RU2758098C1 - Method for manufacturing indicator microcapsules using magnetic and plasmon nanoparticles - Google Patents

Abstract

FIELD: medicine; biology; ecology.

SUBSTANCE: invention relates to microcapsules for use in medicine, biology, ecology and various industries. The method for manufacturing indicator microcapsules using magnetic and plasmon nanoparticles that enhance the luminescent properties of carbon points connected to their carrier consists in the use of centrifugation to clean carbon points that are not connected to the carrier and the addition of plasmon nanoparticles during the final procedure. A porous inorganic microsphere of calcium carbonate doped with magnetic nanoparticles of iron oxide Fe₃O₄ is used as a carrier. The connection of carbon points with the carrier is made by their introduction into alternating polymer layers of polyelectrolytes of polyallylamine hydrochloride (PAAC) and poly(4-styrene sulfate) sodium (PSS) on the surface of calcium carbonate microspheres. During the final procedure, plasmon nanoparticles are added.

EFFECT: invention provides a reduction in toxicity due to isolation by a polymer shell and the possibility of subsequent removal of the indicator microcapsule together with the analyzed particle using a magnetic field.

1 cl, 1 ex

Description

Semiconductor analyzers based on changes in electrical conductivity are widely used to monitor various toxic compounds (including aldehydes). Such sensors are portable and have good stability, but suffer from limited selectivity. Modification of the metal oxide or selective membrane is required. Commercially available assay kits offer a convenient method for the semi-quantitative determination of aldehydes. Compared to colorimetry, fluorometry has become a highly sensitive method for the determination of various toxic aldehydes and their derivatives. As an excellent fluorescent material, carbon dots (C-dots) are inexpensive and environmentally friendly.

Metal-enhanced fluorescence (MFF) is a phenomenon in which fluorescence is enhanced when a fluorophore is near plasmonic metallic nanomaterials. The introduction of fluorescent C-dots and metal nanoparticles into silica spheres or polymers noticeably enhances the fluorescence of C-dots by tens of times and, thus, is an effective approach for obtaining a more intense fluorescence response. UMP assays are "signaling" and have a high fluorescence response limit. However, modern UFM analyzers usually have complex modifications and limited operational requirements. The development of new analyzers based on MFMs, as well as their practical application, remains a challenge.

Obtaining new hybrid structures based on carbon dots with both optical and magnetic properties is an urgent task and has many solutions. Nanoparticles of metals (gold, silver, platinum, palladium), which have a pronounced effect of surface plasmon resonance, are ideal reagents for analyzing the presence of toxic aldehyde compounds and their derivatives in various media based on metal-enhanced fluorescence. The relatively low cytotoxicity of the C-dots used, the dependence of their luminescence band on the excitation wavelength and their size make carbon dots an excellent material for the manufacture of analytical systems and instruments. C-dots can be easily incorporated into inorganic micro-sized capsules to produce hybrid luminescent nanocomposite materials. Compared to commercial capsules encoded with organic dyes and semiconductor quantum dots, C-dot markers have different unique properties. First, compared to semiconductor quantum dots, C-dots exhibit superior stability and biocompatibility. Secondly, since the surface of the C-points is usually rich in hydrophilic groups such as carboxyl, amino and hydroxyl groups, they show good solubility and stability in water. Thirdly, C-points are quite cheap and easy to prepare. As a more environmentally friendly alternative to metal-containing quantum dots, it is planned to use C-dots, which have a dependence of photoluminescence on the excitation wavelength and high optical stability, which plays an important role in fluorescence imaging both in vitro and in vivo or in situ. The ability to modify the surface of C-points also expands the area of their application for analysis. By changing the surface groups, it is possible to prepare carbon dots for delivery of various drugs and change the optical properties of the dots. Also, the presence of magnetite in the capsule makes it possible for the capsules to have the ability to magnetotaxis - movement associated with the reaction of the material to a magnetic field. Due to the presence of plasmonic metal nanocrystals, not only an additional opportunity appears for spectrometric determination of the concentration of microcapsules, but also the enhancement of luminescence by plasmonic metal nanocrystals.

