MICROFLUIDIC DEVICE FOR DETECTING BIOMOLECULES IN SWEAT AND WEARABLE BIOSENSOR PATCH USING THE SAME

Disclosed are apparatuses, methods, and method of manufacture of a microfluidic device for detecting biomolecules in sweat, and a biosensor patch for detecting biomolecules in sweat, in which the microfluidic device and a biosensor are combined, the device and the patch being capable of detecting various target molecules present in sweat by electrochemical signals and also detecting the concentration of a target molecule. The microfluidic device includes a fluidic passing layer including an inlet part, a sensing element in communication with the inlet part, a reagent storage part in communication with the sensing element part, and a disposer in communication with the sensing element, and a fluidic connection layer including a microfluidic tube arranged in a vertical direction to the inlet part, a sensing space provided between the sensing element and the biosensor, and a reagent storage space positioned below the reagent storage part, the fluidic connection layer being disposed below the fluidic passing layer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(a) of Korean Patent Application No. 10-2020-0010235 filed on Jan. 29, 2020, in the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference for all purposes.

BACKGROUND

Field

The following description relates to a microfluidic device for detecting biomolecules in sweat, a biosensor patch including the same, a method of manufacturing the microfluidic device, and a method of detecting biomolecules in sweat by using the microfluidic device.

2. Description of Related Art

As interest in personal health increases, personal testing is becoming more common, and such personal diagnostic devices can play an important role in the detection of biomolecules using sweat or interstitial fluid. However, due to the difficulty in sampling for the detection of actual proteins, small molecules, or the like and detecting measurement reactions, the use of these devices is currently limited to enzyme sensor technology for glucose measurement. Therefore, to address these problems, there is a gradually increasing need for a wearable device that is capable of sampling and detecting measurement reactions.

As a wearable device, for sweat sampling, technology for transferring sweat to a measurement device using cotton fabrics, fiber tissues, absorption pads, or the like is common, and research on sweat sampling using a microfluidic device is been conducted. However, this technology is limited to transferring sweat samples to a measurement device, and wearable devices using the same are also limited to detecting ions (sodium and potassium) and biomolecules (glucose and lactic acid) using a color conversion sensor or an electrochemical sensor. For detecting proteins, small molecules, or the like in sweat, to use an affinity-based biosensor in the form of skin attachment, a technology that enables the detection of measurement reactions as well as sweat sampling is required. In the affinity-based biosensor, element technologies such as labeling, washing, and reagent solution injection may be needed, and there is a need to develop a technology for a skin-attachment-type system equipped with all these functionalities.

SUMMARY

In one general aspect, there is provided a microfluidic device provided above a biosensor, the microfluidic device including a fluidic passing layer including an inlet part configured to collect sweat secreted from the skin, a sensing element in communication with the inlet part via a first microchannel and being configured to receive sweat from the inlet part, a reagent storage part in communication with the sensing element part via a second microchannel and being configured to supply a detection reagent to the sensing element part, and a disposer in communication with the sensing element part via a third microchannel and being configured to accommodate sweat of the sensing element part, and a fluidic connection layer including a microfluidic tube arranged in a vertical direction to supply sweat secreted from the skin to the inlet part, a sensing space provided between the sensing element and the biosensor to transfer sweat of the sensing element to the biosensor, and a reagent storage space positioned below the reagent storage part to store the detection reagent, wherein the fluidic connection layer is disposed below the fluidic passing layer.

The first microchannel may include an arch-type flap valve configured to prevent fluid from flowing towards the inlet part.

The second microchannel may include a check valve.

The third microchannel may include a burst valve.

The reagent storage part may include a button having a tapered shape.

The sensing element may include a microchannel configured to induce a capillary phenomenon.

The fluidic passing layer or the fluidic connection layer may have a thickness of 500 μm or less.

The microfluidic device may include a hydrophilic polymer formed on an elastic polymer.

The elastic polymer may include polydimethylsiloxane (PDMS) or polyurethane (PU), and the hydrophilic polymer comprises polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), or (3-mercaptopropyl) trimethoxysilane (MPTMS).

