl orthosilicate to the aqueous solution of step (1) successively, and reacting at room temperature for 3 to 5 hours to obtain fluorescent particles;

(3) Add an amination, carboxylation or mercaptolation reagent to the alcohol solution of fluorescent particles in step (2), and react at 25-60° C. for 1-6 hours to obtain surface biofunctionalized fluorescent nanoparticles.

The present invention also provides another method for preparing the above-mentioned surface biofunctionalized core-shell fluorescent nanoparticles, which includes the following steps:

(1) Provide microemulsions of TritonX-100, n-hexanol, cyclohexane and fluorescent substances;

(2) adding ammonia water and tetraethyl orthosilicate to the aqueous solution of step (1) successively, and reacting at room temperature for 8 to 15 hours to obtain fluorescent particles;

(3) Add an amination, carboxylation or mercaptolation reagent to the alcohol solution of fluorescent particles in step (2), and react at 25-60° C. for 1-6 hours to obtain surface biofunctionalized fluorescent nanoparticles.

The preferred version of the preparation method of the above-mentioned two kinds of surface biofunctionalized core-shell fluorescent nanoparticles is that the amination, carboxylation or mercaptolation reagents are N-(2-aminoethyl)-3-amino Propyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, or mercaptoacetic acid or mercaptopropionic acid.

Those skilled in the art can understand without any special experiments that the modifier is a silane modifier, but is not limited to the above-mentioned ones. For the selected shell composition, a person of ordinary skill can select a suitable shell forming agent according to the prior art. For example, when the shell component is silica, tetraethyl orthosilicate or other suitable shell formers may be used.

A preferred version of the above method for preparing biofunctional fluorescent nanoparticles is that the microemulsion system contains TritonX-100, n-hexanol, and cyclohexane, and its volume ratio is 1:1～3:4～6 .

The second content of the present invention is to provide the application of the above-mentioned biofunctionalized fluorescent nanoparticles, wherein the organic functional groups are combined with nucleic acids, proteins, nucleotides, amino acids or their derivatives through chemical bonds.

Another application of the above-mentioned biofunctional fluorescent nanoparticles is that the organic functional groups are combined with animal and plant cells or subcellular structures or virus particles.

Beneficial effect

1. Compared with the fluorescent substances in the prior art that are only modified on the surface of nanomaterials, the biologically functionalized fluorescent nanoparticles of the present invention can make nanomaterials not only have the properties that can be easily indicated by fluorescent labels, but also have organic functional groups. The biological application function of combining chemical bonds with nucleic acids, proteins, nucleotides, amino acids or their derivatives, and even with animal and plant cells or subcellular structures or virus particles.

2. The experiments of the present invention show that after the fluorescent substance is wrapped in nanoparticles, its luminescent property is stable, the particles are evenly distributed, and the surface is smooth. It is a new type of nanometer material for ultramicro detection.

3. The shell layer of the biologically functionalized fluorescent nanoparticles of the present invention is made of non-toxic and biocompatible materials such as silicon dioxide and agarose, which makes it possible to be applied in the field of biochemistry.

4. The preparation method of the present invention adopts low-cost organic fluorescent substances, shell-forming agents, etc., so that the actual large-scale production is feasible.

5. The use of nanoparticles to wrap fluorescent substances and biological functional groups can effectively take advantage of the surface area of nanomaterials, improve the efficiency of biological reactions, speed up the reaction time, and reduce the total amount of the reaction system, enabling trace detection and trace reactions Simple and easy.

6. Biologically functionalized nanoparticles provide a basis for the study of the physical and chemical properties of biological materials at the nanometer level, and further provide the possibility of expanding applications.

7. Linking nucleic acids, proteins, nucleotides, amino acids, antibodies, polypeptides, animal and plant cells, subcellular structures, viruses, bacteria, etc. to the nanoparticles of the present invention can be widely used in the detection of various molecules and the detection of diseases Tracer therapy.

Description of drawings

Figure 1 is a transmission electron micrograph of silica-coated fluorescent nanoparticles. It can be seen from the figure that the nanoparticles wrapped with the fluorescent substance have a very regular shape and good monodispersity, indicating that the silica has well wrapped the fluorescent substance in it.

Fig. 2 is a fluorescent micrograph of nanoparticles coated with fluorescent substances by silica. It can be seen from the figure that the encapsulated fluorescent substance nanoparticles have good fluorescent properties, and it further proves that the silica has successfully encapsulated the fluorescent substance in it.

Fig. 3 is the fluorescence spectrum of nanoparticles coated with fluorescent substances by silica. It can be seen from the spectrum that the nano-particles encapsulated with fluorescent substances have better fluorescence intensity, which further proves that the silica has successfully encapsulated the fluorescent substances in it.

