RU2543631C2 - Method for functionalising surface of magnetic nanoparticles - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: according to the method magnetic nanoparticles and glutaric dialdehyde are added to a polymer in a phosphate buffer under conditions of an alternating magnetic field, then, peptised and activated particles are three times washed with a phosphate buffer solution, depositing them under the influence of a constant magnet, the activated particles are mixed with an anti-analite solution for conjugation under conditions of the alternating magnetic field, after which the conjugate is washed three times by deposition-resuspension in the phosphate buffer solution, alternating the action of constant and alternating magnetic fields.

EFFECT: obtaining strong functional conjugates which can serve as a basis for the creation of effective instrument-free systems for simplified diagnostic testing.

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Description

The invention relates to the field of molecular immunobiotechnology and can be used in the production of highly sensitive test systems for (qualitative and semi-quantitative) determination of any compounds capable of stereospecific interactions (antigens, antibodies, nucleic acids, biologically active macromolecules), as well as in the technology of manufacturing functionally modified particles suitable for use in pharmacology, cosmetology, medicine.

To date, analysis systems with direct labeling of analyte-specific compounds (anti-analytes) with optically dense particles or water-soluble color polymers of various nature and sizes are quite well known. Among these labels are latexes, including color (Tarcha et al., Abbot Labs., Eur.pat.appl. 89116594.6), red blood cells (Guesdon JL., Avrameas S., Ann. Immunol., 1980, v. 131 C, p. 389-396), non-metallic colloids of sulfur, selenium, tellurium (Yost et al., Eur. pat. appl. 88110459.0), polymerized dyes Congo red, Trypan blue, Lissamine blue (Henry Robert P., US Pat. 4.452. 886), colloidal gold (Roth J., Heitz PU, Immunolabeling with the Protein A - Gold Technique: An Overview, Ultrastructural Pathology, 1989, 13: 467-484.). Using these labels allows you to visually determine the presence of the analyte in homogeneous analysis systems (without separation of the reacting components) - for example, by agglutination of rather large particles (with sizes from 0.2 to several micrometers) or by changing the spectral characteristics (color) of specific agglutinating colloids, and in numerous heterogeneous systems - Erythroimmunoadsorption and, more broadly, Sedimentation-Adsorption (Immuno) Analysis; dot, Western, Southern, Northern blotting; immuno-and hybridofiltration (Flow-Through analyzes), immunomigration (immunochromatography) and a number of others - by staining the specific binding region of the analyte on a non-porous or porous solid-phase carrier. Despite the possibility of quantitative recording using simple colorimeters, reflectometers, densitometers, the main advantage of these methods is the direct visual determination of the analyte, which conditionally distinguishes them in the group of so-called tool-less methods.

The closest to the claimed object in terms of technical nature and the achieved result is a method for producing conjugates based on colloidal carbon particles (D.Yu. Plaksin, MB Raev, ET Gromakovskaya. Method for stereospecific analysis and method for producing conjugate for stereospecific analysis. RF patent No. 2089912 dated 09/10/1997).

In the prototype, the task of increasing the reliability of the analysis and the stability of the detecting reagents used in the analysis is solved by covalently conjugating an affinity compound (anti-analyte) with colloidal carbon particles. For this, the particles are first coated with a water-soluble polymer, which is an inert amphipathic compound capable of relatively strong adsorption on the surface of the particles. Sorption of the polymer allows to obtain a suspension stable in solutions. Sorbed polymer exposes to the aqueous phase functional groups capable of covalently reacting with homo- and / or heterobifunctional conjugating compounds. Subsequent treatment of the resulting complex with a bifunctional reagent, taken in large molar excess relative to the functional groups of the polymer, leads to covalent crosslinking between the sorbed polymer molecules and, at the same time, to the introduction of reactive groups of the bifunctional reagent onto the surface of the complex. The next step is to release the activated label from excess conjugating reagent. The subsequent compound with an intact (unmodified) anti-analyte (affinity compound) is completed by covalently attaching anti-analyte molecules to the surface of the carbon label. As a result, the inventive method allows to obtain conjugates of a strong covalent structure that are stable in solutions, including in the presence of high concentrations of detergents.

However, the multi-stage nature of the conjugation creates a number of technological difficulties. First of all, this concerns the need for a number of chromatographic purification procedures: from excess sorbed polymer in the first stage, and, as a consequence, the need for concentration; from an excess of activating bifunctional reagent, followed by the concentration procedure again, from unbound anti-analyte molecules. Such multi-stage not only lengthens the method described in the prototype, but also makes it time-consuming, low-tech, costly.

The objective of the invention is to develop a method for producing durable functional conjugates based on magnetic nanoparticles.

The basis for solving this problem is the use of magnetic fields created by rotating and / or fixed magnets.

