Patch-type body fluid collection and testing system with porous microneedles and method for manufacturing the same

Abstract

[Problem] To provide an inspection device provided with a porous microneedle that has high mechanical strength and that allows more rapid sampling of interstitial fluid, etc., with capillary force; and to provide a microneedle manufacturing method. [Solution] An inspection device which includes a porous microneedle and a paper substrate sensor that has at least one measurement region, wherein the microneedle is formed from microspheres of a biodegradable material.

Description

[Technical field]

[0001] The present invention relates to a patch-type bodily fluid sampling and testing system having a porous microneedle, and a method for manufacturing the microneedle. The present invention also relates to a patch-type bodily fluid sampling and testing system having a structure in which a porous microneedle and a paper-based sensor are integrated. [Background technology]

[0002] In recent years, point-of-care testing (POCT) devices that can provide health monitoring through real-time rapid diagnosis and testing have been attracting much attention (Non-Patent Document 1). For example, there are hopes for diagnoses using microchips for health monitoring that can diagnose lifestyle-related diseases, known as adult diseases, such as diabetes, high blood pressure, cancer, stroke, and heart disease, as well as infectious diseases such as influenza and COVID-19, from minute amounts of blood.

[0003] The number of diabetes patients, a representative lifestyle-related disease, is estimated at 10 million in Japan, and 20 million including those at risk, and it has also become a major health issue worldwide. Diabetes is a disease that cannot be cured completely with current medical technology, and managing blood glucose levels and preventing the onset of complications are important strategies for managing patients' risks. Therefore, continuous monitoring of blood glucose levels in daily life is essential for those at risk of diabetes or those with diabetes.

[0004] However, the currently commercially available self-monitoring blood glucose (SMBG) kits are painful because they measure blood glucose levels by pricking a finger with a needle to draw blood. To reduce the burden on patients, research has been conducted on diagnostic devices that track blood glucose levels using media such as tears, urine, and sweat, but there are many issues such as the inconvenience of wearing the devices, low measurement accuracy, and the fact that dirt can affect the measurement results.

[0005] Meanwhile, interstitial fluid (ISF) inside the skin has become a promising alternative to blood samples because ISF contains abundant biomarkers (such as glucose, cholesterol, and proteins) that can accurately reflect their concentrations in blood (Non-Patent Document 2). Therefore, it is necessary to develop a simple and minimally invasive approach to extract skin ISF for routine self-medication monitoring in routine preventive medicine.

[0006] In addition, minimally invasive biosensors using needles less than 1 mm long called microneedles are attracting attention. Microneedle (MN) arrays are an effective approach to puncture the dermis layer to painlessly extract ISF, and so far, microneedles with fine hollow structures, microneedles made of hydrogel, and porous microneedles have been reported. However, MNs with hollow structures made of metal or silicon are easily broken, and fragments of MNs may remain in the skin and cause damage to the human body. In addition, a biodegradable polymer, MN, with a porous structure has been attracting much attention in recent years. However, various issues remain to be overcome before it can be put to practical use, such as the complex processing required, the time required for production, and the difficulty in obtaining sufficient mechanical strength (e.g., strength that allows easy puncture of the skin). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] B. Lei and T. Prow, “A review of microsampling techniques and their social impact”, Biomed. Microdevices, 21, 4 (2019), pp1-30. [Non-Patent Document 2] B. Martin and H. Derendorf, “Clinical microdialysis: Current applications and potential use in drug development”, Trends Anal. Chem, 25, 7 (2006), pp674-680. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0008] The present invention aims to provide a testing device equipped with a porous microneedle that has high mechanical strength and is capable of quickly collecting interstitial fluid or the like by capillary force, and a method for manufacturing the microneedle. Another object of the present invention is to provide a testing device that includes a porous microneedle and a paper-based sensor and is capable of measuring components in interstitial fluid in multiple measurement areas. [Means for solving the problem]

[0009] The present inventors have been investigating a method for producing porous microneedles made of biodegradable polymers using the "salt leaching method" (a method for obtaining pores by dissolving particles of mixed salt such as NaCl). Although the pore structure (pore size and porosity) can be easily controlled by the salt leaching method, the processing is complicated and it is difficult to obtain sufficient mechanical strength.

[0010] Therefore, after extensive research, the inventors discovered that porous microneedles can be produced from microspheres of biodegradable polymers such as polylactic acid by heat treatment, and that the porous microneedles obtained in this manner have high mechanical strength, thus completing the present invention. The inventors have also discovered that by directly connecting such a porous microneedle array to a paper substrate sensor, it is possible to provide a testing device that is capable of quickly measuring blood glucose levels, etc. in a minimally invasive manner.

[0011] That is, the present invention provides: [1] Porous microneedles, and Paper-based sensor having at least one measurement area - Patents.com Including, The microneedles are formed from microspheres of a biodegradable material. Inspection equipment. [2] The testing device described in [1], wherein the microneedle is made of microspheres of a biodegradable material bonded to each other to form a network of interconnected pores. [3] The inspection device according to [1] or [2], wherein the biodegradable material includes at least one of polylactic acid, polyglycolic acid, poly(lactide-co-glycolide) copolymer, PEG copolymer, polyhydroxybutyric acid, and ethyl cellulose. [4] The inspection device according to any one of [1] to [3], further comprising a microneedle substrate, the microneedles being joined to the microneedle substrate. [5] The inspection device according to any one of [1] to [4], further comprising a flow path layer between the paper substrate sensor and the microneedle. [6] The inspection device according to any one of [1] to [3], wherein the microneedles and the paper base sensor are integrated. [7] The inspection device according to any one of [1] to [6], wherein the microneedle has a breaking compressive strength of 0.5 N or more measured under the following conditions: Conditions: A compressive load is applied in the axial direction to a single microneedle, and the load at the yield point obtained from the load-displacement curve is measured as the breaking strength. [8] (a) preparing a biodegradable material microsphere solution or suspension containing microspheres of a biodegradable material; (b) injecting the solution or suspension into a female mold; (c) drying the solution or suspension to obtain a microneedle precursor; and (d) heating the microneedle precursor at a predetermined temperature so that the microspheres are partially in a liquid phase or rubbery state and bonded together; A method for producing a porous microneedle comprising the steps of: [9] The manufacturing method described in [8], wherein the biodegradable material microsphere solution or suspension is prepared by preparing solution A by dissolving a biodegradable material in an organic solvent, mixing the solution A with an aqueous solution containing a surfactant, then evaporating the organic solvent and stirring.