Figure 4 is the Raman spectrum of nanoparticles modified with thiol groups. It can be seen from the spectrum that the Raman spectrum of SH is at 2167cm ^-1 . 1328cm ^-1 is assigned to the out-of-plane rocking vibration spectrum of CH ₂ , 1388cm ^-1 and 1464cm ^-1 are the stretching vibration peaks of CH ₂ , and 1594cm ^-1 can be assigned to the stretching vibration peak of CC chain, and the peak around 2934cm ^-1 The bands belong to the antisymmetric stretching vibration of CH ₂ , and the peak at 395cm ^-1 is produced by the CC chain deformation vibration. The symmetrical stretching vibrations of localized modes of (CH ₂ )n and Si-O ^- appear around 1075cm ^-1 . There are corresponding symmetrical stretching vibrations of ^-O -Si-O ^- localized modes at 1003cm ^-1 and 502cm ^-1 . The peak at 1231cm ^-1 is the stretching vibration of C-Si, and the Raman peaks at 668cm ^-1 and 730cm ^-1 are caused by the stretching vibration of CS bond. The appearance of Raman peaks of carbon-silicon bonds, silicon-oxygen bonds, carbon-carbon bonds, carbon-hydrogen bonds, and carbon-sulfur bonds indicates that mercapto compounds have been successfully attached to the surface of nanoparticles, and some effective mercapto groups are exposed on the surface of nanoparticles. on the surface of nanoparticles.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

For the experimental methods that do not specify specific conditions in the following examples, generally follow conventional conditions, such as: chemical product manuals, or according to the conditions suggested by the manufacturer. All inorganic chemical reagents and organic solvents were purchased from Shanghai Chemical Reagent Factory, fluorescein isothiocyanate, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, mercaptoacetic acid, 3-mercaptopropyltrimethyl Oxygensilane (MPTS) was purchased from Sigma.

Example 1

Preparation Method 1 of Core-shell Fluorescent Nanoparticles

Mix isopropanol and water uniformly at a ratio of 5:1, and sonicate for 5 minutes. Weigh 0.1-0.8 mg of fluorescent substance fluorescein isothiocyanate FITC, configure it into an aqueous solution, and process it ultrasonically for 10 min. Pour the upper clear layer into a three-necked flask, and keep stirring to keep it in a dispersed state. Take 1mL of concentrated ammonia water and slowly add it into the constantly stirring solution system. Then take 1-3 mL of tetraethyl orthosilicate, and also slowly add it into the constantly stirring solution system. The system was reacted at room temperature for 3 to 5 hours. The reaction product was poured out, and the particles were washed 3 to 5 times with twice distilled water. The cleaned particles were vacuum-dried for 5 hours at a temperature of 20-50° C., and the particles were collected for future use. From Figure 1, we can see that the silicon dioxide has well wrapped the fluorescent substance in it.

Example 2

Preparation Method 2 of Core-shell Fluorescent Nanoparticles

Mix TritonX-100, n-hexanol and cyclohexane uniformly at a ratio of 1:2:5 to form a transparent and stable microemulsion system. Put the above microemulsion system in ultrasonic treatment for 30-60 minutes, add fluorescent substance fluorescein isothiocyanate FITC 0.5mg to it, use ultrasonic treatment for 6 minutes, take out the supernatant liquid and pour it into a three-necked flask, stir for 30 minutes It's even. Take 1mL of concentrated ammonia water and dilute it with 2mL double-distilled water, slowly add it into the microemulsion under constant stirring after 30 minutes, and keep stirring for 30 minutes to disperse the ammonia water evenly in the microemulsion. After 1 hour, 1-3 mL of tetraethyl orthosilicate was added dropwise to the microemulsion, while stirring continuously for 10 hours, and the temperature of the system was kept between 15-30°C. Add acetone to the system to precipitate the particles, or let the system stand overnight to allow the particles to settle naturally, and use ethanol to wash the particles. Dry the particle sample under vacuum.

Example 3

Amino modification on the surface of fluorescent nanoparticles

Take 20 mg of the fluorescent nanoparticles prepared in Example 1 or 2, add 30-50 mL of methanol and glycerol in a 5:3 mixture, and use ultrasonic treatment for 20-60 minutes; weigh 1-3 mL of AEAPS [N-(2-aminoethyl)-3-aminopropyltrimethoxysilane], ultrasonic treatment for 10 to 60 minutes; mix the two solutions evenly, react at 60°C for 5 hours, and then take out The particles were washed with methanol for 3 times, followed by vacuum drying at 40-80° C. for 2 hours, and the particles were collected to obtain biofunctionalized fluorescent nanoparticles with amino groups modified on the surface.

