BG111169A - Highly sensitive immunochromatographic systems with magnetic nanoparticles and fluorescent markers - Google Patents

Abstract

The invention relates to an immunochromatographic system for determining the concentration of different components in liquid media, which is intended for an analysis of one or several components (antigens) in one and the same food sample, biological fluids, waters, soil extracts, and others. The sample antigen connects with a surplus from a specific antibody, which is an immobilized in advance on modified magnetic nanoparticles. The immunochromatographic test strip is immersed in the mixture, the formed conjugates are moved around on a chromatographic mechanism, and reach the adsorbed conjugates antigen-fluorescent dyes of the conjugate pad. The latter bind to the unfilled active centers of the antibody, the obtained conjugates continue to move on the modified membrane, and at the end of the membrane are retained by a permanent magnet. The held conjugates of the test line are excited by an emission of laser or LED sources with a corresponding exciting wavelength, corresponding to the exciting frequency of the fluorescent dyes used, connected to the conjugates. The emitted light is treated optically and is filtered by interferential thin-layer strip filters. The filtered monochromatic luminous flux falls on a sensitive photodiode or other type of highly sensitive photo receptive element. The signal is transformed and fed into a computer system for expressing the concentration of antigen present in the sample. The immunoreaction with antigens with low molecular mass (antibiotics) goes on a competitive basis and the response is inversely proportional to the concentration of the analyzed component (i.e. in the presence of more antigen the signal is lower, the absence of an antigen creates a high signal). 1 claims, 5 figures

Description

Title of the invention

High-sensitivity immunochromatographic systems with magnetic nanoparticles and fluorescent markers

Field of technology

Manufacture of instruments and appliances for measuring, testing and navigation № 26.51 of the Classification of Economic Activities in the Republic of Bulgaria.

Prior art

A number of color immunochromatographic tests based on visual and absorption measurements have been developed, but they provide poor sensitivity and accuracy. It is known that fluorescence measurement improves sensitivity by 1-2 orders of magnitude compared to color measurement. In fluorescence measurement, the limit of detection depends on the number of fluorescent photons generated, instead of the color change of nanoparticles as in absorption test strips.

One of the most important elements of an immunochromatographic system is the markers. The markers are colored or fluorescent nanoparticles with a size of 15-800 nm, allowing unimpeded passage through the membrane carrier. Colored nanoparticles are colloidal gold or colored latex, less commonly used, carbon or liposomes [1,2]. Enzymes are also used as markers, but additional chromogen needs to be added [3]. Fluorescent dyes directly attached to the antibody [4] or latex particles impregnated with fluorescent dyes [4] are also used. There are no data in the literature on the simultaneous use of magnetic nanoparticles and fluorophore in the preparation of immunochromatographic tests. Preliminary modification of the magnetic nanoparticles with a suitable polymer (chitosan, polyethyleneimine) will lead to the introduction of active groups on the surface of the particles necessary for the immobilization of the antibody on them. The application of magnetic nanoparticles will provide perfect separation and purification of the labeled antibodies using a magnetic field, high immobilization yield of the antibody on the large specific C surface of the nanoparticles and, accordingly, to improved test sensitivity. The addition of the fluorescent dye as a marker will result in accurate quantitative measurement and improved sensitivity of the assay.

The membrane carrier is also an important element of immunochromatographic tests. They are made of nitrocellulose, polyamide, polysulfone and polyethylene [1,2]. The hydrophilicity and pore size of the membrane substrate significantly affect the flow flow. For this purpose, in a number of publications, C membranes are pre-treated with surfactants or other chemical reagents [1,2]. The improvement of these two parameters can be achieved by two new modifications. Plasma modification increases the hydrophilicity and preserves the porous structure of the membrane. The preparation of a nanostructured membrane by applying a second polymer layer provides controlled pore size, good hydrophilicity and a significant amount of active groups leading to a high degree of immobilization of bioagents. These two modifications will reduce the analysis time due to the increased flow rate, reduce the non-specific adsorption of the membrane surface and improve the immobilization yield of the test and control line reagents of the immunochromatographic systems, as well as improve the sensitivity. of analysis.