The use of non-encapsulated C-points poses a number of problems that can be solved by the creation of heterostructures based on polymer or inorganic capsules. Compared to polymer capsules, inorganic capsules used for analytical response to the presence of toxic compounds have good mechanical strength, however, the permeability of these inorganic capsules is not controlled, which severely limits their use. Therefore, a polymer-inorganic composite capsule is needed, in which the inorganic spheres have mechanical strength to withstand, for example, drying and osmotic pressure, and the polymer fraction allows control of the permeability of the shell. In addition, the embedded inorganic particles can also impart specific optical and magnetic properties. Given these properties, the inclusion of functional inorganic nanocomposites in polyelectrolyte shells is one of the simple ways to solve the aforementioned disadvantage for both pure organic capsules and inorganic capsules, for example, the ability to encapsulate small molecules and adjust the permeability of the shell.

The closest to the claimed invention and adopted as a prototype is the heterostructure of nanocomposite materials

"Metal-enhanced fluorometric formaldehyde assay based on the use of in-situ grown silver nanoparticles on silica-encapsulated carbon dots" ( article Yang W., Zhang G., Ni J. et al. Microchim Acta 187, 137 (2020) https://doi.org/10.1007/s00604-019-4105-21 where C-points were obtained as follows: 0, 5 g of sodium citrate was dissolved in 10 ml of ultrapure water, followed by the addition of 10 ml of N- (β-aminoethyl) -γ-aminopropylmethyldimethoxysilane, the mixture was shaken until a homogeneous solution was formed.Then the solution was transferred into a 50 ml polytetrafluoroethylene autoclave and heated at 160 ° C for After cooling the mixture to room temperature, a clear yellow solution was obtained.The resulting products were purified by precipitation with petroleum ether three times and then dialyzed for 24 h (porosity 1 kDa) to remove unreacted reagents, fluorescent carbon dots were dispersed in ethanol to prepare a stock solution (1 g / L). Fluorescent silica SiO ₂ spheres were obtained by the Steber method: with magnetic stirring, 1 ml of the initial solution of C-points, 0.5 ml of ammonia solution and 0.2 ml of tetraethylorthosilicate were successively added to 50 ml of ethanol. After about 2 hours, the solution turned cloudy with the appearance of a white precipitate indicating the formation of a C-point @ SiO ₂ composite. Then 2 ml of (3-aminopropyl) triethoxysilane was added to the C-points @ SiO ₂ to obtain the C-points @ SiO ₂ -NH ₂ . The solution was vigorously stirred at 60 ° C for 4 h. The resulting precipitate was separated from the solution by centrifugation at 10,000 rpm and washed three times separately with ethanol and water. A solution of C-points @ SiO ₂ -NH ₂ (1 g / L) and 100 μL of fresh AgNO ₃ solution (0.5 mm) were mixed and kept for 30 min, then 100 μL of formaldehyde in various concentrations and 700 μL of ultrapure water were added and the solutions were incubated at room temperature for an additional 30 min. Fluorescence spectra were recorded with excitation at a wavelength of 370 nm. Solutions of C-points @ SiO ₂ -NH ₂ and AgNO _{3 were} mixed in an optimal ratio (0.1 g / L C-points @ SiO ₂ -NH ₂ and 50 μM Ag ⁺ ) and incubated for 30 min to obtain fluorescent analyzers C -dots @ SiO ₂ -NH ₂ -Ag ⁺ . Then, a square piece of nano-sponge (1 cm × 1 cm × 1 cm) was completely immersed in the absorption solution for 5 minutes. The excess solution in the nano-sponge piece was squeezed out, and the wet nano-sponge piece was used directly for subsequent detection. Samples of formaldehyde gas were prepared using the heating evaporation method. One hundred microliters of aqueous formaldehyde at various concentrations (0 ~ 6.67 mM) were injected into gas sampling bags (1 L) filled with air. The air-filled sampling bags were then gently heated to obtain a homogeneous test gas (0 ~ 20 ppm). Pieces of nano-sponge were placed under a UV lamp (excitation 365 nm) and the corresponding luminescence spectra were recorded.