Example 4

Carboxyl modification on the surface of fluorescent nanoparticles

Take the fluorescent nanoparticles prepared in Example 1 or 2 and add them into a buffer solution composed of ethanol acetate (pH=4.5), and mix them uniformly by ultrasonic; add 3-mercaptopropyltrimethoxysilane (MPTS) into the same buffer solution in solution. The two buffer solutions are mixed, reacted under normal temperature conditions for 2-4 hours, centrifuged to separate the particles, washed three times with the same buffer solution, dried in vacuum at 60° C., and collected the particles.

Disperse the collected particles in the same buffer solution, add thioglycolic acid to the solution system, disperse the solution evenly, react the system at room temperature for 2 to 4 hours, centrifuge to collect the particles, wash several times and then vacuum dry to obtain Biofunctionalized fluorescent nanoparticles with surface-modified carboxyl groups.

Example 5

Thiol group modification on the surface of fluorescent nanoparticles

Take the fluorescent nanoparticles prepared in Example 1 or 2 and add them to ethanol acetate buffer (pH=4.5), and mix them uniformly by ultrasonic; at the same time, add 3-mercaptopropyltrimethoxysilane (MPTS) to another acetic acid in ethanol buffer. Mix the above two solutions, react at 25°C for 1 hour, take out the particles, wash with ethanol acetate buffer solution (pH=4.5) three times, and dry them in vacuum at 60°C for 2 hours, collect the surface particles to obtain sulfhydryl groups biofunctionalized fluorescent nanoparticles. It can be seen from the Raman characterization spectrum in Figure 4 that the sulfhydryl groups have been modified to the surface of the fluorescent nanoparticles.

Example 6

Amino-modified fluorescent nanoparticles for protein separation

Take 1-3 mg of biofunctional fluorescent nanoparticles with surface-modified amino groups prepared as in Example 3, and add them to a phosphate buffer system with a pH of 7.0-8.0, and add a cross-linking agent such as glutaraldehyde in an amount of 50-200 μL , forming a mixed solution. The mixed solution is treated with ultrasonic wave for 10-30 minutes. Under appropriate temperature conditions, react for 4 to 6 hours. After centrifuging to remove the supernatant, and then ultrasonically washing with phosphate buffer saline for 2-3 times, the particles were re-dispersed in the phosphate buffer solution.

Weigh a certain amount of traditional Chinese medicine (ganoderma lucidum, angelica, astragalus, etc.), process according to the decoction method of traditional Chinese medicine, centrifuge to remove impurities in the solution, and collect the supernatant containing protein components for later use.

Take a certain amount of supernatant solution, add a certain amount of particle solution to it, shake and mix, react at room temperature for 1 to 3 hours, centrifuge to remove the supernatant, wash with phosphate buffer solution for 1 to 2 times, and finally wash the obtained The protein-linked particles were redispersed in phosphate buffer solution for later use.

Add the above-mentioned protein-linked particle solution to cancer cells or normal cells cultured in vitro, and observe the effect of different protein components in various traditional Chinese medicines on the apoptosis of cancer cells and the proliferation ability of normal cells.

Example 7

Carboxyl-modified fluorescent nanoparticles for RNA interference

A certain amount of the carboxyl-modified biofunctionalized fluorescent nanoparticles prepared in the above-mentioned Example 4 was added to a phosphate buffer system with a pH of 7.0-8.0 to form a mixed solution. The mixed solution is treated with ultrasonic wave for 10-30 minutes.

Take a sufficient amount of double-stranded interfering small RNA (siRNA) modified with amino groups in a specific sequence, and add it to the mixed solution containing about 2 mL of the above-mentioned particles. React at room temperature for 3 hours, and keep vibrating continuously, so that the amino group on the siRNA and the carboxyl group on the outer shell react under the action of a fixative, and are directly connected to the outer shell. After the reaction, the particles were separated by ultrafiltration and washed three times with phosphate buffer. The thus obtained fluorescent nanoparticles linked with double-stranded interfering small RNA are stored in phosphate buffer.

The above-mentioned particle solution linked with siRNA is added to cells with phagocytosis cultured in vitro to observe the inhibitory effect of different siRNA sequences on cell-specific genes, and can also be used for cell transfection tracking and real-time monitoring.

Example 8

Fluorescent nanoparticles modified with amino groups for cell monitoring

Take 1-3 mg of biofunctional fluorescent nanoparticles with surface-modified amino groups prepared as in Example 3, and add them into a phosphate buffer system with a pH of 7.0-8.0, and add 50-200 μL of cross-linking agent to form a mixed solution. The mixed solution is treated with ultrasonic wave for 10-30 minutes. Under appropriate temperature conditions, react for 4 to 6 hours. After centrifuging to remove the supernatant, and then ultrasonically washing with phosphate buffer saline for 2-3 times, the particles were re-dispersed in the phosphate buffer solution.

