KR20230023177A - Microfluidic immunoassay chip and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a microfluidic-based immunodiagnostic chip and a manufacturing method thereof, wherein the immunodiagnostic chip includes a detection unit for fluorescence detection of a target protein from an injected sample. In this case, the detection unit includes: a substrate unit; a coated unit coated on the substrate unit and containing polyethyleneimine; a first capturing protein immobilized on the coated unit and binding to the target protein; and a non-specific blocker (NSB) dispersed between the coated unit and the first capturing protein. Accordingly, the microfluidic-based immunodiagnostic chip is capable of performing highly sensitive and rapid diagnosis using polyethyleneimine.

Description

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

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

A conventional rapid immunodiagnostic strip is a diagnostic platform using a laminar flow-based immunochromatographic principle, and has mainly used a nitrocellulose membrane as a material for a detection layer. Such a nitrocellulose membrane is a porous paper capable of stably and easily immobilizing capture proteins, such as capture antibodies and antigens, at a high density, and thus is an important material in the manufacture of rapid immunodiagnostic strips. However, recently, as the demand for the development of a highly sensitive quantitative diagnostic platform increases, a rapid immunodiagnostic strip using a fluorescence reader is being developed, and the need for a substrate that is transparent and has low autofluorescence is increasing. Therefore, the nitrocellulose membrane, which is relatively opaque and has a high amount of self-fluorescence emitted from the material itself, has limitations in being applied to a technology using a fluorescence reader.

Therefore, recently, interest in the development of a rapid immunodiagnostic platform for high-sensitivity quantitative diagnosis using microfluidic technology has increased. A study using the material is being conducted.

However, in the case of materials such as PDMS or glass, mass productivity is not high and the material is expensive, so there are limitations that are not suitable for commercialization.

Republic of Korea Patent No. 10-1210899

Accordingly, the technical problem of the present invention is conceived in this respect, and an object of the present invention is to provide a microfluidic-based immunodiagnostic chip capable of performing high-sensitivity and rapid diagnosis using polyethyleneimine.

In addition, 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 detector for detecting 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, before coating the coating unit, further includes a surface treatment polymer formed on the substrate unit, wherein the surface treatment polymer is APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) ) can be.

In one embodiment, a filter unit for removing signal interfering substances from the injected sample, and a first detection protein coupled with a fluorescent dye are dispensed, and the injected sample reacts with the first detection protein for a predetermined time Conjugation It may further include a chamber unit, and in the detection unit, a conjugate in which the target protein and the first detection protein are bound in the conjugation chamber unit may bind to the first capture protein.

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

In the method for manufacturing an immunodiagnostic chip according to an embodiment for realizing the above object of the present invention, the step of manufacturing a detection unit for detecting fluorescence of a target protein from an injected sample includes cleaning the substrate unit, the substrate unit Activating the surface, coating the activated substrate with a solution containing polyethyleneimine, treating the coated substrate to improve bonding strength, and first capturing protein molecules on the treated substrate A step of reacting with the contained solution, and a step of coating a non-specific binding blocker (NSB) on the substrate portion reacted with the first capture protein molecule.

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

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

In one embodiment, the step of improving the bonding force by treating the coated substrate portion is to treat the coated substrate portion with GA (glutaraldehyde) or EDC / NHS ((3-dimethylaminopropyl) carbodiimide / Nhydroxysuccinimide) to obtain a first capture protein molecule and bonding strength can be improved.

In one embodiment, in the step of coating a non-specific binding blocker (NSB) on the substrate, the aldehyde residue or the EDC/ Residues of the 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 the first detection protein, the second detection protein, and the control protein in the conjugation chamber unit, forming a microfluidic channel, and the prepared detection unit and the conjugation chamber unit and the A step of combining the microfluidic channels may be further included.

In one embodiment, in the step of manufacturing the detection unit, an inspection unit for detecting a conjugate in which the target protein and the first detection protein are combined, and a control unit for detecting a conjugate in which the control protein and the second detection protein are combined are included. It can be manufactured separately.

According to the embodiments of the present invention, in the conventional nitrocellulose membrane-based immunochromatographic technique, it has high sensitivity by overcoming the limitations in fluorescence quantitative detection or sensitivity improvement due to high autofluorescence and opacity. It is possible to improve usability and fabrication as a rapid immunodiagnostic kit capable of performing fluorescence quantitative detection while maintaining

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 inducing higher fluorescence detection. In this case, after coating the solution, GA (glutaraldehyde) or EDC / NHS ((3-dimethylaminopropyl) carbodiimide / Nhydroxysuccinimide) treatment can further improve the binding force with the first capture protein molecule, through which high fluorescence detection Of course, high-sensitivity detection is possible.

