KR20090006607A - Target capturing method, microfluidic channel system for capturing target and target assaying method - Google Patents

Abstract

A microfluidic channel system, a method for capturing a target by using the system, and a method for analyzing a target are provided to improve the reusability of the microfluidic channel system by removing the captured beads by the pressure control of the microfluidic channel. A microfluidic channel system comprises a base(10) where at least one microfluidic channel is formed for the capture of a target; a first microfluidic channel(20) which is formed on the base and where the target to be separated and the sensing material capable of bonding or reacting with the target are inputted; and a compression part(30) which is formed on the base and applies the pressure to at least some part of the first microfluidic channel to compress it.

Description

 Target capturing method, microfluidic channel system for capturing target and target assaying method}

The present invention relates to a target capture method, a microfluidic channel system for target capture, and a target analysis method, and in particular, a microfluidic channel system capable of capturing an antigen, DNA, protein, and the like, which is a target substance, and performing the captured substance. (microfluidic channel system).

The development of microfluidics in biochemical detection systems for disease diagnosis and drug development has led to faster and more convenient sample analysis. In microfluidics, the sample to be analyzed has a state of solution, and microfluidics is a research field that controls the flow of microfluids. One of the important issues in microfluidics is the development of systems for capturing target materials and analyzing captured target materials. Recently, various devices have been developed for capturing and analyzing cells by magnetic beads and carriers bound to antibodies. The enzyme-linked immunosorbent assay (ELISA), an immunoassay that is one of the important analytical methods in medicine and biochemistry, takes one day or more to perform immunoassay and is complicated to operate. In addition, there is a problem that the reagent cost is burdened.

FIG. 1 is a conceptual diagram illustrating a main algorithm of Choi, Jin-Woo et al., An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capability (Royal Society of Chemistry, 2000). In this paper magnetic beads are used as carriers for the solid material, the captured target antigens, to immobilize the antibodies.

An analysis device common to FIGS. 1A to 1G is a sandwich immunoassay device provided with an electromagnet. Figure 1 (a) shows that the magnetic beads coated with the antibody is introduced into the electromagnet, (b) shows that the magnetic beads are fixed by the electromagnet. (C) of FIG. 1 shows that a plurality of antigens including an antigen, which is a target substance, are introduced while magnetic beads are fixed, and (d) shows that other antigens except for an antigen, which is a target substance, are discharged. Indicates. Figure 1 (e) shows the introduction of enzyme-labeled second antibodies, (f) shows the electrochemical detection process after the second antibodies are bound to the antigen, (g) is another additional Shows the process of removing magnetic beads for analysis.

Fig. 2 is a conceptual diagram showing the "Immunosorbent Assay System (American Chemical Society, 2000)" proposed by Sato et al. In the system proposed by Sato, beads are first introduced into a microchannel having a barrier through which beads cannot pass, and then polystyrene. After adsorption of s-IgA (antigen) to the surfaces of the beads, the antibody against labeled s-IgA is immobilized on the beads via an antigen-antibody reaction, and then the remaining antigens are washed and then subjected to thermal lens microscopy. The analysis system proposed by Sato requires only a relatively simple configuration, a barrier for bead capture, but the system proposed by Sato is vulnerable in terms of reusability in that it is difficult to remove the captured beads after the analysis is completed. Do.

As a related patent document, Korean Patent Laid-Open Publication No. 2006-94416 discloses a bioanalytical device capable of observing a path change of microparticles according to the intensity and amount of change of a specific magnetic field in order to know the concentration of a specific detection target biomolecule.

The present invention alleviates the problem that the system is structurally complicated by using materials such as magnetic nanoparticles and electromagnets as compared with the conventional magnetic-based cell trapping system, and reduces the problem that the conventional microfluidic channel system is not reusable. In view of the above, the implementation of the system is simple, which is suitable for miniaturization, shortens the reaction time, and facilitates the removal of the trapped target materials and the excellent reusability of the microfluidic channel system, the target capture method, and the target analysis method. It aims to provide.

According to an aspect of the present invention, there is provided a microfluidic channel system for target capture, including: a base on which at least one microfluidic channel for target capture is formed; A first microfluidic channel formed on the base and receiving a target to be separated and a sensing material capable of binding or reacting with the target; And a compression unit compressing the first microfluidic channel to capture the target by applying pressure to at least a portion of the first microfluidic channel.

