Paper-based microfluidic device including plurality of puddles and hydrophobic barriers and manufacturing method thereof

Abstract

The present invention relates to: a paper-based microfluidic device including a plurality of puddles and hydrophobic barriers; and a manufacturing method thereof. Specifically, the paper-based microfluidic device according to the present invention can move a fluid without external power due to the porous structure of a substrate made of paper, and thus can provide a simple and highly portable analysis platform. In addition, unlike conventional μPADs, the paper-based microfluidic device is fully compatible with 96-well plates and the like and thus can be easily integrated with existing laboratory equipment or smartphones, can simultaneously process multiple samples on a single device (chip), can reduce experimental costs by using small amounts of reagents, and is highly durable and stable and thus can be widely used in various biological, clinical, and environmental analysis environments, such as chromaticity, absorbance, and fluorescence analysis environments.

Description

Paper-based microfluidic device comprising multiple puddles and hydrophobic barriers and method for manufacturing the same

[0001] The present invention relates to a paper-based microfluidic device for biological, clinical, and environmental analysis, more specifically, to a paper-based microfluidic device comprising a plurality of puddings and hydrophobic barriers and a method for manufacturing the same.

[0002] Paper-based microfluidic devices (μPADs) or lab-on-paper are designed to enable various biochemical analyses using small sample volumes through a miniaturized paper structure. Specifically, by utilizing the porous structure of paper to enable fluid movement without external power, they provide a simple and highly portable analysis platform. These devices are primarily utilized for various analyses by forming hydrophilic and hydrophobic regions on the paper surface using technologies such as wax printing or inkjet printing.

[0003] Meanwhile, conventional μPADs have been used for the detection of small molecules, proteins, and pathogens through colorimetric, electrochemical, and fluorescence analysis; however, they have faced limitations such as difficulties in analytical standardization, low productivity, and a limited analytical range. In particular, the need for customized analytical equipment based on various designs resulted in low versatility, and poor analytical reproducibility made it difficult to ensure data reliability. Furthermore, existing μPADs generally provided only a limited number of reaction zones, making them unsuitable for high-throughput analysis.

[0004] Meanwhile, commercially available 96-well plates today offer high productivity and reproducibility, but they are primarily made of plastic materials, which cause environmental pollution upon disposal and are not compatible with existing μPADs, making it difficult to leverage the advantages of paper-based platforms.

[0005] The inventors have confirmed that if a pattern layer having multiple puddles and hydrophobic barriers is formed on a paper substrate using a relatively simple silk screen printing method, and a microfluidic device is manufactured in a form compatible with existing 96-well plates, etc., so that it can be directly applied to microplate readers or fluorescence microscopes widely used in laboratories or smartphones, it can become an innovative platform suitable for multi-purpose analysis that is eco-friendly.

[0006] Specifically, the paper-based microfluidic device prepared according to the present invention can be utilized for colorimetric, absorbance, and fluorescence analysis, reduces experimental costs by using small amounts of reagents, and can be used in various environments due to its high durability and stability. Through this, the problems of existing μPADs are resolved, and by maximizing the advantages of the paper-based platform, environmentally friendly, high-productivity, high-reproducibility, and multi-purpose analysis can be enabled.

[0007] The present invention aims to provide a paper-based microfluidic device comprising multiple puddles and hydrophobic barriers and a method for manufacturing the same, wherein a pattern layer having multiple puddles and hydrophobic barriers is formed on a paper substrate using a relatively simple silk screen printing method, and the microfluidic device is manufactured in a form compatible with existing 96-well plates, etc., thereby allowing multiple samples to be processed simultaneously on a single chip (device) unlike existing paper-based analysis, reducing experimental costs by using a small amount of reagents, and enabling utilization in various environments such as color, absorbance, and fluorescence analysis with high durability and stability.

[0008] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0009] The present specification provides a paper-based microfluidic device comprising: a porous substrate; and a pattern layer formed on the porous substrate, the pattern layer comprising a plurality of puddles and a hydrophobic barrier formed in a region other than the region where the puddles are located.

[0010] For example, the porous substrate may be a paper material comprising one selected from cellulose, cellulose acetate, cellulose nanofibrils, carboxymethylcellulose, hydroxypropylcellulose, and combinations thereof.

[0011] For example, the above puddles have a concave structure so that 0.01 to 90 µl of solution can be contained within each of the above puddles.

[0012] For example, the above puddle may have one shape selected from circular, elliptical, ring-shaped, polygonal, linear, spiral, branched, and combinations thereof.

[0013] For example, the hydrophobic barrier may include one selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof.

[0014] For example, a protective film may be further provided on the pattern layer.

[0015] For example, the microfluidic device may have a thickness in the range of 0.275 to 0.325 mm.

[0016] For example, if an adhesive tape is brought into contact with the microfluidic device one or more times to reduce the puddle depth to 65% or less of the initial depth, the color sensitivity and sensing efficiency can be improved by more than 200% compared to the initial value.

[0017] For example, the microfluidic device described above can be used for one or more types of analysis selected from color analysis, absorbance analysis, and fluorescence analysis.

[0018] For example, the microfluidic device is compatible with one type of micro-well plate selected from a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, and a 384-well plate, and can be applied to a smartphone, a micro-plate reader, or a fluorescence microscope.

[0019] In addition, the present specification provides a method for manufacturing a paper-based microfluidic device, comprising: a) preparing a porous substrate; b) placing a silk screen having impermeable patterns corresponding to a plurality of puddle shapes on the substrate and applying a hydrophobic solution to form a hydrophobic barrier in an area other than the area where the plurality of puddles are located; and c) after step b, heating at least once to a temperature within the range of 100 to 180 ℃ to cure the hydrophobic barrier.

[0020] For example, the hydrophobic solution of step b above may be a mixture of one polymer selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof, and a curing agent in a weight ratio of 8.5 : 1.5 to 9.5 : 0.5.

[0021] For example, the above step c may further include a process of attaching a protective film to the pattern layer of the microfluidic device.

[0022] The paper-based microfluidic device according to the present invention enables fluid movement without external power due to the porous structure of the paper substrate, thereby providing a simple and highly portable analysis platform.

