In-vitro diagnostic biosensor for health care and manufacturing method thereof

Abstract

An in-vitro diagnostic biosensor for health care is provided. The in-vitro diagnostic biosensor for health care comprises: a substrate; an electrode layer provided on the substrate; and a sensor layer, made of a nanocomposite, provided on the electrode layer, wherein the nanocomposite may comprise: a latticed metal oxide nanosheet; and metal nanoparticles, dispersed on the metal oxide nanosheet, modified to respond to target biomaterial.

Description

In vitro diagnostic biosensor for healthcare and method of manufacturing the same

[0001] The present invention relates to an in vitro diagnostic biosensor for healthcare and a method for manufacturing the same, and more specifically, to an in vitro diagnostic biosensor for healthcare having high selectivity and excellent sensitivity toward a target biomaterial and a method for manufacturing the same.

[0002] Recently, cholesterol monitoring technology linked with wearable devices has been advancing. Portable cholesterol meters that connect with smartphones have been developed, enhancing user convenience, and this technology is establishing itself as a core element of personalized healthcare services.

[0003] In particular, the Asia-Pacific region is attracting attention as the fastest-growing market due to rapid economic growth and the development of medical infrastructure.

[0004] However, conventionally, there has been a lack of sensor applications that respond specifically to cholesterol. In other words, since conventional cholesterol sensors respond to blood glucose in addition to cholesterol, blood glucose acted as noise from a cholesterol perspective. Consequently, the sensitivity of conventional cholesterol sensors to cholesterol is not very high.

[0005] In addition, conventional cholesterol sensors consisted mainly of enzyme-based electrochemical sensors.

[0006] These enzyme-based sensors detect cholesterol using cholesterol oxidase. However, these enzyme-based sensors have problems such as enzyme instability, complex manufacturing processes, and high costs.

[0007] Accordingly, research is being conducted on non-enzymatic sensors that do not use enzymes. Since these non-enzymatic sensors can resolve issues of stability and cost, continuous growth is expected, driven by the increase in hypercholesterolemia and cardiovascular diseases, growing interest in personal health management, and technological advancements.

[0008] Therefore, there is an urgent need to develop non-enzymatic sensors with high selectivity and excellent sensitivity for cholesterol.

[0009] The technical problem that the present invention aims to solve is to provide an in vitro diagnostic biosensor for healthcare having high selectivity and excellent sensitivity toward target biomaterials, and a method for manufacturing the same.

[0010] Another technical problem that the present invention aims to solve is to provide a non-enzyme-based in vitro diagnostic biosensor for healthcare and a method for manufacturing the same.

[0011] Another technical problem that the present invention aims to solve is to provide an in vitro diagnostic biosensor for healthcare that can be simply manufactured and a method for manufacturing the same.

[0012] The technical problems that the present invention aims to solve are not limited to those described above.

[0013] To solve the above-mentioned technical problem, the present invention provides an in vitro diagnostic biosensor for healthcare.

[0014] According to one embodiment, the in vitro diagnostic biosensor for healthcare comprises: a substrate; an electrode layer provided on the substrate; and a sensor layer provided on the electrode layer and composed of a nanocomposite, wherein the nanocomposite may comprise a metal oxide nanosheet forming a lattice structure; and metal nanoparticles dispersed on the metal oxide nanosheet and modified to respond to a target biomaterial.

[0015] According to one embodiment, when the target biomaterial is cholesterol, the metal nanoparticles can be modified into digitonin.

[0016] According to one embodiment, the digitonin can induce a change in the size of the metal nanoparticles depending on the concentration of the cholesterol.

[0017] According to one embodiment, the metal nanoparticles may be composed of gold (Au) nanoparticles.

[0018] According to one embodiment, the digitonin can be immobilized on the surface of the gold (Au) nanoparticles by sulfuric acid (H2SO4) which promotes an esterification reaction between the carboxyl group (-COOH) on the surface of the gold (Au) nanoparticles and the hydroxyl group (-OH) of the digitonin.

[0019] According to one embodiment, the electrode layer is made of gold (Au), and the metal oxide nanosheet may be made of nickel oxide (NiO).

[0020] According to one embodiment, the metal oxide nanosheet may have X-ray diffraction peaks for the (200) and (220) crystal planes.

[0021] Meanwhile, the present invention provides a method for manufacturing an in vitro diagnostic biosensor for healthcare.

[0022] According to one embodiment, the method may include the steps of: preparing a substrate having an electrode layer, a metal oxide nanosheet, and metal nanoparticles modified to respond to a target biomaterial, respectively; slurrying and mixing the prepared metal oxide nanosheet and the modified metal nanoparticles, respectively, to prepare a mixture; and forming a sensor layer composed of a nanocomposite on the electrode layer from the prepared mixture.

