RU2528885C2 - Method for detecting analyte from particle solution and device for implementing it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biology and medicine, namely to qualitative and/or quantitative indication of analytes. A device for analyte collection from a solution by concentrating it on magnetic particles comprises a flow chamber consisting of upper and lower passages comprising electrodes for generating an electric field perpendicular to a fluid flow in a fluid passage from a semi-permeable dialysis membrane arranged between the upper and lower passages, a magnetic field concentrator and a magnet for generating a magnetic moment in the concentrator.

EFFECT: ensured accelerated and higher detection sensitivity.

3 cl, 1 dwg, 1 ex

Description

Technical field

The invention relates to biology and medicine, namely to a qualitative and / or quantitative indication of analytes in solution, in particular in an immunobiological sample containing an analyte of protein and / or nucleic origin (proteins, peptides, DNA, viruses, cells, cell membranes). The device and method allow acceleration of analyte detection, which is achieved by concentrating a charged analyte on magnetic particles in the analyte solution stream and placed due to the action of a magnetic or electric field in the channel of the device with semipermeable walls.

The device and method allow a fundamentally new method to accelerate the detection of analytes in samples, including those containing a low concentration of analyte, or in the case of antibodies with low affinity, including those with low specificity. The invention improves the detection limit not only by accelerating the detection time of the analyte in solution, but also by increasing the sensitivity of diagnostic tests.

State of the art

A known method for the diagnosis of analyte in samples, based on a magnetic recognition system (application RU 2009120688). The invention provides for the use of a label for an analyte that is attached to a magnetic or magnetizable substance, comprising a recognition component for attaching a label to an analyte, and a component for binding or encapsulating a magnetic or magnetizable substance, a metal-binding protein, polypeptide or peptide. In the present invention, there is no possibility of analyte concentration, which reduces the possibility of applying the method in the case of a low concentration of the latter. For this reason, this invention does not accelerate analyte indication.

A device is known for highly sensitive magnetic detection of analytes in a biological sample (patent application RU 98120854; publication WO 97/40377). In the described invention, the detection of protein analytes, based on the binding of receptors and ligands, is carried out using two devices - one for magnetization in order to create a magnetic field at the site of the sample, the second - a detector device for measuring the magnetic properties of the sample. Despite the use of the magnetic field generated by the device for magnetization, the invention does not provide for the use of magnetic particles, as well as a method of accelerating the collection of analyte in the measured sample. In addition, the invention is configured to perform in vivo measurements.

Known multi-analytic immunoassay using microparticles (Application RU 2008127246). It proposes the mixing of various categories of microparticles coated with biospecific reagents to bind various desired analytes and labeled with one or more fluorochromes in various concentrations. Detection is based on the difference in the ratios of concentrations of long-term fluorescent fluorochromes in microparticles that differ in different types of analytes. The invention does not provide for the concentration of the analyte and, therefore, does not allow to obtain a time gain when it is indicated.

Closest to the claimed invention is the invention of US 7960184 and US 7824927 (prototype). The prototypes characterized a different type of device used for the analysis of biological samples. The invention describes the detection of biological analytes in a sample, which, unlike the claimed one, is based on the use of immobilized test molecules using microarrays. These methods involve the immobilization of detectable molecules at the interface near which the analyte is concentrated. The reaction in the prototype is between the reagent immobilized on the surface and the free analyte, which does not allow to achieve acceleration of detection of the analyte.

Thus, despite the fact that individual methods and devices for detecting analytes in samples using certain detection methods are known from the prior art, a device for detecting analyte, which allows the analyte to be concentrated on particles and thereby accelerate its indication, was proposed by the authors for the first time .

The possibility of carrying out the invention

The rate of adsorption of analytes on the surface of activated particles is determined by the product of their concentrations in solution or suspension. Therefore, the collection time can be significantly reduced by concentrating both the analyte solution and the suspension of particles. The need to implement this method can be dictated, for example, in order to determine a low concentration antigen, or in order to use antibodies with low affinity.