[0023] In addition, unlike conventional μPADs, it is fully compatible with 96-well plates and other devices, making it easy to integrate with existing laboratory equipment or smartphones. It can process multiple samples simultaneously on a single device (chip), reduces experimental costs by using small amounts of reagents, and can be widely used in various biological, clinical, and environmental analysis environments, such as color, absorbance, and fluorescence analysis, due to its high durability and stability.

[0024] FIG. 1 schematically illustrates a method for manufacturing a paper-based microfluidic device according to an embodiment of the present invention.

[0025] FIG. 2 shows, when manufacturing a paper-based microfluidic device according to an embodiment of the present invention, (a) to (c) optical images of the microfluidic device according to the number of hydrophobic solution coatings (2, 4, 6), (d) line intensity profiles, and (e) puddle radius (r Puddle ), (f) PDMS coating depth (D pdms (g) shows the results of the analysis of the circularity value (R %) and (h) the uniformity coefficient (u) of the puddle.

[0026] FIG. 3 shows (a) a side view of a paper-based microfluidic device according to an embodiment of the present invention with various volumes of colorimetric solutions (PAni-NPs) dispensed into the puddles, (b) gray values ​​of the puddle rings, (c) description of the maximum gray value and average gray value of the inner circle, (d) a value obtained by subtracting the average gray value from the maximum gray value in different volumes and dividing by 1 (1 / η), (e) an optical image of the microfluidic device with adsorbed PAni-NPs, (f) gray values ​​of each of the 96 puddles with adsorbed PAni-NPs (error rate 0.8%), (g) an optical image of the microfluidic device for acetone detection, and (h) a* color change of the microfluidic device for acetone concentration detection.

[0027] FIG. 4 shows the results of a comparative analysis of a paper-based microfluidic device and a 96-well plate according to an embodiment of the present invention, including (a) the required solution volume and image of the microfluidic device with various pH solutions (1 to 12) dispensed, (b) absorbance, (c) the required solution volume and image of the 96-well plate with various pH solutions (1 to 12) dispensed, (d) absorbance, (e) analysis of the absorbance ratio (λ540 / λ610) of the universal pH indicator colors of the microfluidic device and the 96-well plate, and (f) a correlation plot.

[0028] FIG. 5 illustrates the detection of various molecules using a paper-based microfluidic device according to an embodiment of the present invention, wherein (a) Mg of various concentrations 2+ Optical image of 96-PPP dispersed, (b) ascorbic acid, (c) acetoacetic acid, (d) Mg 2+ Absorbance as a function of concentration, (e) ascorbic acid concentration, (f) acetoacetic acid concentration, (g) Mg 2+ (h) a plot fitted using a semi-linear fit as a function of concentration, (i) a plot fitted using a sigmoid fit as a function of ascorbic acid concentration, and (i) a plot fitted using a standard curve fit as a function of acetoacetic acid concentration.

[0029] FIG. 6 shows the results of amyloid fluorescence analysis using a paper-based microfluidic device according to an embodiment of the present invention, including: (a) 96-PPP with precipitated amyloid and enlarged optical and fluorescence images; (b) a schematic diagram of the microfluidic device before and after amyloid adsorption; (c) analysis of fluorescence intensity of puddles with precipitated amyloid (n=480); (d) a schematic diagram of amyloid amplified after treatment with amyloid degrading agent and after time has passed; (e) a graph of analysis of the increase in amyloid fluorescence sensitivity in solution according to the concentration of amyloid degrading agent; (f) a graph of analysis of the increase in puddle fluorescence sensitivity according to the concentration of amyloid degrading agent; (g) fluorescence and 3D images of puddles according to the concentration of amyloid degrading agent; (h) a graph of cross-sectional analysis of puddles; (i) a schematic diagram of the puddle shape without treatment with amyloid degrading agent; and (j) a schematic diagram of the puddle shape containing amyloid degrading agent.

[0030] FIG. 7 illustrates the leakage test results according to the hydrophobic barrier material (coating polymer: PVA, resin, PDMS) and the type of paper in a paper-based microfluidic device according to one embodiment of the present invention.

[0031] FIG. 8 shows an analysis of regions with and without a PDMS coating (hydrophobic barrier) in a paper-based microfluidic device according to an embodiment of the present invention, including (a) an image of the top of a smartphone, (b) a microscope image, (c) a scanning electron microscope (SEM) image of the microfluidic device, and (d) X-ray diffraction (XRD) patterns of bare filter paper (purple) and PDMS-coated filter paper (blue).

[0032] FIG. 9 illustrates the advantages of using a paper-based microfluidic device according to an embodiment of the present invention, including (a) flexibility, (b) storage advantages, and (c) equipment compatibility.

[0033] FIG. 10 illustrates a schematic form of a paper-based microfluidic device according to an embodiment of the present invention, comprising various forms of puddings and hydrophobic barriers.

[0034] FIG. 11 illustrates whether color sensitivity and sensing efficiency are improved when an adhesive tape is contacted several times, in a paper-based microfluidic device according to an embodiment of the present invention, the shape of the cut surface of the puddle, a method for controlling the shape, and the color sensitivity and sensing efficiency.

[0035] FIG. 12 schematically illustrates a sampling method for a paper-based microfluidic device according to an embodiment of the present invention.

[0036] FIG. 13 is the result of a leakage test under heating (60°C) of a paper-based microfluidic device according to one embodiment of the present invention and a measurement of the chemical compatibility of the hydrophobic barrier to various chemical solutions using the paper-based microfluidic device.

[0037] FIG. 14 is an experimental result confirming the reproducibility of a paper-based microfluidic device according to an embodiment of the present invention.

[0038] Hereinafter, embodiments of the present invention are described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments disclosed below. Furthermore, in order to clearly disclose the present invention in the drawings, parts unrelated to the present invention have been omitted, and identical or similar reference numerals in the drawings indicate identical or similar components.

[0039] The purpose and effects of the present invention may be naturally understood or become clearer from the following description, and the purpose and effects of the present invention are not limited to the description below alone.

[0040] The objectives, features, and advantages of the present invention will become clearer through the following detailed description. Furthermore, in describing the present invention, if it is determined that a detailed description of known technology related to the present invention may unnecessarily obscure the essence of the invention, such detailed description will be omitted. Hereinafter, embodiments according to the present invention will be described in detail with reference to the attached drawings.