[0023] According to one embodiment, when the target biomaterial is cholesterol, the metal nanoparticles are modified into digitonin, and the metal nanoparticles may be composed of gold (Au) nanoparticles.

[0024] According to one embodiment, the electrode layer is made of gold (Au), and the metal oxide nanosheet may be made of nickel oxide (NiO).

[0025] According to one embodiment, the preparation step comprises: a process of synthesizing the gold (Au) nanoparticles; a process of obtaining an initial modified gold (Au) nanoparticle from the synthesized gold (Au) nanoparticles; and a process of adding digitonin to a solution containing the obtained initial modified gold (Au) nanoparticles to obtain gold (Au) nanoparticles modified with digitonin, wherein sulfuric acid (H2SO4) may be further added in the process of obtaining the gold (Au) nanoparticles modified with digitonin.

[0026] According to one embodiment, in the step of preparing the mixture, the slurried metal oxide nanosheet and the modified metal nanoparticle can be mixed in a 1:1 ratio.

[0027] According to one embodiment, in the step of forming the sensor layer, a sensor layer composed of a nanocomposite can be formed on the electrode layer from the prepared mixture through drop casting.

[0028] According to an embodiment of the present invention, the apparatus comprises a substrate; an electrode layer provided on the substrate; and a sensor layer provided on the electrode layer and composed of a nanocomposite, wherein the nanocomposite may include a metal oxide nanosheet forming a lattice structure; and metal nanoparticles dispersed on the metal oxide nanosheet and modified to respond to a target biomaterial.

[0029] Accordingly, an in vitro diagnostic biosensor for healthcare having high selectivity for target biomaterials and a method for manufacturing the same can be provided.

[0030] Furthermore, an in vitro diagnostic biosensor for healthcare, which is a cholesterol sensor based on a particle size change mechanism through specific binding, can also be applied to targets such as N-acetylglucosamine, a cancer-related glycoprotein biomarker, and D-mannose, an immunological glycostructure-based biomarker, by binding Wheat Germ Agglutinin (WGA), Concanavalin A (ConA), etc., to gold (Au) nanoparticles instead of digitonin based on the same mechanism.

[0031] That is, according to an embodiment of the present invention, digitonin bound to metal nanoparticles has a high affinity for cholesterol, which is a target biomaterial, so the selectivity for cholesterol of an in vitro diagnostic biosensor for healthcare can be improved, and through this, interference from non-target biomaterials can be minimized.

[0032] In addition, according to an embodiment of the present invention, an in vitro diagnostic biosensor for healthcare having excellent sensitivity to a target biomaterial and a method for manufacturing the same may be provided.

[0033] That is, according to an embodiment of the present invention, even minute changes in cholesterol concentration can be precisely detected by utilizing the electrochemical properties of gold nanoparticles modified with digitonin and nickel oxide nanosheets.

[0034] In addition, according to an embodiment of the present invention, an in vitro diagnostic biosensor for healthcare that can be simply manufactured and a method for manufacturing the same may be provided.

[0035] That is, according to an embodiment of the present invention, the synthesis process of gold nanoparticles and nickel oxide and the digitonin binding process can be carried out relatively simply, and through this, mass production can be easily achieved.

[0036] Thus, according to an embodiment of the present invention, a non-enzyme-based in vitro diagnostic biosensor for healthcare can be provided, which overcomes the limitations of existing enzyme-based cholesterol sensors and has high sensitivity and selectivity toward target biomaterials, thereby having high potential for use in the field of medical diagnosis.

[0037] FIG. 1 is a schematic diagram illustrating an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention.

[0038] FIG. 2 is a schematic diagram illustrating metal nanoparticles modified with digitonin for an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention.

[0039] FIG. 3 is a flowchart showing the process sequence of a method for manufacturing an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention.

[0040] FIGS. 4 to 6 are schematic diagrams for explaining step S110 of FIG. 3.

[0041] Figure 7 is a schematic diagram illustrating step S130 of Figure 3.

[0042] Figure 8 is an electron microscope image of a nickel oxide film prepared according to Comparative Example 1.

[0043] Figure 9 is a scanning electron microscope image of a nanocomposite prepared according to Example 1.

[0044] Figure 10 shows the XRD measurement results for the nanocomposite prepared according to Example 1.

[0045] Figures 11 and 12 are the results of analyzing a nanocomposite prepared according to Example 1 using energy-dispersive X-ray spectroscopy.

[0046] Figure 13 shows the CV analysis results in Potassium Ferricyanide-Ferrocyanide solution for each sample.

[0047] Figure 14 is a CV graph showing the change in cholesterol concentration of the nanocomposite prepared according to Example 1.

[0048] Figure 15 is a graph showing the change in current density according to the cholesterol concentration of the nanocomposite prepared according to Example 1.