Description of the developed method

The problem is solved by introducing magnetic nanoparticles into a polymer solution adsorbed on their surface, in the immediate vicinity of a rotating magnet. In this case, the particles of the introduced material acquire a rotational motion, which contributes to almost instantaneous peptization of the particles. The introduction into the suspension of peptized particles of a bifunctional reagent promotes surface activation for further conjugation of particles with molecules of affinity compounds (anti-analytes). The only intermediate stage is the removal of excess sorbed polymer and a bifunctional reagent, which is carried out during the sequential deposition-resuspension of particles on a fixed magnet. The last procedure is repeated after conjugation of the activated particles with anti-analyte molecules.

The inventive method of conjugation provides the opportunity for a very wide selection of specific adsorbed polymers and activating / conjugating reagents. This circumstance compels the applicant to submit a formula of invention drawn up in principle terms, an attempt to specify which would inevitably lead to a narrowing of the scope of the claimed object.

The following specific examples describe a method for producing detecting conjugates carried out using glutaraldehyde as a crosslinking (homo) bifunctional reagent. The use of this substance is not essential for the implementation of the method. Instead of glutaraldehyde, other well-known homo- and heterobifunctional compounds can be used which, in second-order reactions, are capable of reacting preferably with primary amino groups that are most universally represented in possible anti-analytes and anti-anti-analytes (although, for covalent conjugation with suspension forms of such anti- analytes, such as Fab'-fragments of antibodies, may be more optimal, for example, maleimide reagents involved in thiol-disulfide exchange reactions).

The use of glutaraldehyde is preferred, since it additionally meets the requirement of universality of the proposed method and is a homo-bifunctional reagent that reacts relatively specifically with primary amino groups of proteins. In addition, an important circumstance is the availability and practically the lowest cost of glutaraldehyde, which also compares favorably with other possible ones, the use of which in the framework of the proposed method from an economic point of view, is obviously a less profitable opportunity.

EXAMPLES OF SPECIFIC IMPLEMENTATION

The following examples are provided only to demonstrate the capabilities of the present invention, but in no way limit both the range of possible technological methods for the synthesis of specific reagents and the scope of the possible application of the invention as a whole.

The specific quantitative parameters given in the examples, even in the range of the indicated ranges, are not fixed by the claims, because they are not fundamental conditions for the implementation of the proposed method.

1. Obtaining activated magnetic nanoparticles.

To 20 ml of a solution of bovine serum albumin (1-50 mg / ml in 0.01-0.2 M phosphate buffer solution (PBS) pH 6.0-8.5) add 1-2 grams of magnetic particles. Mixing is carried out in a container previously placed above the rotating element of the magnetic stirrer. Due to the rotation of the particles themselves, their peptization time is several minutes. An equal volume of glutaraldehyde is added to the suspension of peptized particles at a concentration of 10-25% and after 40-60 minutes the rotation of the magnet is stopped. Due to the magnetic field, the particles settle to the bottom for 5 minutes, after which the supernatant is removed and the FBI is added. The inclusion of rotation of the magnet leads to a rapid resuspension of particles in the buffer solution. The washing procedure is repeated three times, which leads to an exhaustive release of excess polymer and a bifunctional reagent.

2. Obtaining conjugates of magnetic nanoparticles with anti-analytes (Streptavidin, Protein G, Antibodies)

After the last decontamination, activated particles resuspended in 4.5 ml of PBS are introduced into 0.5 ml of PBS containing 5 mg of human monoclonal anti-alpha-fetoprotein (McAtAFP) antibodies. The procedure is carried out in a magnetic field of a rotating magnet. The conjugation time is 30-60 minutes, after which the excess anti-analyte is removed by washing the particles three times during their sedimentation-resuspension using a fixed magnet. The washed particles are resuspended in an FBI containing 20% glycerol, 1% BSA and 0.1% sodium azide.

Conjugates of carbon particles to Protein G, streptavidin, and other antibodies are obtained in a similar manner.

The effectiveness of the reagents obtained using the developed technology was demonstrated in analytical models in comparison with reagents similar in affinity compounds, the labels of which were non-magnetic carbon particles with a size of 160 nm.

Example 1. Two-site determination of AFP in the blood serum of pregnant women on a non-porous solid phase using the conjugate MkAtAFP - MCH.

Plates for serial dilution from polystyrene were used as a solid-phase reagent, the inner surface of the holes of which were sensitized with monoclonal antibodies to human AFP sorbed in the form of pillboxes from drops of 5 microliters from the FBI with a protein concentration of 0.01 mg / ml. The blood serum of a pregnant woman (gestational age - mid-third trimester) was added to the wells in serial ten-fold dilution in the FBI containing 0.05% Tween 20 (FBT). After a 30-minute incubation, the MCPAtAFP-MH conjugate was added to the wells washed with PBS and the plate was placed on a fixed magnet. The results of the analysis were observed after 15 minutes in the form of colored spots at the sites of sorption of the first antibodies with a gradual quenching of the color as the serum was diluted. The last visualized point was observed when diluting serum 1000 times.

A similar study with the detection of a specific complex by conjugate MkAtAFP-carbon demonstrated the sensitivity of the determination at the level of 100-fold dilution of serum, which is an order of magnitude lower than the sensitivity of the proposed method.