[0041]

[0042] Conventional μPADs, used for the detection of small molecules, proteins, and pathogens through colorimetric, electrochemical, and fluorescence analysis, have faced limitations such as difficulties in analytical standardization, low productivity, and a limited analytical range. In particular, the need for customized analytical equipment based on various designs has resulted in low versatility, and poor analytical reproducibility has made it difficult to ensure data reliability. Furthermore, existing μPADs generally provide only a limited number of reaction zones, making them unsuitable for high-throughput analysis. Meanwhile, while commercially available 96-well plates offer high productivity and reproducibility, they are primarily made of plastic, causing environmental pollution upon disposal. Additionally, their incompatibility with existing μPADs makes it difficult to leverage the advantages of paper-based platforms.

[0043]

[0044] The inventors confirmed through experiments that if a microfluidic device is manufactured in a form compatible with existing 96-well plates, etc., and can be directly applied to equipment such as smartphones, microplate readers, and fluorescence microscopes widely used in laboratories, by forming a pattern layer equipped with multiple puddles and hydrophobic barriers on a paper substrate using a relatively simple silk screen printing method, it can be utilized as an innovative platform suitable for eco-friendly and multi-purpose analysis.

[0045]

[0046] Hereinafter, a paper-based microfluidic device including a plurality of puddles and hydrophobic barriers and a method for manufacturing the same will be described in more detail.

[0047]

[0048] Method for manufacturing a paper-based microfluidic device

[0049] A method for manufacturing a paper-based microfluidic device according to an embodiment of the present invention comprises: a) a step of preparing a porous substrate; b) a step of positioning a silk screen having impermeable patterns corresponding to a plurality of puddle shapes formed thereon on the substrate and applying a hydrophobic solution to form a hydrophobic barrier in an area other than the area where the plurality of puddles are located; and c) a step of curing the hydrophobic barrier by heating at least once at a temperature within the range of 100 to 180 ℃ after step b (see FIG. 1).

[0050]

[0051] First, prepare a porous substrate (step a).

[0052] The substrate of the present invention can absorb and move fluid without separate external power through a porous structure and capillary action, and may serve as a support for a microfluidic device according to the present invention.

[0053] Specifically, the porous substrate according to one embodiment of the present invention may be a paper material comprising one selected from cellulose, cellulose acetate, cellulose nanofibrils, carboxymethylcellulose, hydroxypropylcellulose, and combinations thereof, and more specifically, a paper material comprising high-purity α-cellulose, and more specifically, a Whatman filter paper.

[0054] Meanwhile, the pore size in the porous substrate according to one embodiment of the present invention may be in the range of 18 to 22 μm, and for example, polydimethylsiloxane (PDMS) is stably absorbed into the pores within the above pore range, so that the effect of preventing leakage is excellent compared to cases where it is less than or greater than the above pore range.

[0055]

[0056] Next, a silk screen having opaque patterns corresponding to a plurality of puddle shapes is placed on the above substrate, and a hydrophobic solution is applied to form a hydrophobic barrier in an area other than the area where the plurality of puddles are located (step b).

[0057] The above step is a step of providing a pattern layer on the above-described substrate, wherein the pattern layer may include a plurality of puddles and a hydrophobic barrier formed in an area other than the region where the plurality of puddles are located.

[0058] As a specific example, step b above can be performed by a silk screen printing method, and can be performed by placing a silk screen having opaque patterns corresponding to a plurality of puddle shapes formed on the substrate and applying a hydrophobic solution. At this time, after applying the hydrophobic solution, a wiper can be used to move back and forth in the front, back, left, and right directions of the silk screen to achieve a more uniform application.

[0059] The application process of the above hydrophobic solution may be repeated one or more times. Specifically, depending on the number of times the application process of the hydrophobic solution is repeated, the amount of the component constituting the hydrophobic barrier formed on the substrate may vary, and accordingly, the shape and depth of the multiple puddles described below may vary.

[0060] The hydrophobic solution used in the above step may be a mixture of one polymer selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof, and a curing agent in a weight ratio of 8.5:1.5 to 9.5:1.5, specifically in a weight ratio of 9:1. The curing agent may be, for example, one selected from vinyl-terminated polydimethylsiloxane, silica filler, platinum catalyst, and combinations thereof. Meanwhile, when the hydrophobic solution is prepared by mixing in the above weight ratio, it may be properly cured to ensure flexibility and chemical resistance, and at the same time, have the effect of stably imparting hydrophobicity.

[0061] Meanwhile, the hydrophobic barrier formed by being applied to the substrate through the above steps can be formed in a shape that surrounds the area where each pudding is located by curing and fixing according to step c described later.

[0062]

[0063] Next, after step b above, the hydrophobic barrier is cured by heating at least once to a temperature within the range of 100 to 180 ℃ (step c).

[0064] The above step is a step for curing and fixing the hydrophobic barrier formed on a substrate by a silk screen printing method in step b, and can be performed by heating at a temperature within the range of 100 to 180 ℃ one or more times. Specifically, the above step can be performed by using a hot plate to heat at a temperature of 100 to 180 ℃, more specifically 110 to 170 ℃, even more specifically 120 to 160 ℃, for example, 150 ℃ for 10 minutes. Meanwhile, if heating is performed below the above temperature range, there may be a problem of reduced mechanical strength and hydrophobicity due to incomplete curing, and if heating is performed above the above temperature range, there may be a problem of deterioration of physical properties due to thermal decomposition and bubble generation.

[0065] Furthermore, the above steps may further include a process of attaching a protective film to the pattern layer to protect the pattern layer, which includes the plurality of puddles and hydrophobic barriers, from external contaminants. As a specific example, in step c, the hydrophobic barrier may be cured and fixed by heating at a temperature of 150°C for 10 minutes, and then a film made of polyvinyl chloride (PVC) may be attached to the pattern layer as a protective film. Then, the hydrophobic barrier may be further cured and fixed by heating again with a hot plate at a temperature of 100 to 180°C, specifically 110 to 170°C, more specifically 120 to 160°C, for example, 150°C for 10 minutes.

[0066]

[0067] Meanwhile, according to one embodiment of the present invention, after step b and before step c, a step of applying a predetermined range of pressure to the area where the plurality of puddles are located to deform the cross-sectional shape of the puddles into a desired shape may be further included. At this time, the means of applying pressure is not particularly limited, but as an example, fine-sized beads may be used.

