KR102585794B1 - Microfluidic immunoassay chip and method for manufacturing the same - Google Patents

Abstract

In the microfluidic-based immunodiagnostic chip and its manufacturing method, the immunodiagnostic chip includes a detection unit that fluorescently detects the target protein from the injected sample. In this case, the detection unit includes a substrate portion, a coating portion coated on the substrate portion and containing polyethyleneimine, a first capture protein immobilized on the coating portion and binding to the target protein, and the coating portion. and a non-specific blocker (NSB) dispersed between the first capture proteins.

Description

Microfluidic-based immunodiagnostic chip and manufacturing method thereof {MICROFLUIDIC IMMUNOASSAY CHIP AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a microfluidic-based immunodiagnostic chip and a manufacturing method thereof, and more specifically, to a microfluidic-based immunodiagnostic chip capable of performing high-sensitivity rapid diagnosis using polyethyleneimine and a manufacturing method thereof. will be.

Conventionally, rapid immunodiagnostic strips are diagnostic platforms using laminar flow-based immunochromatographic principles and have mainly used nitrocellulose membranes as detection layer materials. This nitrocellulose membrane is a porous paper that can easily and stably immobilize capture proteins (capture antibodies and antigens) at high density, making it an important material in the production of rapid immunodiagnostic strips. However, as the demand for development of highly sensitive quantitative diagnostic platforms has recently increased, rapid immunodiagnostic strips using fluorescence readers are being developed, and the need for substrates that are transparent and have low autofluorescence is increasing. Accordingly, the nitrocellulose membrane, which is relatively opaque and has a large amount of autofluorescence emitted by the material itself, has limitations in being applied to technology using a fluorescence reader.

Therefore, recently, there has been increasing interest in the development of a high-sensitivity quantitative diagnostic rapid immunodiagnostic platform using microfluidic technology, and to develop such a microfluidic-based rapid immunodiagnostic chip, a substrate such as PDMS (polydimethylsiloxane) or glass is used. Research using the material is being conducted.

However, in the case of materials such as PDMS or glass, mass production is not high and the materials are expensive, so there are limitations that make them unsuitable for commercialization.

Republic of Korea Patent No. 10-1210899

Accordingly, the technical problem of the present invention was conceived in this regard, and the purpose of the present invention is to provide a microfluidic-based immunodiagnostic chip that can perform high-sensitivity rapid diagnosis using polyethyleneimine.

Additionally, another object of the present invention is to provide a method for manufacturing the microfluidic-based immunodiagnostic chip.

An immunodiagnostic chip according to an embodiment for realizing the object of the present invention described above includes a detection unit that detects fluorescence of a target protein from an injected sample. In this case, the detection unit includes a substrate portion, a coating portion coated on the substrate portion and containing polyethyleneimine, a first capture protein immobilized on the coating portion and binding to the target protein, and the coating portion. and a non-specific blocker (NSB) dispersed between the first capture proteins.

In one embodiment, the detection unit further includes a surface treatment polymer formed on the substrate before coating the coating unit, and the surface treatment polymer includes (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA). ) can be.

In one embodiment, a filter unit that removes signal interference substances from the injected sample, and a conjugation unit in which a first detection protein bound to a fluorescent dye is dispensed, and the injected sample reacts with the first detection protein for a predetermined period of time. It may further include a chamber unit, and in the detection unit, a conjugate combining the target protein and the first detection protein may bind to the first capture protein in the conjugation chamber unit.

In one embodiment, a second detection protein and a control protein are additionally dispensed into the conjugation chamber, and the detection unit includes a second capture protein that binds to a conjugate of the control protein and the second detection protein. It may further include a contrast unit.

In the method of manufacturing an immunodiagnostic chip according to an embodiment for realizing another object of the present invention described above, the step of manufacturing a detection unit that fluorescesly detects a target protein from an injected sample includes cleaning the substrate unit, activating the surface, coating the activated substrate with a solution containing polyethyleneimine, treating the coated substrate to improve binding force, and forming the treated substrate with a first capture protein molecule. It includes reacting with a solution contained therein, and coating a non-specific binding blocker (NSB) on the substrate portion on which the first capture protein molecule has been reacted.