[0049] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the technical concept of the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0050] In this specification, when a component is described as being on another component, it means that it may be formed directly on the other component or that a third component may be interposed between them. Additionally, in the drawings, shapes and sizes are exaggerated for the effective description of the technical content.

[0051] Additionally, although terms such as first, second, third, etc., have been used to describe various components in the various embodiments of this specification, these components should not be limited by such terms. These terms are used merely to distinguish one component from another. Accordingly, what is referred to as the first component in one embodiment may be referred to as the second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiment. Furthermore, in this specification, "and / or" is used to mean including at least one of the components listed before and after it.

[0052] In the specification, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "include" or "have" are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof. Additionally, in this specification, "connection" is used to include both indirectly connecting multiple components and directly connecting them.

[0053] Additionally, terms such as “…part,” “…unit,” and “module” described in the specification refer to a unit that processes at least one function or operation, and this may be implemented in hardware, software, or a combination of hardware and software.

[0054] Furthermore, in describing the present invention below, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted.

[0055]

[0056] FIG. 1 is a schematic diagram illustrating an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention, and FIG. 2 is a schematic diagram illustrating metal nanoparticles modified with digitonin of an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention.

[0057]

[0058] As illustrated in FIGS. 1 and 2, an in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention is a sensor that can be applied to disease management by detecting the concentration of a target biomaterial (T) in the body through an examination of a body fluid (C).

[0059] At this time, the in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention is a non-enzyme-based sensor, which overcomes the limitations of existing enzyme-based sensors and can have high sensitivity and selectivity for target biomaterials (T).

[0060] Accordingly, the in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention can have high utility in the field of medical diagnosis.

[0061]

[0062] An in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention may be formed by including a substrate (110), an electrode layer (120), and a sensor layer (130).

[0063]

[0064] The above substrate (110) can provide a forming surface for the electrode layer (120). Accordingly, the above substrate (110) can support a sensor layer (130) provided on the electrode layer (120).

[0065] According to one embodiment of the present invention, such a substrate (110) may be provided as a silicon (Si) substrate. However, the present invention does not limit the substrate (110) to only a silicon substrate.

[0066]

[0067] The electrode layer (120) may be provided on the substrate (110). At this time, the electrode layer (120) may be formed on one surface of the substrate (110) through electron beam deposition or sputtering deposition.

[0068] According to one embodiment of the present invention, the electrode layer (120) may be made of gold (Au). However, this is merely an example, and the electrode layer (120) may be made of any one metal or two or more alloys selected from a group of metal candidates that have good bonding properties with the material forming the sensor layer (130).

[0069]

[0070] The sensor layer (130) may be provided on the electrode layer (120). The sensor layer (130) may be bonded to the electrode layer (120).

[0071] According to one embodiment of the present invention, the sensor layer (130) may be made of a nanocomposite.

[0072] Here, the nanocomposite forming the sensor layer (130) may include metal oxide nanosheets (131) and metal nanoparticles (132).

[0073] The metal oxide nanosheet (131) can be formed on the electrode layer (120). The metal oxide nanosheet (131) can be bonded to the electrode layer (120).

[0074] At this time, according to one embodiment of the present invention, the metal oxide nanosheet (131) may be made of a material that has good adhesion to the electrode layer (120) made of gold (Au). In addition, the metal oxide nanosheet (131) may be made of a material that has good adhesion to the metal nanoparticle (132).

[0075] Accordingly, according to one embodiment of the present invention, the metal oxide nanosheet (131) may be made of nickel oxide (NiO).

[0076] Accordingly, the bonding force between the electrode layer (120) and the metal nanoparticle (132) can be strengthened by the metal oxide nanosheet (131).

[0077] According to one embodiment of the present invention, a metal oxide nanosheet (131) made of nickel oxide (NiO) can form a lattice structure. That is, the metal oxide nanosheet (131) made of nickel oxide (NiO) has high crystallinity and exhibits X-ray diffraction peaks for crystal planes (200) and (220), which means that it forms a lattice structure.

[0078] Due to the lattice structure of the metal oxide nanosheet (131) made of such nickel oxide (NiO), the metal nanoparticles (132) can be uniformly dispersed on the metal oxide nanosheet (131).

[0079] Accordingly, the sensor layer (130) can secure a large specific surface area, and through this, the reaction area of ​​the sensor layer (130) to the target biomaterial (T) can be increased.

[0080] According to one embodiment of the present invention, the metal oxide nanosheet (131) made of such nickel oxide (NiO) can strengthen the bonding force between the electrode layer (120) and the metal nanoparticle (132), provide a large specific surface area, increase the reaction area of ​​the sensor layer (130) for the target biomaterial (T), improve electrochemical stability, and increase electron transfer efficiency.