[0068]

[0069] Paper-based microfluidic device

[0070] The paper-based microfluidic device of the present invention manufactured according to the method described above may be provided with: a porous substrate; and a pattern layer formed on the porous substrate, comprising a plurality of puddles and a hydrophobic barrier formed in a region other than the region where the puddles are located.

[0071] Meanwhile, as described above, the porous substrate may be a material comprising cellulose, cellulose acetate, cellulose nanofibrils, carboxymethylcellulose, hydroxypropylcellulose, and one selected from combinations thereof.

[0072] Meanwhile, the puddles have a concave structure that can accommodate the solution when dispensing the sampling solution, and multiple puddles may be provided. For example, the puddles have a concave structure so that 0.01 to 90 µl of solution can be accommodated in each puddle.

[0073] Meanwhile, the number of the above-mentioned multiple puddles can be matched to the number of wells of each well plate so as to be compatible with one type of micro-well plate selected from a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, and a 384-well plate.

[0074] In addition, the puddle according to one embodiment of the present invention may have one shape selected from circular, elliptical, ring-shaped, polygonal, linear, spiral, branched, and combinations thereof, and its size may be appropriately adjusted according to the need and purpose (see FIG. 10).

[0075] Meanwhile, the hydrophobic barrier formed around the periphery of the puddles and surrounding each puddle may include one selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof, and the height of the hydrophobic barrier (i.e., the depth of the puddles) may, for example, range from 0.2 to 0.325 mm.

[0076] Meanwhile, when the hydrophobic barrier is composed of the above-mentioned material, the strength and durability of the hydrophobic barrier are enhanced compared to general polymers such as polyvinyl alcohol (PVA) or resin, thereby minimizing leakage of the solution (see FIGS. 7 to 8).

[0077]

[0078] Meanwhile, the paper-based microfluidic device of the present invention not only exhibits flexible characteristics but also has a thickness in the range of 0.275 to 0.325 mm, which can provide storage advantages (see FIG. 10).

[0079] In addition, the paper-based microfluidic device of the present invention may have its color sensitivity and sensing efficiency improved by more than 200% compared to the initial value when the adhesive tape is brought into contact with the microfluidic device at least once, specifically within the range of 1 to 5 times, thereby reducing the puddle depth to 65% or less of the initial depth (see FIG. 13).

[0080]

[0081] Meanwhile, in a paper-based microfluidic device according to one embodiment of the present invention, the hydrophobic barrier can exhibit barrier properties for 18 hours or more against one or more solutions selected from methanol, ethanol, acetonitrile, sodium dodecyl sulfate (SDS), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, cetrimonium bromide (CTAB), polyethylene glycol tert-octylphenyl ether (Triton X-100), polysorbate 20, formaldehyde, sodium hydroxide (NaOH), deionized water, and solutions with pH 1, 4, 7, and 12. In addition, the microfluidic device has excellent durability and structural stability, as no structural damage or collapse of the hydrophobic barrier occurs even when immersed in deionized water for 3 days or more, or left in a 100°C oven for 3 days.

[0082]

[0083] The microfluidic device according to the present invention described above can be utilized for one or more types of analysis selected from colorimetric analysis, absorbance analysis, and fluorescence analysis. Specifically, it is compatible with one type of micro-well plate selected from 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, and 384-well plates, and can be applied to a smartphone, a microplate reader, or a fluorescence microscope, making it usable in various biological, clinical, and environmental analysis environments (see FIG. 9).

[0084]

[0085] Examples

[0086] The present invention is capable of various modifications and may take various forms, and specific embodiments are illustrated and described in detail below. However, this is not intended to limit the invention to the specific disclosed forms, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention.

[0087]

[0088] Examples 1-1, 1-2 and 1-3

[0089] A Whatman filter paper (material: Whatman No. 41, manufactured by Cytiva) was prepared, and a silk screen having an impermeable pattern of 96 circular areas arranged in the same configuration as a 96-well plate to be compatible with a 96-well plate was placed on the Whatman filter paper. Next, 5 ml of a hydrophobic solution, prepared by mixing polydimethylsiloxane (PDMS) and a polydimethylsiloxane curing agent in a weight ratio of 9:1, was applied onto the silk screen. Next, a wiper was moved in the forward, backward, left, and right directions of the silk screen to pass the hydrophobic solution onto the silk screen. Meanwhile, the case where the above process was repeated twice is hereinafter defined as S2 (Example 1-1), the case where it was repeated four times is defined as S4 (Example 1-2), and the case where it was repeated six times is defined as S6 (Example 1-3).

[0090] Next, the previously formed hydrophobic barrier was cured and fixed by heating for 10 minutes using a hot plate at 150°C, and a protective film (made of PVC) was attached to the pattern layer. Finally, the paper-based microfluidic device of the present invention (hereinafter referred to as 96-PPP) was prepared by heating for an additional 10 minutes on the hot plate after attaching the protective film (see FIG. 1).

[0091]

[0092] [Experiment 1. Verification of Puddle Shape According to Number of Hydrophobic Solution Applications]

[0093] The inherent absorption and capillary phenomena of porous substrates, such as paper, affect the distribution and reaction time of the solution, and these characteristics can significantly impact the performance and consistency of 96-PPP. Meanwhile, when a hydrophobic solution containing PDMS is applied to a porous substrate, the amount of solution absorbed by the substrate varies depending on the number of applications, which directly affects the size and uniformity of the puddle regions. Generally, increasing the number of applications increases the contact time between the substrate and the hydrophobic solution, leading to increased absorption of the solution. This phenomenon can not only reduce the size of the puddles but also compromise their uniformity.

[0094] Figure 2 shows the results of observing the puddle regions according to each coating cycle model through optical images. Although no significant visual differences were observed between models S2, S4, and S6, it was confirmed that some puddles in the S6 model tended to fail to maintain their circular shape due to excessive absorption of the hydrophobic solution. This suggests that excessive absorption of the hydrophobic solution can deform the concave structure and shape of the puddles. To quantitatively evaluate this, the gray value (line-intensity) profiles of each microfluidic device model were analyzed. The analysis results showed that the puddle region of the S2 model was the largest, while that of the S6 model was the smallest. This was also reflected in the size of the hydrophobic barrier region, with the hydrophobic barrier height (D) of the S2 model PDMS It was found that ) was the smallest and the S6 model was the largest.

