KR20220157269A - Immunoassay biochip with multi-layer structure and immunoassay measuring apparatus using the same - Google Patents

Abstract

Through a biochip that implements a color ELISA technique, which is a standard immune response measurement method, on a chip, the present invention is intended to solve a problem of sensitivity degradation while quickly obtaining results using a small amount of sample. To this end, sensitivity is improved by increasing an optical path for measuring absorbance through a biochip in which a plurality of films are laminated to form a plurality of reaction chambers. In this way, in order to stack a plurality of layers of reaction chambers in which an immune response to an input sample occurs, a plurality of reaction film layers dividing a boundary of each reaction chamber; and a bonding film layer intervening between the respective reaction film layers and adhering the respective reaction film layers are used.

Description

Multi-layered immune response biochip and immune response measuring device using the same {Immunoassay biochip with multi-layer structure and immunoassay measuring apparatus using the same}

The present invention relates to an immune response measurement technique used to measure the presence or absence and amount of a specific antigen or antibody in a sample through an antigen-antibody reaction.

An immune response is an antigen-antibody binding reaction in which an antigen and an antibody selectively bind, and is used to measure the presence or absence of a specific antigen or antibody in a sample and its amount. As a representative immune response measurement method, there is an enzyme-linked immunosorbent assay (ELISA), which measures the degree of antigen-antibody reaction by using an enzyme as an indicator in the antigen-antibody binding reaction. A color ELISA technique that induces a color reaction between the enzyme and a substrate and measures the degree of color development by absorbance is established as a standard immune response measurement method. Immunoassay methods have been developed that replace markers with fluorescent substances, nanomaterials, and luminescent substances, etc., by applying enzyme immunoassay, and generally measure the amount of antigen-antibody reaction and marker-induced reaction regardless of marker is collectively referred to as ELISA.

This ELISA technique is widely used in protein analysis research in bio laboratories, and for this purpose, ELISA substrates and ELISA readers have been developed and used. For ELISA, antigen-antibody immobilization, antigen-antibody reaction, and marker-substrate reaction are sequentially performed, and a washing process is accompanied between these reactions to minimize non-specific reactions. These multiple reaction processes are generally laboriously performed by skilled personnel in laboratories.

Recently, a miniaturized chip (biochip) that causes an immune response by applying microfluidic control technology has been developed in order to minimize labor force and rapidly perform an immune response using a small amount of sample with high sensitivity. The immune response using this biochip can provide benefits such as securing user convenience, reproducibility and reliability through automation, application of on-site diagnosis through miniaturization of the device and quick diagnosis result, and low cost of diagnostic analysis. In addition, in order to effectively realize this technology, various microfluidic control technologies, measurement technologies, biochemical reaction technologies, chip material technologies, nanomaterial technologies, automation technologies, and small device technologies that can be applied to immunodiagnostic biochips are being developed.

In addition, various biochips have been developed in which the color ELISA technique, which is a standard method for measuring an immune response, is implemented on a chip. However, with the miniaturization of the reaction chamber accompanying the miniaturization of the biochip, when the color reaction is measured using absorbance, an optical path is reduced and sensitivity is lowered.

In order to overcome this problem, a method of installing a waveguide in the reaction chamber has been proposed. However, difficulties arise in the manufacturing process of additionally installing optical waveguides, the problem of limited materials for optical waveguides for refractive index and immobilization of antibodies, and indirectly measuring the effect of immune reactions on the surface of optical waveguides on light inside the waveguides Complexity issues arise.

As a more direct solution, a method of making both ends of the reaction chamber a reflective structure and passing light through the reaction chamber has been developed in order to secure a large optical path in the reaction chamber. However, in this case, precise alignment between the light source, the chip, and the measuring unit is required, and light is scattered by unknown factors in the reaction chamber, such as microbubbles, so that measurement results may vary greatly.

As described above, due to the miniaturization of the reaction chamber of the biochip, there is a problem of a decrease in sensitivity due to a decrease in the light path for measuring the absorbance of the color ELISA.