The hydrophilic polymer may be formed in any one or any combination of the inlet part of the fluidic passing layer, the microfluidic tube of the fluidic connection layer, the sensing element of the fluidic passing layer, and the sensing space of the fluidic connection layer.

In another general aspect, there is provided a skin-attachment-type biosensor patch to detect a biomolecule in sweat, the biosensor patch including a fluidic passing layer including an inlet part configured to collect sweat secreted from the skin, a sensing element in communication with the inlet part via a first microchannel and being configured to receive sweat from the inlet part, a reagent storage part in communication with the sensing element part via a second microchannel and being configured to supply a detection reagent to the sensing element part, and a disposer in communication with the sensing element part via a third microchannel and being configured to accommodate sweat of the sensing element part, and a fluidic connection layer including a microfluidic tube arranged in a vertical direction to supply sweat secreted from the skin to the inlet part, a sensing space provided between the sensing element and a biosensor to transfer sweat of the sensing element to the biosensor, and a reagent storage space positioned below the reagent storage part to store the detection reagent, wherein the fluidic connection layer is disposed below the fluidic passing layer, and the biosensor is provided below the fluidic connection layer and comprises a probe configured to detect the biomolecule.

The biosensor may be an affinity-based nanostructure in which a probe may be fixed to a sensor.

The biosensor patch may be stretchable.

In another general aspect, there is provided a method of manufacturing a microfluidic device, the method including fabricating a three-dimensional mold for manufacturing a microfluidic device using a 3D printer, forming a fluidic passing layer and a fluidic connection layer by pouring an elastic polymer solution into the mold and thermally curing the solution, treating the fluidic passing layer and the fluidic connection layer with a hydrophilic polymer, and combining the fluidic passing layer and the fluidic connection layer, subsequent to the treating with the hydrophilic polymer.

DETAILED DESCRIPTION

Hereinafter, examples will be described in detail with reference to the accompanying drawings. The scope of the examples is not limited to the descriptions provided in the present specification. The examples are not construed as limited to the disclosure and should be understood to include all changes, equivalents, and replacements within the idea and the technical scope of the disclosure.

When describing the examples with reference to the accompanying drawings, like reference numerals refer to like constituent elements and a repeated description related thereto will be omitted. In the description of examples, detailed description of well-known related structures or functions will be omitted when it is deemed that such description will cause ambiguous interpretation of the present disclosure.

FIG. 1is a diagram illustrating an example of a biosensor patch10disposed on a skin500of a subject, andFIG. 2is a diagram illustrating an example of the biosensor patch10illustrated inFIG. 1.

Referring toFIGS. 1 and 2, the biosensor patch10may include a microfluidic device100having a double layer and a biosensor200.

In an example, the microfluidic device100is arranged above the biosensor200and includes a fluidic passing layer (“FPL”)110and a fluidic connection layer (“FCL”)120. InFIGS. 1 and 2, I is the inlet unit, S is the sensing element unit, R is the reagent storage unit, and D is the disposal unit.

The fluidic passing layer110is disposed above the fluidic connection layer120, and includes an inlet part111through which sweat secreted from the skin is collected, a sensing element part112that is in communication with the inlet part111via a microchannel to receive sweat from the inlet part111, a reagent storage part113that is in communication with the sensing element part112via a microchannel to supply a detection reagent to the sensing element part112, and a disposal part114that is in communication with the sensing element part112via a microchannel to accommodate sweat of the sensing element part112.

The fluidic connection layer120is disposed below the fluidic passing layer110and above the biosensor200. In an example, the fluidic connection layer120includes: a microfluidic tube121arranged in a vertical direction to supply sweat secreted from the skin, a sensing space122that is provided between the sensing element part112and the biosensor200to transfer sweat of the sensing element part112to the biosensor200, and a reagent storage space123that is positioned below the reagent storage part113to store a reagent.