[0081] The metal nanoparticles (132) can be dispersed on the metal oxide nanosheet (131). At this time, the metal nanoparticles (132) can be uniformly dispersed on the metal oxide nanosheet (131) as the metal oxide nanosheet (131) forms a lattice structure.

[0082] In this way, as the metal nanoparticles (132) are uniformly dispersed on the metal oxide nanosheet (131), a large specific surface area of ​​the sensor layer (130) can be secured, and thereby, the reaction area of ​​the sensor layer (130) for the target biomaterial (T) can be increased.

[0083] According to one embodiment of the present invention, these metal nanoparticles (132) can be modified to respond to a target biomaterial (T).

[0084] Here, the target biomaterial (T) may be cholesterol. In this way, when the target biomaterial (T) is cholesterol, the metal nanoparticle (132) may be modified into digitonin (133).

[0085] That is, if the target biomaterial (T) is cholesterol, the digitonin (133) can be bound to the surface of the metal nanoparticle (132).

[0086] Thus, the digitonin (133) that modifies the metal nanoparticle (132) so that the metal nanoparticle (132) responds to cholesterol, which is the target biomaterial (T), is a complex molecule composed of a steroid nucleus and several saccharides. The digitonin (133) is a type of saponin and is extracted from plants, particularly digitalis.

[0087] These digitonins (133) have a high affinity for cholesterol molecules, which allows the digitonins (133) to easily bind to cholesterol-rich cell membranes.

[0088] The above digitonin (133) selectively dissolves cell membranes containing cholesterol. Through this, digitonin (133) is used to release cytoplasm and intracellular organelles. Due to this characteristic of digitonin (133), only intracellular organelles can be selectively separated without destroying the cell membrane. This can be useful for studying internal structures such as mitochondria and ribosomes.

[0089] In addition, the digitonin (133) is frequently used to fractionate cells by their components. The digitonin (133) frees up the cytoplasm and unifies organelles, allowing only specific parts to be analyzed.

[0090] And in the process of extracting and purifying cell membrane proteins, digitonin (133) can be used to effectively dissolve the cell membrane and separate the proteins.

[0091] These digitonins (133) are also utilized in medical and pharmacological research and play an important role in the development of treatment methods for various diseases related to cholesterol.

[0092] At this time, digitonin (133) may exhibit toxicity at high concentrations. Additionally, the digitonin (133) has the characteristic of selectively dissolving cell membranes that are high in cholesterol.

[0093] In particular, since digitonin (133) binds strongly to cholesterol, it has specificity for structures high in cholesterol.

[0094] That is, according to one embodiment of the present invention, digitonin (133) bound to metal nanoparticles (132) has a high affinity for cholesterol, which is a target biomaterial (T), so the selectivity for cholesterol of the in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention can be improved, and through this, interference from non-target biomaterials can be minimized.

[0095] In this way, digitonin (133) has the ability to specifically bind to cholesterol and binds to metal nanoparticles (132), and can induce a change in the size of the metal nanoparticles (132) depending on the concentration of cholesterol.

[0096] In this way, when the size of the metal nanoparticle (132) changes due to digitonin (133) that specifically binds to cholesterol, the electrochemical signal changes, and through this, the concentration of cholesterol can be precisely measured.

[0097] Meanwhile, according to one embodiment of the present invention, the metal nanoparticles (132) may be made of a metal that has good bonding properties with a metal oxide nanosheet (131) made of nickel oxide (NiO). The metal nanoparticles (132) may be made of, for example, gold (Au) nanoparticles.

[0098] The above digitonin (133) can be bonded to the surface of a metal nanoparticle (132) made of gold (Au) nanoparticles.

[0099] The above digitonin (133) can be fixed to the surface of the metal nanoparticle (132) made of gold (Au) nanoparticles through an esterification reaction between the carboxyl group (-COOH) on the surface of the metal nanoparticle (132) made of gold (Au) nanoparticles and the hydroxyl group (-OH) of the digitonin (133).

[0100] At this time, the digitonin (133) can be effectively fixed to the surface of the metal nanoparticle (132) made of gold (Au) nanoparticles by sulfuric acid (H2SO4) that promotes the esterification reaction.

[0101] According to one embodiment of the present invention, metal nanoparticles (132) made of gold (Au) nanoparticles can be surface-treated to generate a carboxyl group (-COOH) that combines with the hydroxyl group (-OH) of the digitonin (133).

[0102] In this way, according to one embodiment of the present invention, the metal nanoparticle (132) can effectively bind to digitonin (133) to form a cholesterol-sensing substance modified with digitonin (133).

[0103] In addition, according to one embodiment of the present invention, metal nanoparticles (132) modified with digitonin (133) can improve reaction sensitivity through a high surface area to volume ratio and promote selective binding with cholesterol.