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

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

Since the present invention can be applied with 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 form disclosed, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals have been used for like elements throughout the description of each figure. Terms such as first and second may be used to describe various components, but the components should not be limited by the terms.

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

In this application, the terms "comprise" or "consisting of" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

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

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

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

That is, in the prior art, the detection unit was manufactured in a state in which the capture protein 20 was simply coated on the substrate unit 10, and accordingly, the target protein 40 - the first detection protein 50 was applied to the capture protein 20. ) It was characterized in that the conjugate was bound and fluorescence was detected.

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

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

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

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

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

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

In this case, since the substrate 210 of the immunodiagnosis chip 200 may include thermoplastics such as PMMA (polymethyl methacrylate), PC (poly carbonate), COC (cyclic olefin copolymer), etc. Unit 110 may also be constructed from the same material.

The coating portion 120 is formed by coating the substrate portion 110 . The coating portion 120 includes polyethyleneimine (PEI), and in this embodiment, as the coating portion containing polyethyleneimine is coated on the substrate portion 110, the capture protein 140 is more It can be immobilized at high density, thereby improving detection sensitivity.

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

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

Meanwhile, the NSB 130 is dispersed in the substrate portion 110 where the coating portion 120 and the capture protein 140 are formed, and the non-specific binding blocker (NSB) 130 captures the provided capture protein 130. It reacts with remaining aldehyde residues or EDC/NHS residues that are not bound to protein molecules.

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

As described above, as confirmed through 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 conjugates of the target protein 150-detection protein 160 bound to the capture protein 140 increases, and consequently, the sensitivity of fluorescence detection can be improved.

That is, in this embodiment, polyethyleneimine (PEI) is used as a cross-linking molecule for immobilizing the capture protein 140 on the coating portion 120, which is plastic. 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. can

2B is a schematic diagram showing an 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 the present embodiment will be described.

In this case, in the immobilized state in this embodiment, 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 to more easily perform the adsorption of the coating portion 120 adsorbed on the substrate portion 110, for example, APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde).

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

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

FIG. 3 is a plan view illustrating a microfluidic-based immunodiagnostic chip in which the immobilized state of FIG. 2A is applied to a detection unit. FIG. 4A is a schematic diagram showing the immobilization state of the inspection unit of the detection unit of FIG. 3 , and FIG. 4B is a schematic diagram showing the immobilization state of the control unit of the detection unit of FIG. 3 .

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

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

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

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

The inlet part 220 is formed at one end of the substrate part 210 and is a part into which a sample to be detected is injected, and the outlet part 270 is the substrate opposite to the inlet part 220. It is formed at the other end of the part 210, and is a part from which the detection-completed sample 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. can

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

In the immunodiagnostic chip 200 in FIG. 3, as will be described later, the detection unit 250 further includes a control unit 252 in addition to the inspection unit 251, and the capture protein described in FIGS. 2A and 2B ( 140), a target protein 150-detection protein 160 conjugate is generated in the inspection unit 251.

Therefore, the detection protein 160 in the reaction performed in the inspection unit 251 is hereinafter referred to as the first detection protein 160 in order to distinguish it from the reaction in the control unit 252 . On the other hand, the first detection protein 160, the second detection protein 161, and the control protein 151 dispensed through the conjugation chamber part 240 pass through the filter part 230 and flow through the sample will react 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 240 for a predetermined time. time will be located.

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

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

In this case, as has already been described, the inspection unit 251 can perform the fluorescence detection as in the immobilized state 100 of the detection unit 250 described above 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 1 combined with the conjugate of the detection protein 160, fluorescence detection is performed.

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

Furthermore, as described above, the detection unit 250 further includes a matching unit 252 . As shown in FIG. 4B, the control unit 252 except that the conjugate to be bound is the 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 same configuration as the detection unit 250 described above 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 control protein 151-second detection protein 161 complex. The only difference is being.

The immobilization state in the control unit 252 can be confirmed through FIG. 4B showing the immobilization state in the control unit 252, 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.

4B, in the control part 252, the second capture protein 141 binds to the control protein 151-second detection protein 161 complex, but the substrate part 110 ) It was illustrated that the coating unit 121 is coated directly on. However, as in the previous inspection unit 251, even in the case of the control unit 252, the surface of the substrate unit 110 is preliminarily treated with a polymer and then the coating unit 121 is coated. (see FIG. 2B), and in the control part 252 prepared in this way, the control protein 151-second detection protein 161 complex may be bound to the second capture protein 141.