Example 2. Direct determination of trophoblastic β-1-glycoprotein (TBH) in the blood serum of pregnant women on a non-porous solid phase using conjugate McAtTBH-MCH.

Plates for serial dilutions from polystyrene were used as the solid phase, on the inner surface of the holes of which the blood serum of a pregnant woman (gestation - mid-third trimester) was adsorbed in the form of pillboxes from drops of 5 microlitres from 0.02 M carbonate-bicarbonate buffer pH 9, 6 in serial ten-fold dilution. After a 30-minute incubation, the droplets were aspirated, and the McAtTBG-MH conjugate was added to the wells washed with PBS and the plate was placed on a fixed magnet. The results of the analysis were observed after 15 minutes in the form of colored spots at the sites of sorption of the studied samples with a gradual quenching of the color as the serum was diluted. The last visualization point was observed when diluting the serum 100,000 times.

Detection by conjugate McAtTBG-carbon demonstrated a sensitivity of protein determination in serum dilution of 10,000 times.

Example 3. Direct determination of immunoglobulins G (IgG) using conjugate G protein - MF on a non-porous solid phase.

As a solid phase, polystyrene serial dilution plates were used, on the inner surface of the wells of which human IgG was adsorbed in the form of pillboxes from drops of 5 microliters from 0.02 M carbonate-bicarbonate buffer pH 9.6 in serial double dilution. After a 30-minute incubation, the droplets were aspirated, and protein-MCH conjugate G was added to the wells washed with PBS and the plate was placed on a fixed magnet. The results of the analysis were observed after 15 minutes in the form of colored spots at the sites of sorption of the studied samples with a gradual quenching of the color as the serum was diluted. The concentration of the last visualized point was 7.5 ng / ml.

When detecting conjugate G protein-carbon, the concentration of the last visualized point was 32 ng / ml, which indicates a four-fold difference in sensitivity in favor of the proposed method.

Example 4. Direct determination of biotinylated proteins using the Streptavidin-MH conjugate on a non-porous solid phase.

As a solid phase, polystyrene serial dilution plates were used, on the inner surface of the wells of which biotinylated BSA was adsorbed in the form of dots from drops of 5 microlitres from 0.02 M carbonate-bicarbonate buffer pH 9.6 in serial double dilution. After a 30-minute incubation, the droplets were aspirated, and Streptavidin-MH conjugate was added to the wells washed with PBS and the plate was placed on a fixed magnet. The results of the analysis were observed after 15 minutes in the form of colored spots at the sites of sorption of the studied samples with a gradual quenching of the color as the serum was diluted. The concentration of the last visualized point was 1 ng / ml.

The sensitivity of the determination of biotinylated BSA during detection by Streptavidin-carbon conjugate is an order of magnitude inferior to the proposed method and amounted to 10 ng / ml.

In order to preliminary assess the stability of the obtained reagents, four conjugates were stored for three months at a temperature of 22 ° C. A second study of the detecting properties, in the arrangements of the examples described above, demonstrated the same analytical qualities. This indicates a strong and, therefore, stable structure of the synthesized reagents, which, in turn, guarantees the reliability and reproducibility of the testing system.

The technological result from the use of the present invention consists, first of all, in that the technology for producing conjugates of magnetic particles is independent of unique or expensive equipment and objectively deficient or expensive reagents. The proposed conjugates can serve as the basis for the creation of effective tool-free systems for simplified diagnostic testing with high sensitivity and reliability. Practically any biologically active molecules carrying at least one free amino group in their structure can be used as compounds imparting the functional qualities of MF. The application of the developed method can find its implementation not only in the field of biotechnology (separation of proteins, nucleic acids, cells, etc.), but also in solving such urgent problems as targeted drug delivery, contrasting in MRI, etc. The widespread use of various magnetic carriers for the purpose of targeted delivery of biologically active compounds in modern practice is limited by the instability of conjugates. The basis of this instability is primarily problems associated with the nature of the connection of biomolecules with particles. In most cases, these are non-covalent bonds, which is the main limitation in the use of such conjugates. The proposed development solves the problem of instability, substantially ensuring the principle of translationality and the prospects of its use in real practice.

Claims (1)

A method of functionalizing the surface of magnetic particles, including sorption of a water-soluble polymer containing functional groups on the surface of magnetic nanoparticles, which is then covalently attached to the surface of the particles and simultaneously activated by treatment with glutaraldehyde, after which the resulting activated complex is covalently conjugated with an anti-analyte, characterized in that Magnetic nanoparticles and glutaraldehyde are added in phosphate buffer as a bifunctional reagent under alternating conditions. magnetic field, after which the peptized and activated particles are washed three times with phosphate buffered saline from excess polymer and glutaraldehyde, precipitating particles under the influence of a permanent magnet, the activated particles are mixed with an anti-analyte solution for conjugation under conditions of an alternating magnetic field, and then washed again three times conjugate from excess anti-analyte by precipitation-resuspension in a phosphate buffer solution, alternating the action of constant and alternating magnetic fields.