RU2510509C1 - Microfluid system for immunoassay - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: microfluid system comprises an analysed fluid passage and another four passages perpendicular to the analysed fluid passage, and with its one end connected thereto; one of these passages is measuring and comprises fluid receptors; the other passage is reference and comprises a fluid only, while the other two passages accommodate fluorescent tags with a substrate immobilised in the fluid medium.

EFFECT: higher reliability and simplified operation.

7 cl, 1 ex, 3 dwg

Description

The invention relates to devices for immunoassays and can be used for laboratory diagnosis of viral infections.

For laboratory diagnosis of viral infections, enzyme-linked immunosorbent, or immunofluorescence analysis methods are most often used.

Enzyme-linked immunosorbent assay is designed to detect antibodies and is based on the use of specific viral proteins - antigens isolated from infected cells, or obtained by genetic engineering. Viral antigens are sorbed on the walls of a plastic cell and incubated with the test blood serum containing the antibodies to be detected. After washing the non-specifically bound mating antibodies, the second antibodies are added to the cells covalently bound to the enzyme, usually peroxidase or alkaline phosphatase. An excess of the second antibodies is washed, and the amount of bound, depending on the amount of antiviral antibodies in the serum, is estimated by the intensity of the enzymatic reaction.

The method of immunofluorescence analysis is used to detect both antigens and antibodies. This method is based on the use of reagents labeled with a fluorescent dye. Antibodies are most often labeled with fluorescein with isothiocyanate. Labeled antibodies bind to the antigen, forming complexes that can be detected using fluorescence microscopy. There are three modifications of an immunofluorescence assay:

- The direct immunofluorescence method is used to detect antigens. It is based on the direct determination of an antigen bound to a solid support using fluorescently labeled antibodies. The reaction is evaluated using a fluorescence microscope.

- The method of indirect immunofluorescence allows the detection of antibodies to a known antigen. An antigen sorbed on a solid support binds to detectable antibodies. Antigen-antibody complexes are detected using labeled antibodies to immunoglobulins.

- The method of competitive immunofluorescence is based on the addition of a known concentration of labeled antigen to the test sample and the reaction with antibodies adsorbed on a solid support. Since labeled and unlabeled antigens compete for binding to antibodies, the concentration of antigen in the test sample can be determined by the amount of labeled antigen bound.

Microfluidic technologies are one of the new, rapidly developing areas of convergence of sciences and technologies, based on the integration of high-tech and high-tech methods of microsystems, electronics, physics, chemistry, optics and hydraulics, molecular and cellular biology, computer science and other sciences.

The main advantage of microfluidic technologies is that they allow you to work with extremely small volumes of reagents - 10 ^-9 -10 ^-15 liters and picograms of substances. The considered working media can be liquids of various nature containing insoluble micro- and nanoparticles, polymer molecules, cell suspensions, emulsions consisting of micelles, drops of non-mixing liquids or air bubbles, etc.

Microfluidic systems are characterized by extremely large surface / volume ratios and high mass and heat transfer rates, which makes them extremely effective for intensifying the course of target processes.

A number of basic characteristics of microfluidic technologies make them extremely valuable and promising for solving a wide range of fundamental and applied problems:

- microfluidic technologies operate with micro / femto volumes of analyzed liquids;

- the fluid flow in microfluidic channels is laminar, because of this, it is possible to accurately calculate the transported masses of substances to various points of the microfluidic system, depending on the geometry of the channels, pressure distribution, and properties of the fluid;

- due to laminarity, mixing of fluid flows is carried out as a result of diffusion;

- laminarity of flows and diffusion in combination can provide the creation of gradients of substances in microfluidic systems;

- in the microfluidic system, the ratio of surface to volume increases, which creates optimal conditions for diffusion and heat exchange;

- capillary systems make it possible to implement the most important transport methods of modern analytical chemistry at the micro level: flow-injection analysis based on pressure differences and highly efficient capillary electrophoresis;

- the unique possibilities of creating microchannels of complex configuration, embedding various types of microreactors, controlling microflows make it possible to realize complex multi-stage analytical reactions;

- highly sensitive systems for detecting analysis results, for example, fluorescence microscopy and spectroscopy make it possible to detect the desired components at the level of trace amounts (up to single molecules);

- the volumes of liquids with which microfluidic devices are able to operate are comparable with cell sizes or orders of magnitude smaller than them.

Microfluidic systems are integrated devices - laboratories on chips formed by a system of channels with a laminar flow of liquids, which creates optimal and easily controlled conditions for the flow of transport and diffusion processes; the functioning of bacterial and eukaryotic cells; molecular-biological, molecular-genetic, biochemical, chemical reactions.

The free immunodiffusion reaction in microfluidic systems is designed to detect antibodies or antigens in the studied liquids and is based on the fact that the antigen-antibody complex forms during the interaction of the antigen with the antibody, resulting in a significant change in the size of the analyzed particles. The size of the antigen and fluorescent dye conjugates is much smaller than the size of the resulting antigen-antibody complex. An increase in particle size leads to a change in the diffusion rate. The change in the diffusion rate of labeled particles in the channels of the microfluidic system can be estimated by the distance traveled by the dye in the channel. Modern detection methods make it possible to determine the characteristics of the diffusion process of fluorescently-labeled particles. The method is limited in its ability to determine antigens due to the comparability of the sizes of the formed complexes and antibodies.