The inlet part111of the fluidic passing layer110and the microfluidic tube121of the fluidic connection layer120may be in communication with each other to form one space when the layers are attached to each other to constitute the microfluidic device100. In addition, when the biosensor200is attached to the microfluidic device100to constitute the biosensor patch10, the inlet part111, the microfluidic tube121, and an inlet220of the biosensor may be in communication with one another to form one space, and sweat may be introduced into and stored in the space. For convenience of description, the inlet part111, the microfluidic tube121, and the inlet220are given different names and reference numerals, but when the layers are attached to constitute the biosensor patch10, these parts may function as an inlet unit I, which is a single space. In an example, the shape of the inlet part111, the microfluidic tube121, or the inlet220may be a circular shape. Other shapes of the inlet unit I, such as, for example, a polygonal shape such as a triangular, tetragonal, pentagonal, or hexagonal shape may be appropriately used without departing from the spirit and scope of the illustrative examples described. In the biosensor patch10, the inlet unit I (seeFIG. 3) formed as a single space may have a volume of 5 μL to 50 μL, such as, for example, 7 μL to 20 μL and 32 μL to 50 μL.

The sensing element part112of the fluidic passing layer110and the sensing space122of the fluidic connection layer120may be in communication with each other to form one space when the layers are attached to constitute the microfluidic device100, and a probe210attached to the biosensor200may be provided below the space. For convenience of description, the sensing element part112and the sensing space122are given different names and reference numerals, but when the layers are attached to constitute the microfluidic device100, may function as a sensing element unit S, which is a single space. The shape of the sensing element part112or the sensing space122may be a circular shape. Other shapes of the sensing element unit S, such as, for example, a polygonal shape such as a triangular, tetragonal, pentagonal, or hexagonal shape may be appropriately used without departing from the spirit and scope of the illustrative examples described. In the biosensor patch10, the sensing element unit S (seeFIG. 3) formed as a single space may have a volume of 20 μL to 50 μL, such as, for example, 33 μL to37μL and 34 μL to 36 μL.

The reagent storage part113of the fluidic passing layer110and the reagent storage space123of the fluidic connection layer120may be in communication with each other to form one space when the layers are attached to constitute the microfluidic device100, and a detection reagent may be pre-stored in the space in a process of manufacturing the biosensor patch10. For convenience of description, the reagent storage part113and the reagent storage space123are given different names and reference numerals, but when the layers are attached to constitute the microfluidic device100, may function as a reagent storage unit R (seeFIG. 3), which is a single space. The shape of the reagent storage part113or the reagent storage space123may be a circular shape. Other shapes of the reagent storage unit R, such as, for example, a polygonal shape such as a triangular, tetragonal, pentagonal, or hexagonal shape may be appropriately used without departing from the spirit and scope of the illustrative examples described. In the biosensor patch10, the reagent storage unit R (seeFIG. 3) formed as a single space may have a volume of 30 μL to 100 μL, such as, for example, 75 μL to 85 μL and 78 μL to 82 μL.

The disposal part114may be provided only in the fluidic passing layer110. In other specific embodiments, the disposal part114of the fluidic passing layer110and a disposal space124of the fluidic connection layer120may be attached to each other to form the microfluidic device100, and in this case, may be in communication with each other to form one space. The space allows fluid, e.g., sweat, remaining in the sensing element unit S (seeFIG. 3) to be transferred so that the detection reagent can be introduced into the sensing element unit S. For convenience of description, the disposal part114and the disposal space124are given different names and reference numerals, but when the layers are attached to constitute the microfluidic device, may function as a disposal unit D (seeFIG. 3), which is a single space. The shape of the disposal part114or the disposal space124may be a circular shape. Other shapes of the disposal unit D, such as, for example, a polygonal shape such as a triangular, tetragonal, pentagonal, or hexagonal shape may be appropriately used without departing from the spirit and scope of the illustrative examples described.

The inlet part111, the reagent storage part113, and the disposal part114of the fluidic passing layer110, or the microfluidic tube121, the reagent storage space123, and the disposal space124(when included) of the fluidic connection layer120are each independently connected to the sensing element part112of the fluidic passing layer110or the sensing space122of the fluidic connection layer120via microchannels115to117.