[0095] In addition, when checking for leakage in the puddle areas using ethanol and acetone, leakage of the solution into adjacent puddle areas was observed in some regions of the S2 model. On the other hand, the shape of the puddle areas in the S6 model was non-uniform due to excessive absorption of the hydrophobic solution. In contrast, the S4 model demonstrated the best performance, maintaining a circular shape without leakage in all 96 puddle areas.

[0096] Roundness values ​​were analyzed to evaluate the shape of the puddle area. Roundness indicates the degree to which the puddle area is close to a perfect circle, and the closer the value is to 100%, the closer it is to a circle. As a result of the analysis, the roundness values ​​of models S2, S4, and S6 were 97.94 ± 1.33, 97.29 ± 1.11, and 95.26 ± 1.33, respectively, showing a tendency for uniformity to decrease due to excessive interaction with the hydrophobic solution as the number of applications increased.

[0097] Finally, the uniformity index (u) was calculated to evaluate the performance of each 96-PPP device model. The uniformity index (u) is the hydrophobic barrier height (D PDMS ) and puddle area radius (r Puddle The standard deviation (S) of r It is calculated as follows in Equation 1 based on ):

[0098]

[0099] (Equation 1)

[0100]

[0101] Meanwhile, according to Equation 1 above, the u values ​​for models S2, S4, and S6 were 150.34, 367.55, and 238.93, respectively. This confirmed that model S4 exhibited the highest uniformity, which implies that model S4 has the optimal number of hydrophobic solution applications. In model S2, leakage occurred because there was insufficient time for the hydrophobic solution to penetrate sufficiently into the substrate, and in model S6, the uniformity of the puddle region deteriorated due to an excessive amount of hydrophobic solution. Therefore, it was concluded that model S4 maintains the most appropriate uniformity and shape, making it suitable for future research and applications. These analysis results suggest that the number of hydrophobic solution applications significantly influences the size, uniformity, and leakage prevention of the puddle region during 96-PPPP fabrication.

[0102]

[0103] [Experiment 2. Characterization of Paper-Based Microfluidic Devices]

[0104] To analyze the characteristics of 96-PPP according to the example, the results after dispensing the solution were analyzed. Most studies to date have focused on colorimetric analysis suitable for paper-based platforms that are easy to interpret visually. Conventional μPADs are typically prepared by applying or printing a reagent solution directly to a specific test area of ​​the device, and the required reagent volume varies depending on the size and shape of the test area.

[0105] On the other hand, in this study, various experiments were conducted to determine the solution capacity of 96-PPP and the changes in characteristics and reliability after drying.

[0106] First, the volume of solution that a single puddle region can accommodate was tested. Referring to Fig. 3 (a), various volumes of color nanoparticle solutions (2 to 90 µL) were dispensed, and the puddle region was analyzed immediately after dispensing and after drying. At 2 µL, the puddle region was not sufficiently filled, but at volumes of 10 µL or more, it was sufficiently filled. Interestingly, due to the hydrophobic properties of the hydrophobic barrier, solutions up to 90 µL were stably maintained without overflowing into adjacent puddle regions. This means that the 96-PPP according to the present invention can accommodate not only small volume experiments but also experiments requiring large volumes, such as protein analysis and enzyme kinetics studies, and can be utilized under various experimental conditions.

[0107] In Figure 3 (b), we further tested the optimal volume for 96-PPP. Higher volumes increase the coffee ring effect, which refers to the phenomenon where particles migrate to the edges during the evaporation process, leaving ring-shaped precipitates. The performance indicators used to ensure uniform analysis within the puddle region were the gray value of the puddle region's outline and the standard deviation of the inner circle. The results showed that at a volume of 10 µL, the gray value of the outline and the standard deviation of the inner circle were 5.50 and 3.24, respectively, which were the lowest among the tested volumes. This indicates that this volume exhibits the most uniform distribution and minimal coffee ring effect, making it optimal for analysis with 96-PPP.

[0108] Referring to Figure 3 (c), the line intensity of the puddle region was analyzed according to the volume of the solution containing the color nanoparticles. This analysis was intended to understand the effect of solution volume on the distribution and uniformity of nanoparticles within the puddle region. The results showed that as the volume increased, the gray values ​​of both the puddle region and the outline decreased. Furthermore, cross-sectional analysis of the circular puddle region in various directions (horizontal, vertical, and diagonal) showed uniform gray values ​​in all directions, indicating that the distribution of nanoparticles and the formation of the puddle region were uniform. This uniformity is essential for ensuring reliable and reproducible analysis results, suggesting that consistent features can be generated through effective control of the nanoparticle solution.

[0109] Referring to Fig. 3 (d), it was observed that after the solution was dried, the value of the dried puddle region (1 / η = an indicator of the uniformity of the color distribution) decreased as the volume increased. This indicates that as the solution volume increases, the aggregation of nanoparticles becomes more dense, resulting in lower uniformity. The saturation point (τ) was determined to be 5 μL. However, to optimize the stability and absorption of the solution in the 96-PPP according to the present invention, it is preferable to use the closest volume (10 μL), as using a volume close to the saturation point can increase uniformity and reduce the risk of overflow or incomplete absorption.

[0110] Obtaining uniform results across all 96 puddle regions is critical to the effectiveness of microfluidic devices in analytical applications. Achieving consistent results when the same solution is dispensed into all 96 puddle regions ensures the reliability and reproducibility of analytical results in high-throughput screening and other applications requiring consistent and accurate measurements of multiple samples. Uniformity of results also minimizes variability, thereby preventing significant errors in quantitative analysis.

[0111] Figure 3 (e) shows the results of dispensing a colorimetric solution (PAni-NPs, 10 µL) into each of the 96 puddle regions and analyzing the gray values ​​of each puddle region. As a result of the analysis in Figure 3 (f), the gray values ​​of the 96 puddle regions adsorbed with PAni-NPs were 203.07 ± 1.33, with an error range of only 0.8%. This indicates that the colorimetric solution is uniformly distributed across all puddle regions, demonstrating the accuracy and reliability of the 96-PPP adsorbed with PAni-NPs. This level of uniformity highlights the superiority of the 96-PPP adsorbed with PAni-NPs in applications requiring high throughput and accurate analysis, and verifies its effectiveness and reliability as a microfluidic analysis device.

