RU2644466C2 - Method for obtaining nanoparticles of colloid gold with average diameter 25-30 nm - Google Patents

Abstract

FIELD: technological processes.

SUBSTANCE: invention can be used in manufacture of markers in immunochromatography. For the production of colloidal gold nanoparticles, the reduction of the hydrochloric acid with sodium citrate is carried out. On a magnetic stirrer, a temperature of 300 °C and a mixing regime of 375 rpm. To the Erlenmeyer flask add 49.23 ml of deionized water at room temperature and add the stirring bar of the magnetic stirrer. Flask with water is placed on a magnetic stirrer, after 6 minutes, 0.058 ml of a 10 % solution of hydrochloric acid are introduced. Then boil for 2 minutes and add 0.72 ml of a 1 % solution of 5,5-aqueous sodium citrate. When the color of the solution changes from blue to red, the stirring regime with a magnetic stirrer is changed from 375 rpm to 500 rpm and lower the temperature from 300 °C to 200 °C. Solution is boiled for 20 minutes, the flask is disconnected from the reflux condenser and removed from the magnetic stirrer. Flask with a solution of colloidal gold nanoparticles is left at room temperature for 18 hours. Obtained preparation of colloidal gold nanoparticles is stored in a refrigerator at a temperature of 4 °C.

EFFECT: invention makes it possible to obtain particles of colloidal gold with average diameter of 25–30 nm.

1 cl, 1 dwg, 4 tbl, 1 ex

Description

The invention relates to colloid chemistry and immunochemistry, and in particular to the field of producing colloidal gold nanoparticles (NchKZ) of size 25-30 nm, which can be used as markers in immunochromatography.

There is a known method of obtaining NCHK of a given size by the citrate method of Frens [Frens G. // Nat. Phys. Sci. - 1973. - V. 241. - No. 105. P. 20-22]. To a boiling 0.01% aqueous solution of hydrochloric acid HAuCl ₄ ⋅ 3H ₂ O (ZHVK), a 1% aqueous solution of sodium citrate is added in a volume depending on the desired particle size. The volume of a 1% solution of sodium citrate, which must be added per 100 ml of sol to obtain a given size NCHKZ, calculated by the following formula

,

,

where d is the average diameter of NChKZ, nm; V is the volume of a 1% sodium citrate solution [Titov, A.A. Development and optimization of immunochromatographic tests for the detection of botulinum toxins / A.A. Titov, I.V. Shilenko, A.A. Morozov, S.P. Yarkov, V.N. Zlobin // Applied Biochemistry and Microbiology. - 2012. - T. 48, No. 2. - C. 249-256].

The Franc method allows one to obtain relatively monodisperse gold nanoparticles with a diameter of 10-60 nm [Utochkin SD Synthesis of gold, silver and Au / Ag alloy nanoparticles by the citrate method / S.D. Utochkin, T.A. Sherstneva, M.Yu. Queen // Advances in chemistry and chemical technology. - 2010. - T. 24, No. 7 (112). - C. 122-126].

Franc's citrate method was used in the work of N.V. Byzova [Byzova N.V. Development of an immunochromatographic test system for the detection of Helicobacter pylori antigens / N.A. Byzova, A.V. Zherdev, P.G. Sveshnikov, E.G. Sydykhov, B.B. Dzantiev // Applied Biochemistry and Microbiology. - 2015. - T. 51, No. 5. - S. 520-530]. The methodology used in this work is as follows. To 97.5 ml of deionized water, add 1.0 ml of a 1% solution of hydrochloric acid, bring to a boil and add 1.5 ml of a 1% solution of sodium citrate with stirring. The mixture is boiled for 25 minutes, cooled and stored at a temperature of 4-6 ° C. The sizes and uniformity of the synthesized NCHCs are evaluated using a transmission electron microscope of the CX-100 type (Jeol, Japan). Using this technique, it is possible to obtain a preparation of nanoparticles that does not contain conglomerates. The shape of the NchCZ is close to spherical; the average diameter is 34 nm.