As described above, the detection unit 250 is formed by being divided into the inspection unit 251 and the control unit 252, and through the inspection unit 251, the first capture protein 140 is the target protein ( 150)-first detection protein 160 conjugate is detected and fluorescence detection is performed, and through the control unit 252, the second capture protein 141 passes through the control protein 151-second detection protein A conjugate of (161) is detected, and fluorescence detection in the inspection unit 251 is additionally performed as a control group or a control group.

Meanwhile, the waste chamber part 260 is a flow line formed on the substrate part 210, and the inlet part 220, the filter part 230, the conjugation chamber part 240, the A flow line of the sample is formed by passing 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 separately manufactured, respectively, and separately After manufacturing, the immunodiagnostic chip 200 can be finally manufactured in a form of being connected or combined with each other.

That is, the microfluidic channel 201 is formed on the substrate portion 210, the conjugation chamber portion 240 is formed on the substrate portion 210, and finally, the separately manufactured detection unit ( 250) to the substrate portion 210 on which the microfluidic channel 201 and the conjugation chamber portion 240 are formed, the immunodiagnostic chip 200 can be manufactured.

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

FIG. 5 is a flowchart illustrating 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, first, the detection unit 250 is manufactured. Hereinafter, the manufacturing of the detection unit 250 will be first described.

The substrate part 100 may be substantially the same as the substrate part 210 shown in FIG. If it is formed through a process to be formed, it is sufficient to perform the process to be described subsequently with respect to the part forming the detection unit 250 that follows.

In contrast, if the detection unit 250 is separately manufactured and combined with the substrate unit 210 on which the microfluidic channel 201 is formed, the substrate unit 110 is for manufacturing the detection unit 250. , It can be understood that the subsequent manufacturing process of the detection unit 250 is performed over the entire area of the substrate unit 110 .

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

After that, the surface of the substrate part 100 is activated (step S20).

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

Thereafter, coating is performed on the activated substrate portion 110 (step S40). Prior to performing the coating, a step of optionally surface treating the activated substrate portion 100 with a polymer (step S30) is performed. can be performed

That is, the surface treatment step (step S30) using the polymer is selectively 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 is done, the detection unit described with reference to FIG. 2B is manufactured.

In the step of treating the surface 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, adsorption of the coating portion 120 following the substrate portion 110 may be further improved through surface treatment using the polymer.

Thereafter, a solution containing polyethyleneimine (PEI) is coated on the activated substrate portion (110, step S20 only) or the substrate portion additionally subjected to surface treatment (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 unit 120, and polyethyleneimine forms a high-density amine group. Therefore, it has the advantage of high-density immobilization on various solid surfaces such as cells, nucleic acids, and proteins. As a result, the first capture protein 140 can be immobilized at high-density.

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

In this case, in the step of improving the bonding force (step S50), the coated substrate 110 is treated with GA (glutaraldehyde) or EDC / NHS ((3-dimethylaminopropyl) carbodiimide / Nhydroxysuccinimide) to form a first capture The bonding force with the protein 140 molecule is improved.

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

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

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

Thereafter, a non-specific binding blocker (NSB) 130 is coated on the substrate 110 on which the first capture protein molecules are 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 EDC/NHS residues remaining on the substrate 110 without binding of the first capture protein molecules are removed. 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 finished.

On the other hand, the detection unit 250 includes an inspection unit 251 and a control unit 252 as shown in FIG. 3, and the manufacturing process of the detection unit 250 described above is eventually the manufacturing and The same can be applied to the manufacture of contrast portion 252 .

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

In this case, as described above, the second capture protein 141, not the first capture protein 140, should be stabilized and immobilized at a high density on the control portion 252.

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

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

In this case, the sample forms a conjugate between the target protein included in the sample and the first detection protein while passing through the conjugation chamber unit 240. To this end, the conjugation chamber unit 240 The first detection protein 160 is dispensed and dried. In this case, if the control part 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 part 240 And it should 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 directly formed or formed separately and mounted on the substrate part 210 .