One of the main ways to accelerate the sorption of the analyte is the traditional intensive mixing, which helps to reduce the thickness of the immiscible layer around the particles. A variant of the same mixing is implemented in the flow of the analyte solution, passed through a column of particles (for example, through a chromatographic column). However, in this case there is no possibility of concentration of the analyte itself.

Trace analysis of various analytes, such as toxins and viruses, faces a number of physical limitations. First, the reaction rate is almost always proportional to the concentration of the analyte, so it is so small that it takes many hours and even days to bind the analyte.

Secondly, any test molecule with which the analyte is "recognized" (a specific antibody or a complementary nucleotide sequence) has a certain affinity for the analyte, determined by the dissociation constant, the meaning of which is that when the analyte concentration is equal to the dissociation constant, half The test molecules will be bound to the analyte, and at lower analyte concentrations, the fraction of such complexes will decrease approximately in proportion to the ratio of the concentration to the dissociation constant. This directly implies the requirement of a very strong binding (small dissociation constant) for test molecules intended for the analysis of trace amounts of analyte. Molecules of immunoglobulins have a dissociation constant of the order of 10 ^-9 - 10 ^-11 M, which limits the limiting concentration of protein analytes to 10 pg / ml. Given that many biological toxins act in significantly lower concentrations, this circumstance seriously complicates the use of standard methods for their analysis.

Thus, the task is to create a more versatile and efficient device for collecting analyte from solution, which allows for acceleration and concentrated particles.

The inventive task is solved by the fact that a device is proposed for the rapid detection and collection of analyte (natural or recombinant proteins, an infectious agent or an infectious agent component — viruses or bacteria, DNA, a cell or a cellular component, a cell membrane) on magnetic particles in a channel with semipermeable walls in the flow of an analyte solution mixed with a suspension of particles on the surface of the particles, consisting of a channel with semipermeable walls, electrode flow chambers, a magnetic concentrator.

The technical result consists, firstly, in accelerating the time of detection of the analyte in the solution, and secondly, in increasing the sensitivity of diagnostic tests. It is known that any antibody can bind an analyte only if its concentration is greater than a certain value at which the formation of analyte-antibody complexes is faster than their decay. With the described approach, by increasing the local concentration of the analyte, the analyte determination limit is significantly reduced, which minimizes the cost of the reactive mixture, and also contributes to the time gain needed to determine the analysis.

Brief Description of the Drawings

Figure 1. Schematic representation of the cross section of the flow chamber for collecting poured onto magnetic particles. 1 - electrode channels, 2 - channel from a semipermeable membrane; 3 - electrodes; 4 - magnet; 5 - a magnetic field concentrator.

The implementation of the invention

The proposed device consists of a channel formed by a dialysis membrane, on both sides of which are adjacent electrode chambers (Fig 1). The analyte is collected from a fluid stream containing particles and analyte. To carry out detection, magnetic particles must fall into the gutter, for which they apply a magnetic or electric field, gravitational (inertial) force. In particular, to create a magnetic field, a magnetic field concentrator is attached to one side of the channel, creating an inhomogeneous magnetic field that causes the movement of magnetic particles to the channel wall. The electric and magnetic field can vary both in magnitude and in direction, which will contribute to a better concentration of particles. For example, a field is applied in such a way as to cause both analyte and particles to move to one channel wall, concentrate near this wall and react quickly. This invention uses particles with a density higher than the density of the liquid. The resulting complexes of analyte and magnetic particles are removed by the fluid flow in the channel.