Another example of the use of sodium citrate ZHVK reduction is the work of A.A. Titova et al. [Titov A.A. Development and optimization of immunochromatographic tests for the detection of botulinum toxins / A.A. Titov, I.V. Shilenko, A.A. Morozov, S.P. Yarkov, V.N. Zlobin // Applied Biochemistry and Microbiology. - 2012 - Volume 48. - No. 2. - S. 249-256]. NCHCs with a diameter of 25-47 nm are obtained by the method of Francis by reduction of HAuCl _{4 with} sodium citrate. To 100 ml of deionized water, 1 ml of 1% HAuCl _{4 was} added, the solution was brought to a boil, and 1% sodium citrate was added with stirring in a volume depending on the target diameter of the nanoparticles. For NchKZ with a diameter of 25 nm, 2.0 ml, 31 nm - 1.25 ml, 47 nm - 1.0 ml of a 1% sodium citrate solution are added. After which the solutions are boiled for another 5 minutes and cooled to room temperature. NchKZ preparations are stored at a temperature of plus 4-6 ° C in the dark. To conduct electron microscopy, NchKZ samples are applied to copper grids coated with a film - a substrate made of polyvinyl formal. Pictures of the NchKZ preparations are obtained on an electron transmission microscope of the JEM1011 type (Jeol, Japan) at an accelerating voltage of 80 kV and an increase of 320,000.

Closest to the claimed method is a method for producing colloidal gold particles with a diameter of 30 nm using citrate reduction [Hermanson G. Bioconjugate Techniques. Second Edition / Greg.Hermanson // Academic Press. - 2008. - P. 924]. Prepare 1 ml of a 4% solution of ZHVK in deionized water, 0.5 ml of the resulting solution is added to 200 ml of deionized water, brought to boiling with stirring. 3 ml of a 1% sodium citrate solution are added to boiling water with rapid stirring and boiled for another 30 minutes. A sign of the formation of colloidal gold particles is the transition of the color of the solution from blue to red.

In common with the claimed method is the restoration of ZHVK with sodium citrate, as well as the target diameter of the nanoparticles is 30 nm. However, in the above method, there is no series of data necessary for its reproduction: the mixing mode is not set (the number of revolutions of the “mixing” rod per minute and the temperature of heating the solution at different stages of obtaining NChKZ), as well as the time and conditions of introducing reagents, which does not allow one to read the indicated method relevant to the proposed.

An analysis of the scientific literature also revealed that there are various modifications of the Frances method. For example, in some sources, hydrochloric acid is added immediately, and sodium citrate is added during boiling, in others, both reagents are added sequentially during boiling [L. Dykman. Gold nanoparticles: synthesis, properties, biomedical application / L.A. Dykman, V.A. Bogatyrev, S.Yu. Schegolev, N.G. Khlebtsov. - M .: Science. - 2008. - 319 p.].

There are discrepancies in the volume of sodium citrate introduced into the ZHVK solution to obtain particles with a given size. Table 1 presents a comparative analysis of indicators from several research projects that contain information on the volume of sodium citrate added to the ZHVK solution to obtain particles with a given size when preparing it according to the Franc method, and the data on the calculation of the amount of sodium citrate to be added to ZHVK solution to obtain particles of the corresponding size according to the formula also recommended for use in individual works [Dressier D. Botulinum toxin antibody testing: comparison between the immunoprecipitation assay and the mouse diaphragm assay / D. Dressie r, G. Dirnberger / European Neurology. - 2001. - V. 45, No. 4. - P. 257-260].

From the data presented in table 1, it follows that when preparing larger diameter particles of NchCZ using the Franc method, the amount of sodium citrate added to the solution decreases both according to various literature sources and when calculated according to the formula. However, when comparing the literature data and the data obtained when calculating the amount of sodium citrate added to the solution using the formula for one particle diameter, it is observed that in the first case, sodium citrate is recommended to be added more than in the second.

In immunochromatography (ICA), both small NCHC preparations of 3-5 nm and large diameters up to 50 nm are used. However, NchKZ with a diameter of 25 ± 5 nm are of the greatest interest for ICA. Particularly, particles of this size, according to the articles, are the most optimal for effective sorption of antibodies, provide adequate migration of colloidal gold-labeled antibodies through the porous structure of the nitrocellulose membrane and visual signal detection in the test zone [N. Byzova The effect of the composition of the conjugate of colloidal gold with proteins on the effectiveness of their use in immunochromatographic analysis / N.V. Byzova, D.V. Sotnikov // Modern problems of science and education. - 2013 - No. 3. - S. 1-8].