[0112] Figure 3 (g) shows the results of an acetone analysis performed using a single microfluidic device to further verify the effect of 96-PPP according to the present invention. This analysis was selected because acetone is a volatile solvent with a high evaporation rate, making accurate measurement difficult. The fact that the microfluidic device demonstrates the ability to handle such volatile substances highlights its robustness and reliability in various analytical applications. In Figure 3 (h), 1×10 -6 from 1x10 0 Sigmoid curve fitting analysis was performed on acetone concentrations of (%). The analysis was R 2 It showed high accuracy of 0.94. The above results demonstrate that the 96-PPP according to the present invention can provide accurate and reliable quantitative analysis even for volatile substances such as acetone.

[0113]

[0114] [Experiment 3. Comparative Analysis Between Paper-Based Microfluidic Devices and 96-Well Plates]

[0115] While conventional μPADs require dedicated applications, smartphones, or custom devices, the 96-PPP according to the present invention provides quantitative analysis using a smartphone and perfect compatibility with standard microplate readers. The 96-PPP according to the present invention is designed to be similar in size to a standard 96-well plate, allowing for rapid measurement by simply inserting it into a reader or using a smartphone.

[0116] Figure 4 (a) shows that absorbance analysis was performed using a universal pH indicator capable of measuring various pH values ​​to verify the efficacy of the 96-PPP according to the present invention. This verification included cross-checking with a standard 96-well plate. In the experiment, 10 µl of pH indicator solution was dispensed into each puddle area and dried for use. The total amount of pH solution used across all 96 puddle areas was only 0.96 ml. The 96-PPP according to the present invention exhibited uniform absorbance at wavelengths between 300 nm and 800 nm, with the largest variation observed at 540 nm (see Figure 4 (b)).

[0117] Figure 4 (c) shows the results of using a 96-well plate for comparison. In the 96-well plate, a total of 200 µl of solution (100 µl of indicator and 100 µl of pH solution) was required in each well, and a total of 9.6 ml of solution was required for all 96 wells combined. Referring to Figure 4 (d), the absorbance measurements of the 96-well plate also showed the largest differences at 450 nm, 540 nm, and 610 nm depending on the pH value.

[0118] Referring to the results above, the 96-PPP according to the present invention demonstrates efficiency and cost-effectiveness by reducing reagent usage to one-tenth while showing results comparable to a 96-well plate. Maintaining accuracy and reliability while significantly reducing reagent usage highlights the practical advantages of the paper-based microfluidic device according to the present invention, particularly in resource-constrained environments.

[0119] Figure 4 (e) shows the results of comparing the absorbance ratios of the two platforms at the two most prominent wavelengths to verify the consistency of the 96-PPP and 96-well plate according to the present invention. For comparison, wavelengths of 540 nm and 610 nm were selected. The results showed that the absorbance ratio (λ540 / λ610) decreased in both platforms as pH increased. However, at pH 11 and 12, a slight increase in the absorbance ratio was observed in both platforms. This is because the universal pH indicator contains various colorimetric indicators and interacts differently at high pH levels.

[0120] Figure 4 (f) is a graph showing the correlation between the absorbance ratios of the 96-PPP according to the present invention and the 96-well plate at various pH levels. The correlation graph is based on the linear equation y = 0.997x + 0.054 and R 2 The value was 0.973, and the p-value was <0.0001. This high correlation indicates strong agreement between the 96-PPP according to the present invention and the 96-well plate, confirming that the 96-PPP according to the present invention can reliably reproduce results obtained with a traditional 96-well plate.

[0121] Taken together, these results suggest that the 96-PPP according to the present invention improves the practicality and efficiency of paper-based microfluidic devices (μPADs) by eliminating the need for dedicated equipment and ensuring compatibility with existing microplate reader systems. This innovation has the potential to simplify high-speed processing analysis in various applications, including environmental monitoring and clinical diagnostics.

[0122]

[0123] [Experiment 4. Detection of Various Molecules]

[0124] Referring to FIG. 5, the 96-PPP according to the present invention detects not only pH but also magnesium ions (Mg 2+The ability to detect important analytes such as ), ascorbic acid, and acetoacetic acid was also evaluated. These three substances have significant biochemical and clinical implications, and their detection plays a crucial role in demonstrating the versatility and reliability of 96-PPPP according to the present invention. Mg 2+ It is an essential mineral involved in various physiological processes, ascorbic acid (vitamin C) is a powerful antioxidant, and acetoacetic acid is an important metabolite in ketone body metabolism. Detecting these analytes serves as an important indicator for evaluating the wide range of applications of 96-PPP according to the present invention.

[0125] The versatility of this platform allows Mg to be utilized in various fields such as clinical diagnostics, metabolic research, and environmental monitoring. 2+ This was verified by detecting relevant analytes such as ascorbic acid and acetoacetic acid. This is explained in detail in Figures 5 (a), (b), and (c). The change in color intensity in the puddle region corresponds to the concentration of the analyte, and Mg 2+ The higher the concentration of acetoacetic acid, the more distinct the color change becomes, and ascorbic acid exhibits strong coloring as the concentration increases. These visual differences demonstrate that the 96-PPP according to the present invention has the ability to visually distinguish various concentrations.

[0126] The absorption spectra of these analytes were measured using a microplate reader. Mg 2+ In the case of (see Fig. 5 (d)), the absorption spectrum is in the concentration range (5×10 -4 Distinct peaks are observed at different wavelengths reflecting varying absorption intensities over the range of ~ 2×10 mg / mL. In particular, a significant difference was observed at 570 nm and 700 nm. In the case of ascorbic acid (see Fig. 5 (e)), 1×10 -4Significant differences in absorbance were observed at 510 nm and 660 nm in the concentration range of ~ 1×10 mg / mL. Finally, acetoacetic acid (see Fig. 5 (f)) 1×10 -4 Significant changes in absorption were observed at 450 nm and 550 nm at a concentration of ~1X10 mg / mL, further demonstrating the detection capability of the platform.