[0104]

[0105] As described above, an in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention may have a sensor layer (130) made of a nanocomposite comprising a metal oxide nanosheet (131) made of nickel oxide (NiO) forming a lattice structure and a metal nanoparticle (132) made of gold (Au) nanoparticles modified with digitonin (133) to respond to cholesterol, which is a target biomaterial (T).

[0106] Accordingly, the in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention can have high selectivity for cholesterol, which is a target biomaterial (T).

[0107] That is, according to an embodiment of the present invention, digitonin (133) bound to metal nanoparticles (132) has a high affinity for cholesterol, which is a target biomaterial (T), so the selectivity for cholesterol of the in vitro diagnostic biosensor (100) for healthcare can be improved, and through this, interference from non-target biomaterials can be minimized.

[0108] In addition, an in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention may have excellent sensitivity to cholesterol, which is a target biomaterial (T).

[0109] That is, the in vitro diagnostic biosensor (100) for healthcare according to one embodiment of the present invention can precisely detect even minute changes in cholesterol concentration by utilizing the electrochemical properties of metal nanoparticles (132) made of gold (Au) nanoparticles modified with digitonin (133) and metal oxide nanosheets (131) made of nickel oxide (NiO).

[0110] In this way, according to an embodiment of the present invention, a non-enzyme-based in vitro diagnostic biosensor (100) for healthcare can be provided, which overcomes the limitations of existing enzyme-based cholesterol sensors and has high sensitivity and selectivity for target biomaterials (T), e.g., cholesterol, thereby having high potential for use in the medical diagnostic field.

[0111]

[0112] Hereinafter, a method for manufacturing an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention will be described with reference to FIGS. 3 to 7.

[0113]

[0114] FIG. 3 is a flowchart showing a method for manufacturing an in vitro diagnostic biosensor for healthcare according to an embodiment of the present invention in process order, FIGs. 4 to 6 are schematic diagrams for explaining step S110 of FIG. 3, and FIG. 7 is a schematic diagram for explaining step S130 of FIG. 3.

[0115]

[0116] Referring to FIG. 3, a method for manufacturing an in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention may include steps S110, S120, and S130.

[0117]

[0118] S110 step

[0119] The above step S110 is a step of preparing a substrate (110) having an electrode layer (120 in FIG. 4), a metal oxide nanosheet (131 in FIG. 7), and metal nanoparticles (132 in FIG. 7) modified to respond to a target biomaterial (T in FIG. 2), respectively.

[0120] Referring to FIG. 4, in step S110, a metal can be deposited on one surface of the substrate (110) to form an electrode layer (120). At this time, in step S110, gold (Au) can be deposited on one surface of the substrate (110) to form an electrode layer (120) made of gold (Au) on one surface of the substrate (110).

[0121] To this end, in step S110, gold (Au) can be deposited on one surface of the substrate (110) through electron beam deposition or sputtering deposition, and thereby, an electrode layer (120) made of gold (Au) can be formed on one surface of the substrate (110).

[0122] However, this is merely an example, and the metal forming the electrode layer (120) in the present invention is not necessarily limited to gold (Au).

[0123] Meanwhile, in the above S110 step, a silicon (Si) substrate may be used as the above material (110), for example.

[0124] In the above S110 step, a substrate (110) having an electrode layer (120) on one side can be cleaned.

[0125] To this end, in step S110, a substrate (110) having an electrode layer (120) on one side may first be immersed in, for example, a 0.1M hydrochloric acid (HCl) solution and stirred approximately 30 times, and then washed with distilled water.

[0126] Next, in step S110, the substrate (110) washed with distilled water can be sterilized in 99.99% anhydrous ethanol and washed with distilled water.

[0127] Through this, the contamination level of the surface of the electrode layer (120) is reduced, and a more stable state can be maintained in the long term.

[0128] In addition, on the other hand, referring to FIG. 5, a metal oxide nanosheet (131 in FIG. 7) can be manufactured in step S110. At this time, according to one embodiment of the present invention, a metal oxide nanosheet (131 in FIG. 7) made of nickel oxide (NiO) can be manufactured in step S110.

[0129] To this end, in step S110, a mixed solution of nickel(II) nitrate hexahydrate and sodium hydroxide can be prepared first (S11).

[0130] For example, in step S110 above, 0.006M nickel(II) nitrate hexahydrate and 0.12M sodium hydroxide solution can be mixed in a ratio of 45.5:4.5 and then mixed with a stirrer at 600 rpm for 30 minutes to prepare a mixed solution of nickel(II) nitrate hexahydrate and sodium hydroxide.

[0131] Next, in step S110, hydrothermal synthesis of the prepared mixed solution can be carried out (S12).

[0132] For example, in step S110 above, the prepared mixed solution can be placed in an autoclave and hydrothermal synthesis can be carried out at 200°C for 10 hours.