Known microfluidic system in the form of a microfluidic chip for rapid multiple enzyme-linked immunosorbent assay, which is immobilized antigens in one or multiple channels [Application US No. 20090123336, IPC: B01J 19/00, B01J 019/00; G01N 33/00]. Each channel includes a winding path section or a plurality of winding path sections. The receptor for detecting the analyte can be fixed in various sections of the winding path, for example by adsorption. Different receptors can be immobilized in different sections of the winding paths of each channel or in different channels to simultaneously detect many analytes. This chip is especially useful for enzyme-linked immunosorbent assay.

A microfluidic system in the form of a microfluidic chip for detecting the presence of an analyte in a liquid sample is also known, which is a monolith and a channel in this monolith. The duct in the channel has a direction between the inlet and the outlet and includes at least one section of a winding path [US Patent No. 8075854 IPC G01N 30/00]. On this chip, the winding path section includes a receptor that is complementary to the substance whose presence will be tested.

This microfluidic system is adopted as a prototype of the invention.

Its disadvantage is the need to carry out washing - to remove the analyte unbound from the receptor from the channel.

The invention solves the problem of creating a system for determining the substrate, capable of carrying out and testing the reaction between the substrate and the receptor, without immobilizing the receptor and subsequent washing.

The problem is solved in that a microfluidic system is proposed that includes a channel for the analyzed fluid, characterized in that it also contains four channels located perpendicular to the channel for the analyzed fluid, and one end connected to it, while one of these channels is a measuring and receptors are placed in it in a liquid medium, another channel is a reference one and contains a liquid medium, and fluorescent labels with a substrate immobilized on them in a liquid medium are placed in two other channels.

The system channels are made in monolith.

The channels are closed at the second end, which is not connected to the channel for the analyzed fluid.

The channels perpendicular to the channel with the analyzed fluid have the same dimensions.

The measuring and reference channels contain the same liquid medium.

The liquid medium is usually a buffer solution.

Fluorescent labels can be in the form of quantum dots.

Fig. 1 shows a diagram of the proposed microfluidic system in the initial position before the analysis, where: 1 - measuring channel, 2 - reference channel, 3 - channel with fluorescent labels, 4 channel with a channel with fluorescent labels, 5 - channel with the analyzed liquid, 6 - receptors, 7 - fluorescent label, 8 - substrate.

Figure 2 shows a diagram of the proposed microfluidic system in the final position after analysis, where: 9 are the complexes of the substrate with receptors, L is the distance from channel 5, where the fluorescence intensity is measured.

Figure 3 shows the growth curves of the fluorescence intensity in the system during the analysis described in the example.

As shown in Figs. 1 and 2, the proposed system has 5 channels. Channel 1 is measuring and in the initial state, receptors 6 are placed in it in a liquid medium. The channel 2 adjacent to it is the reference one and it is filled with the same liquid medium as channel 1, but in the initial state it does not contain any particles, including receptors. Two other channels 3 and 4 are located opposite the measuring and reference channels. These channels in the initial state contain fluorescent labels 7 with substrate 8 immobilized on them. Their size is larger than the size of the substrate and the size of the receptor. The central channel 5 is perpendicular to the channels 1, 2, 3, and 4 and communicates with each of them. Channel 5 is for the analyzed fluid.

The analysis is carried out in the following order.

Channels 1, 2, 3, 4 are filled with a liquid, for example, buffer solution, while receptors 6 are placed in channel 1, and fluorescent labels 7 with a substrate immobilized on them are placed in channels 3 and 4. Fluorescent labels are, for example, quantum dots with antigens immobilized on their surface. The sizes of antigens are significantly smaller than the sizes of antibodies specific to them and the sizes of fluorescent labels. In the channel 5 serves the analyzed fluid, which contains the substrate 8. The width and depth of the channels can vary from 10 μm to 1 mm, the most preferred dimensions are less than 500 μm in width. The supply of reagents is carried out using standard syringes or in another way, any method can be used to advance the liquid: pressure, gravity, capillary forces or others.

After the channel 5 is filled with the analyzed liquid, its flow is stopped. Under stationary conditions, the diffusion process begins from channel 5 of the substrate 8 to channels 1, 2, 3, and 4, and the process of diffusion of labels 7 from channels 3 and 4 through channel 5 to channels 1 and 2, respectively. In this case, the diffusion coefficient of the substrate in the channels is significantly greater than the diffusion coefficient of the labels. In channel 1, complexes of substrate 8 and receptors 6 are formed, as a result of which the number of free receptors in channel 1 decreases.