The large number of modifications of the citrate method of Francis, as well as the lack of clear recommendations on the preparation of colloidal gold, revealed the expediency of developing a step-by-step method for producing a low-pass NKH of an average diameter of 25-30 nm, optimal for effective sorption of antibodies.

The technical result of the invention is to obtain colloidal gold nanoparticles with a diameter of 25-30 nm.

The technical result is achieved by practicing the stages of preparing colloidal gold, taking into account all the necessary temperature and time parameters, as well as the mixing modes established at each stage, providing conditioned NCHZ with an average particle diameter of 25-30 nm, and includes the following stages: install on a magnetic stirrer a temperature of 300 ° C and a mixing mode of 375 rpm; 49.23 ml of room temperature deionized water are added to the Erlenmeyer flask and a “stirring” rod of the magnetic stirrer is introduced; a flask of water is mounted on a magnetic stirrer; after 6 minutes (time is fixed by a stopwatch from the moment the flask with the contents is placed on the stove and the heating starts to drain off the condensate on the walls of the reflux condenser and / or flask), 0.058 ml of a 10% solution of ZHVK are added, boiled for 2 minutes and 0.72 ml is added 1 % solution of 5.5-aqueous sodium citrate; when the color of the solution changes from blue to red, the mixing mode on the magnetic stirrer is changed from 375 rpm to 500 rpm and the temperature is reduced from 300 ° C to 200 ° C, the solution is boiled for another 20 minutes; after 20 min, disconnect the flask from the reflux condenser and remove from the magnetic stirrer; the flask with the NchKZ solution was left at room temperature for 18 hours in order to visually assess the quality of the prepared preparation.

At the stages of preparation of NCHKZ preparations with a given particle size, parameters affecting the particle size are taken into account, such as the number of revolutions of the stirrer, temperature, time of introduction of reagents regarding the appearance of the first signs of boiling of deionized water, conditions for making a solution of ZHVK and boiling time (from the moment of appearance of a red color until the end of the experiment).

In order to confirm the objectivity of choosing the optimal conditions for preparing NchKZ with an average particle diameter of 25-30 nm, we present the results of the evaluation of a series of NchKZ preparations obtained by working out individual stages.

In the course of the invention, the following conditions were determined to influence the quality of NchKZ preparation:

1) determination of the temperature of the water at which it is necessary to introduce the cold water (cold water) (in cold, heated or boiling water);

2) the establishment of the time of the appearance of the first signs of boiling (3 or 6 min), when it is necessary to add the necessary reagents (ZHVK and / or sodium citrate);

3) determination of the optimal value of the number of revolutions of the mixer at the beginning of the experiment (375 or 800 rpm) and when a red color of the solution (500 or 800 rpm) appears.

HAuCl ₄ золото 3H ₂ O (Sigma, USA), distilled water (GOST 6709-72), deionized water (GOST 11.029.003-80), hydrochloric and nitric acid, sodium citrate 5.5 were used in the work - water ("Reahim", Russia).

For weighing reagents, we used Adventurer electronic analytical balance (OHAUS, USA). All reagents were prepared on deionized water. To obtain deionized water, the UF Arium 611 UF system (Sartorius, Germany) was used.

Colloidal gold nanoparticles were prepared using a heated magnetic stirrer (Heidolph, Germany). The optical parameters of nanoparticle preparations were evaluated on a SPEX SPP-705-4 scanning spectrophotometer (ZAO Spectroscopic Systems, Russia). When spectrophotometry was evaluated, the optical density of the solution in the wavelength range up to 300 to 600 nm, determining the value of the wavelength at which there is a maximum absorption peak. The sizes, dispersion properties, and electron density of particles were analyzed using a JEM-1011 electron transmission microscope (Jeol, Japan).

To prepare a 10% solution of hydrochloric acid, 10 ml of deionized water was added to 1 g of ZHVK. The finished solution was stored in a bottle of dark glass in the refrigerator at a temperature of 4 ° C.

A solution of 1% sodium citrate was prepared immediately before each experiment, for this, 0.69 g of sodium citrate was added to 50 ml of deionized water, taking into account the water content (Na ₃ C ₆ H ₅ O ₇ × 5.5H ₂ O), and filtered through a syringe filter nozzle with a pore size of 0.22 μm.