More specifically, as illustrated inFIG. 4A, the inlet part111and/or the microfluidic tube121are/is in communication with the sensing element part112and/or the sensing space122via a first microchannel115so that sweat introduced into the inlet part111can be transferred to the sensing element part112or the sensing space122. The first microchannel115may include a venous valve-mimicking arch-type flap valve (see the left image ofFIG. 4B) that allows fluid to flow only towards the sensing element part112or the sensing space122and prevents fluid from flowing towards the inlet part111or the microfluidic tube121.

The reagent storage part113and/or the reagent storage space123are/is in communication with the sensing element part112and/or the sensing space122via a second microchannel116so that the detection reagent stored in the reagent storage part113or the storage space123can be transferred to the sensing element part112or the sensing space122. The second microchannel116may include a check valve (see the middle image ofFIG. 4B) that does not allow the detection reagent to flow bidirectionally at a certain pressure or less.

In an example, the reagent storage part113may be a button having a tapered shape. The button having a tapered shape is a device for transferring the detection reagent to the sensing element part112or the sensing space122by applying a pressure at a desired time point, and as illustrated inFIG. 5, may include a protrusion and a groove, unlike buttons having a general shape, and accordingly, when the button is pushed once, the protrusion and the groove are fastened, thus preventing the button from returning to its original state. Thus, it is possible to prevent the backflow of a reagent into the reagent storage part113or the reagent storage space123, caused by a negative pressure generated by the button returning to its original state.

The sensing element part112and/or the sensing space122are/is in communication with the disposal part114and/or the disposal space124via a third microchannel117so that sweat present in the sensing element part112or the sensing space122can be transferred to the disposal part114or the disposal space124. The third microchannel117may include a burst valve (see the right image ofFIG. 4B) that allows the remaining fluid to flow when a certain pressure or higher is applied.

In an example, the sensing element part112may include, therein, a microchannel with a zigzag pattern that facilitates the flow of collected sweat and allows the collected sweat to uniformly spread over the entire area of the sensing element part (seeFIG. 7).

Each of the layers constituting the microfluidic device100may be formed to a thickness of 500 μm or less through a mold fabricated using a 3D printer.

In an example, the microfluidic device100may have a hydrophilic polymer for imparting hydrophilicity formed on an elastic polymer for imparting elasticity, and thus is capable of efficiently absorbing and transferring sweat. The elastic polymer may be, for example, polydimethylsiloxane (PDMS), polyurethane (PU), or the like, and the hydrophilic polymer may be, for example, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), (3-mercaptopropyl)trimethoxysilane (MPTMS), or the like. In particular, the inlet part of the fluidic passing layer and the microfluidic tube of the fluidic connection layer, the sensing element part of the fluidic passing layer and the sensing space of the fluidic connection layer, or all of them may be treated with the hydrophilic polymer so that sweat secreted from the skin of a subject can be satisfactorily introduced into the microfluidic device.

In an example, the microfluidic device100may be coupled to the biosensor200including the probe210for detecting a biomolecule, thereby constituting the skin-attachment-type biosensor patch10for detecting a biomolecule in sweat.

In an example of the biosensor patch10, the biosensor200may include an affinity-based three-dimensional nanostructure in which the probe210capable of detecting a target biomolecule is fixed to a sensor, and through this structure, biomolecules present in sweat, e.g., cortisol, may be measured in units of picomoles (PM).

As used herein, the term “biomolecule” refers to a molecule that constitutes a living organism and is needed for the structure, function, signal transduction, and the like of a living organism.

In an example, the biosensor200may further include the inlet220through which sweat can be introduced.

In an example, the biosensor patch10may be attached to the skin of a subject to be analyzed, and more particularly, may be positioned and attached such that the biosensor200is in direction contact with the skin, such as arms, legs, or the torso, of a subject. The subject may include animals, for example, mammals (e.g., humans, apes, monkeys, mice, cows, dogs, and cats) to analyze a biomolecule in sweat naturally secreted from the subject, but the present invention is not limited thereto.

In an example, the biosensor patch10is stretchable.

The biosensor patch10according to an embodiment of the present invention may be disposable.