As shown in Figure 1, the flow chamber consists of electrode channels (1), between which there is a flow channel with a groove (2) made of a semipermeable membrane. Electrodes (3) are placed in the channels (1), which make it possible to create an electric field perpendicular to the fluid flow in the channel (2). A magnetic field concentrator (5) made of soft magnetic material is placed in the lower electrode channel (1). The magnet (4) creates a magnetic moment in the hub (5). The flow channel (2) is made in the form of a trench with a recess located above the magnetic field concentrator (5), which allows the collection of magnetic particles in the trench, significantly increasing their concentration compared to the flow channel with a flat bottom.

The collection of a charged analyte is carried out by applying voltage to the electrodes (3), leading to the appearance of an electric field across the flow. The direction of the field is chosen so that the analyte is displaced downstream and collected in the channel groove (2). The field can be magnetic, electric, gravitational.

Removal of magnetic particles from the channel can be carried out in a continuous mode by a fluid flow, selecting the flow rate and the magnitude of the magnetic field. In another mode, the magnetic field is periodically turned off by removing the magnet (4). This mode is conveniently implemented by replacing the permanent magnet (4) with an electromagnet and periodically turning off the current in the electromagnet.

An example embodiment of the invention

The invention is illustrated (but not limited to) by the following example.

Example 1. Rapid detection of trace amounts of staphillococcus enterotoxin in a sample using a device.

The following solutions were prepared for the experiment: Working buffer: 10 mM imidazole, 20 mM glycine, 1% polyvinylpyrrolidone, 1% polyvinyl alcohol, pH = 9.0, and a suspension of magnetic particles (MyOne, Invitrogen, with streptavidin immobilized on the surface) with immobilized on the surface with monoclonal antibodies to staphylococcal enterotoxin. Biotinylated antibodies were bound to the surface via streptavidin molecules.

Solutions of different concentrations of staphillococcus enterotoxin A in a working buffer solution.

Solutions No. 2 and No. 3 were placed in syringe pumps, which fed them into the mixer at a speed of 30 and 10 μl per minute, respectively. The mixer was a T-shaped adapter. Immediately after the mixer, the mixture of solutions entered the device. A voltage of 100 V was applied to the platinum electrodes of the device, which was accompanied by the appearance of a current of 6 mA. To cool the reactor and remove hydrolysis products, solution No. 1 was pumped through the electrode channels at a rate of 6 ml / min. The liquid flowing out of the reactor was collected in small portions into a microtube. To exclude the possibility of the reaction of enterotoxin with magnetic spheres after passing through the reactor, a special collection procedure was applied with the immediate separation of the enterotoxin solution from the magnetic microspheres. To do this, a density gradient was created in microtubes by placing a 50% glycerol solution at the bottom of the tubes and layering solution No. 1 on it. The end of the tube from the reactor was placed in the upper layer, and a strong rare-earth permanent magnet was brought to the bottom of the tube, so that the magnetic particles emerging from the end of the tube were attracted to the bottom of the tube, while the free enterotoxin remained in the upper solution. Then the upper solution was removed, and the magnetic particles were washed with solution No. 1.

The collected washed samples of magnetic particles were analyzed on a flowing biochip in scanning mode. The biochip surface was scanned by pumping a suspension of magnetic particles at a rate of 20 microliters per minute. The registration of magnetic particles associated with spots of secondary antibodies was carried out under a microscope equipped with a dark-field illuminator and a digital camera.

It was found that using 30,000 magnetic particles, 10 femtograms of enterotoxin (or 200,000 molecules) can be recorded in a sample with a volume of 100 μl.

It should be noted that at the limit of detection using an active reactor (extremely low concentration was 0.1 picogram per 1 ml), the molar concentration of enterotoxin was approximately 3 × 10 ^-15 M. In the absence of a device, the antigen-antibody complex dissociation constant typical for monoclonal antibodies To _d = 10 ^-9 M, the proportion of enterotoxin-binding antibody molecules on the surface of magnetic spheres would be only 3 × 10 ^-15 / 10 ^-9 = 3 × 10 ^-6 , which is insufficient for their detection. Electroconcentration on a dialysis membrane can increase the local concentration near the membrane by 10 ⁴ times, which accordingly will increase the proportion of bound antigens to a completely measurable value of 3%.