All laboratory glassware intended for the preparation of NCHKZ preparations is pretreated with “royal vodka” (a mixture of concentrated hydrochloric and nitric acids in a ratio of 3: 1) for 5 minutes, followed by washing the glassware twice in distilled and twice in deionized water, drying in air and processing in a dry heat cabinet of all bottles (flasks, etc.) with necks closed with foil. Processing mode in a dry oven: 180 ° C, 60 min.

To prepare 50 ml of colloidal gold of series No. 1-6, 0.058 ml of a 10% solution of ZHVK was taken. In the course of work, the conditions for introducing a solution of ZHVK were developed. In the preparation of a series of colloidal gold No. 1-4, 10% ZHVK was added to heated water, boiled for 2 minutes, then 0.72 ml of sodium citrate was added. In the preparation of a series of colloidal gold No. 5, 6, a 10% ZHVK solution was added directly with boiling water and boiled for 2 min until sodium citrate was added. In addition, at this stage, the necessary time interval was also established for introducing reagents regarding the appearance of the first signs of boiling deionized water. The need to develop this parameter is associated with the absence of visible signs of boiling in deionized water with proper preparation. 2 time intervals for boiling deionized water were chosen — 3 min (the appearance of condensate) and 6 min (the beginning of condensate draining along the walls of the reflux condenser and / or flask). Moreover, in the preparation of series No. 1 and No. 3, a 1% solution of sodium citrate was added after 3 minutes, series No. 2 and No. 4 after 6 minutes. In the preparation of series No. 5 and No. 6, a 10% solution of ZHVK was introduced, respectively, after 3 minutes and 6 minutes. In addition, ceteris paribus, in the preparation of colloidal gold used different modes of mixing speed of the solutions during the cooking process: for series No. 1 and No. 2 — the entire period of gold cooking was carried out at 800 rpm; for series No. 3 and No. 4 - 375 rpm, followed by an increase in the number of revolutions of the mixer from the moment of the appearance of red color to 500 rpm. For series No. 5 and No. 6, a mixing mode of 375 rpm was used with a subsequent increase in the number of revolutions of the mixer from the moment of the appearance of a red color to 500 rpm, while the volume of a 1% sodium citrate solution introduced into the ZHVK solution was also changed from 0, 72 ml per 1.44 ml to ensure that a change in the volume of sodium citrate added to the solution affects the diameter of the nanoparticles. For all series of nanoparticle preparations, the temperature of water heating at the beginning of the experiment was 200 ° C; when a red color appeared, the temperature was raised to 300 ° C.

The conditions for obtaining preparations of colloidal gold nanoparticles are presented in table 2.

The obtained preparations of colloidal gold nanoparticles were evaluated visually by spectrophotometry and electron microscopy, the results of the study are presented in table 3.

From the data presented in table 3, we can conclude that NchKZ preparations of series No. 1 and No. 2 are substandard and cannot be used for further work. A visual assessment of preparations of series No. 1 and No. 2 revealed that the solution is colloidal, has a blue color. According to electron microscopy, the obtained particles of colloidal gold are heterogeneous in shape and size, form a large number of conglomerates. The graphs obtained by spectral analysis of preparations of nanoparticles of series No. 1 and No. 2 do not have a clearly formed absorption peak, it is impossible to estimate its width, which also indicates the heterogeneity and poor dispersion properties of the NCH.

The most conditioned preparations were series No. 3-6. Nanoparticles in these series of preparations, according to electron microscopy, are uniform in shape and size. According to the results of spectrophotometry, isosceles absorption peaks were obtained, confirming the good dispersion composition of nanoparticles. The preparations of the indicated series in terms of particle sizes (from 15 to 30 nm) turned out, in accordance with the literature data, suitable for further development of immunochromatographic test systems. The preparation of the series No. 3 from the preparation of the series No. 4 and the preparation of the series No. 5 from the preparation of the series No. 6 are distinguished by a high uniformity of particles and an optimal particle size close to 1 unit. the optical density of the solution of colloidal gold for series No. 4 and No. 6, which, according to literature [Dykman, L.A. Gold nanoparticles: synthesis, properties, biomedical application / L.A. Dykman, V.A. Bogatyrev, S.Yu. Shchegolev, N.G. Khlebtsov // Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of Sciences. - M .: Science. - 2008. - 319 p.], Is also one of the criteria for the condition of KZ and the possibility of its successful use for the development of immunochromatographic test systems.