The prototype has the following disadvantages: in addition to the complexity of manufacturing, it is impossible to easily vary the size of silica microspheres, and the resulting microspheres dissolve only in the presence of hydrofluoric acid, which, in turn, is completely unsuitable for analytical use in biological media.

The technical problems to be solved by the alleged invention are to simplify the manufacturing technology and reduce the effect of the substances used on the environment, as well as the possibility of withdrawing the obtained indicator microcapsules from the system after use.

The present invention proposes the preparation of microcapsules based on calcium carbonate containing luminescent C-dots, magnetic nanoparticles and plasmonic metal nanocrystals, a method for their preparation by mixing magnetic nanoparticles, C-dots, plasmonic metal nanocrystals and polyelectrolytes, thereby preparing the microcapsule for detection toxic aldehydes and their derivatives with high sensitivity and selectivity with a minimum number of sample processing steps; allowing to control the microcapsule by a magnetic field with simultaneous detection of luminescence and localized plasmon resonance with the ability to determine the concentration.

Example.

Preparation of indicator microcapsules using magnetic and plasmonic nanoparticles

Synthesis of C-points:

Luminescent carbon dots (C-dots) are carbon dots additionally stabilized by aminoethylaminopropylisobutyl of a polyhedral oligomeric silsesquioxane (POSS) of the main amine, 50 nm in size, obtained by high-temperature autoclavable solvothermal synthesis. 5.5 mmol of citric acid and 5 mmol of the basic amine POSS precursor are dissolved in 10 ml of o-xylene in an autoclave with a Teflon beaker. Next, the autoclave is heated for 5 hours at a temperature of 200 ° C. After cooling the autoclave to room temperature, the reaction products are filtered off and centrifuged at 5000 rpm for 10 minutes in order to separate the reaction product from agglomerates of large particles.

Synthesis of spheres of calcium carbonate (CaCO ₃ ) doped with magnetic nanoparticles of the composition Fe ₃ O ₄ :

In a 1: 1 ratio, an aqueous 0.33 CaCl ₂ solution and an aqueous 0.33 Μ Na ₂ CO ₃ solution are mixed with the addition of 200 μl of an aqueous solution of magnetic nanoparticles of the composition Fe ₃ O ₄ . After 30 seconds of reaction, the resulting CaCO ₃ spheres are centrifuged for 40 seconds at 2500 rpm. The formed precipitate is washed twice with distilled water and precipitated by centrifugation for 40 seconds at a speed of 2500 rpm, the supernatant liquid is removed.

Obtaining indicator microcapsules: 1 ml of a 0.5 Μ NaCl solution with a PAGE polyelectrolyte concentration of 6 mg / ml (pH 6.5) is added _{to the deposited CaCO 3} spheres doped with magnetic Fe ₃ O _{4 nanoparticles.} The resulting dispersion is shaken for 10 minutes. Then the mixture is centrifuged for 30 seconds at 4000 rpm, the supernatant with an excess of PAGE polyelectrolyte is removed, the precipitate is washed twice with distilled water. Next, 1 ml of 0.5 Μ NaCl solution with a PSS polyelectrolyte concentration of 6 mg / ml (pH 6.5) is added to the spheres. The resulting dispersion is shaken for 10 minutes and centrifuged for 30 seconds at 4000 rpm, the supernatant with an excess of PSS polyelectrolyte is removed, the precipitate is washed twice with distilled water. Layer-by-layer formation of the shell occurs due to alternating layers of oppositely charged polyelectrolytes. After coating with layers of polyelectrolytes (PAGE-PSS), 200 μL of an aqueous solution of carbon dots in PSS are added to the spheres, which are attached to the outer negatively charged layer of PSS polyelectrolyte due to electrostatic interaction. The solution is shaken for 10 minutes and then centrifuged for 30 seconds at 4000 rpm to remove unreacted C-points along with the supernatant. To the resulting composition is added 1 ml of a 0.5 Μ NaCl solution with a PAAG polyelectrolyte concentration of 6 mg / ml (pH 6.5). The resulting dispersion is shaken for 10 minutes. Excess polyelectrolyte is removed by two stages of washing: centrifugation for 30 seconds at 4000 rpm and addition of 1 ml of distilled water, removal of the supernatant. The last stage in the preparation of indicator microcapsules is the addition of 200 μl of a colloidal solution of plasmonic metal nanocrystals, which attach to the polyelectrolyte due to electrostatic interaction. The resulting dispersion is shaken for 10 minutes. Unreacted plasmonic metal nanocrystals are removed by centrifugation for 30 seconds at 4000 rpm, followed by removal of the supernatant.