In one embodiment, in the step of activating the surface of the substrate, the surface of the substrate may be treated with oxygen plasma or a sodium chloride (NaOH) solution.

In one embodiment, before coating the activated substrate portion, the method includes surface treating the activated substrate portion with a polymer, wherein the surface-treated polymer is (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA). ) can be.

In one embodiment, the step of improving the binding force by treating the coated substrate portion includes treating the coated substrate portion with GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide) to form the first capture protein molecule. can improve the bonding strength.

In one embodiment, in the step of coating a non-specific binding blocker (NSB) on the substrate, aldehyde residues remaining without the first capture protein molecule on the substrate or the EDC/ Residues of NHS can react with the NSB.

In one embodiment, the non-specific binding blocker (NSB) may include ethanolamine, bovine serum albumin (BSA), or whole milk powder.

In one embodiment, dispensing and drying a first detection protein, a second detection protein, and a control protein in a conjugation chamber unit, forming a microfluidic channel, and mixing the manufactured detection unit and the conjugation chamber unit with the The step of combining microfluidic channels may be further included.

In one embodiment, in the step of manufacturing the detection unit, an inspection unit that detects a complex of the target protein and the first detection protein, and a control unit that detects a complex of the control protein and the second detection protein. It can be manufactured separately.

According to embodiments of the present invention, in the conventional nitrocellulose membrane-based immunochromatographic technology, the limitations in quantitative fluorescence detection or improvement of sensitivity due to high autofluorescence and opacity are overcome, thereby providing high sensitivity. This can improve usability and manufacturability as a rapid immunodiagnostic kit that can perform quantitative fluorescence detection.

In particular, by coating and reacting a solution containing polyethyleneimine on the substrate, the number per unit area of the first capture protein immobilized on the substrate increases, thereby leading to higher fluorescence detection. In this case, after coating the solution, the binding force with the first capture protein molecule can be further improved by treatment with GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide), through which high fluorescence detection can be achieved. Of course, detection with high sensitivity is possible.

Furthermore, so that the solution containing polyethyleneimine can be more easily adsorbed and reacted to the substrate, surface treatment can be performed in advance using APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) polymers, This can ultimately lead to higher fluorescence detection.

Figure 1 is a schematic diagram showing a physical adsorption method in a microfluidic-based micro immunodiagnostic chip according to the prior art.
Figure 2a is a schematic diagram showing the immobilization state in a microfluidic-based immunodiagnostic chip according to an embodiment of the present invention.
Figure 2b is a schematic diagram showing the immobilization state in a microfluidic-based immunodiagnostic chip according to another embodiment of the present invention.
Figure 3 is a plan view showing a microfluidic-based immunodiagnostic chip with the immobilized state of Figure 2a applied to the detection unit.
Figure 4a is a schematic diagram showing the fixed state of the inspection part of the detection part of Figure 3, and Figure 4b is a schematic diagram showing the fixed state of the control part of the detection part of Figure 3.
FIG. 5 is a flowchart showing a method of manufacturing the microfluidic-based immunodiagnostic chip of FIG. 3.

Since the present invention can be subject to various changes and can have various forms, embodiments will be described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention. While describing each drawing, similar reference numerals are used for similar components. Terms such as first, second, etc. may be used to describe various components, but the components should not be limited by the terms.

The above terms are used only for the purpose of distinguishing one component from another. The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise.

In this application, terms such as “comprise” or “consist of” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the attached drawings.

Figure 1 is a schematic diagram showing a physical adsorption method in a microfluidic-based micro immunodiagnostic chip according to the prior art.

Referring to Figure 1, in a microfluidic-based micro immunodiagnostic chip according to the prior art, an example of applying the physical adsorption method as the adsorption method in the detection unit is shown.