[0133] Next, in step S110, centrifugation of the nickel oxide (NiO) solution produced through hydrothermal synthesis of the mixed solution can be performed (S13).

[0134] In the above S110 step, for example, the nickel oxide (NiO) solution can be centrifuged at 4,000 rpm for 10 minutes, and through this, the supernatant can be removed from the nickel oxide (NiO) solution.

[0135] At this time, in step S110, the nickel oxide (NiO) removed from the supernatant can be washed approximately three times with deionized water.

[0136] Next, in step S110, the washed nickel oxide (NiO) can be dried (S14).

[0137] For example, in the above S110 step, the washed nickel oxide (NiO) can be dried in an oven at 60°C for 30 minutes.

[0138] Next, in step S110, the dried nickel oxide (NiO) can be annealed (S15).

[0139] For example, in the above S110 step, the dried nickel oxide (NiO) can be annealed at 400°C for 1 hour.

[0140] When the annealing of the nickel oxide (NiO) is completed, a metal oxide nanosheet (131 in Fig. 7) made of nickel oxide (NiO) can be completed.

[0141] As such, the metal oxide nanosheet (131 in FIG. 7) made of nickel oxide (NiO) has high crystallinity and can form a lattice structure that exhibits X-ray diffraction peaks for the (200) and (220) crystal planes.

[0142] Due to the lattice structure of the metal oxide nanosheet (131 in FIG. 7) made of such nickel oxide (NiO), metal nanoparticles (132 in FIG. 7) prepared on the other hand can be uniformly dispersed on the metal oxide nanosheet (131 in FIG. 7) through a subsequent process.

[0143] Accordingly, through a subsequent process, a sensor layer (130 in Fig. 7) formed as a nanocomposite consisting of metal oxide nanosheets (131 in Fig. 7) and metal nanoparticles (132 in Fig. 7) can secure a large specific surface area, and thereby, the reaction area of ​​the sensor layer (130 in Fig. 7) for the target biomaterial (T in Fig. 2) can be increased.

[0144] According to one embodiment of the present invention, a metal oxide nanosheet (131 in FIG. 7) made of such nickel oxide (NiO) can strengthen the bonding force between the electrode layer (120) and the metal nanoparticle (132 in FIG. 7), provide a large specific surface area, increase the reaction area of ​​the sensor layer (130 in FIG. 7) for the target biomaterial (T in FIG. 2), improve electrochemical stability, and increase electron transfer efficiency.

[0145] On the other hand, referring to FIG. 6, in step S110, metal nanoparticles (132 in FIG. 7) modified to respond to a target biomaterial (T in FIG. 2) can be prepared.

[0146] In this case, according to one embodiment of the present invention, when the target biomaterial (T in FIG. 2) is cholesterol, the metal nanoparticle (132 in FIG. 7) can be modified into digitonin (133 in FIG. 7) having excellent affinity for cholesterol, and the metal nanoparticle (132 in FIG. 7) can be made of gold (Au) nanoparticles.

[0147] To this end, according to one embodiment of the present invention, in step S110, gold (Au) nanoparticles forming metal nanoparticles (132 in FIG. 7) can first be synthesized (S21).

[0148] For example, in step S110 above, gold (Au) nanoparticles forming metal nanoparticles (132 in FIG. 7) can be synthesized by dissolving 5 mg of gold (III) chloride hydrate in 95 ml of distilled water, then adding 5 ml of 1% sodium citrate dihydrate at 90°C and heating for 30 minutes. Afterward, when the synthesized gold (Au) nanoparticles are cooled to room temperature, the gold (Au) nanoparticle solution is completed.

[0149] Next, in step S110, initial modified gold (Au) nanoparticles can be obtained from the synthesized gold (Au) nanoparticles (S22).

[0150] For example, in step S110 above, 1 ml of the synthesized gold (Au) nanoparticle solution may be treated with nitrogen (N) gas for 10 minutes, then 1 ml of polysorbate 20 (Tween 20), a nonionic surfactant, may be added and left at 4°C for at least 30 minutes.

[0151] Here, the polysorbate 20 can be adsorbed onto the surface of gold (Au) nanoparticles to stabilize the dispersion and prevent aggregation between gold (Au) nanoparticles, thereby helping the solution maintain a colloidal state.

[0152] Furthermore, in step S110 above, after leaving for 30 minutes, 1 ml of 3-mercaptopropionic acid (3-MPA) at a concentration of, for example, 0.0005 M can be added to the gold (Au) nanoparticle solution and left for an additional 3 hours at 4°C.

[0153] In this process, the 3-mercaptopropionic acid forms a strong gold-sulfur bond (Au-S bond) on the surface of the gold (Au) nanoparticles and can introduce a carboxyl group (-COOH) on the surface of the gold (Au) nanoparticles.