Therefore, the present invention aims to solve the problem of reduced sensitivity by improving a biochip in which a color ELISA technique, which is a standard immunoreaction measurement method, is implemented on a chip.

In order to solve the above problems, the present invention provides a multi-layered immune response biochip in which a plurality of reaction chambers are formed by stacking a plurality of films in order to increase an optical path for measuring absorbance. That is, a sensitivity improvement effect is obtained by increasing an optical path for measuring absorbance through a biochip in which a plurality of reaction chambers are formed by stacking a plurality of films.

Specifically, according to one feature of the present invention, in order to stack a plurality of layers of reaction chambers in which an immune response to the input sample occurs, a plurality of reaction film layers separating the boundaries of each reaction chamber; and a bonding film layer intervening between the respective reaction film layers and adhering the respective reaction film layers.

In addition, the plurality of stacked reaction chambers may include an inlet into which a sample enters a first reaction chamber of the plurality of stacked reaction chambers; Connection holes connecting flow paths of the plurality of stacked reaction chambers; and an outlet through which samples are discharged from the last reaction chamber of the plurality of stacked reaction chambers, which are selectively formed on the reaction film layer or the bonding film layer.

Meanwhile, according to another feature of the present invention, a device for measuring an immune response using the multi-layered immune response biochip is provided.

The configuration and operation of the present invention introduced above will become more clear through specific embodiments to be described later along with the drawings.

In conventional ELISA-implemented biochips, the light path for measuring the absorbance of color ELISA is reduced due to the miniaturization of the reaction chamber, resulting in low sensitivity. On the other hand, in the biochip according to the present invention, multiple reaction chambers are formed by stacking multiple films. The effect of improving the sensitivity can be obtained by increasing the optical path for measuring absorbance. In addition, the plurality of reaction chambers formed by the laminated film are miniaturized compared to the reaction wells of the conventional ELISA substrate, and the effect of using a small amount of sample and the effect of deriving rapid immune response results as the diffusion distance is shortened there is In addition, since a plurality of low-cost films can be stacked, simple and automated mass production is possible, thereby reducing the price of the biochip.

In addition, convenience of use can be obtained by applying an immune response measuring device capable of sequentially injecting a sample and a reaction solution to the multilayered biochip of the present invention and causing a color reaction.

1 is a cross-sectional view illustrating a multi-layered immune response biochip and an accessory device according to the present invention.
2 is an exploded view of the multi-layered immune response biochip.
Figure 3a shows the bioreaction in the reaction well of conventional ELISA.
3B shows a bio reaction in the reaction chamber of the multi-layered biochip according to the present invention.
4 shows a theoretical formula for the size of emitted light of the multi-layered immunoreactive biochip according to the present invention.
5 shows measurement data of emitted light of the multi-layered immunoreactive biochip according to the present invention.

Advantages and features of the present invention, and methods of achieving them, will become clear with reference to the detailed description of preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described below and may be implemented in various other forms. The examples are only provided to completely disclose the present invention and to completely inform those skilled in the art of the scope of the invention to which the present invention belongs, the present invention is defined by the description of the claims will be. In addition, terms used in this specification are for describing embodiments and are not intended to limit the present invention. In this specification, singular forms also include plural forms unless otherwise specified. Also, as used herein, the term "comprises" means the presence or absence of one or more other elements, steps, operations, and/or elements other than the mentioned elements, steps, operations, and/or elements; It is used in the sense of not excluding additions. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of the embodiments, if a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted.

1 is a cross-sectional view illustrating an embodiment of a multi-layered immunoreactive biochip and an accessory device, and FIG. 2 is an exploded view of the biochip. 3A shows a bioreaction in a reaction well of a conventional ELISA, and FIG. 3B shows a bioreaction in a reaction chamber 200 of a multilayered biochip 100 according to an embodiment of the present invention.