During diffusion, fluorescent labels 7 enter channel 5, and then into channel 1 and 2, and some of them form complexes with the remaining free receptors 6, because labels are a complex of a fluorescent particle with a substrate immobilized on the surface. In this case, the diffusion coefficient of the resulting complexes is substantially less than the diffusion coefficient of the labels. The presence of substrate in channel 5 is determined by the difference in fluorescence intensity at a distance L in channels 1 and 2. The rate of increase in intensity depends on the ratio of the number of fluorescence labels and the resulting complexes of labels with receptors. The increase in intensity in channel 2 at a distance L gives a normalization coefficient for determining the presence of antigens, provided that the concentration of labels in channels 3 and 4 is the same.

The operation of this system is based on a change in the diffusion rate of fluorescent nanocomplexes compared with the diffusion rate of fluorescent labels in the channels of the microfluidic system. As is known from information sources, the free immunodiffusion reaction is designed to determine the presence in the analyzed fluid of only particles whose sizes are significantly larger than the fluorescent labels used. The change in the diffusion rate is based on an increase in the Stokes radius during the formation of fluorescent nanocomplexes during the interaction of large analyte particles with small labels. This limits the possibility of determining the presence of small particles in the analyzed liquid due to the almost unchanged Stokes radius during the interaction of large marks with small analyzed particles.

The work of the proposed system is based on the binding of a standard labeled (immobilized on the surface of a fluorescent label) and unlabeled substrate present in the test sample to the receptor in channel 1. Since the labeled and unlabeled substrate compete for binding to the receptor, the diffusion rate of the fluorescent complex in channel 1 can be determined the presence in channel 5 of a competing substrate. In the absence of a competing substrate in channel 5, the fluorescence intensity that forms in channels 1 and 2 at a distance L from the intersection with channel 5 due to diffusion of the fluorescent complexes will differ after time t. This difference is due to an increase in the Stokes radius of the fluorescent complex due to binding to a receptor located in channel 1. In the case when a substrate (unlabeled small particles) is present in channel 5, all (or partially) the receptor from channel 1 binds to it before channel 1 diffuse fluorescent complexes from channel 3. Thus, there is no free receptor to bind to the fluorescent complex, and the diffusion rate of fluorescent complexes in channels 1 and 2 becomes the same. As a result, the fluorescence intensity that forms in channels 1 and 2 at a distance L from the intersection with channel 5 due to the diffusion of the fluorescence complexes will be the same after time t.

By measuring the fluorescence intensity in channels 1 and 2 at a distance L for a time t and normalizing the fluorescence intensity in channel 1 to the fluorescence intensity in channel 2, we can determine the presence of a substrate in the test liquid.

The measurement of fluorescence intensity is carried out in the absence of fluid movement in the system.

It is possible to normalize the system for quantitative determination of the substrate in the analyzed fluid, however, in practice it is enough to conduct a qualitative analysis to determine the presence or absence of the disease.

Thus, the proposed microfluidic system for determining the substrate is able to carry out and test the reaction between the substrate and the receptor without immobilizing the receptor and subsequent washing, which greatly facilitates the analysis.

Among other things, the system allows analyzes when the sizes of antigens are significantly smaller than the sizes of antibodies specific to them and the sizes of fluorescent labels.

Example

Fluorescent labels are Qdots 625 quantum dots with the p24 protein of HIV, the human immunodeficiency virus antigen, immobilized on their surface. Fluorescent labels are placed in channels 3 and 4 of the microfluidic system.

Antibodies to HIV p24 protein were placed in channel 1, channel 2 was left filled with TBS buffer solution (50 mM TRIS, 0.9% NaCl, pH 7.2). In the channel 5 serves the analyzed fluid - HIV p24 protein at a concentration of 0.08 mg / ml in a buffer solution.

To register a signal, the system is positioned on a stage of an inverted fluorescence microscope and the diffusion process is shot frame by frame in channels 1 and 2 at a distance of 3 mm from the intersection of channels 1 and 2 with channel 5.

Figure 3 shows a graph of the intensity of the fluorescent signal according to the results of frame-by-frame shooting versus time after supplying the analyzed liquid to the system. Curve 1 shows the increase in signal intensity in the reference channel 2, and curve 2 shows the increase in signal intensity in the measuring channel 1. The increase in fluorescence intensity indicates the presence of HIV p24 protein in the tested liquid sample.

Claims (7)

1. Microfluidic system, including a channel for the analyzed fluid, characterized in that it also contains four channels located perpendicular to the channel for the analyzed fluid and one end communicating with it, while one of these channels is a measuring one and receptors are placed in it in a liquid medium , the other channel is a reference one and contains only a liquid medium, and fluorescent labels with a substrate immobilized on them in a liquid medium are placed in two other channels. 2. The microfluidic system according to claim 1, characterized in that the channels are made in a monolith. 3. The microfluidic system according to claim 1, characterized in that the channels are closed from the second end. 4. The microfluidic system according to claim 1, characterized in that the channels perpendicular to the channel with the analyzed fluid have the same dimensions. 5. The microfluidic system according to claim 1, characterized in that the measuring and reference channels contain the same liquid medium. 6. The microfluidic system according to claim 1, characterized in that the liquid medium is a buffer solution. 7. The microfluidic system according to claim 1, characterized in that the fluorescent labels are in the form of quantum dots.

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