About 3 μg of monoclonal antibody was added to the mixed solution containing about 2 mL of the above-mentioned particles. React at a reaction temperature of 2-10°C for 3 hours, so that the amino groups on the antibody and the amino groups on the shell layer react under the action of a fixative, so that the antibody is directly connected to the shell layer through Schiff Bond. After the reaction, the particles were separated by centrifugation and washed three times with phosphate buffer. The thus obtained fluorescent nanoparticles modified with monoclonal antibodies are stored in phosphate buffer, and the fluorescent nanoparticles containing antibody molecules can be used to quickly and accurately detect extremely small amounts of harmful bacteria. When this nanoparticle is added to the solution of the test sample, it will stick to the bacteria; then, by centrifugation, according to the size difference between the nanoparticle and the bacterial cell, the bacteria and the nanoparticle can be precipitated together; separated Nanoparticles are attached to the bacteria, and the existence of the bacteria to be detected can be determined according to the luminescent phenomenon of these particles. This detection method uses specific antibodies against target bacteria to make fluorescent particles, which makes the detection specificity very high; in addition, since thousands of fluorescent particles are attached to bacteria at the same time, even a single cell can be detected.

Claims (10)

1. the hud typed fluorescent nano particles of a surface biological functionalization possesses the hud typed structure of kernel and shell: contain fluorescent material in its inner nuclear layer; Outer shell is made of the penetrating material of fluorescence; The outer shell surface is the decorative layer of organo-functional group.

2. the hud typed fluorescent nano particles of surface biological functionalization according to claim 1 is characterized in that, said organo-functional group is amino, carboxyl or sulfydryl, perhaps its combination.

3. the hud typed fluorescent nano particles of surface biological functionalization according to claim 1 is characterized in that, said fluorescent material is fluorescein isothiocynate, RB 200, TRITC, phycoerythrin, perhaps its combination.

4. the hud typed fluorescent nano particles of surface biological functionalization according to claim 1 is characterized in that, said outer shell composition is silicon dioxide, agarose, olefin polymer, polyacrylonitrile or epoxy compound, perhaps its combination.

5. the hud typed fluorescent nano particles of surface biological functionalization according to claim 4 is characterized in that, said outer shell composition is a silicon dioxide.

6. the preparation method of the hud typed fluorescent nano particles of the described surface biological functionalization of claim 1 comprises the steps:

(1) provides the aqueous solution of isopropyl alcohol and fluorescent material;

(2) ammoniacal liquor and ethyl orthosilicate are taken up in order of priority add in the said aqueous solution of step (1), under the condition of room temperature, react and obtained fluorescent particles in 3～5 hours;

(3) in the alcoholic solution of the fluorescent particles of step (2), add amination, carboxylated or sulfhydrylization reagent,, promptly obtain the fluorescent nano particles of surface biological functionalization 25～60 ℃ of reactions 1～6 hour down.

7. the preparation method of the hud typed fluorescent nano particles of the described surface biological functionalization of claim 1 comprises the steps:

(1) provides the microemulsion of TritonX-100, n-hexyl alcohol, cyclohexane and fluorescent material;

(2) ammoniacal liquor and ethyl orthosilicate are taken up in order of priority add in the said aqueous solution of step (1), under the condition of room temperature, react and obtained fluorescent particles in 8～15 hours;

(3) in the alcoholic solution of the fluorescent particles of step (2), add amination, carboxylated or sulfhydrylization reagent,, promptly obtain the fluorescent nano particles of surface biological functionalization 25～60 ℃ of reactions 1～6 hour down.

8. according to the preparation method of the hud typed fluorescent nano particles of claim 6 or 7 described surface biological functionalization, it is characterized in that, said amination, carboxylated or sulfhydrylization reagent are respectively N-(2-amino-ethyl)-3-TSL 8330,3-sulfydryl propyl trimethoxy silicane, 3-sulfydryl propyl trimethoxy silicane, also can be mercaptoacetic acid or mercaptopropionic acid.

9. the application of the hud typed fluorescent nano particles of the described surface biological functionalization of claim 1 is characterized in that, the functional group that organises of said particle surface combines with nucleic acid, protein, nucleotide, amino acid or its derivant by chemical bond.

10. the application of the hud typed fluorescent nano particles of the described surface biological functionalization of claim 1 is characterized in that, organise functional group and the animal and plant cells or subcellular structure or virion of said particle surface combine.

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