FIG. 3is a diagram illustrating an example of a process of operating the biosensor patch10. The process of operating the biosensor patch10will be described in detail with reference toFIG. 3.

The biosensor patch10is manufactured in the state where a detection reagent is stored in the reagent storage unit R, and sweat is introduced via the inlet unit I and stored. Hydrophilic treatment facilitates the absorption of sweat and the transfer thereof to the sensing element unit S, and enables a certain amount of sweat to be maintained in the sensing element unit S for a latent time for binding with a target biomolecule. After a certain period of time has elapsed, a button of the reagent storage unit R is pressed with a finger, allowing the detection reagent to be transferred to the sensing element unit S and by detecting an electrochemical signal, a target biomolecule is detected. In this case, sweat remaining in the sensing element unit S is transferred to the disposal unit D.

The term “detection reagent” as used herein refers to a reagent containing a redox species required to obtain an electrical signal from a biosensor, or another biomolecule, other chemicals, or the like capable of specifically binding to a biomolecule to be detected, and the detection reagent may be, for example, one or more selected from potassium hexacyanoferrate(III), phosphate-buffered saline (PBS), a potassium chloride (KCI) solution, an antibody, RNA, DNA, a hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin, a radioisotope marker, an aptamer, and a substance capable of specifically binding to a tumor marker. However, other detection reagent may be used without deviating from the spirit and scope of the illustrative examples described.

In an example, the detection of a biomolecule using the biosensor patch10includes not only determining whether a biomolecule is present or not but also measuring the concentration thereof.

In an example, a method of measuring the concentration of a biomolecule present in sweat of a subject, includes attaching the biosensor patch10to a surface of the skin of the subject, allowing sweat absorbed via the inlet unit I to be maintained in the sensing element unit S, pressing a reagent storage unit button to transfer a detection reagent to the sensing element unit S, and detecting an electrochemical signal to detect a target biomolecule. The operations of the method of measuring the concentration of a biomolecule present in sweat of a subject may be performed in the sequence and manner as described, although the order of some operations may be changed or some of the operations omitted without departing from the spirit and scope of the illustrative examples described.

In an example, a method of detecting a biomolecule present in sweat, the method includes attaching the biosensor patch according to the present invention to a surface of the skin, allowing sweat absorbed via an inlet unit to be maintained in a sensing element unit, pressing a reagent storage unit button to transfer a detection reagent to the sensing element unit, and detecting an electrochemical signal to detect a target biomolecule.

Hereinafter, a method of manufacturing the microfluidic device100will be described, and the detailed description of the same parts as those of the above-described microfluidic device100are incorporated herein by reference. Thus, the above description may not be repeated here.

The method of manufacturing the microfluidic device100according to an example includes fabricating a three-dimensional mold for manufacturing the microfluidic device100, by using a 3D printer, forming the fluidic passing layer110and the fluidic connection layer120by pouring an elastic polymer solution into the mold and thermally curing the same, treating the fluidic passing layer110and the fluidic connection layer120obtained in the above process, with a hydrophilic polymer, and combining the fluidic passing layer110and the fluidic connection layer120that have been subjected to hydrophilic treatment.

In an example, a mold including a pattern of a microfluidic device is fabricated using a 3D printer. The fabrication of a microfluidic device pattern using existing photolithography may be inconvenient because a plurality of two-dimensional pattern layers may have to be stacked to form a three-dimensional shape after the formation of a two-dimensional shape, which may be addressed by fabricating a mold using a 3D printer. In addition, a microfluidic device manufactured through the mold can completely maintain the pattern, and enables the manufacture of a microfluidic device within a short time. Each of layers of the manufactured microfluidic device is treated with a hydrophilic polymer, thereby facilitating the absorption of sweat. The microfluidic device100manufactured using the above-described method and the biosensor200are coupled to manufacture the biosensor patch10.

Hereinafter, some examples of manufacture of biosensor patch with microfluidic device, and its various components are described below. However, these examples are provided only to facilitate the understanding of the disclosure and are not intended to limit the scope of the illustrated examples described.