That is, in the prior art, the detection unit was manufactured by simply coating the capture protein 20 on the substrate 10. Accordingly, the capture protein 20 was comprised of a target protein 40 and a first detection protein 50. ) was characterized in that the conjugate was combined and detected by fluorescence.

However, as the detection unit is manufactured by coating only the capture protein 20 on the substrate 10, the density and number of capture proteins 20 coated and remaining on the substrate 10 are relatively low, Accordingly, there is a problem in that the number of target protein (40)-first detection protein (50) complexes detected is not large, which causes problems of lowering the sensitivity and precision of fluorescence detection.

Accordingly, this embodiment overcomes the problems in the prior art of FIG. 1 and will be described in detail below.

Figure 2a is a schematic diagram showing the immobilization state in a microfluidic-based immunodiagnostic chip according to an embodiment of the present invention.

First, with reference to FIG. 2A, the immobilization state 100 of the detection unit 250 in the microfluidic-based immunodiagnostic chip 200 shown in FIG. 3 according to this embodiment will be described.

That is, the detection unit 250 includes a substrate unit 110, a coating unit 120, an NSB 130, and a capture protein 140.

The substrate portion 110 not only forms the base substrate of the detection unit 250, but is also coupled to the substrate portion 210 of the immunodiagnostic chip 200, which will be described later, or is integrated with the substrate portion 210. It can be extended and formed.

In this case, since the substrate portion 210 of the immunodiagnostic chip 200 may include thermoplastic plastics such as PMMA (polymethyl methacrylate), PC (poly carbonate), COC (cyclic olefin copolymer), etc., the substrate of the detection unit similarly Part 110 may also be made of the same material.

The coating portion 120 is formed by coating the substrate portion 110. The coating portion 120 contains polyethyleneimine (PEI). In this embodiment, as the coating portion containing the polyethyleneimine is coated on the substrate portion 110, the capture protein 140 becomes more active. It can be immobilized at high density, which improves detection sensitivity.

In addition, the capture protein 140 is coated on the substrate portion 110 coated with the coating portion 120 to prepare for final binding to the target protein.

In this case, the capture protein 140 is immobilized at high density on the substrate 110 by providing a solution containing the capture protein molecule onto the substrate 110 coated with the coating portion 120. You can.

Meanwhile, the NSB 130 is dispersed in the coating part 120 and the substrate part 110 on which the capture protein 140 is formed, and the NSB (non-specific binding blocker) 130 is the provided capture protein. The protein molecule reacts with the remaining unbound aldehyde residue or the residue of EDC/NHS, which will be described later.

In this case, the NSB 130 may include ethanolamine, bovine serum albumin (BSA), or whole milk powder.

As described above, as confirmed by the immobilization state 100 of the detection unit 250 in this embodiment, the capture protein 140 can be immobilized at a higher density on the substrate unit 110, Accordingly, the number of target protein 150-detection protein 160 complexes bound to the capture protein 140 increases, ultimately improving the sensitivity of fluorescence detection.

That is, in the case of this embodiment, polyethyleneimine (PEI) is used as a cross-linking molecule to immobilize the capture protein 140 on the plastic coating portion 120. In this case, since polyethyleneimine has a high density of amine groups, it has the advantage of being able to be immobilized at high density on various solid surfaces such as cells, nucleic acids, and proteins. Ultimately, the capture protein 140 can be immobilized at high density. You can.

Figure 2b is a schematic diagram showing the immobilization state in a microfluidic-based immunodiagnostic chip according to another embodiment of the present invention.

Referring to FIG. 2B, the immobilization state 101 of the detection unit 250 in the microfluidic-based immunodiagnostic chip 200 shown in FIG. 3 according to this embodiment will be described.

In this case, the immobilized state in this embodiment is one in which a polymer 170 layer is additionally formed in the immobilized state in FIG. 2A, and the polymer 170 will be further described.