In the microfluidic channel system of the present invention, the compression unit is disposed to overlap with the first microfluidic channel, and the second microfluidic channel compresses or decompresses the first microfluidic channel through a pressure control inside the channel, and a second microfluidic channel; The apparatus may further include a fluid supply unit configured to supply a fluid for regulating pressure inside the microfluidic channel. In the microfluidic channel system of the present invention, the sensing material is immobilized on the beads and the target material is immobilized on the beads via the sensing material. In particular, it is preferable to use beads of a size such that beads do not pass between the compressed spaces when the first microfluidic channel is compressed. In addition, the microfluidic channel system of the present invention may further include a target supply for supplying a target material to the first microfluidic channel, a sensing material supply for supplying a sensing material, and a discharge part for discharging the supplied materials.

The target capture method according to the present invention for solving the other technical problem is

a) introducing a target material to be captured and a sensing material capable of binding or reacting with the target material to the first microfluidic channel; And b) applying pressure to at least a portion of the first microfluidic channel to compress the first microfluidic channel to capture a target material.

Target analysis method according to the present invention for solving the other technical problem is

Measuring the electrical or optical properties of the target captured in the first microfluidic channel according to the target capture method of the present invention described above.

According to the present invention, since materials such as magnetic nanoparticles and electromagnets are unnecessary as compared with the conventional magnetic-based cell capture system, the implementation of the system is simple, suitable for miniaturization, and shorten the reaction time. In addition, according to the present invention, trapped beads can be easily removed through pressure control on the microfluidic channel, which is advantageous in terms of reusability of the microfluidic channel system.

Hereinafter, a target capture method, a microfluidic channel system for target capture, and a target analysis method will be described in detail with reference to the accompanying drawings and embodiments.

3 is a schematic diagram illustrating a microfluidic channel system for target capture according to an embodiment of the present invention.

The base 10 is formed with at least one microfluidic channel for target separation, for example a glass, silicone or plastic (acrylic) based base. Here, the target is a substance to be detected or analyzed, for example, antigens, DNA, proteins, and the like.

The first microfluidic channel 20 is formed on the base 10 and receives a target to be separated and a sensing material that can be combined or react with the target. The first microfluidic channel 20 preferably has transparency so that the reaction can be observed through equipment such as a thermal lens microscope and an electron microscope from the outside. In addition, since it must be compressed by the pressure from the compression unit 30, it is preferably made of a stretchable polymer material. For example, polymer materials that can be used in the first microfluidic channel 20 include polymethyl methacrylate (PMMA), polycarbonate, polycyclic olefine, polyimide, Polyurethanes and the like, and polydimethylsiloxane (PDMS) is particularly preferred.

The first microfluidic channel 20 is integrally or separately from the target material inlet 21 receiving the target material, the sensing material inlet 22 receiving the sensing material, and the washer fluid inlet 23 receiving the washer fluid. It may further include. When the inlets 21 to 23 are included separately, for example, three fused silica capillaries corresponding to each of the inlets 21 to 23 are connected to the input side of the first microfluidic channel. It is also possible to configure so that.

In addition, as shown in FIG. 4B, when the target material is a specific antigen, and the sensing material is used as an antibody that reacts with the specific antigen and fixed to the beads, the first microfluidic channel 20 performs the color reaction. Labeled entibody bound to the enzyme causing the binding and binding to the antigen is introduced through the sensing material inlet 22 or an enzyme causing the reaction is generated through a separate enzyme material inlet (not shown). I can receive it. In this case, the target material may be analyzed by analyzing the color reaction according to the enzyme. An example of a substance that can replace the enzyme substance is antibody-colloidal gold. Colloidal gold can be used for photothermal immunoassay.

The target material, the sensing material and the washer fluid may be introduced into the first microfluidic channel through external storage means, and may also be introduced through the storage means provided in the microfluidic channel system of the present invention. In FIG. 3, the target material storage 41 stores the target material and supplies the target material to the first microfluidic channel, the sensing material storage 42 stores / supplies the sensing material, and the washer fluid storage 43 Stores / supplies washer fluid. Here, examples of the washer liquid include pure water or phosphate buffer.