An example of a manufacture of biosensor patch with microfluidic device coupled thereto is described below.

An example of fabrication of mold using 3D printer is as follows.

A mold having a three-dimensional shape is fabricated using a 3D printer as illustrated inFIG. 6(minimum pattern: 10 μm). The mold is coated with an anti-adhesion material to allow the microfluidic device100made of a PDMS material to be easily detached therefrom.

An example of manufacture of Microfluidic Device is as follows. In an example, Plasma or Polymer Treatment for imparting Hydrophilicity is applied.

To perform hydrophilic treatment only on the inlet part111and the sensing element part112of the fluidic passing layer, oxygen plasma treatment is performed for 30 seconds while the reagent storage part113and the disposal part114are masked. Oxygen plasma treatment makes a PDMS material hydrophilic for a very short period of time, but the effect does not last for a long time, and thus a long-lasting hydrophilic treatment technique is needed to efficiently collect sweat even after being coupled to the biosensor200. Thus, a technique for maintaining hydrophilicity for a long time using PVP, which is a hydrophilic polymer, is applied. An appropriate amount of a solution in which PVP is dissolved in ultrapure water to a concentration of 20 wt % is added dropwise to the inlet part111and the sensing element part112, and then the solution is maintained at room temperature for 3 minutes. Thereafter, the resulting structure is washed clean with ultrapure water and stored in an airtight place.

An example of formation of Microchannel in Sensing Element Part is as follows.

When a microchannel is formed, the flow of fluid is facilitated along the microchannel by a capillary phenomenon. As illustrated inFIG. 7, a microchannel having a diameter of 200 μm and a zigzag pattern is formed in the sensing element part112, so that the entire surface of the sensing element part112can be filled with the collected sweat sample.

An example of fabrication of Reagent Storage Unit Button is as follows.

In the case where connection between the reagent storage part113and the reagent storage space123of the fluidic connection layer120is a vertical type (see the left image ofFIG. 5), when a button provided above the reagent storage part113is pressed with a finger, the button is restored by the elastic force of PDMS, and thus there was a problem in that a reagent was unable to be efficiently transferred to the sensing element part112and the sensing space122. To address the above-described problem, the reagent storage part113or a button therefor of the fluidic passing layer110is designed to have an angular shape (see the right image ofFIG. 5). Accordingly, when the button provided above the reagent storage part113is pressed with a finger, the angular shape is engaged with and fixed to the reagent storage space123of the fluidic connection layer120, thus preventing the button from being restored by an elastic force, so that the backflow of a reagent transferred to the sensing element part112and the sensing space122is prevented.

An example of fabrication of Check Valve and Burst Valve is as follows.

To confine sweat in the sensing element unit for a certain period of time, a burst valve (FIG. 8) is positioned in the third microchannel117that allows the sensing element unit S to be in communication with the disposal unit D, and a check valve (FIG. 9) is positioned in the second microchannel116that allows the sensing element unit S to be in communication with the reagent storage unit R. Each microchannel has a width of 100 μm and a height of 200 μm, and the burst valve or the check valve is formed an angle of 120° with respect to the microchannel, thus enabling sweat to be confined in the sensing element unit before a pressure is applied from outside.

An example of fabrication of Flap Valve for preventing Backflow is as follows.

To allow sweat to flow only from the inlet unit I towards the sensing element unit S, as illustrated inFIG. 10, a non-return valve (an arch-type flap valve) that mimics a vascular valve structure and prevents backflow is positioned in a first microchannel that allows the inlet unit I to be in communication with the sensing element unit S. A very thin wall (thickness of 50 μm) is formed in a microchannel portion of the mold fabricated using a 3D printer to form a bent valve capable of independently moving in the microchannel. The valve is designed to have a thickness of 200 μm and a height of 700 μm.

An example of manufacture of Biosensor Patch with Microfluidic Device coupled thereto is as follows.