[0127] To quantify detection capability and ensure accuracy, the absorption ratio was calculated at specific wavelengths where the most pronounced difference occurs. Each Mg 2+ It shows the absorption ratios with respect to the concentrations of , ascorbic acid, and acetoacetic acid. In Fig. 5 (g), Mg 2+ The absorption ratio of (λ570 / λ700) is 5X10 -3 Strong linear correlation with concentration (R²) over the range of ~ 2×10 mg / mL 2 It shows (= 0.98), indicating reliable detection over a wide concentration range. In Fig. 5 (h), the absorbance ratio of ascorbic acid (λ510 / λ660) exhibits a sigmoid nonlinear relationship with concentration, with a high correlation coefficient (R 2 = 0.97). Finally, in Fig. 5 (i), the absorption ratio of acetoacetic acid (λ550 / λ450) increases noticeably with increasing concentration and shows a strong correlation (R 2 = 0.97) represents.

[0128] Compared to traditional methods, the 96-PPP according to the present invention demonstrated similar performance while using significantly fewer reagents. Strong correlations and consistent results for different analytes highlight the efficiency and cost-effectiveness of the 96-PPP according to the present invention. Maintaining accuracy and reliability while significantly reducing reagent usage highlights the practical advantages of the 96-PPP according to the present invention, particularly in resource-constrained environments. Overall, the 96-PPP according to the present invention is effective against Mg 2+It demonstrated robust performance in the detection of ascorbic acid and acetoacetic acid, thereby confirming its potential for high-throughput analysis in various applications, including clinical diagnosis and environmental monitoring.

[0129]

[0130] [Experiment 5. Amyloid Fluorescence Analysis]

[0131] The fluorescence analysis performance of 96-PPP according to the present invention was evaluated using amyloid as the target analyte. Amyloid is a protein that plays a clinically important role in the pathology of neurodegenerative diseases such as Alzheimer's disease; it is insoluble and exists in various structural forms (monomers, oligomers, fibers), making it difficult to accurately measure its concentration and aggregation state. Many existing studies have relied on analyzing amyloid in solution or observing it on a planar substrate, but this approach has limited physiological relevance as it fails to reproduce complex three-dimensional structures and microenvironments such as the extracellular matrix (ECM) in vivo. In contrast, 96-PPP according to the present invention provides a microstructural environment similar to the ECM, which can simulate actual amyloid deposition behavior. Furthermore, it is advantageous for evaluating therapeutic efficacy as it allows for the simultaneous analysis of changes following treatment with amyloid degrading agents or inhibitors.

[0132] First, 96-PPPs adsorbed with amyloid were prepared and subjected to fluorescence analysis. Figure 6 (a) illustrates the amyloid-deposited 96-PPPs prepared by dispensing the amyloid solution. Figure 6 (b) shows the results of the fluorescence analysis, revealing a dome-shaped pattern where the strongest fluorescence signal is observed in the center of the puddle region and gradually decreases toward the edges. This is interpreted as the result of a concentration gradient being formed as amyloid is adsorbed at high density in the center, where initial contact with the solution occurs, and then diffuses outward. Additionally, it appears that the edges dried first due to the combined effects of capillary action and evaporation, leading to a greater concentration of amyloid in the center. These observations demonstrate the ability to visually identify deposition patterns that are difficult to confirm using conventional 96-well plate-based solution analysis.

[0133] Next, the reproducibility and uniformity of the 96-PPP according to the present invention were verified. Figure 6 (c) shows the results of fluorescence analysis of five 96-PPP sheets prepared under the same conditions. The respective fluorescence intensities were measured as 46,732 ± 1,466, 48,361 ± 1,330, 46,397 ± 1,265, 46,665 ± 1,636, and 46,953 ± 1,258, with an overall average value of 47,295 ± 1,656. The error rate was very low at approximately 3.5%, confirming that amyloid was uniformly adsorbed throughout the 96-PPP. Additionally, when the amyloid solution was diluted 10-fold and dispensed equally, the fluorescence intensity decreased by approximately 8,371 units to approximately 38,924 ± 1,039. This suggests that the 96-PPP platform can quantitatively reflect differences in amyloid signals according to changes in concentration.

[0134] Next, the response to amyloid-degrading enzymes was analyzed. Figures 6 (d) and 6 (e) schematically illustrate the degradation and amplification patterns when amyloid was treated with pepsin. At a pepsin concentration of 0.05 mg / mL, amyloid showed a gradual degradation pattern, while at 0.25-0.5 mg / mL, it showed a tendency to increase again after a temporary decrease. At high concentrations of 1-2 mg / mL, amyloid was instead continuously amplified. This is consistent with reports in the existing literature and implies that amyloid-degrading enzymes do not simply degrade proteins but can induce new fibrillation and amplification under specific conditions.

[0135] Figure 6 (f) shows the results of fluorescence analysis when pepsin solutions of various concentrations were dispensed onto amyloid-deposited 96-PPP. It was observed that the fluorescence intensity increased relative to the baseline as the pepsin concentration increased, which demonstrates that the amplification phenomenon reported in solution is reproduced on the 96-PPP platform. Additionally, amyloid amplification was minimized when heat-denatured pepsin was used, confirming that amyloid amplification occurs selectively due to enzymatic activity.

[0136] Figure 6 (g) illustrates a fluorescence image of pepsin-treated amyloid, demonstrating that the fine spatial distribution of amyloid, which cannot be confirmed by conventional 96-well plate analysis, can be observed at high resolution. Figure 6 (h) shows a graph of the cross-sectional fluorescence intensity analysis results, confirming that the signal distribution at the center and edges differs significantly depending on the pepsin concentration. Figure 6 (i) shows the result of amyloid accumulating in a ring shape at the edges due to Marangoni flow under untreated conditions (0 mg / mL), indicating that this is a natural pattern formation caused by solution evaporation and differences in surface tension. On the other hand, Figure 6 (j) illustrates the phenomenon where the fluorescence intensity at the center of the puddle increases again as the pepsin concentration increases, and amyloid is re-amplified and accumulates. This suggests that pepsin degrades amyloid while simultaneously promoting a new fibrillation process, resulting in an effect that reinforces central accumulation.

[0137] Additionally, a similar amplification trend to that of pepsin was observed when trypsin was used, although there were variations in the initial basal intensity due to differences in the buffer conditions of the two enzymes (pH 2 DW vs. pH 7 DW). These results demonstrate that the patterns of amyloid amplification and degradation can vary depending on the type of enzyme and conditions, proving that 96-PPP is a useful platform for comparing and analyzing these patterns at high throughput.