Another side of the first microfluidic channel 10 may further include an exhaust material storage 44. The discharge material reservoir 44 stores target material, discharge material supplied from the sensing material and washer fluid reservoirs and finally discharged through the first microfluidic channel.

The compression unit 30 applies pressure to at least a portion of the first microfluidic channel 20 and compresses the first microfluidic channel 20. The compression unit 30 is disposed to overlap the first microfluidic channel 20, and the second microfluidic channel 31 and the second microfluidic channel 31 compressing or decompressing the first microfluidic channel through pressure adjustment inside the channel. And a fluid supply part 32 for supplying a fluid for regulating the internal pressure of the two microfluidic channels.

The second microfluidic channel 31 is formed above the base 10 and is disposed such that a portion thereof overlaps with the first microfluidic channel 20. The second microfluidic channel 31 expands and contracts according to the internal pressure change, and in particular, compresses the first microfluidic channel 20 when the second microfluidic channel 31 is expanded. In the present invention, the expansion of the second microfluidic channel due to the overlapping structure of the second microfluidic channel 31 and the first microfluidic channel 20 and the increase of the internal pressure of the second microfluidic channel, and thus the second microfluidic channel The algorithm of target capture through the contraction control of is a major feature. For example, the fluid supply 32 has a pneumatic control valve or a hydraulic control valve capable of contracting or expanding the second microfluidic channel 31 through pneumatic control. An end of the second microfluidic channel located opposite the fluid supply 32 may have a closed structure. In this case, the second microfluidic channel 31 may be expanded and contracted by adjusting the fluid pressure flowing into the second microfluidic channel 32 through the fluid supply part 32.

In addition, the microfluidic channel system of the present embodiment protects the first microfluidic channel 20 and the second microfluidic channel 31, and a protective layer (not shown) that guides the expansion direction of the second microfluidic channel 31. ) May be further provided. The first microfluidic channel when the direction of expansion of the second microfluidic channel 31 at the overlapping point of the second microfluidic channel and the first microfluidic channel is not directed toward the first microfluidic channel 20 but toward the opposite side. The compression of 20 may not be performed effectively, in which case the problem may be solved if the protective layer guides the expansion direction of the second microfluidic channel.

The microfluidic channel system according to the embodiment having the above-described structure may be manufactured, for example, in the form of an enzyme immunoassay chip.

On the other hand, as a manufacturing method of the microfluidic channel system for capturing the target of the present invention, for example, a glass glass is provided as a base, and a target to be separated and a sensing material capable of combining or reacting with the target are introduced. After forming the first microfluidic channel on the base, a microfluidic channel system is fabricated by forming a compression part on the base, the compression unit arranged to contact at least a portion of the first microfluidic channel and compressing the first microfluidic channel. There is a way.

4A to 4E are reference diagrams conceptually illustrating a target capture method using the microfluidic channel system of the present invention. In particular, FIGS. 4A to 4E show cross-sectional views in the A-A 'direction.

4A is a schematic diagram showing an A-A 'cross section when the internal pressure of the second microfluidic channel is the same as the outside.

4B shows that the antibody 52 bound to the beads 51 is introduced in the state in which the first microfluidic channel 20 is compressed as the internal pressure of the second microfluidic channel 31 increases. In this embodiment, the bead is a surface fixed with a sensing material that binds to the target to be analyzed. For example, a specific antigen can be targeted and the antibody which binds the said antigen can be used as a sensing substance. Examples of the beads include polystyrene beads and polymethylmethacrylate beads. The beads are introduced into the beads adsorbed by the antibody through the sensing material inlet 22 of the first microfluidic channel. At this time, a phosphate buffer may be injected to move the introduced beads to the intersection point.

FIG. 4C illustrates that a plurality of antigens are introduced in FIG. 4B, and the antigen 53, which is a target substance, is bound to the antibody 52 immobilized by the beads 51.

FIG. 4D shows that the washer solution is introduced in FIG. 4C to discharge unbound antigens. The washer fluid is introduced through the washer fluid storage 43, and antigens that do not bind to the antibody 52 are stored in the discharge material storage 44 according to the inflow of the washer fluid.

FIG. 4E shows that the antibody 54 that binds to the enzyme causing the color reaction in FIG. 4D and is capable of binding to the antigen, which is the target substance, is introduced to bind to the antigen. Unbound antibody is stored in the discharge material storage 44 as the washer solution is introduced.