[10] A porous microneedle obtained by the manufacturing method according to [8] or [9].

[11] A microneedle formed from microspheres of a biodegradable material, the microspheres being bonded to each other to form a network of interconnected pores.

[12] The microneedle according to

[11] , having a breaking compressive strength of 0.5 N or more measured under the following conditions: Conditions: A compressive load is applied in the axial direction to a single microneedle, and the load at the yield point obtained from the load-displacement curve is measured as the breaking strength.

[13] A microneedle array comprising a plurality of the microneedles according to any one of

[10] to

[12] arranged upright on a microneedle substrate. This provides: Effect of the Invention

[0012] The present invention can provide a porous microneedle that has high mechanical strength and is capable of rapidly collecting interstitial fluid or the like by capillary force. In addition, the porous microneedle manufacturing method of the present invention can easily obtain a porous microneedle without requiring complicated steps such as those in the conventional manufacturing method using the salt leaching method. Furthermore, in the porous microneedle manufacturing method of the present invention, the microspheres are bonded together, so that a flow path can be formed with a smaller porosity than that obtained by forming pores such as in the salt leaching method. In other words, the porous microneedle obtained by the manufacturing method of the present invention has a high-density structure, and can achieve both the above-mentioned mechanical strength and fluid performance.

[0013] The present invention also provides a testing device that includes a porous microneedle and a paper substrate sensor and is capable of measuring components in interstitial fluid in multiple measurement regions. Such a testing device can simultaneously detect and measure multiple biomarkers such as blood glucose levels and cholesterol. During the measurement, the reaction in the paper substrate of the sensor structure attached to the skin can be visualized by a color change or the like. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a schematic diagram for comparing the morphology of microneedles obtained by the salt leaching method and the microneedles of the present invention. [Diagram 2] 1 shows a non-limiting example of an inspection device of the present invention. [Diagram 3] FIG. 1 shows a schematic diagram of how porous microneedles can be molded directly onto a paper-based sensor. [Figure 4] 1 shows an overview of a glucose concentration measuring microneedle patch equipped with a testing device of the present invention. [Diagram 5] 1 shows the fabrication process of the porous polylactic acid microneedle (PLA MN) of the present invention. [Figure 6] The (a) shape and (b) dimensions of the porous PLA MN after heat treatment are shown (n=5 for the dimension measurements). [Figure 7] 1 shows the results of investigating the porous structure of the porous PLAMN obtained in Example 1. [Figure 8] 1 shows the results of measuring the porosity of the porous PLA MN obtained in Example 1. [Figure 9] The diagram on the left shows an outline of a test method for measuring the absorption volume of a sample fluid using the porous PLA MN prepared in Example 1, and the measurement results (right) are shown. [Figure 10] FIG. 1 shows a schematic diagram of extraction of glucose-loaded ISF from 1% agarose gel and evaluation of blue color development. [Figure 11] 1 shows the results of evaluating the extraction and sensing performance of glucose-loaded sample fluid using the porous PLA MN prepared in Example 1. [Figure 12] The diagram on the left shows an outline of the test method for measuring the mechanical strength of the porous PLA MN prepared in Example 1, and the obtained load-displacement curve (right) is shown. [Figure 13] The figure shows the shape (left) of the porous PLA MN prepared in Example 1 after a test to measure its mechanical strength, and the obtained mechanical strength (right). [Figure 14] The schematic diagram of the insertion test method of porous PLA MN using pig skin (top diagram) and the results of the insertion test using porous PLA MN after heat treatment at different temperatures (middle and bottom diagrams) are shown. [Figure 15] An overview of connecting a porous microneedle substrate and a paper-based sensor using transparent tape (top image) and an overview of connecting via a flow path layer (bottom image) are shown. [Figure 16] The graph on the left shows the blue color development of the paper-based sensor in response to the glucose concentration in a phosphate-buffered saline solution extracted from a 1% agarose gel by a porous microneedle, and the results of quantitative measurement of the color intensity using an image processing program (right). [Figure 17] This shows a comparison of the detection limit concentration of glucose between a patch-type body fluid sampling and testing system with porous microneedles and a paper-based colorimetric sensor and a microneedle electrochemical sensor that do not use microneedles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] One embodiment of the present invention is a testing device (hereinafter also referred to as the "test device of the present invention") that includes a porous microneedle and a paper-based sensor having at least one measurement area, the microneedle being formed from microspheres of a biodegradable material. Each component of the inspection device of the present invention will be described in detail below.

[0016] 1. Microneedling (1) Structure and characteristics of microneedles The microneedle used in the testing device of the present invention (hereinafter also referred to as the "microneedle of the present invention") is porous and formed from a microsphere of a biodegradable material. Specifically, the microneedle of the present invention is manufactured using a microsphere of a biodegradable material. In this specification, the term "microsphere" refers to spherical fine particles having an average particle size on the order of μm (preferably 1 to 100 μm, more preferably 5 to 30 μm, and even more preferably 10 to 20 μm). The average particle size is usually determined by measurement using an optical microscope. In the microneedle of the present invention, preferably, microspheres of a biodegradable material are bonded to each other to form a network of interconnected pores. Although it is not intended to be bound by theory, conventional porous microneedles formed from biodegradable resins have been manufactured by a salt-leaching method using water-soluble particles such as sodium chloride, but the microneedles of the present invention have a different morphology from microneedles obtained by such a method. That is, in the salt-leaching method, a biodegradable material and water-soluble particles are usually mixed, the mixture is filled into a dispenser or the like, droplets are discharged, and the mixture is shaped into a microneedle, which is then immersed in water to dissolve the water-soluble particles. When the water-soluble particles are removed, the site where the water-soluble particles were present becomes a void, and a porous microneedle is obtained (see, for example, WO2019 / 176126). In contrast, the microneedle of the present invention is produced by injecting a microsphere solution of a biodegradable material into a female mold, drying the microneedle precursor, and heating the microspheres to about 150 to 250° C. to bond the microspheres together, forming a network of interconnected (communicating) continuous pores, which results in a robust pore structure in the microneedle of the present invention.