That is, referring to FIG. 2B, before coating the coating portion 120 on the substrate portion 110, the polymer 170 may be formed for surface treatment.

In this case, the polymer 170 is used to more easily adsorb the coating portion 120 on the substrate portion 110, for example, APTES ((3-aminopropyl)triethoxysilane). and GA (glutaraldehyde).

Thereafter, after the polymer 170 is formed for surface treatment, the coating portion 120, the capture protein 140, and the NSB 130 are formed in the same manner as described with reference to FIG. 2A.

As described above, in this embodiment, by performing surface treatment using a polymer on the substrate portion 110, subsequent adsorption of the coating portion 120 can be performed more easily, and ultimately, the substrate portion ( The density of the capture protein 140 immobilized on 110) can be further increased.

Figure 3 is a plan view showing a microfluidic-based immunodiagnostic chip with the immobilized state of Figure 2a applied to the detection unit. Figure 4a is a schematic diagram showing the fixed state of the inspection part of the detection part of Figure 3, and Figure 4b is a schematic diagram showing the fixed state of the control part of the detection part of Figure 3.

Referring to FIG. 3, the immunodiagnostic chip 200 includes an inlet part 220, a filter part 230, a conjugation chamber part 240, a detection part 250, and an inlet part 220 formed on the substrate part 210. It includes a waste liquid chamber part 260 and an outlet part 270.

In this case, the inlet part 220, the filter part 230, the waste liquid chamber part 260, and the outlet part 270 are referred to as the microfluidic channel 201 for convenience of explanation.

That is, the microfluidic channel 201 is formed on the substrate 210.

As described above, the substrate portion 210 forms the base of the chip 200 and may include thermoplastic plastics such as PMMA (polymethyl methacrylate), PC (poly carbonate), COC (cyclic olefin copolymer), etc. there is.

The inlet portion 220 is formed at one end of the substrate portion 210 and is a portion into which the sample to be detected is injected, and the outlet portion 270 is a portion of the substrate on the opposite side of the inlet portion 220. It is formed at the other end of the part 210, and is a part where the sample after detection is removed to the outside.

The filter unit 230 removes signal interference substances that may cause signal interference related to the fluorescence signal from the sample injected into the inlet unit 220, thereby improving the accuracy of the fluorescence signal and minimizing noise. You can.

The conjugation chamber unit 240 is located at the rear end of the filter unit 230, and the first detection protein 160, the second detection protein 161, and the control protein 151 to which a fluorescent dye is bound are dispensed. .

As will be described later, the immunodiagnostic chip 200 in FIG. 3 includes the detection unit 250 additionally including a control unit 252 in addition to the test unit 251, and the capture protein described in FIGS. 2A and 2B ( A complex of the target protein 150 and the detection protein 160, which binds to 140), is generated in the inspection unit 251.

Therefore, hereinafter, in order to distinguish it from the reaction in the control unit 252, the detection protein 160 in the reaction performed in the test unit 251 is referred to as the first detection protein 160. Meanwhile, the first detection protein 160, the second detection protein 161, and the control protein 151 dispensed through the conjugation chamber unit 240 are the samples flowing through the filter unit 230. reacts with.

That is, in order for the sample to react with the first detection protein 160, the second detection protein 161, and the control protein 151 for a predetermined time, the sample is placed on the conjugation chamber unit 240 for a predetermined time. time is located.

Accordingly, the target protein 150 included in the sample may bind to the first detection protein 160 to form a complex of the target protein and the first detection protein. In this case, the second detection protein 161 and the control protein 151 react in the control section 252 to be described later to form a complex, and therefore do not react separately from the sample. As described above, the detection unit 250 includes a test unit 251, and the test unit 251 detects the target protein 150 provided through the conjugation chamber 240 - the first detection protein ( The first capture protein 140 is bound to the conjugate of 160), and fluorescence detection is performed.