Immunochromatographic test strips are universal in nature and only with the change of the antibody or receptor they can be applied for analysis of various components in biological fluids, in food, water and others. Determining low concentrations of antibiotics in milk is a very important issue. Antibiotics are low molecular weight compounds and the competitive immunological method is suitable for them. A number of rapid immunochromatographic assays are known to determine the concentration of antibiotics in milk with labeled antibodies or receptors. Receptor assays are slower and require incubation at 50 ° C [5,6]. All these immunochromatographic tests use colloidal gold particles or colored latex particles as markers [5-7]. In addition, these tests are only available with visual and semi-quantitative detection via an SSD camera. There are very few immunochromatographic tests for multianalysis. For the simultaneous determination of three types of antibiotics (beta-lactams, sulfonamides and tetracyclines) in the same sample, the β-STAR immunochromatographic system is known, from USB Bioproducts, Belgium described in www.wikipatents.com/US-Patent- 5434053, [6] based the response to specific antisera and receptors conjugated to gold particles and the corresponding antibiotics. The remainder of unbound antibodies and receptors travel across the membrane and are then retained on the antigen-protein conjugate test line. For better visualization, there is also a control line near the test line containing protein-gold particles. The main disadvantage of all published immunochromatographic systems is the inability to provide quantitative measurements, as the detection is visual. In addition, all immunochromatographic tests have a test and control line. In our version with magnetic nanoparticles and fluorescent dye, it is not necessary to have test and control lines with reagents and this significantly reduces the cost of the test.

The aim of the present patent is to develop new highly sensitive nanoimmunochromatographic systems with precise quantitative detection using magnetic nanoparticles and fluorescent markers, a modified polymer membrane and a sensitive fluorescent module.

Technical essence of the invention

The problem is solved by creating a method based on the immobilization of several - N number (in the case of clarity of the statement three) types of antibodies or receptors (1), (2) and (3) according to figure 1, on the surface (4). ) of modified magnetic nanoparticles (5) with a polymer film containing active groups necessary for the immobilization of antibodies or receptors. The amount of dry magnetic nanoparticles with immobilized antibodies or receptors in the cuvette (9) of Figure 1 is determined to be greater than the expected maximum concentration of test components in the fixed sample volume (7) of Figure 2. The magnetic nanoparticles are then mixed with the immobilized antibodies or receptors and the liquid sample (8) to be analyzed. The latter bind to the respective test components (10), (11) and (12), called antigens in the sample. Mixing takes place in a cuvette (9). The immunochromatographic system is composed of several components, including a sample support (13), a conjugate support (14), a plastic holder (16), a membrane (17) containing capture reagents and an adsorbent support (18), (Fig. 3). . The support (14) contains lyophilized conjugates of identical three antigens bound to three different fluorescent dyes located in the field (15). The test is immersed in the cuvette (9) and the conjugates formed (25) in the cuvette are moved to the second support containing the three conjugates (antigen-fluorescent dye), (15). (figure 4). The latter bind to the free active sites of the antibodies and move along the modified membrane (26, 27, 28). At the end of the membrane, a permanent magnet (29) retains the magnetic conjugates (30) and is illuminated by three different laser or LED sources with corresponding excitation lengths (31, 32, 33) corresponding to the excitation wavelengths of the three different fluorescent dyes connected. with conjugates of magnetic nanoparticles. The latter begin to phosphoresce with wavelengths corresponding to the bound dye conjugates to the magnetic nanoparticles. The greater the amount of antigen in the sample, the less amount of antigen-fluorescent dye conjugate binds to the immobilized receptor or antibody and the lower the light emission. The radiation emitted by the phosphorescence is filtered and focused by a special optical system (34) on three separate photodiodes (35) or another type of highly sensitive photodetector elements for the respective three wavelengths. Analog signals are converted into digital and entered into a computer system for interpretation through specialized software.

The advantages of the invention are:

1. Ideal separation of the formed conjugates modified magnetic nanoparticles-antibody or modified magnetic nanoparticles-receptor due to the magnetic nanoparticles and the applied magnetic field.

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2. Avoid the use of test and control lines containing expensive reagents due to the simplified retention of magnetic conjugates with a permanent magnet.

3. High sensitivity of the immunochromatographic test due to the large specific surface area of the magnetic nanoparticles, leading to high immobilization yield of the antibody (receptor).

4. Low limit of detection and high sensitivity of immunochromatographic systems, dominating over all other tests, due to the use of markers with fluorescent dyes.