Claims (1)

A method of manufacturing indicator microcapsules using magnetic and plasmonic nanoparticles that enhance the luminescence properties of carbon dots attached to their carrier, using centrifugation to remove carbon dots that have not adhered to the carrier and adding plasmonic nanoparticles at the final procedure, characterized in that it is used as a carrier porous inorganic microsphere of calcium carbonate CaCO ₃ , doped with magnetic nanoparticles of iron oxide Fe ₃ O ₄ , obtained by mixing in a 1: 1 ratio of an aqueous 0.33 Μ CaCl ₂ solution and an aqueous 0.33 Μ Na ₂ CO ₃ solution with the addition of 200 μl of an aqueous solution magnetic nanoparticles of the composition Fe ₃ O ₄ , the obtained CaCO ₃ spheres are centrifuged after 30 seconds of the indicated reaction of introduction for 40 seconds at a speed of 2500 rpm, the formed precipitate is washed twice with distilled water and precipitated by repeated centrifugation for 40 seconds at a speed of 2500 rpm min, supernatant liquid removed, carbon points are obtained by the solvothermal method from 5.5 mmol of citric acid and 5 mmol of polyhedral oligomeric silsesquioxane (POSS) polyhedral oligomeric silsesquioxane (POSS) precursor of aminoethylaminopropylisobutyl, dissolved in 10 ml of o-xylene in an autoclave with a Teflon beaker, heated for 5 hours at a temperature of 200 ° C , the reaction products are filtered and centrifuged at a speed of 5000 rpm for 10 minutes in order to separate the reaction product from agglomerates of large particles; sodium (PSS) on the surface kaltsiykarbonatnyh microspheres, centrifugation for purifying not joined to the carrier carbon dots produce four times after the formation of each polyelectrolyte layer, the deposited areas to CaCO ₃ nanoparticles doped magnetic Fe ₃ O _4, added 1 mL of 0.5 Μ NaCl solution with concentration of polyelectrolyte PAGE 6 mg / ml at pH 6.5, the resulting dispersion is shaken for 10 minutes, then the mixture is centrifuged for 30 seconds at a speed of 4000 rpm, the supernatant liquid with an excess of polyelectrolyte PAGE is removed, the precipitate is washed twice with distilled water, then 1 ml of 0.5 Μ NaCl solution with a PSS polyelectrolyte concentration of 6 mg / ml at pH 6.5 is added to the spheres, the resulting dispersion is shaken for 10 minutes and centrifuged for 30 seconds at 4000 rpm, the supernatant is in excess PSS polyelectrolyte is removed, the precipitate is washed twice with distilled water, 200 μl of an aqueous solution of carbon dots in POSS is added, the solution is shaken for 10 minutes, centrifuged for 30 seconds at 4000 rpm in order to remove unreacted carbon dots together with the supernatant liquid to the obtained 1 ml of a 0.5 Μ NaCl solution with a PAAG polyelectrolyte concentration of 6 mg / ml at pH 6.5 is added to the composition, the dispersion is shaken in a stream 10 minutes, the excess polyelectrolyte is removed using two stages of washing - centrifugation for 30 seconds at 4000 rpm, adding 1 ml of distilled water and removing the supernatant; at the final procedure for adding plasmonic nanoparticles to the resulting dispersion, add 200 μl of colloidal solution of plasmonic metal nanocrystals, it is shaken for 10 minutes, after which unreacted plasmonic metal nanocrystals are removed by centrifugation for 30 seconds at a speed of 4000 rpm, followed by removal of the supernatant.