[0154] In this way, the carboxyl group (-COOH) generated on the surface of the gold (Au) nanoparticles by 3-mercaptopropionic acid can provide a basis for immobilizing digitonin (133 in Fig. 7) on the surface of the gold (Au) nanoparticles forming the metal nanoparticles (132 in Fig. 7) through an esterification reaction with the hydroxyl group (-OH) of digitonin (133 in Fig. 7).

[0155] Furthermore, in the above S110 step, centrifugation can be performed on the gold (Au) nanoparticle solution to which 3-mercaptopropionic acid has been added.

[0156] For example, in step S110 above, a gold (Au) nanoparticle solution with added 3-mercaptopropionic acid can be centrifuged at 6,000 rpm for 20 minutes, the supernatant is removed, and 0.01 M sodium phosphate buffer is added to wash the solution three times under the same conditions.

[0157] After washing is complete and the supernatant is removed, the initially modified gold (Au) nanoparticles can be obtained.

[0158] Next, according to one embodiment of the present invention, in step S110, gold (Au) nanoparticles modified with digitonin (133 in FIG. 7) can be obtained (S23).

[0159] For example, in step S110 above, digitonin (133 in Fig. 7) and a small amount of high-concentration concentrated sulfuric acid (H2SO4) can be added to the initial modified gold (Au) nanoparticles, i.e., a solution of gold (Au) nanoparticles with carboxyl groups (-COOH) formed on the surface, and left at 4°C for 5 hours.

[0160] At this time, a small amount of high-concentration concentrated sulfuric acid (H2SO4) promotes the esterification reaction between the hydroxyl group (-OH) of digitonin (133 in Fig. 7) and the carboxyl group (-COOH) on the surface of the initially modified gold (Au) nanoparticles, thereby effectively immobilizing digitonin (133 in Fig. 7) onto the gold (Au) nanoparticles.

[0161] Furthermore, in step S110 above, centrifugation can be performed on a gold (Au) nanoparticle solution to which digitonin (133 in Fig. 7) and a small amount of high-concentration concentrated sulfuric acid (H2SO4) have been added.

[0162] For example, in step S110 above, a gold (Au) nanoparticle solution containing digitonin (133 in Fig. 7) and a small amount of high-concentration concentrated sulfuric acid (H2SO4), which has been left for 5 hours, is centrifuged at 15,000 rpm for 20 minutes using a centrifuge, the supernatant is removed, and 0.01 M sodium phosphate buffer is added to wash the solution three times under the same conditions.

[0163] Finally, after washing is complete, the supernatant is removed, and gold (Au) nanoparticles stably conjugated with digitonin (133 in Fig. 7) can be obtained.

[0164] That is, metal nanoparticles (132 in Fig. 7) composed of gold (Au) nanoparticles modified with digitonin (133 in Fig. 7) can be obtained.

[0165]

[0166] S120 step

[0167] Step S120 above is a step of slurrying the metal oxide nanosheet (131 in FIG. 7) prepared through Step S110 and the metal nanoparticle (132 in FIG. 7) modified to respond to the target biomaterial (T in FIG. 2), respectively.

[0168] In addition, the above S120 step is a step of preparing a mixture by mixing a slurryed metal oxide nanosheet (131 in FIG. 7) and a metal nanoparticle (132 in FIG. 7) modified to respond to a target biomaterial (T in FIG. 2).

[0169] To this end, in step S120, for example, 3 mg of nickel oxide (NiO) forming a metal oxide nanosheet (131 in FIG. 7) and 1 ml of 0.1% Nafion are mixed and sonicated for 20 minutes to make a slurry of a metal oxide nanosheet (131 in FIG. 7) made of nickel oxide (NiO).

[0170] On the other hand, in step S120 above, for example, 30 mg of gold (Au) nanoparticles modified with digitonin (133 in Fig. 7), which form metal nanoparticles (132 in Fig. 7) modified to respond to a target biomaterial (T in Fig. 2), can be mixed with 0.01 ml of 0.1% Nafion to make a slurry of gold (Au) nanoparticles modified with digitonin (133 in Fig. 7).

[0171] Next, in step S120, a mixture of gold (Au) nanoparticles modified with nickel oxide (NiO) and digitonin (133 in Fig. 7) can be prepared by mixing metal oxide nanosheets (131 in Fig. 7) made into slurries and gold (Au) nanoparticles modified with digitonin (133 in Fig. 7), respectively.

[0172] At this time, in step S120, a metal oxide nanosheet (131 in FIG. 7) made of slurry and gold (Au) nanoparticles modified with digitonin (133 in FIG. 7) can be mixed in a 1:1 ratio.

[0173]

[0174] S130 step

[0175] Referring to FIG. 7, step S130 is a step of forming a sensor layer (130) made of a nanocomposite on the electrode layer (120) from the mixture prepared through step S120, namely, a mixture of nickel oxide (NiO) and gold (Au) nanoparticles modified with digitonin (133).