A PDMS solution is poured into the mold fabricated using a 3D printer and thermally cured at 80° C., and then the cured product is detached therefrom to form the fluidic passing layer110and the fluidic connection layer120to a thickness of 500 μm or less. To combine the two layers, an appropriate amount of a PDMS solution is added dropwise to the fluidic connection layer120, and then very thinly coated at 2,000 rpm for 30 seconds in a spin coater. Subsequently, the fluidic connection layer120is semi-cured on a hot plate at 85° C. for 15 minutes, and then aligned with and coupled to the fluidic passing layer110, thereby completing the manufacture of a microfluidic device. The microfluidic device is coupled to a biosensor patch using the same method as described above.

An example of Fluid Evaluation under Polyvinylpyrrolidone (PVP) Treatment Conditions is as follows.

After oxygen plasma treatment or PVP treatment for imparting hydrophilicity to PDMS according to the method described above, artificial sweat mixed with a red dye is added dropwise to compare the cross-sections of fluid after 5 hours and 6 days.

As illustrated inFIG. 11, in the case of PDMS subjected to oxygen plasma treatment (top ofFIG. 11), the cross-section of fluid exhibits a convex shape after 5 hours, showing a loss of hydrophilicity. In contrast, in the case of PDMS (bottom ofFIG. 11) subjected to PVP treatment, the fluid showed a shape of being widely spread on the surface of PDMS even after 6 days, through which it is confirmed that hydrophilicity is maintained for a long period of time.

An example of Fluid Transfer Evaluation according to shape of Reagent Storage Unit Button is as follows.

To prevent the reagent transferred from the reagent storage unit to the sensing element unit from flowing back into the reagent storage unit, in addition to a check valve in a microchannel connecting the two spaces, a button having a tapered shape is used to prevent the formation of a negative pressure generated by the reagent storage unit button being restored by an elastic force and backflow due to this (see the right side ofFIG. 5).

As illustrated inFIG. 12, it is confirmed that a button having a general shape returned to its original shape after being pressed by a pressure, and thus the reagent transferred to the sensing element unit flowed back into the reagent storage unit (see top ofFIG. 12), whereas, when a button having a tapered shape is used, a reagent can be stably maintained in the sensing element unit without backflow into the reagent storage unit (see bottom ofFIG. 12).

An example of Measurement of Concentration of Molecule in Sweat Using Biosensor Patch is as follows.

Artificial sweat including cortisol at a concentration of 1 pg/ml to 1 pg/ml is prepared as a target molecule, and then the impedance of the artificial sweat is measured using the biosensor patch10. As a result, as illustrated inFIG. 13, a graph showing that the greater the concentration of cortisol, the greater the impedance value is obtained.

In addition, the biosensor patch10was attached to a human body, and then the impedance of a sweat sample obtained through exercise was measured, and the measurement results thereof are illustrated inFIG. 14.

The embodiments described above disclose a microfluidic device that is attached to an affinity-based biosensor attached to the skin and capable of detecting biomolecules in sweat to transfer sweat for detection and a detection reagent to the biosensor, and a testing apparatus using a biosensor to which the microfluidic device is coupled, thus completing the present invention. The microfluidic device that supplies sweat discharged from the skin to a biosensor.

Also disclosed is a skin-attachment-type biosensor patch for detecting a biomolecule in sweat, a method of manufacturing the microfluidic device, and a method of detecting a biomolecule present in sweat using the biosensor patch.

The embodiments described above relates to a microfluidic device for detecting biomolecules in sweat, and a biosensor patch for detecting biomolecules in sweat, in which the microfluidic device and a biosensor are combined, the device and the patch being capable of detecting various target molecules present in actual sweat by electrochemical signals and also detecting the concentration of a target molecule. Furthermore, the microfluidic device may be coupled to various high-accuracy affinity-based biosensors, which have low detection limitations, and is a system that has various capabilities from sampling to the transfer and measurement of a sample in the state of being attached to a body, and thus can also be applied as a real-time monitoring system. Thus, the microfluidic device is expected to be widely used as a platform that is applicable to a testing apparatus for detecting various biomolecules.

The microfluidic device can be coupled to various high-accuracy affinity-based biosensors, which have low detection limitations, and thus is expected to be used as a platform that is applicable to a testing apparatus for detecting various biomolecules.