[0017] In the microneedle of the present invention, it is not necessary for all of the microspheres to be bonded to each other, but it is preferable that the microspheres constituting the microneedle precursor are in a state in which it can be confirmed by an electron microscope or the like that they are bonded to each other by heating, as shown in Figure 7.

[0018] The biodegradable material constituting the microneedle of the present invention contains at least one of polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), PEG copolymer, polyhydroxybutyric acid, and ethyl cellulose. Here, the microneedle of the present invention may be composed only of the above-mentioned biodegradable materials, or may contain trace amounts of raw materials used in the method for producing the microneedle of the present invention described below (e.g., polyvinyl alcohol, methyl cellulose, sorbitan fatty acid esters, sorbitan monooleate, surfactants such as sodium dodecyl sulfate and hexadecyltrimethylammonium bromide, etc.) and other additives (e.g., carboxymethyl cellulose (CMC), hyaluronic acid) within a range that does not impair the function of the microneedle of the present invention. In addition, the microneedle of the present invention may be at least partially coated with a coating agent without impairing its function. As the coating agent, materials commonly used in this technical field (CMC, hyaluronic acid, etc.) can be used.

[0019] The shape of the microneedle of the present invention may be an approximately conical or pyramidal shape, but a polygonal shape (for example, an approximately pyramidal shape) is preferred as it penetrates the skin more easily than an approximately conical shape.

[0020] The diameter of the tip of the microneedle of the present invention is usually 10 μm to 60 μm, and the diameter or maximum dimension of the base is, for example, about 50 μm to 800 μm. The height of the microneedle determines the depth of penetration into the skin. In the microneedle of the present invention, the height is preferably 300 μm or more and 1500 μm or less, so as to reach the dermis and not stimulate pain sensation.

[0021] When multiple microneedles are provided, the smaller the intervals are, the better for absorbing a sample of interstitial fluid, and an interval of 500 to 5000 μm is preferable.

[0022] Regarding the angle of the tip of the microneedle, the larger the angle, the greater the mechanical strength, but the greater the force required for penetration. A tip angle of 15 to 30° is preferable, since the force required for the microneedle to penetrate is less than 0.2 N.

[0023] The microneedle of the present invention has a strength of 0.1 N or more, preferably 0.5 N or more, in terms of the load at the yield point measured under the following conditions. Conditions: A compressive load is applied in the axial direction to a single microneedle, and the load at the yield point obtained from the load-displacement curve is measured as the breaking strength. The microneedle of the present invention has such high mechanical strength, which makes it possible to realize the testing device of the present invention. As described above, the microneedle of the present invention has a robust pore structure formed by forming a continuous pore network that is interconnected (communicated) by bonding microspheres together. As a result, the microneedle of the present invention is porous and can have higher mechanical strength than microneedles obtained by the conventional salt-leaching method. This point will be explained in more detail with reference to the schematic diagram of FIG. 1. That is, in the salt leaching method, a certain porosity or more is required to connect the pores, and therefore a porosity of about 60% is required. Therefore, when a flow path is formed, the porosity becomes high and the mechanical strength becomes low. On the other hand, in the microneedle of the present invention obtained by the microsphere method, even if the porosity is low, continuous voids are formed in the gaps between the microspheres. Therefore, it has high mechanical strength while maintaining the flow path.

[0024] The porosity of the microneedle of the present invention is usually 10 to 40%, preferably 20 to 30%. Here, the porosity is measured by the water absorption method using a porous membrane, comparing the mass before and after fluid extraction to measure the porosity of the porous microneedle according to the following procedure (see P. Liu, et al., J Mater Chem B, 2020). First, the dry mass (W dry ) is recorded, and then the membrane is immersed in deionized (DI) water and the surface water is removed after the absorption is saturated. Then, the mass is immediately measured and W wet Record as . Calculate the porosity using the following formula:

[0025]

number

[0026] In formula (1), ρ p is the density of the biodegradable material, ρ 0 is the density of DI water (1.0 g / cm 3 ).

[0027] The microneedles of the present invention have excellent water absorption capabilities such as water absorption speed, etc. As shown in the examples, when the microneedle precursor is heated at a high temperature, the microspheres are partially transformed into a liquid phase or rubbery state by heat treatment, and are bonded to form a strong interconnected micropore network, which can efficiently extract skin interstitial fluid by capillary force.

[0028] One index of water absorption capacity is absorption volume, and the microneedle of the present invention has an absorption volume of usually 10 to 150 μL, preferably 60 to 120 μL. Here, the absorption volume is measured by piercing a microneedle array having 169 porous PLA MNs erected in a 1% agarose gel, removing it from the gel after 2 minutes, and measuring its weight.

[0029] Furthermore, the absorption rate of the microneedle of the present invention is usually 0.01 to 0.3 μL / min, and preferably 0.2 to 0.3 μL / min per microneedle. Here, the absorption rate is measured by piercing a microneedle array having 169 porous PLA MNs upright in a 1% agarose gel, removing it from the gel after 2 minutes, and measuring its weight.

[0030] The microneedles of the present invention may themselves be provided on a paper-based sensor.

[0031] A plurality of the microneedles of the present invention may be arranged upright on a microneedle substrate to form a microneedle array, which may then be joined to a paper-based sensor. That is, another aspect of the present invention is a microneedle array in which a plurality of microneedles of the present invention are arranged upright on a microneedle substrate (hereinafter also referred to as "the microneedle array of the present invention").