In this case as well, with reference to FIGS. 2A and 2B, it has been explained that the capture protein 140 is bound to the complex of the target protein 150 and the detection protein 160 in the detection unit 250, but in FIG. The immunodiagnostic chip 200 further includes a control unit 252 in addition to the test unit, and a capture protein bound to the control unit 252 (hereinafter referred to as the test unit 251 to distinguish it from the second capture protein 141). The capture protein bound in is named the first capture protein (140).

In this case, as already described, the inspection unit 251 can perform the fluorescence detection in the same manner as the immobilized state 100 of the detection unit 250 described with reference to FIG. 2A.

That is, as shown in FIG. 4A, the inspection unit 251 is manufactured with the same configuration as the detection unit 250 of FIG. 2A, and accordingly, the first capture protein 140 is the target protein 150-product. 1 By combining with the conjugate of detection protein 160, fluorescence detection is performed.

Of course, although not shown, the inspection unit 251 may perform the fluorescence detection in the same manner as the immobilized state 101 of the detection unit 250 described with reference to FIG. 2B.

Furthermore, as described above, the detection unit 250 further includes a contrast unit 252. As shown in FIG. 4B, the control unit 252 except that the bound conjugate is a conjugate of the control protein 151 and the second detection protein 161, and the capture protein is the second capture protein 141. And, substantially, the configuration of the comparison unit 252 is the same as that of the detection unit 250 previously described with reference to FIGS. 2A and 2B.

That is, the configuration of the control unit 252 is the same as that of the inspection unit 251, and the second capture protein 141 binds to the conjugate of the control protein 151 and the second detection protein 161. The only difference is what happens.

This immobilization state in the control unit 252 can be confirmed through FIG. 4B, and the immobilization state in the control unit 252 is substantially the same as the immobilization state in the inspection unit 251 in FIG. 4A. do.

In addition, through FIG. 4B, in the control unit 252, the second capture protein 141 binds to the complex of the control protein 151 and the second detection protein 161, and the substrate unit 110 ) It is exemplified that the coating portion 121 is coated directly on the top. However, like the inspection part 251, the control part 252 is manufactured in a way that the substrate part 110 is first surface treated with a polymer and then the coating part 121 is coated. (see FIG. 2B), and in the control unit 252 manufactured in this way, the second capture protein 141 can be combined with the control protein 151-second detection protein 161 complex.

As described above, the detection unit 250 is formed separately into the inspection unit 251 and the control unit 252, and the first capture protein 140 is transmitted through the inspection unit 251 to the target protein ( 150) - The conjugate of the first detection protein 160 is detected and fluorescence detection is performed, and through the control unit 252, the second capture protein 141 is detected by the control protein 151 - the second detection protein. The conjugate of (161) is detected, and an additional test for fluorescence detection in the inspection unit 251 is performed as a control group or control group.

Meanwhile, the waste liquid chamber part 260 is a flow line formed on the substrate part 210, and includes the inlet part 220, the filter part 230, the conjugation chamber part 240, and the A flow line of the sample is formed through the detection unit 250 and the outlet unit 270.

Meanwhile, in the immunodiagnostic chip 200 according to this embodiment, as will be described later, the detection unit 250, the conjugation chamber unit 240, and the microfluidic channel 201 may be manufactured separately. After being manufactured, the immunodiagnostic chip 200 can be finally manufactured by being connected or combined with each other.

That is, the microfluidic channel 201 is formed on the substrate 210, the conjugation chamber 240 is formed on the substrate 210, and finally, the separately manufactured detection unit ( The immunodiagnostic chip 200 can be manufactured by combining the microfluidic channel 201 and the substrate 210 on which the conjugation chamber 240 is formed.

Below, the manufacturing method of the microfluidic-based immunodiagnostic chip 200 of FIG. 3 will be described in detail.

FIG. 5 is a flowchart showing a method of manufacturing the microfluidic-based immunodiagnostic chip of FIG. 3.