Referring first to FIGS. 1 and 2 , the multi-layered biochip 100 of this embodiment is formed by stacking a plurality of reactive film layers 110 and a bonding film layer 120 therebetween. A pattern of a specific shape is perforated in the reaction film layer 110 and the bonding film layer 120 (see FIG. 2 ), and by stacking the pattern perforated layers, a plurality of stacked reaction chambers 200 are formed inside the biochip 100. , Connection hole 210 connecting the passages of the stacked reaction chambers 200, an inlet 220 into which the sample 50 is stacked, the inlet 220 and the first reaction chamber are connected, and the last A connection channel 225, which is a passage connecting the reaction chamber to the outlet 230, and an outlet 230 through which the sample 50 is discharged from the last reaction chamber are formed. Although this embodiment shows an example in which three reaction chambers 200 are stacked in layers, the number of reaction chambers 200 in the stacked thickness direction has no limit.

Referring to FIG. 2 , the bonding film layers 120a to 120c are installed between the reaction film layers 110a to 110d and serve to adhere the reaction film layers 110a to 110d so as not to leak. In addition, patterns for the reaction chamber 200, the connection channel 225, and the outlet 230 are perforated in the bonding film layers 120a to 120c. Meanwhile, the reaction film layer 110a on the uppermost layer of the biochip has a pattern for the inlet 220 and the outlet 230, and the reaction film layers 110b and 110c in the middle layer have the connection hole 210 and the outlet 230. The pattern for is formed by perforation, and the perforation pattern is not formed in the reaction film layer 110d on the lowermost layer.

Referring back to FIG. 1 , an interfacing structure 300 in which a reaction solution injection well 310 and an outlet connector 320 are formed is installed above the biochip 100 . This structure facilitates the sample 50 being injected into the multi-layered biochip 100 from the outside and being discharged from the biochip 100 to the outside through the tubing 330 by the suction operation of the suction pump 500. It is a structure for In the multi-layered biochip 100 and the interfacing structure 300, the hole 312 and the inlet 220 formed at the bottom of the reaction liquid injection well 310, and the outlet connector 320 and the outlet 230 are aligned so that there is no leakage. should be glued When the sample 50 is dropped into the reaction solution injection well 310, it moves through the inlet 220 of the multi-layered biochip 100, the connection channel 225, and the plurality of reaction chambers by the suction operation of the suction pump 500. 200) and a plurality of connection holes 210, it is transported to the outlet 230, and enters the suction pump 500 through the outlet connector 320 and the tubing 330.

The sample 50 includes an antibody, a blocking solution, an antigen, a secondary antibody, a substrate, and a washing solution, and is sequentially injected one by one. After being stored for a certain period of time in the multi-layered bio-immuno-reaction biochip 100, it can be discharged to the outside again by the suction pump 500. Depending on the purpose of the reaction, the type of sample, storage time, transfer rate and amount can be adjusted. Through this bio-reaction process, an immune reaction is performed in the reaction chamber 200 of the multi-layered biochip 100, and finally a color development reaction occurs.

1 and 3B, the light source element 400 is above the reaction chamber 200 and the light receiving element 460 is below it. The light emitted from the light source device 400 enters the reaction chamber 200 as the incident light 420 and passes through the reaction chamber 200 as the transmitted light 430 and exits as the emitted light 440 . The emitted light 440 is detected by the light receiving element 460 . The light source device 400 may be a light emitting diode (LED) or a laser diode (LD), and the light receiving device 460 may be a photodiode (PD), an avalanche photodiode (APD), or a photo multiplier tube (PMT).

With this structure, the color reaction of the sample is measured from the emitted light 440 transmitted through the reaction chamber 200 of the multi-layered biochip 100. That is, the incident light 420 incident from the top of the multi-layered biochip 100 loses its intensity due to light absorption while passing through the biochip 100 and comes out as the outgoing light 440, and the absorbance is measured according to the intensity attenuation rate. can do. In order to improve measurement accuracy, it is preferable to allow a certain amount of light to be incident from the light source device 400 as the incident light 420 through the incident light pinhole 410 . In addition, it is preferable to measure the emitted light 440 passing through a certain area of the pinhole by the light receiving element 460 by the emitted light pinhole 450 .