[0032] In the microneedle array of the present invention, the microneedles can be arranged vertically and horizontally as appropriate. The spacing between the microneedles is preferably small in order to absorb samples of interstitial fluid, and is preferably 500 to 5000 μm.

[0033] The microneedle substrate may be formed from the same material as the microneedles or from a different material.

[0034] In one embodiment, the microneedle substrate is composed of a film or a hydrocolloid film containing at least one of polylactic acid resin, polyvinyl alcohol resin, polymethyl methacrylate resin, and polyurethane resin.

[0035] In another embodiment, the microneedle substrate is formed from a biodegradable material, the biodegradable material including at least one of polylactic acid, polyglycolic acid, poly(lactide-co-glycolide) copolymer, PEG copolymer, polyhydroxybutyric acid, and ethyl cellulose. In one preferred embodiment, the microneedle substrate is formed from the same biodegradable material as the microneedles, and the two are integrally constructed.

[0036] (2) Manufacturing method of microneedles Another embodiment of the present invention is (a) preparing a biodegradable material microsphere solution or suspension containing microspheres of a biodegradable material; (b) injecting the solution or suspension into a female mold; (c) drying the solution or suspension to obtain a microneedle precursor; and (d) heating the microneedle precursor at a predetermined temperature so that the microspheres are partially in a liquid phase or rubbery state and bonded together; This is a method for producing a porous microneedle, which includes the steps of:

[0037] The biodegradable material includes at least one of polylactic acid, polyglycolic acid, poly(lactide-co-glycolide) copolymers, PEG copolymers, polyhydroxybutyric acid, and ethylcellulose.

[0038] The particle size of the microspheres of the biodegradable material is preferably 5 to 30 μm. If the particle size of the microspheres is within this range, it is preferable in terms of achieving both mechanical strength and fluid performance.

[0039] The solution or suspension of biodegradable microspheres means a liquid in which the biodegradable microspheres are dissolved or dispersed in water or an organic solvent, preferably a suspension of biodegradable microspheres, more preferably a suspension in which the biodegradable microspheres are dispersed in water.

[0040] In the manufacturing method of the present invention, the biodegradable material microsphere solution is preferably prepared by preparing solution A by dissolving a biodegradable material in an organic solvent, mixing the solution A with an aqueous solution containing a surfactant, and then evaporating the organic solvent.

[0041] The organic solvent is not particularly limited as long as it dissolves the biodegradable material, and examples thereof include dichloromethane and acetone.

[0042] The concentration of the biodegradable material in solution A is, for example, 0.05 to 0.1% (w / v).

[0043] The type of surfactant is preferably polyvinyl alcohol (PVA), CMC (carboxymethyl cellulose), etc. These surfactants can reduce the surface tension of the solution obtained by mixing solution A with an aqueous solution, and stabilize the resulting microspheres. In addition, solution A and / or the aqueous solution containing a surfactant may contain other additives (e.g., carboxymethylcellulose (CMC), hyaluronic acid) to the extent that the function of the resulting porous microneedle is not impaired.

[0044] The solution obtained by mixing solution A with the aqueous solution can be stirred at about room temperature at 500 to 1500 ppm using a magnetic stirrer or the like to evaporate the organic solvent.

[0045] In step (b), a solution or suspension of biodegradable microspheres is poured into a female mold. The mold used here is a female micromold prepared from a metal master mold consisting of a large number of microneedles, and its material is preferably polydimethylsiloxane (PDMS), SUS, or the like. The shape and size of the microneedles of the metal master mold can be appropriately determined according to the shape and size of the desired microneedle.

[0046] The mold may have only the mold shape of the microneedle to be prepared. In this case, a single microneedle can be obtained. In addition, when manufacturing the inspection device of the present invention in which the microneedle and the paper base sensor are integrated as described below, it is preferable to provide such a cavity in the female micro mold. The female micromold can have a desired number of cavities. The female micromold can have cavities arranged vertically and horizontally as appropriate. The interval between the cavities is usually 500 to 5000 μm, preferably 1000 to 3000 μm.

[0047] The cavity may have a shape in which a plurality of microneedles are joined to a microneedle substrate. In this case, a microneedle array in which a plurality of microneedles are joined to a microneedle substrate and erected can be obtained. The cavity of the micromold itself may have a desired number of cavities. The cavities of the micromold itself may be appropriately arranged vertically and horizontally. The interval between the cavities is preferably 500 to 5000 μm.

[0048] It is preferable to inject a solution or suspension of biodegradable microspheres into the cavity of the female mold, and then place the mold in a vacuum or apply centrifugal force to fill the cavity with the microspheres.

[0049] In step (c), the solution or suspension of biodegradable microspheres is dried to evaporate the water, solvent, and dispersant. As a drying method, a piping may be provided in the female micromold to control the temperature, or the entire micromold may be placed in a dryer such as a convection oven to dry it. The drying temperature is preferably 25 to 100° C., and the drying time can be appropriately determined, but is, for example, 1 to 24 hours.

[0050] After drying, water evaporates from the solution or suspension of biodegradable microspheres to obtain a microneedle precursor composed of biodegradable microspheres. At this stage, the microneedle precursor may be removed from the mold and subjected to the next step (d). Alternatively, at this stage, the microneedle precursor may be left in the mold and subjected to the next heating step (d) without being removed from the mold.

[0051] In step (d), the microneedle precursor is heated at a predetermined temperature. In the microneedle precursor, each microsphere maintains its shape and is not bonded to each other. In the manufacturing method of the present invention, it is important to heat the microneedle precursor at a high temperature to bond the microspheres to each other. The heating temperature must be such that the microspheres are deformed and bonded to each other, and varies depending on the type of biodegradable resin. For example, in the case of polylactic acid, the heating temperature is preferably 170 to 200°C, more preferably 170 to 190°C. In the case of polyglycolic acid, the temperature is preferably 170 to 250°C. In the case of a poly(lactide-co-glycolide) copolymer, the temperature is preferably 50 to 200°C. In the case of a PEG copolymer, the temperature is preferably 30 to 200°C. In the case of polyhydroxybutyric acid, the temperature is preferably 100 to 200°C. In the case of ethyl cellulose, the temperature is preferably 80 to 300°C. The drying time is, for example, 1 to 24 hours.