Referring to FIG. 5, in the method of manufacturing the immunodiagnostic chip 200, the detection unit 250 is first manufactured. Hereinafter, the manufacturing of the detection unit 250 will first be described.

The substrate portion 100 may be substantially the same as the substrate portion 210 shown in FIG. 3, and accordingly, the microfluidic channel 201 may be pre-formed on the substrate portion 210 or subsequently formed on the substrate portion 210. If it is formed through a process, it is sufficient to perform the process described later on the part forming the detection unit 250.

On the other hand, if the detection unit 250 is manufactured separately and combined with the substrate unit 210 on which the microfluidic channel 201 is formed, the substrate unit 110 is used for manufacturing the detection unit 250. , it can be understood that the subsequent manufacturing process of the detection unit 250 is performed in the entire area of the substrate unit 110.

In manufacturing the detection unit 250, first, the substrate unit 100 is cleaned to remove foreign substances remaining on the substrate unit 110 (step S10).

Afterwards, the surface of the substrate 100 is activated (step S20).

In this case, the surface activation of the substrate 100 is performed in advance to smoothly perform the coating process described later, and the surface of the substrate 100 is treated with oxygen plasma or sodium chloride (NaOH) solution. The process can be performed.

Afterwards, coating is performed on the activated substrate portion 110 (step S40). Before performing the coating, a step of selectively surface treating the activated substrate portion 100 with a polymer (step S30) is performed. It can be done.

That is, the surface treatment step using the polymer (step S30) is optionally performed. If the step (step S30) is omitted, the detection unit described with reference to FIG. 2A is manufactured, and the step (step S30) is performed. When this happens, the detection unit described with reference to FIG. 2B is manufactured.

In the surface treatment step of the activated substrate 110 using the polymer (step S30), APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) polymer surface treatment may be performed (steps S31 and S32). ).

In this case, the adsorption of the coating part 120 following the substrate part 110 can be further improved through surface treatment using the polymer.

Afterwards, a solution containing polyethyleneimine (PEI) is coated on the activated substrate portion (110, step S20 only) or the additionally surface-treated substrate portion (110, step S30 is also performed). (Step S40).

That is, coating is performed using polyethyleneimine (PEI) as a cross-linking molecule for immobilizing the first capture protein 140 on the coating portion 120, and polyethyleneimine has a high density of amine groups. Therefore, it has the advantage of being able to immobilize cells, nucleic acids, proteins, etc. at high density on various solid surfaces, and ultimately, the first capture protein 140 can be immobilized at high density.

Afterwards, the substrate portion 110 coated with polyethyleneimine (PEI) is treated to improve bonding strength (step S50).

In this case, in the step of improving the binding force (step S50), the first capture is subsequently formed by treating the coated substrate portion 110 with GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide). Improves binding force with protein (140) molecules.

That is, as the polyethyleneimine is coated on the substrate portion 110, a high density of amine residues exists on the substrate portion 110, and the subsequently formed first capture protein 140 molecule has an amine group and a carboxyl group. In order to further improve the binding force between this high density of amine residues and the amine group or carboxyl group of the first capture protein molecule, GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide) treatment as described above. Perform.

Afterwards, the treated substrate portion 110 is reacted with a solution containing the first capture protein molecule (step S60). That is, a solution containing the first capture protein molecule is provided onto the treated substrate portion 110.

Thus, the first capture protein 140 is immobilized on the substrate 110 at high density.

Afterwards, a non-specific binding blocker (NSB) 130 is coated on the substrate 110 on which the first capture protein molecule is reacted and immobilized (step S170).

As described above, the NSB 130 includes, for example, ethanolamine, bovine serum albumin (BSA), or whole milk powder.

As the NSB 130 is coated on the substrate 110, aldehyde residues or residues of the EDC/NHS that are not bound to the first capture protein molecule remain on the substrate 110. It reacts with the NSB (130). Thus, the first capture protein 140 is stabilized and immobilized at high density on the substrate 110.