Referring to FIG. 3A, in the case of the conventional ELISA reaction well 600, the antibody 60 is immobilized on the surface of the reaction well 600 containing the sample 50, and the antigen-antibody interaction with the antigen 65 The reaction is performed and the result is measured as absorbance of light 430 passing in the depth direction of the reaction well. On the other hand, in the case of the multi-layer immunoreactive biochip 100 of the present invention, the reaction film layer 110 forms the top and bottom surfaces of the reaction chamber 200, and the antibody 60 is immobilized on the inner surface thereof to form an antigen. (65) and an antigen-antibody reaction are performed, and the result is measured as absorbance of light transmitted in a vertical direction through the layered biochip 100.

The ELISA reaction well 600 usually has a diameter of about 7 to 8 mm and a depth L of the sample is about 3 to 5 mm, whereas the height of the reaction chamber 200 in this embodiment is the height H of the bonding film layer, which is about 10 to 200 micrometers. The rate of the antigen-antibody reaction is mainly affected by the diffusion rate of the sample, and according to the law of diffusion, it is inversely proportional to the square of the distance between the antibody and the antigen on the surface. That is, in the case of the multilayer immunoreactive biochip of this embodiment, the diffusion distance is equal to the height (H) of the reaction chamber, which is relatively very small compared to conventional ELISA reaction wells, so that the speed of antigen-antibody reaction is very fast.

In the case of the ELISA reaction well, the light path length for measuring absorbance is the depth L of the sample, and in the case of the multi-layered immune response biochip of this embodiment, the light path length of one reaction chamber is H, relative to the ELISA reaction well. short. Since the absorbance is proportional to the optical path length according to Beer-Lambert's law, the absorbance is small in the reaction chamber 200 of the biochip of this embodiment, and thus the sensitivity of the measurement is low. As described above, in the multi-layered immunoreactive biochip of the present invention, the reaction chambers 200 are stacked in the vertical height direction to secure an optical path of 'number of layers * H' for the sample, and when manufactured with 'number of layers * H ≒ L' An optical path similar to that of conventional ELISA reaction wells can be secured. That is, it is possible to significantly increase the reaction rate while lowering the loss of absorbance measurement sensitivity.

이다. 이 공식은 유체의 흡광도 A는 유체를 통과하는 광로 길이 d와 유체 내 분해물질의 몰농도 c에 비례한다(ε은 몰흡광계수임)는 의미이다. [The absorbance(A) for a liquid is proportional to the length of light propagated through the liquid (d) and the mole concentration of an analyte(c) in the liquid, where ε is the mole extinction coefficient.] The Beer-Lambert's law is

to be. This formula means that the absorbance A of a fluid is proportional to the optical path length d passing through the fluid and the molar concentration c of the decomposition material in the fluid (ε is the molar extinction coefficient). [The absorbance(A) for a liquid is proportional to the length of light propagated through the liquid (d) and the mole concentration of an analyte(c) in the liquid, where ε is the mole extinction coefficient.]

Meanwhile, the reaction film layers 110a to 110d are thin polymer films having a thickness of about 10 to 200 micrometers, and are preferably made of materials having low surface roughness and high transmittance for light measurement. For example, the transmittance of the reactive film layers 110a to 110d is preferably 0.95 or more. In addition, it is preferable that the reaction film layers 110a to 110d have high surface bioreactivity in order to immobilize the antibody 60 on the surface. For example, polystyrene (PS), polycarbonate (PC), PMMA, COC (cyclo-olefin-copolymer) materials may be used. In order to increase the surface bioreactivity and immobilize the antibody 60, the surface of the film may be modified through surface treatment, application of a coupling agent, and immobilization of a biomaterial. To this end, techniques such as oxygen plasma, self-assembly monolayer (SAM), and (3-aminopropyl)triethoxysilane (APTES) may be applied.