[0052] The porous microneedles obtained by the above-mentioned manufacturing method of the present invention have a network of interconnected (communicating) continuous pores formed by partially converting the microspheres into a liquid phase or rubber state and bonding them together, thereby forming a robust pore structure. The porous microneedles obtained by the manufacturing method of the present invention have higher mechanical strength and superior water absorption capacity than microneedles obtained by conventional methods.

[0053] 2.Paper-based sensor The testing device of the present invention includes a paper substrate sensor having at least one measurement area. The capillary action of a liquid on a porous medium can be described by the following Washburn equation (2), and based on this, the inventors have investigated a method for reducing the analysis time of components in interstitial fluid.

[0054]

number

[0055] In equation (2), L is the flow distance of the liquid, γ is the surface tension of the liquid, R is the radius of the pore, μ is the viscosity of the liquid, θ is the contact angle between the liquid and the porous material, and t is the flow time.

[0056] Since there is a practical limit to increasing the hydrophilicity and pore size in a porous medium, the present inventors focused on the flow distance (L). Since microneedles are usually fabricated on a microneedle substrate, it is possible to reduce the thickness of the microneedle substrate to reduce the movement distance. However, the thickness of the microneedle substrate is affected by the manufacturing method, and it is difficult to strictly control the thickness. Therefore, the inventors thought that paper could be a suitable substrate because it is a porous medium with strong water absorption, and came up with a structure that integrates microneedles and a paper substrate sensor. Paper can be several hundred micrometers thick, and the flexibility of paper increases its usefulness as a patch type for human skin. And once the porous microneedles absorb the analyte, the paper substrate can rapidly transport it to the sensing area.

[0057] The paper substrate sensor has a paper substrate and at least one measurement area. As the paper substrate, a filter paper is preferably used. As the filter paper, a filter paper for quantitative analysis as specified by JIS P3801 is preferable, and a filter paper or a nitrocellulose membrane having a thickness of 100 to 500 μm is more preferable.

[0058] The paper-based sensor has at least one measurement area in a paper substrate such as filter paper, and detects the reaction with components in the interstitial fluid such as glucose. The measurement is mainly a colorimetric measurement using enzymes, and can determine the concentration and detection of components in the interstitial fluid.

[0059] The measurement areas include an area for measuring the target components (glucose, cholesterol, cortisol, etc.) in the interstitial fluid, and a body fluid reaction area for confirming collection of body fluid. The region for measuring the component to be detected contains an enzyme, a peroxide reacting substance, or a color-developing dye that reacts with the component.

[0060] For example, the area for measuring glucose includes glucose oxidase (GOx), peroxidase (HRP), and a color-developing dye such as tetramethylbenzidine (TMB). In the area of ​​measuring cholesterol, cholesterol oxidase is included. The body fluid reaction area, which confirms collection of body fluid, contains cobalt chloride, etc.

[0061] A paper-based sensor may have one measurement area, or may have two or more measurement areas. The inspection device of the present invention may be provided with a plurality of paper substrate sensors each having one measurement area.

[0062] 3. Inspection Equipment The testing device of the present invention essentially comprises the microneedle of the present invention and a paper-based sensor having at least one measurement area.

[0063] The testing device of the present invention may further comprise a microneedle substrate, in which case the microneedles are joined to the microneedle substrate and the testing device comprises a microneedle array. The microneedle array and the paper-based sensor can be attached by adhesion or pressure bonding.

[0064] The testing device of the present invention may further comprise a flow path layer between the paper substrate sensor and the microneedle (or microneedle array). The flow path layer is made of a water-absorbent material such as cellulose or filter paper, and has the function of limiting the exudation range of interstitial fluid according to the measurement area of ​​the paper-based sensor. In particular, when the paper-based sensor has multiple measurement areas, it is preferable to provide a flow path layer.

[0065] The flow path layer can be formed by first attaching a double-sided tape with holes (e.g., about 2 mm in diameter) to the microneedle substrate and filling the holes with cellulose powder, and then attaching the paper-based sensor layer to the other side of the double-sided tape to form a fluid channel from the microneedle to the paper-based sensor.

[0066] The inspection device of the present invention may have one or more measurement areas in one paper substrate sensor, and may also have two or more measurement areas. The inspection device of the present invention may be provided with a plurality of paper substrate sensors each having one measurement area.

[0067] A non-limiting example of a test device of the present invention is shown in Figure 2. The test device shown in this figure is provided with three paper-based sensors having measurement areas for glucose, cholesterol, and cortisol, respectively.

[0068] Such a testing device with multiple measurement areas can simultaneously detect and measure multiple biomarkers such as glucose, cholesterol, etc. During the measurement, the reaction in the paper substrate of the sensor structure attached to the skin can be visualized by color changes, etc., and information such as the concentration of the biomarkers can be obtained and analyzed on the spot by combining optical measurements such as with a camera and analysis of color concentration by software.

[0069] For example, blood glucose levels can be confirmed from a standard color chart or by comparing with a standard color change. If necessary, the blood glucose level can be quantified using an application on a mobile device, etc. For example, if a person wants to know their blood glucose level in detail, they can take a photo using an application with image processing capabilities and have the data quantified. Furthermore, it is possible to link with other healthcare monitoring systems and perform monitoring at medical institutions.

[0070] In one embodiment of the test device of the present invention, the porous microneedles may be provided directly on the paper substrate sensor. That is, in one embodiment of the testing device of the present invention, the microneedle and the paper-based sensor are integrated together.