Through the above process, manufacturing of the detection unit 250 is completed.

Meanwhile, the detection unit 250 includes an inspection unit 251 and a control unit 252 as shown in FIG. 3. The manufacturing process of the detection unit 250 described above ultimately involves manufacturing the inspection unit 251 and the control unit 252. The same can be applied to the manufacturing of the control unit 252.

In addition, if the detection unit 250 includes the inspection unit 251 and the control unit 252, the manufacturing process of the detection unit 250 is applied in two adjacent areas, respectively, so that the inspection unit 251 ) and the control unit 252 can be manufactured simultaneously.

In this case, as described above, the second capture protein 141, rather than the first capture protein 140, must be stabilized and immobilized at high density on the control unit 252.

As described above, once the manufacturing of the detection unit 250 is completed, the conjugation chamber unit 240 and the microfluidic channel 201 are formed through an additional process.

First, the conjugation chamber unit 240 may be formed directly on the substrate unit 210, or may be formed separately and mounted on the substrate unit 210.

In this case, while the sample passes through the conjugation chamber unit 240, a complex is formed between the target protein contained in the sample and the first detection protein. For this purpose, the conjugation chamber unit 240 The first detection protein 160 is dispensed and dried. In this case, if the control unit 252 is included, in addition to the first detection protein 160, the second detection protein 161 and the control protein 151 are simultaneously dispensed into the conjugation chamber unit 240. and must be dried as described above (step S80).

That is, after the first detection protein, the second detection protein, and the control protein are dispensed and dried in the conjugation chamber unit 240, the conjugation chamber unit 240 is placed on the substrate unit 210. It is formed directly or separately and mounted on the substrate 210.

Afterwards, a microfluidic channel 201, that is, the inlet part 220, the filter part 230, the waste liquid chamber part 260, and the outlet part 270, is formed on the substrate part 210. (Step S90).

As described above, the substrate portion 210 on which the microfluidic channel 201 is formed is manufactured separately and then combined with the substrate portion 110 on which the detection portion 250 is formed to produce the immunodiagnostic chip 200. It can be finally manufactured. In this way, if the substrate portion 210 on which the microfluidic channel 201 is formed is manufactured separately, the substrate portion 210 on which the microfluidic channel 201 is formed may be manufactured before the detection unit 250. , As shown with reference to FIG. 5, it may be manufactured after the detection unit 250 is manufactured.

Alternatively, the detection unit 250 may be formed in a predetermined area of the substrate 210 where the microfluidic channel 201 is formed. In this way, even when the detection unit 250 is formed in a predetermined area of the substrate 210, the microfluidic channel 201 is formed even after the detection unit 250 is formed or before the detection unit 250 is formed. can be formed.

Through the above process, the manufacturing of the immunodiagnostic chip 200 is completed.

According to the above-described embodiments of the present invention, limitations in quantitative detection of fluorescence or improvement in sensitivity due to high autofluorescence and opacity in conventional nitrocellulose membrane-based immunochromatographic technology are overcome, It is possible to improve usability and manufacturability as a rapid immunodiagnostic kit that can perform quantitative fluorescence detection while maintaining sensitivity.

In particular, by coating and reacting a solution containing polyethyleneimine on the substrate, the number per unit area of the first capture protein immobilized on the substrate increases, thereby leading to higher fluorescence detection. In this case, after coating the solution, the binding force with the first capture protein molecule can be further improved by treatment with GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide), through which high fluorescence detection can be achieved. Of course, detection with high sensitivity is possible.

Furthermore, so that the solution containing polyethyleneimine can be more easily adsorbed and reacted to the substrate, surface treatment can be performed in advance using APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) polymers, This can ultimately lead to higher fluorescence detection.

Although the present invention has been described above with reference to preferred embodiments, those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the following patent claims. You will understand that it is possible.