According to the results of the experiment, it was found that:

1) the addition of ZHVK in heated or boiling water does not fundamentally affect the quality of the obtained NchKZ;

2) the best results were obtained by adding ZHVK after 6 minutes from the start of heating the reaction mixture (placing the flask on a magnetic stirrer) until the first signs of boiling appear - the beginning of condensate draining on the walls of the flask;

3) the optimal mode of mixing the solution is as follows: at the beginning of the experiment, 375 rpm with a subsequent increase in the number of revolutions of the stirrer to 500 rpm from the moment the red color appears;

4) the exposure time to stabilize the particles after the appearance of a red color of the solution during the synthesis of the NchCW should be 20 minutes

Thus, the procedure for producing colloidal gold preparations with a nanoparticle diameter of 25-30 nm in a volume of 50 ml can be represented as follows:

1) pour 49.23 ml of deionized water at room temperature into the Erleinmeyer flask and add the “stirring” rod of the magnetic stirrer;

2) put a reflux condenser on the flask, place the flask with the contents on a magnetic stirrer and set the mode to 375 rpm and 300 ° C;

3) after 6 minutes (from the beginning of placing the flask on a magnetic stirrer until the first signs of boiling appear - the beginning of condensate draining along the walls of the reflux condenser and / or flask), add 0.058 ml of a 10% solution of ZHVK, boil for 2 minutes, add 0.72 ml of 1% - sodium citrate solution;

4) when changing the color of the solution from blue to red, change the operation mode of the magnetic stirrer to the following: 500 rpm and 200 ° C;

5) boil the solution for another 20 minutes, turn off the heating, remove the reflux condenser, remove the flask from the magnetic stirrer. Cool the NChKZ solution at room temperature for a day, then store in the refrigerator at 4 ° C.

If you need to get a different volume of the drug NchKZ (more than 50 ml), it is advisable to recount the amount of reagents introduced into it.

The invention is illustrated by the following example.

Example

A temperature of 300 ° C. and a stirring speed of 375 rpm are set on a magnetic stirrer. 49.23 ml of deionized water at room temperature are added to the Erlenmeyer flask; a “stirring” rod of the magnetic stirrer is added to the flask.

After 6 minutes (from the time from the start of heating, placing the flask with the contents onto the magnetic stirrer and turning on the magnetic stirrer, until condensate drains), 0.058 ml of 10% solution of ZHVK are added, boiled for 2 minutes and 0.72 ml of 1% solution is added 5.5 aqueous sodium citrate.

When the color of the solution changes from blue to red, the mixing mode on the magnetic stirrer is changed from 375 rpm to 500 rpm and the temperature is reduced from 300 ° C to 200 ° C, the solution is boiled for 20 minutes.

After 20 minutes, disconnect the flask from the reflux condenser and remove from the mixer. The flask with the NchKZ solution was left at room temperature for 18 hours in order to visually assess the quality of the prepared preparation.

The operability of the method for obtaining NchKZ with a particle diameter of 25-30 nm was confirmed upon receipt of preparations of three more series.

The results of the evaluation of NCHC preparations by electron microscopy and spectrophotometry showed that the preparations of all series obtained by this method are conditional (table 4).

Claims (1)

A method for producing colloidal gold nanoparticles by reducing hydrochloric acid with sodium citrate, and comprising the following steps: a temperature of 300 ° C. and a mixing mode of 375 rpm are set on a magnetic stirrer; 49.23 ml of room temperature deionized water are added to the Erlenmeyer flask and the stirring bar of the magnetic stirrer is introduced; a flask of water is mounted on a magnetic stirrer; after 6 minutes, add 0.058 ml of a 10% solution of hydrochloric acid, and the indicated time is fixed by a stopwatch from the moment the flask with the contents is placed on the tile and the heating starts to drain off the condensate on the walls of the reflux condenser and / or flask, boil for 2 minutes and add 0.72 ml of a 1% solution of 5.5-aqueous sodium citrate; when the color of the solution changes from blue to red, the mixing mode on the magnetic stirrer is changed from 375 rpm to 500 rpm and the temperature is reduced from 300 ° C to 200 ° C, the solution is boiled for another 20 minutes; after 20 min, disconnect the flask from the reflux condenser and remove from the magnetic stirrer; a flask with a solution of colloidal gold nanoparticles is left at room temperature for 18 hours.

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