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

As described above, the substrate portion 210 on which the microfluidic channel 201 is formed is separately manufactured and then coupled to the substrate portion 110 on which the detection unit 250 is formed to form the immunodiagnostic chip 200. can finally be produced. In this way, if the substrate portion 210 on which the microfluidic channel 201 is formed is separately manufactured, 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 unit 210 where the microfluidic channel 201 is formed. As such, even when the detection unit 250 is formed in a predetermined region of the substrate unit 210, the microfluidic channel 201 is detected after the detection unit 250 is formed or even before the detection unit 250 is formed. can form

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

According to the embodiments of the present invention as described above, in the conventional nitrocellulose membrane-based immunochromatographic technique, by overcoming the limitations in fluorescence quantitative detection or sensitivity improvement due to high autofluorescence and opacity, As a rapid immunodiagnostic kit capable of quantitatively detecting fluorescence while having sensitivity, usability and manufacturability can be improved.

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 inducing higher fluorescence detection. In this case, after coating the solution, GA (glutaraldehyde) or EDC / NHS ((3-dimethylaminopropyl) carbodiimide / Nhydroxysuccinimide) treatment can further improve the binding force with the first capture protein molecule, through which high fluorescence detection Of course, high-sensitivity detection is possible.

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

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. You will understand that you can.

100, 101: immobilized state 110, 210: substrate part
120, 121: coating unit 130, 131: NSB
140, 141: capture protein 150: target protein
151: control protein 160: first detection protein
161: second detection protein 170: polymer
200: immunodiagnosis chip 220: inlet
230: filter unit 240: conjugation chamber unit
250: detection unit 251: inspection unit
252: control unit 260: waste chamber unit
270: outlet

Claims (12)

In an immunodiagnostic chip including a detector for detecting fluorescence of a target protein from an injected sample,
The detecting unit,
board part;
a coating portion coated on the substrate portion and containing polyethyleneimine;
a first capture protein immobilized on the coating and binding to the target protein; and
The immunodiagnostic chip comprising a non-specific blocker (NSB) dispersed between the coating unit and the first capture protein. 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,
An immunodiagnostic chip characterized in that APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde). According to claim 1,
a filter unit for removing signal interference substances from the injected sample; and
Further comprising a conjugation chamber unit in which the first detection protein to which the fluorescent dye is bound is dispensed and the injected sample reacts with the first detection protein for a predetermined time;
The immunodiagnostic chip, characterized in that, in the detection unit, the conjugate in which the target protein and the first detection protein are combined in the conjugation chamber unit binds with the first capture protein. According to claim 4,
A second detection protein and a control protein are additionally dispensed into the conjugation chamber,
The immunodiagnostic chip of claim 1 , wherein the detection unit further comprises a control unit including a second capture protein that binds to a conjugate in which the control protein and the second detection protein are combined. In the method of manufacturing an immunodiagnostic chip including a detector for detecting fluorescence of a target protein from an injected sample,
The step of manufacturing the detection unit is,
cleaning the substrate;
activating a surface of the substrate portion;
coating a solution containing polyethyleneimine on the activated substrate;
Processing the coated substrate portion to improve bonding strength;
reacting the treated substrate portion with a solution containing the first capture protein molecules; and
The 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 portion,
The method of manufacturing an immunodiagnostic chip, characterized in that the surface of the substrate is treated with oxygen plasma or treated with a sodium chloride (NaOH) solution. The method of claim 5, before the step of coating the activated substrate portion,
Including the step of surface treatment of the activated substrate portion with a polymer,
The method of manufacturing an immunodiagnostic chip, characterized in that the surface-treated polymer is APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde). The method of claim 5, wherein the step of improving the bonding force by processing the coated substrate portion,
The 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 a non-specific binding blocker (NSB) on the substrate,
The method of manufacturing an immunodiagnostic chip, characterized in that an aldehyde residue or a residue of the EDC/NHS remaining on the substrate without binding of the first capture protein molecule reacts with the NSB. The method of claim 5, wherein the NSB (non-specific binding blocker),
A method of manufacturing an immunodiagnostic chip comprising ethanolamine, bovine serum albumin (BSA) or whole milk powder. According to claim 5,
dispensing and drying the first detection protein, the second detection protein, and the control protein in the conjugation chamber;
Forming a microfluidic channel; and
The method of manufacturing an immunodiagnostic chip further comprising combining the prepared detection unit and the conjugation chamber unit with the microfluidic channel. The method of claim 11, wherein in the step of manufacturing the detection unit,
Manufacturing of an immunodiagnostic chip characterized in that a test unit for detecting a conjugate in which the target protein and the first detection protein are combined and a control unit for detecting a conjugate in which the control protein and the second detection protein are combined are separately manufactured. method.

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