100, 101: fixed state 110, 210: substrate portion
120, 121: coating part 130, 131: NSB
140, 141: capture protein 150: target protein
151: Control protein 160: First detection protein
161: second detection protein 170: polymer
200: Immunodiagnostic chip 220: Inlet
230: filter unit 240: conjugation chamber unit
250: detection unit 251: inspection unit
252: Control unit 260: Waste liquid chamber unit
270: outlet

Claims (12)

A detection unit that detects the fluorescence of the target protein from the injected sample;
a filter unit that removes signal interference substances from the injected sample; and
In an immunodiagnostic chip including a conjugation chamber portion where a first detection protein bound to a fluorescent dye is dispensed and the injected sample reacts with the first detection protein for a predetermined time,
The detection unit,
substrate part;
A coating portion coated on the substrate portion and containing polyethyleneimine;
a first capture protein that is immobilized on the coating portion and binds to the target protein; and
An immunodiagnostic chip comprising an NSB (non-specific blocker) dispersed in the coating portion and the substrate portion where the first capture protein is formed. The method of claim 1, wherein the detection unit,
Before coating the coating portion, further comprising a surface treatment polymer formed on the substrate portion,
The surface treatment polymer is,
An immunodiagnostic chip characterized by APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde). According to paragraph 1,
An immunodiagnostic chip, wherein in the detection unit, the first capture protein binds to a complex of the target protein and the first detection protein in the conjugation chamber unit. According to paragraph 3,
A second detection protein and a control protein are additionally dispensed into the conjugation chamber portion,
The detection unit further includes a control unit including a second capture protein that binds to a complex of the control protein and the second detection protein. In a method of manufacturing an immunodiagnostic chip including a detection unit for fluorescence detection of a target protein from an injected sample,
The step of manufacturing the detection unit is,
Cleaning the substrate portion;
activating the surface of the substrate portion;
coating the activated substrate with a solution containing polyethyleneimine;
Improving bonding strength by surface treating the coated substrate with a surface treatment polymer;
reacting the surface-treated substrate with a solution containing a first capture protein molecule; and
A method of manufacturing an immunodiagnostic chip, comprising the step of coating a non-specific binding blocker (NSB) on the substrate portion reacted with the first capture protein molecule. The method of claim 5, wherein in the step of activating the surface of the substrate,
A method of manufacturing an immunodiagnostic chip, characterized in that the surface of the substrate is treated with oxygen plasma or sodium chloride (NaOH) solution. The method of claim 5, before coating the activated substrate portion,
Comprising the step of surface treating the activated substrate portion with a polymer,
A method of manufacturing an immunodiagnostic chip, characterized in that the surface-treated polymers are APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde). The method of claim 5, wherein the step of improving bonding strength by treating the coated substrate portion includes:
A method of manufacturing an immunodiagnostic chip, characterized in that the coated substrate is treated with GA (glutaraldehyde) or EDC/NHS ((3-dimethylaminopropyl)carbodiimide/Nhydroxysuccinimide) to improve binding force with the first capture protein molecule. The method of claim 8, wherein in the step of coating NSB (non-specific binding blocker) on the substrate,
A method of manufacturing an immunodiagnostic chip, wherein an aldehyde residue or a residue of the EDC/NHS remaining on the substrate without binding the first capture protein molecule reacts with the NSB. The method of claim 5, wherein the NSB (non-specific binding blocker) is,
A method of manufacturing an immunodiagnostic chip comprising ethanolamine, bovine serum albumin (BSA), or whole milk powder. According to clause 5,
Dispensing and drying the first detection protein, the second detection protein, and the control protein into the conjugation chamber;
Forming a microfluidic channel; and
A method of manufacturing an immunodiagnostic chip further comprising combining the manufactured detection unit and the conjugation chamber unit with the microfluidic channel. The method of claim 11, wherein in manufacturing the detection unit,
Manufacture of an immunodiagnostic chip, characterized in that it is manufactured separately from a test unit that detects a complex of the target protein and the first detection protein and a control unit that detects a complex of the control protein and the second detection protein. method.

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