[0071] The integration of the porous microneedles and the paper-based sensor can be achieved by directly molding the porous microneedles onto the paper-based sensor, as shown in the schematic diagram in Figure 3. As shown in the figure, a solution or suspension of a biodegradable material is injected into a female mold and solidified to obtain a microneedle precursor, which is then heated and bonded in the mold. Then, while heating, the paper substrate is pressed against the microneedle precursor to integrate the two. After the heating process is completed, the microneedle is cooled and removed from the mold to obtain a structure in which the microneedle and the paper substrate sensor are integrated.

[0072] The testing device of the present invention is composed of a porous microneedle paper substrate sensor, and is thin, making it possible to provide a patch-type body fluid collection and testing system that can easily measure multiple components (biomarkers) in interstitial fluid. In addition, the testing device of the present invention can quickly collect interstitial fluid, etc., by capillary force, and can measure components in interstitial fluid without external power.

[0073] Another aspect of the present invention is a patch-type body fluid sampling and testing system comprising the testing device of the present invention (hereinafter also referred to as "the patch-type body fluid sampling and testing system of the present invention" or "the system of the present invention").

[0074] When the patch-type body fluid sampling and testing system of the present invention simultaneously detects and measures multiple biomarkers such as glucose and cholesterol, the system can be equipped with a means for visualizing the reaction in the paper substrate of the sensor structure attached to the skin by a color change or the like, and for combining optical measurement by a camera or the like with analysis of color concentration or the like by software to acquire and analyze information such as the concentration of the biomarkers on the spot. As such a means, for example, a standard color chart may be provided to confirm the blood glucose level, or a standard color change may be provided to confirm the blood glucose level by comparison with this. In addition, using the patch-type body fluid collection and testing system of the present invention, quantification can be performed using an application on a portable device or the like, if necessary. For example, if a person wants to know their blood glucose level in detail, they can take a photo using an application with image processing capabilities and then quantify the level. Furthermore, it is possible to link with other healthcare monitoring systems and perform monitoring at medical institutions.

[0075] A non-limiting example of the use of the subject testing device and subject patch-type bodily fluid sampling and testing system is shown in FIG. Fig. 4 shows an overview of a glucose concentration measuring microneedle patch equipped with the test device of the present invention. As shown in the upper left diagram of the figure, the test device has three reaction areas: a body fluid reaction area, a glucose reaction area A, and a glucose reaction area B, and is attached to the skin in this state. Here, the types of coloring agents in areas A and B are different. Thereafter, the measurement area changes color within a predetermined time (within one minute in this example) as shown in the upper right diagram of Figure 4. This confirms that the body fluid has been collected, and the color change in the glucose reaction area is confirmed. Next, the blood glucose level is confirmed using a standard color chart, as shown in the lower left diagram of Figure 4. The blood glucose level is also confirmed by comparing with the standard color change, with a resolution of about 20 mg / dL and a blood glucose range of about 60 to 300 mg / dL. Furthermore, as shown in the lower right diagram of Figure 4, it can be quantified using an application on a mobile device, etc., if necessary. For those who want to know the blood glucose level in detail, it is possible to take a photo using an application with image processing function and quantify it (resolution is about 1 mg / dL). It is also possible to link it with other healthcare monitoring systems, etc., and monitor it at medical institutions, etc. EXAMPLES

[0076] The present invention will be described below with reference to examples, but the present invention is not limited to these.

[0077] 1. Material Polylactic acid (PLA): Ingeo Biopolymer 4032D (NatureWorks) Polyvinyl alcohol (PVA): 363073-500G (Sigma-Aldrich) Dichloromethane (DCM): 135-02446 (FUJIFILM)

[0078] Glucose sensor used in Reference Example 1 D(+)-Glucose: 4000535 (Hayashi Pure Chemical Industries, Ltd.) D-(+)-Trehalose dihydrate: T9531-5G (Sigma-Aldrich) Peroxidase from horseradish: SRE0082-30KU (Sigma-Aldrich) 3,3',5,5'-Tetramethylbenzidine: 860336-1G (Sigma-Aldrich) Glucose oxidase: G7141 (Sigma-Aldrich) Filter paper: Filter paper, Grade 4, Whatman

[0079] 2. Experimental Equipment Optical microscope: VHX-2000(Keyence) Optical microscope (microsphere observation): OMRON VC3000 Force measurement: MX2-500N-FA-V45(IMADA) Vacuum application: Vacuum desiccator (AS ONE); Guiaphragm type dry vacuum pump DA-30D (ULVAC) Hot plate: Digital hot plate / stirrer PMC-720 (DATAPLATE) Electronic balance: SECURA 125-1SJP (Sartorius)

[0080] [Example 1] Fabrication of porous microneedles using polylactic acid microspheres According to the procedure described in the schematic diagram of the method for preparing a porous microneedle of the present invention shown in FIG. 5, a porous PLA microneedle was prepared using polylactic acid (PLA) microspheres. As shown in Figure 5(ac), 6.7% (w / v) of PLA solution was prepared in dichloromethane (DCM) as the organic phase. The organic phase was then blended into an aqueous phase containing 5% (w / v) polyvinyl alcohol (PVA) as a surfactant. The mixture was stirred at 1000 rpm at room temperature until the DCM evaporated, resulting in PLA microspheres in the PVA solution.

[0081] Once the PLA microspheres were formed, their spherical shape could be stably maintained in the surrounding environment. The diameter of the prepared microspheres was 15.5±6.9 μm. The diameter of the microspheres was measured by optical microscopy.

[0082] Next, the PLA microsphere solution was injected into a PDMS female mold prepared from a metal master mold consisting of 169 pyramidal microneedles with a length of 1200 μm (Figure 5(d)). A vacuum was then applied to fill the microspheres within the cavities. The whole mold was then heated in a convection oven at 50 °C for 2 h, and the micromolded array was peeled off from the mold (Figure 5(e)). Finally, heat treatments at different temperatures (170, 180, 190, 200 °C) were applied for 30 min to bond the microspheres to each other.