5. Rapid analysis, due to the high flow rate through the modified membrane and the rapid kinetic reaction due to the good contact surface between the magnetic nanoparticles with immobilized antibody or receptor and the antigen.

6. Reduction of non-specific adsorption due to the applied new membrane modification.

7. Accurate quantitative detection guaranteed by q developed sensitive fluorescent module.

8. Simple manipulation, which does not require washing procedures.

9. Conducting multianalysis due to the simultaneous use of several different antibodies or receptors and different fluorescent markers.

10. The immunochromatographic test and the portable, compact fluorescent detector can be used in the field and performed in real place and in real time.

11. The proposed immunochromatographic system has a universal character and can be used for analysis of antibiotics, microbial cells, hormones, toxins and other components in samples of milk, dairy products, food, biological fluids, water and others.

Description of the attached figures

Figure 1 - The structure of magnetic nanoparticles with immobilized antibodies or receptors is shown.

Figure 2 - The interaction and binding of immobilized antibodies or receptors on magnetic nanoparticles and antigens in the analyzed liquid sample are shown.

Figure 3 - The individual elements of the immunochromatographic test are presented.

Figure 4 - The immersion of the assay in the cuvette, the movement of the partially bound conjugates from the cuvette in the assay, the binding of the antigen-fluorescent dye conjugates to the free active centers of the antibodies and the retention of the magnetic conjugates by a permanent magnet are shown.

Figure 5. Illumination with three different laser or LED sources with the corresponding excitation lengths corresponding to the excitation wavelengths of the three different fluorescent dyes associated with the conjugates-magnetic nanoparticles and signal detection.

Examples of implementation

According to Fig. 1, the lyophilized immobilized three types of antibodies or receptors (1), (2) and (3) on magnetic nanoparticles (5) covered with a chitosan layer (4) are placed in a cuvette (9). According to FIG. 2, the test sample (7) is then added to the volume of the immobilized receptors or antibodies with a micropipette (8) or an automated dispenser. The antigens (10), (11) and (12) contained in the sample bind selectively to the respective receptors or antibodies, but remain unoccupied active centers in the latter. Immediately C is then immersed in the cuvette (9) an immunochromatographic test consisting of a sample support (13), a conjugate support (14), a location (15) of fluorescent dye-antigen conjugates (19.20,21,22,23, 24), a plastic holder (16), a membrane (17) and an adsorption pad (18). The partially bound conjugates move through the support (13), reach the conjugates of antigenfluorescent dyes located in (15) and they bind to the free active centers of the antibodies. The resulting conjugates move along the membrane (17) and are held by the permanent magnet £ (29) at the end of the membrane (30). Laser systems (31), (32) and (33) or

LED sources illuminate the retained conjugates with the corresponding excitation lengths corresponding to the excitation light of the three different fluorescent dyes. The latter begin to emit radiation with wavelengths corresponding to the emissions of fluorescent dyes from the conjugates (magnetic nanoparticles receptor-antigen-fluorescent dye). The greater the amount of antigen in the sample, the less amount of antigen-fluorescent dye conjugate binds to the immobilized receptor or antibody and the lower the light emission. The emitted light passes through an optical unit (34), is filtered and falls on a sensitive one

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photodiode (35). The resulting fluorescent signal is inversely proportional to the concentration of antigen in the sample. The signals from the three photodetectors are amplified and then converted by analog-to-digital converters and fed to the input of the computer system.

Claims (1)

PATENT CLAIMS Development of -on- Immunochromatographic system for determining the concentration of various components- (residues of antibiotics, microbial cells, hormones, pesticides and other toxins liquid media, characterized by the use of three immobilized antibodies on modified magnetic nanoparticles to conduct competitive immunoreaction with three (analyzed components) in the sample and the immobilized antigen-fluorescent dye conjugates, movement of the conjugates along the modified membrane, retention of the latter with a permanent magnet and with subsequent spectrophotometry of the self-luminosity of the phosphorescent-labeled conjugates, resulting in The immunoreaction in low molecular weight antigens (antibiotics) proceeds on a competitive basis and the response is inversely proportional to the concentration of the analyte. in the presence of a larger amount of antigen the signal is lower; the absence of antigen gives a high signal).