[0138] Consequently, the 96-PPP platform according to the present invention can simultaneously perform quantitative analysis through fluorescence intensity and qualitative analysis through fluorescence imaging, and enables high-throughput amyloid analysis with high reproducibility using only a small amount of reagent. This provides a new technical means to comprehensively evaluate the natural deposition patterns of amyloid, degradation and re-amplification processes following enzymatic treatment, and surface interactions based on the microenvironment, and has very high potential for industrial application in verifying the efficacy of amyloid inhibitors and degraders, early diagnosis and monitoring of neurodegenerative diseases, and large-scale screening studies.

[0139]

[0140] [Experiment 6. Modification of Puddle Structure Using Beads]

[0141] A locally concave structure was formed by applying pressure with a steel ball or a convex object to the upper part of the puddle region of the 96-PPP. This structural deformation induces droplets to stably gather towards the center, and the depth and diameter of the concave portion can be controlled according to the size of the pressure tool and the strength of the force. When this structure is applied, the spreading of droplets is suppressed, and the local concentration of reactants increases, concentrating the color change signal and thereby improving sensitivity (see Fig. 11a).

[0142]

[0143] [Experiment 7. Measurement of Color Sensitivity and Sensing Efficiency According to Adhesive Tape Contact]

[0144] A process was performed in which an adhesive tape was attached to and then removed from the surface of 96-PPP. During this process, a cellulose fiber layer of a certain thickness is removed along with the tape; consequently, the fiber density on the surface is lowered, and a structure is formed in which particles of the same volume can be adsorbed at a relatively higher rate. This structural change induces higher signal intensity under the same conditions, which can contribute to improved sensitivity and a lower detection limit (see Fig. 11b).

[0145]

[0146] [Experiment 8. Measurement of Chemical Compatibility]

[0147] No structural collapse or degradation of barrier performance was observed in 96-PPP even when exposed for more than 18 hours to methanol, ethanol, acetonitrile, sodium dodecyl sulfate (SDS), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, cetrimonium bromide (CTAB), Triton X-100, polysorbate 20, formaldehyde, sodium hydroxide (NaOH), deionized water, and solutions with pH 1, 4, 7, and 12, respectively. This implies that solvent penetration or interlayer separation problems, which commonly occur in conventional paper-based structures, are suppressed. Therefore, the device (platform) has technical significance in that it maintains stable barrier properties in various polar and non-polar solvent environments, demonstrating excellent chemical compatibility (see Fig. 13).

[0148]

[0149] [Experiment 9. Measurement of Reproducibility]

[0150] As a result of continuously fabricating five sets of 96-PPPs within 40 minutes, uniform puddle diameter, depth, and hydrophobic barrier properties were achieved in all samples. This fabrication reproducibility is suitable for high-speed mass production and guarantees the reliability of repeated experiments under identical conditions. This has the technical effect of securing industrial applicability as a standardized platform (see Fig. 14).

[0151]

[0152] [Experiment 10. Durability Measurement]

[0153] Even when 96-PPP was immersed in deionized water for 3 days or treated in an oven at 100°C for 3 days, no structural damage or breakdown of barrier properties occurred. In addition, stability was maintained without performance degradation even when stored at room temperature for more than 6 months. These results indicate that the device (platform) can ensure physical and chemical stability even under long-term storage and various harsh conditions, which provides significant advantages for actual field applications and long-term storage and distribution processes (see Fig. 13).

[0154]

[0155] The foregoing description is merely an illustrative explanation of the technical concept of the present invention, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these embodiments. The scope of protection of the present invention shall be interpreted by the claims below, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

Claims

1. Porous substrate; and A paper-based microfluidic device having a pattern layer formed on the porous substrate and comprising a plurality of puddles and a hydrophobic barrier formed in an area other than the region where the puddles are located.

2. In Paragraph 1, A paper-based microfluidic device, wherein the porous substrate is a paper material comprising cellulose, cellulose acetate, cellulose nanofibrils, carboxymethylcellulose, hydroxypropylcellulose, and one combination thereof.

3. In Paragraph 1, A paper-based microfluidic device having a concave structure in which a solution of 0.01 to 90 μL can be contained within each of the puddles.

4. In Paragraph 1, The above puddle is a paper-based microfluidic device having one shape selected from circular, elliptical, ring-shaped, polygonal, linear, spiral, branched, and combinations thereof.

5. In Paragraph 1, A paper-based microfluidic device comprising one selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof.

6. In Paragraph 1, A paper-based microfluidic device having a protective film further provided on the pattern layer.

7. In Paragraph 1, The microfluidic device is a paper-based microfluidic device having a thickness in the range of 0.275 to 0.325 mm.

8. In Paragraph 1, A paper-based microfluidic device in which, when an adhesive tape is brought into contact with the microfluidic device one or more times to reduce the puddle depth to 65% or less of the initial depth, the color sensitivity and sensing efficiency are improved by more than 200% compared to the initial value.

9. In Paragraph 1, The above microfluidic device is a paper-based microfluidic device used for one or more types of analysis selected from colorimetric analysis, absorbance analysis, and fluorescence analysis.

10. In Paragraph 1, The above microfluidic device is a paper-based microfluidic device compatible with one type of micro-well plate selected from 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, and 384-well plates, and applied to smartphones, microplate readers, or fluorescence microscopes.

11. A method for manufacturing a microfluidic device according to claim 1, a) A step of preparing a porous substrate; b) a step of positioning a silk screen having opaque patterns corresponding to a plurality of puddle shapes formed thereon on the above substrate and applying a hydrophobic solution to form a hydrophobic barrier in an area other than the area where the plurality of puddles are located; and c) a step of curing the hydrophobic barrier by heating at least once at a temperature within the range of 100 to 180 ℃ after step b above; comprising a method for manufacturing a paper-based microfluidic device.

12. In Paragraph 11, A method for manufacturing a paper-based microfluidic device, wherein the hydrophobic solution of step b above is a mixture of one polymer selected from polydimethylsiloxane (PDMS), polydiethylsiloxane (PDES), polymethylphenylsiloxane (PMPS), polydiphenylsiloxane (PDPS), silicone resin, fluorosilicone, and combinations thereof, and a curing agent in a weight ratio of 8.5 : 1.5 to 9.5 : 0.

5.

13. In Paragraph 11, A method for manufacturing a paper-based microfluidic device, further comprising the step of attaching a protective film to a pattern layer of the microfluidic device during step c above.

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