On the other hand, according to the present embodiment, the bonding film layers 120a to 120c determine the height of the reaction chamber 200, and have a thickness of about 10 to 200 micrometers, and the reaction film layers 110a to 110d do not leak. It is preferable that the material be adhered without. For example, the bonding film layers 120a to 120c may be, for example, double-sided tape coated with a bonding material on both sides of the film. In addition, the bonding film layers 120a to 120c may be bonded to the reaction film layers 110a to 110d by applying heat through a thermal adhesive on both sides of the film. The material of the bonding film layers 120a to 120c may also be similar to that of the reactive film layers 110a to 110d.

Referring back to FIG. 1 , the suction pump 500 may be a syringe pump, a peristaltic pump, or the like. In addition, in the middle of the tubing 330 connecting the suction pump 500 and the interfacing structure 300, or at the end of the tubing 330 connected behind the suction pump 500, a waste liquid storage container (not shown) for discarding the used sample Containers such as can be additionally installed.

In addition, the size of the reaction chamber 200 is preferably a height H of 10 to 200 micrometers in order to reduce the reaction time. As described above, the height H of the reaction chamber 200 may be determined by the thickness of the bonding film layers 120a to 120c. In addition, the width of the reaction chamber 200 is preferably larger than the diameter of the incident light pinhole 410 so that a sufficient amount of the incident light 420 can pass through the reaction chamber 200 .

In addition, the connection hole 210 is formed to serve to connect the flow paths of the stacked reaction chambers 200, and preferably has a shape and size that allows the sample 50 to flow smoothly and prevent bubbles from forming. do. For example, the connection hole 210 may be formed in a triangular, square, circular, elliptical, or semicircular shape.

In addition, the connection channel 225 is formed to serve to connect the first reaction chamber to the inlet 220 and connect the last reaction chamber to the outlet 230, and the uppermost bonding film layer 120a and the lowermost bonding film layer ( 120c). A plurality of connection channels 225 may be formed in parallel in one reaction chamber 200 according to the type of sample 50 and the purpose of the reaction. In addition, according to the shape of the connection channel 225, a plurality of reaction solution injection wells 310 and a plurality of outlet connectors 320 may be formed in the interfacing structure 300. In addition, a plurality of reaction chambers 200 may be formed in parallel in one connection channel 225 to simultaneously perform a plurality of reactions.

4 shows a theoretical equation for the intensity of final transmitted light ( I _f ) of the emitted light 440 of the multilayer immunoreactive biochip 100 according to the present invention. Accordingly, the magnitude of transmitted light of each layer of the reaction chamber 200 can be predicted. According to this theoretical equation, the absorbance of the sample 50 having the same transmittance (concentration) is linearly proportional to the number of reaction chambers 200, and the reaction film layers 110a to 110d regardless of the transmittance of the sample 50. ), the multi-layered immunodiagnostic biochip of the present invention basically has a certain level of absorbance.

5 shows measurement data of emitted light 440 in the multi-layered immunoreactive biochip of the present invention. When the antigen-antibody reaction is large, a deep color reaction appears in the sample, and accordingly, the size of the emitted light is reduced. In FIG. 5 , it can be seen that the intensity of the emitted light 440 gradually decreases as the color development reaction proceeds with time in the reaction chamber 200 . In addition, it can be seen that the size of the emitted light 440 decreases as the number of reaction chambers 200 (CH) increases for the same antigen-antibody reaction intensity (sample concentration). This coincides with the prediction by the theoretical formula of FIG. 4 .

So far, the present invention has been described in detail through preferred embodiments of the present invention, but those skilled in the art to which the present invention pertains may differ from the contents disclosed herein without changing the technical spirit or essential features of the present invention. It will be appreciated that it may be embodied in other specific forms. It should be understood that the embodiments described above are illustrative in all respects and not restrictive. In addition, the scope of protection of the present invention is determined by the claims described below rather than the detailed description above, and all changes or modifications derived from the scope of the claims and equivalent concepts should be construed as being included in the technical scope of the present invention. do.