Another advantage of the device is a significant reduction in the time required for the reaction due to the convergence of the reagents. So, when the concentration of microsphere suspension used in this example, the average distance between the spheres was about a = 150 μm. With this scattering, for immunoglobulin molecules with a diffusion coefficient D = 5.5 μm ² / sec, the time τ = (a / 2) ² / 2D = 8 minutes is required to complete the binding reaction outside the device. In the device, only an increase in the concentration of enterotoxin by 10 ⁴ times will reduce the reaction time to 50 milliseconds. Given the convergence of the magnetic spheres in the flow channel, the reaction time will be further reduced by several orders of magnitude. Thus, the device can significantly reduce the speed and reduce the redistribution of sensitivity by the concentration of toxins.

Bibliographic data

1. Groves, S., Turell, M. J., Bailey, C., Morozov, V.N. (2008) Rapid active assay of pathogen-specific antibodies in chickens infected with West Nile Virus. Am. J. Trop. Med. Hyg. 78, 63-69.

2. Morozov V.N., Morozova T.Ya. Methods and devices for active bioassay US Patent 7,960,184.

3. Morozov, V., Bailey, C., Evanskey, M. Analyte detection using an active assay. US 7,824,927 B2, November 2, 2010.

4. Morozov, V.N., Evanskey, M., Tan, Y.K., Shaffer, D., Morozova, T.Y., Bailey, C. (2006) Ultra-filtration membrane for electrophoretic capturing of pathogens for AFM imaging. Langmuir 22, 1742-1748.

5. Morozov, V.N., Groves, S., Turell, M. J., Bailey, C., (2007) Three minutes-long electrophoretically assisted zeptomolar microfluidic immunoassay with magnetic beads detection. J. Am. Chem. Soc., 129, 12628 -12629.

6. Morozov, V.N., Groves, S., Turell, M. J., Bailey, C., (2007) Three minutes-long electrophoretically assisted zeptomolar microfluidic immunoassay with magnetic beads detection. J. Am. Chem. Soc., 129, 12628-12629. Morozov, V.N., Morozova, T.Ya. (2003) Electrophoresis-assisted active immunoassay. Anal. Chem. 75, 68-13-6819.

7. Morozov, V.N., Morozova, T.Ya. (2006) Active bead-linked immunoassay on protein microarrays. Anal. Chim. Acta, 564, 40-52.

8. Morozov, V.N., Shlyapnikov, Y.M., Kidd, J., Morozova, T.Y., Shlyapnikova, E.A. (2011) Conic Electrophoretic Concentrator for Charged Macromolecules. Anal. Chem. 83, 5548-5555.

9. Morozova, T.Ya., Morozov, V.N. Recognition of closely related antigens by active immunoassay. (2008) Anal. Biochem. 374, 263-271.

10. Shlyapnikov, Y.M., Shlyapnikova, E.A., Morozova, T.Y., Beletsky, I.P., Morozov, V.N. (2010) Detection of microarray-hybridized oligonucleotides with magnetic beads. Anal. Biochem. 399, 125-131.

Claims (3)

1. A device for collecting analyte from a solution by concentrating it on magnetic particles, including a flow chamber consisting of upper and lower channels containing electrodes to create an electric field perpendicular to the fluid flow in a flow channel from a semipermeable dialysis membrane located between the upper and lower channels, a magnetic field concentrator and a magnet to create a magnetic moment in the concentrator. 2. The device according to claim 1, characterized in that the magnetic field concentrator that creates the magnetic field is placed in the lower electrode channel and is made of soft magnetic material. 3. The device according to claim 1, characterized in that the flow channel is made in the form of a groove with a recess located above the magnetic field concentrator, which allows you to concentrate magnetic particles directly in the channel groove.

Images