[0083] [Example 2] Evaluation of porous PLA microneedles (1) Shape and dimensions of porous PLA microneedles (MN) The shape and dimensions of the porous PLAMN were measured and are shown in FIG. After drying and peeling from the mold, the height and tip diameter of the MNs were 1120.6 ± 47.4 μm and 33.1 ± 14.6 μm, respectively (n = 5). The fabrication results showed that the shape and sharp tip were maintained after heat treatment, but the height and tip diameter of the MNs were slightly reduced. The shrinkage was attributed to the deformation and bonding of the PLA microspheres inside the MNs (see Figure 6).

[0084] (2) Porous structure and porosity of porous PLA microneedles The porous structure of MNs was investigated using scanning electron microscopy (SEM). Single MN and cross-sectional images are shown in Figure 7.

[0085] From Figure 7, it can be seen that after drying at 50 °C for 2 hours, continuous voids were formed, but the PLA microspheres did not bond to each other. Also, after heat treatment at 170 °C for 30 minutes, it can be seen that some of the microspheres melted and began to bond. Since the melting point of the used PLA is 170 °C, it is considered that the PLA microspheres began to melt. Also, when heated at 180 °C for 30 minutes, most of the microspheres were completely bonded, and finally a micron-sized network of interconnected pores was formed. In contrast, when the PLA MN was heated to 200 °C, the microspheres were over-melted and only a few interconnected voids were observed.

[0086] Next, the porosity of the porous PLA MN was measured by comparing the mass before and after fluid extraction using the water absorption method with the porous PLA membrane. First, the dry mass (W dry ) of the porous membrane was recorded. Next, the membrane was immersed in deionized (DI) water, and after absorption was saturated, the surface water was removed. Then, the mass was measured immediately and recorded as W wet . The porosity was calculated by the following formula, and the calculation results were graphed (see Figure 8).

[0087]

Equation

[0088] In the above formula, ρ p is the density of PLA (1.25 g / cm 3 ), and ρ 0 is the density of DI water (1.0 g / cm 3 ).

[0089] The porosity without heat treatment was 27.2 ± 0.9%. As the heat treatment temperature increased, more PLA microspheres melted and bonded, and as a result, the continuous voids inside the micro needles decreased, so the porosity gradually decreased.

[0090] (3) Absorption capacity of the porous PLA micro needles The absorption volume of sample fluid using the porous PLA MNs prepared in Example 1 was investigated using 1% (w / w) agarose gel covered with aluminum foil to mimic human skin. A force of 5 N was applied to the MN array, causing it to penetrate into the agarose gel (see the left diagram in Figure 9). MNs heat-treated at 170–200 °C penetrated the aluminum foil and absorbed the sample fluid. However, MNs dried at 50 °C could penetrate the aluminum foil, but most of the needles remained in the agarose gel and could not maintain the MN structure. This was thought to be due to the weak bonds between the microspheres inside the MNs.

[0091] The right graph in Figure 9 shows the volume of the sample fluid for continuous absorption for 1 and 2 minutes. The MN heated to 180°C absorbed the most sample fluid in 2 minutes.

[0092] Next, the extraction of glucose from the glucose-loaded agarose gel was evaluated. Agarose gel loaded with 5 mM glucose at a ratio of 1% (w / w) was covered with aluminum foil. The glucose detection paper used in the evaluation was prepared by pipetting an enzyme solution containing glucose oxidase (GOx) and peroxidase (HRP) and a color-developing dye solution using tetramethylbenzidine (TMB), and was then dried at room temperature.

[0093] Next, a sensing paper was attached to the backside of the porous MN array (Figure 10). Finger pressure was then applied to make the MNs penetrate the model skin. The glucose-loaded fluid was extracted and delivered to the sensor layer, and the response was visible as a color change of TMB from the GOx-catalyzed oxidation of glucose (see Figure 10).

[0094] The results of the extraction of sample ISF and glucose sensing performance of porous PLA MNs are shown in Figure 11. Heat treatment above the melting point of PLA significantly improved the extraction performance of MNs. Moreover, PLA MNs heat-treated at 180 °C for 30 min extracted the sample fluid, which was transported to the sensor layer the fastest. As a result, the glucose sensing paper turned completely blue within 2 min. The reason for the almost complete absorption of MNs without heat treatment was that the PLA microspheres did not bond with each other and form stable interconnected pores to transport the sample fluid. On the contrary, the heat-treated microspheres melted and bonded to form strong interconnected micropores, which were believed to extract the sample ISFs by the capillary effect.

[0095] (4) Mechanical strength of porous PLA microneedles A compressive load was applied to a single MN in the axial direction, and the displacement-load curve was measured. The point at which the load decreased and the displacement increased was taken as the yield point, and the load at that point was taken as the breaking strength. The results are shown in Figures 12 and 13. Figure 12 shows an outline of the test method for measuring the mechanical strength of the porous PLA MN produced in Example 1 (left diagram) and the obtained load-displacement curve (right diagram). FIG. 13 shows the shape of the porous PLA MN prepared in Example 1 after a test to measure its mechanical strength (left) and the mechanical strength thus obtained (right).

[0096] (5) Insertion test of porous PLA microneedles using pig skin To verify whether the porous PLA MN has sufficient rigidity to puncture the skin, we selected porcine skin, which has a similar structure to human skin consisting of the stratum corneum, epidermis and dermis, to conduct an insertion test (A. Summerfield, et al., “The immunology of the porcine skin and its value as a model for human skin”, Mol. Immunol, 66, 1(2015),pp14-21.). First, the porous PLA MN was inserted into the porcine skin by finger pressure and peeled off from the skin. Then, the porcine skin was stained with 1% (w / v) methylene blue for 15 min, the methylene blue remaining on the skin was wiped off with ethanol, and the penetration site was examined under an optical microscope. As shown in Figure 14, the porous PLA MN could successfully puncture the porcine skin regardless of whether it was heat-treated or not. However, when the non-heat-treated MN patch was peeled off from the pig skin after insertion, the overall structure of the MN patch was not maintained and it separated (middle panel of Figure 14). This was thought to be because the microspheres in the MN patch lacked adhesion to each other, leading to the separation of the patch when peeled off from the pig skin. In contrast, most of the MNs heat-treated at 180-200 °C successfully punctured the skin, demonstrating effective penetration into the skin barrier (see Figure 14). At the same time, the structure of the MNs remained intact after peeling off from the skin.