Reaction solution (50) Antibody (60) Antigen (65) Multi-layer biochip (100) Reaction film layer (110) Bonding film layer (120) Reaction chamber (200) Connection hole (210) Inlet (220) Connection channel (225) Exit 230 Interfacing structure 300 Reactant injection well 310 Exit connector 320 Tubing 330 Light source element 400 Incident light pinhole 410 Incident light 420 Transmitted light 430 Outgoing light 440 Outgoing light Pinhole (450) Light receiving element (460) Suction pump (500) ELISA reaction well (600)

Claims (14)

Stacked in a plurality of layers, including a reaction chamber in which an immune response to the input sample occurs,
A multi-layered immune response biochip used to measure an immune response occurring in the plurality of stacked reaction chambers. The multi-layered immune response biochip according to claim 1, wherein the height of each reaction chamber constituting the plurality of stacked reaction chambers is 10 to 200 micrometers. The method of claim 1, wherein the stacked plurality of reaction chambers
an inlet into which a sample enters a first reaction chamber of the plurality of stacked reaction chambers;
Connection holes connecting flow paths of the plurality of stacked reaction chambers; and
A multi-layered immune response biochip comprising an outlet through which samples are discharged from the last reaction chamber of the plurality of stacked reaction chambers. The method of claim 1, wherein the stacked plurality of reaction chambers
A plurality of reaction film layers dividing the boundary of each reaction chamber; and
A multi-layered immunoreactive biochip comprising a bonding film layer intervening between the respective reactive film layers and adhering the respective reactive film layers. The method of claim 4, wherein the plurality of stacked reaction chambers include: an inlet into which a sample enters a first reaction chamber of the plurality of stacked reaction chambers; Connection holes connecting flow paths of the plurality of stacked reaction chambers; And an outlet through which samples are discharged from the last reaction chamber of the plurality of stacked reaction chambers,
The inlet, the connection hole, and the outlet are multi-layered immune response biochips, characterized in that formed on one of the reaction film layer and the bonding film layer. 5. The multi-layered immune response biochip according to claim 4, wherein the height of the reaction chamber is equal to the thickness of the bonding film layer. The multi-layered immune response biochip according to claim 4, wherein the thickness of the reactive film layer is 10 to 200 micrometers. The multi-layered immune response biochip according to claim 4, wherein the bonding film layer has a thickness of 10 to 200 micrometers. [Claim 5] The multi-layered immune response biochip according to claim 4, wherein the reaction film layer and the bonding film layer are made of a light-transmitting material. According to claim 3,
at least one connection channel connecting a first reaction chamber of the plurality of stacked reaction chambers and the inlet; and
The multi-layered immune response biochip further comprising at least one other connection channel connecting a last reaction chamber of the plurality of stacked reaction chambers and the outlet. An immune response measurement device using the multi-layered immune response biochip according to any one of claims 1 to 10,
The multi-layered immune response biochip;
a reaction solution injection well for injecting an immune response measurement target into the plurality of stacked reaction chambers of the biochip;
an interfacing structure for discharging materials for which immune response measurement has been completed from the plurality of stacked reaction chambers of the biochip;
a light source element for injecting light into the plurality of stacked reaction chambers of the biochip; and
An immune response measuring device comprising a light-receiving element for detecting emitted light transmitted through the plurality of stacked reaction chambers of the biochip. [Claim 12] The apparatus for measuring immune response according to claim 11, further comprising a suction pump connected to the interfacing structure to inhale and discharge the material for which the immune response has been measured. According to claim 11,
an incident light pinhole positioned before light is incident from the light source element to the plurality of stacked reaction chambers; and
The immune response measuring device further comprises an emission light pinhole located before the light emitted from the plurality of stacked reaction chambers is detected by the light receiving element. [Claim 12] The immune response measuring device according to claim 11, further comprising a waste storage container for storing the material for which the immune response has been measured.

Images