At the detection point of FIG. 4E, various analyzes of the reaction between the antigen and the antibody may be performed using a thermal lens microscope or an immunoelectron microscope. There is no particular limitation on the analysis method for the target in the present invention. For example, an antigen analysis may be performed through an immunoassay method using an antigen-antibody binding reaction. It is also possible to analyze biomolecules by utilizing the selective binding ability between a particular molecule and a sensing substance that can accept that particular molecule.

In the present embodiment, an example for measuring a single target substance, for example, only one antigen is disclosed, but according to the present invention, it is also possible to simultaneously measure a plurality of antigens. For example, when three different types of antibodies are bound to three types of beads, and then introduced into the first microfluidic channel, three different types of antigens reacting with each of the three types of antibodies can be measured. Will be. That is, according to the present invention, it is possible to implement a multiplex type microfluidic channel system for capturing and measuring a plurality of targets.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown not in the above description but in the claims, and all differences within the scope should be construed as being included in the present invention.

1 is a conceptual diagram showing a microfluidic biochemical detection method using a conventional electromagnet.

2 is a conceptual diagram illustrating an immunosorbent assay method using a conventional bead barrier.

3 is a schematic diagram illustrating a microfluidic channel system for target capture according to an embodiment of the present invention.

4A to 4E are reference diagrams conceptually illustrating a target capture method and a target analysis method based on the microfluidic channel system of the present invention.

Claims (14)

A base on which at least one microfluidic channel for target capture is formed; A first microfluidic channel formed on the base and receiving a target to be separated and a sensing material capable of binding or reacting with the target; And And a compression portion formed on said base, said compression portion compressing the first microfluidic channel by applying pressure to at least a portion of the first microfluidic channel. The method of claim 1, The compression unit further includes a fluid supply unit supplying a fluid for adjusting an internal pressure of the second microfluidic channel and a second microfluidic channel which compresses or decompresses the first microfluidic channel through pressure adjustment within the channel. And the second microfluidic channel is arranged to overlap with the first microfluidic channel. The method of claim 1, A target material storage for storing and supplying a target material to the first microfluidic channel, a sensing material storage for storing and supplying the sensing material, and an exhaust material storage for storing materials discharged from the first microfluidic channel And a microfluidic channel system for target capture. The method of claim 1, And the sensing material is introduced into the first microfluidic channel fixedly on the beads. The method of claim 1, The sensing material is introduced into the first microfluidic channel in a fixed state on the bead, and the compression unit applies a pressure above the predetermined reference value to the first microfluidic channel so that the first microfluidic channel is compressed and passes through the channel. Is narrowed, the passage height inside the narrowed channel is smaller than the size of the bead. The method of claim 2, Wherein said fluid supply is a pneumatic regulating valve or a hydraulic regulating valve. The method of claim 2, The first microfluidic channel is transparent and stretchable, and comprises a polydimethylsiloxane (PDMS) family of components, the microfluidic channel system for target capture. The method of claim 2, An upper portion of the second microfluidic channel and protecting at least a portion of the first microfluidic channel and the second microfluidic channel, and when the internal pressure of the second microfluidic channel increases, And a protective layer for guiding the direction of expansion of the second microfluidic channel to expand toward the first microfluidic channel. The method of claim 1, And the first microfluidic channel receives a plurality of targets and a plurality of sensing materials for detecting each target. a) introducing a target material to be captured and a sensing material capable of binding or reacting with the target material to the first microfluidic channel; And b) compressing the first microfluidic channel to apply a pressure to at least a portion of the first microfluidic channel to capture the target material. The method of claim 10, Compressing the first microfluidic channel by applying pressure to at least a portion of the first microfluidic channel in step b) increases the internal pressure of the second microfluidic channel disposed to overlap with the first microfluidic channel, And a method of capturing the first microfluidic channel. The method of claim 10, And the sensing material is introduced into the first microfluidic channel in a fixed state on the beads. The method of claim 11, Raising the internal pressure of the second microfluidic channel is performed until the passage height within the channel narrowed as the first microfluidic channel is compressed is smaller than the size of the bead. 14. The method of claim 10, further comprising the step of measuring electrical or optical properties of the target captured in the first microfluidic channel according to the method for capturing the target.

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