[0097] [Reference example 1] Examination of the connection between a porous microneedle substrate and a paper-based sensor We investigated a method for connecting a porous microneedle substrate and a paper-based sensor using porous PLGA MNs manufactured by the salt leaching method (for details, see Medical Devices and Sensors, Vol.3, Issue 4, e10109, 8th.July,2020). We investigated a method for connecting the porous microneedle substrate and the paper-based sensor with transparent tape (top panel of Fig. 15) and a method for connecting the two via a flow path layer (bottom panel of Fig. 15). Here, the flow path layer was formed from cellulose powder. When connecting using transparent tape, the adhesive strength between the paper substrate and the transparent tape decreased due to moisture conditions, and a space was created between the porous microneedle substrate and the paper substrate sensor, preventing the absorbed sample from reaching the sensor (see the right diagram in the top row of Figure 15). In contrast, in a method in which a porous microneedle substrate and a paper substrate sensor were connected via a flow path layer, it was found that when the paper substrate became wet, the cellulose powder maintained stable contact, causing color development throughout the entire reaction area (see the right diagram in the bottom row of Figure 15).

[0098] In this reference example, porous PLGA MN produced by the salt leaching method was used as the porous microneedle. As described in this specification, the porous microneedle of the present invention has a water absorption capacity equal to or greater than that of the microneedle obtained by the salt leaching method, and therefore, even if the porous microneedle of the present invention is used together with the glucose sensor described above, it is possible to obtain the same results as above.

[0099] [Reference example 2] Glucose concentration was measured using the above glucose sensor and porous PLGA MNs produced by the salt leaching method (for details, see Medical Devices and Sensors, Vol. 3, Issue 4, e10109, 8th. July, 2020). The skin model used in the experiment was agarose gel embedded in PBS buffer and covered with aluminum foil. The porosity of the porous PLGA MNs was 65%, and the insertion strength of the MNs was 20N.

[0100] Scanned images and color intensity of the assay response after 2 min insertion into agarose gels with various glucose concentrations are shown in Figure 16. The figure shows the blue color development of the paper-based sensor in response to the glucose concentration in phosphate buffer solution sampled by a porous microneedle from 1% agarose gel (left) and the quantitative measurement of the color intensity by an image processing program (right). As shown in Figure 16, when the concentration of glucose exceeded 5 mM, a dark blue color appeared, which can be used as an alarm to inform the risk of developing prediabetes (blood glucose levels >5.6 mM). Color development was quantified as color intensity, and the color intensity increased linearly with glucose concentrations up to 5 mM, with the detection limit estimated from the correlation equation to be 0.12 mM.

[0101] Based on the above results, Figure 17 shows a comparison of the detection limit concentration of glucose between the patch-type body fluid sampling and testing system equipped with the porous microneedles of Reference Example 2 and a paper-based colorimetric sensor and a microneedle electrochemical sensor that do not use microneedles.

[0102] In this reference example, porous PLGA MN produced by the salt leaching method was used as the porous microneedle. As described in this specification, the porous microneedle of the present invention can obtain higher mechanical strength than the microneedle obtained by the salt leaching method, and has equal or greater water absorption capacity. Therefore, even if the porous microneedle of the present invention is used together with the glucose sensor described above, it is possible to obtain the same results as above.

Claims

1. Porous microneedles, and Paper substrate sensor having at least one measurement area - Patents.com Including, the microneedles are formed from microspheres of a biodegradable material, the microspheres of the biodegradable material being bonded to each other to form a network of interconnected pores; The inspection device, wherein the porosity of the microneedles is 10 to 40%.

2. 2. The assay device of claim 1, wherein the biodegradable material comprises at least one of polylactic acid, polyglycolic acid, poly(lactide-co-glycolide) copolymers, PEG copolymers, polyhydroxybutyric acid, and ethyl cellulose.

3. The inspection device according to claim 1 , further comprising a microneedle substrate, the microneedles being joined to the microneedle substrate.

4. The testing device according to any one of claims 1 to 3, further comprising a flow path layer between the paper substrate sensor and the microneedle.

5. The testing device according to any one of claims 1 to 2, wherein the microneedle and the paper substrate sensor are integrated.

6. The inspection device according to any one of claims 1 to 5, wherein the microneedle has a breaking compressive strength of 0.5 N or more measured under the following conditions. Conditions: A compressive load is applied in the axial direction to a single microneedle, and the load at the yield point obtained from the load-displacement curve is measured as the breaking strength.

7. (a) preparing a biodegradable material microsphere solution or suspension containing microspheres of a biodegradable material; (b) injecting the solution or suspension into a female mold; (c) drying the solution or suspension to obtain a microneedle precursor; and (d) heating the microneedle precursor at a predetermined temperature so that the microspheres are partially in a liquid phase or rubbery state and bonded together; A method for producing a porous microneedle comprising the steps of:

8. The manufacturing method according to claim 7, wherein the biodegradable material microsphere solution or suspension is prepared by preparing a solution A by dissolving a biodegradable material in an organic solvent, mixing the solution A with an aqueous solution containing a surfactant, then evaporating the organic solvent and stirring.

9. A porous microneedle obtained by the method according to claim 7 or 8, wherein the porosity of the microneedle is 10 to 40%.

10. A microneedle formed from microspheres of a biodegradable material, the microspheres being bonded together to form a network of interconnected pores, the microneedle having a porosity of 10-40%.

11. The microneedle according to claim 10, having a breaking compressive strength of 0.5 N or more measured under the following conditions. Conditions: A compressive load is applied in the axial direction to a single microneedle, and the load at the yield point obtained from the load-displacement curve is measured as the breaking strength.

12. A microneedle array comprising a plurality of microneedles according to any one of claims 10 to 11 arranged upright on a microneedle substrate.

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