[0176] At this time, in step S130, a sensor layer (130) can be formed from the mixture through drop casting, that is, a nanocomposite, i.e., a metal oxide nanosheet (131) made of nickel oxide (NiO) and a metal nanoparticle (132) made of gold (Au) nanoparticles modified with digitonin (133) dispersed on the metal oxide nanosheet (131).

[0177] At this time, the metal nanoparticles (132) made of gold (Au) nanoparticles modified with the above-mentioned digitonin (133) can be uniformly dispersed on the metal oxide nanosheet (131) by means of the lattice structure of the metal oxide nanosheet (131) made of nickel oxide (NiO).

[0178] In this way, as the metal nanoparticles (132) are uniformly dispersed on the metal oxide nanosheet (131), a large specific surface area of ​​the sensor layer (130) can be secured, and through this, the reaction area of ​​the sensor layer (130) for cholesterol, which is the target biomaterial (T), can be increased.

[0179]

[0180] Example 1

[0181] A gold-deposited silicon wafer was selected as the working electrode. The working electrode was immersed in a 0.1M hydrochloric acid solution and stirred approximately 30 times, then washed with distilled water, sterilized in 99.99% anhydrous ethanol, and washed with distilled water.

[0182] In addition, 0.006 M nickel(II) nitrate hexahydrate and 0.12 M sodium hydroxide solution were mixed in a ratio of 45.5:4.5, and then mixed with a stirrer at 600 rpm for 30 minutes to prepare a solution for producing nickel oxide nanosheets.

[0183] Then, the above solution was placed in an autoclave and hydrothermally synthesized at 200°C for 10 hours, and the resulting nickel oxide (NiO) solution was centrifuged at 4,000 rpm for 10 minutes to remove the supernatant, the removed nickel oxide (NiO) was washed in the supernatant, dried in an oven at 60°C for 30 minutes, and then annealed at 400°C for 1 hour to produce nickel oxide (NiO) nanosheets.

[0184] Meanwhile, 5 mg of gold (III) chloride hydrate was dissolved in 95 ml of distilled water, and then 5 ml of 1% sodium citrate dihydrate was added and heated at 90°C for 30 minutes to synthesize gold nanoparticles.

[0185] Next, 1 ml of the synthesized gold nanoparticle solution was treated with nitrogen (N) gas for 10 minutes, then 1 ml of the nonionic surfactant polysorbate 20 (Tween 20) was added, and the mixture was left at 4°C for at least 30 minutes.

[0186] After leaving for 30 minutes, 1 ml of 3-mercaptopropionic acid (3-MPA) at a concentration of 0.0005 M was added to the gold nanoparticle solution and left for an additional 3 hours at 4°C.

[0187] Next, the gold nanoparticle solution with added 3-mercaptopropionic acid was centrifuged at 6,000 rpm for 20 minutes, the supernatant was removed, and the solution was washed three times under the same conditions with the addition of 0.01 M sodium phosphate buffer solution to obtain the initially modified gold nanoparticles.

[0188] Next, digitonin and a small amount of high-concentration concentrated sulfuric acid (H2SO4) were added to the initial modified gold nanoparticle solution and left at 4°C for 5 hours. Afterward, the solution was centrifuged at 15,000 rpm for 20 minutes using a centrifuge, the supernatant was removed, and 0.01 M sodium phosphate buffer solution was added and washed three times under the same conditions to obtain gold nanoparticles modified with digitonin.

[0189] Next, the obtained nickel oxide nanosheets and gold nanoparticles modified with digitonin were each prepared into slurries and mixed in a 1:1 ratio. Then, 0.01 ml of this mixed slurry was dropped onto a gold-deposited silicon wafer with an area of ​​0.5 cm × 0.5 cm using a micropipette, and dried in a 50°C oven for 10 minutes to prepare a nanocomposite.

[0190]

[0191] Comparison Example 1

[0192] A nickel oxide (NiO) nanosheet prepared according to Example 1 was made into a slurry, and 0.01 ml of this slurry was dropped onto a gold-deposited silicon wafer with an area of ​​0.5 cm × 0.5 cm using a micropipette, and then dried in a 50°C oven for 10 minutes to prepare a nickel oxide electrode.

[0193]

[0194] Figure 8 is an electron microscope image of a nickel oxide film prepared according to Comparative Example 1, and Figure 9 is a scanning electron microscope image of a nanocomposite prepared according to Example 1.

[0195] Referring to Fig. 8, it can be seen that nickel oxide (NiO) nanosheets form a lattice structure, and referring to Fig. 9, it can be seen that gold nanoparticles modified with digitonin are uniformly distributed on the nanosheets forming the lattice structure.

[0196] Through this, it was confirmed that nickel oxide (NiO) nanosheets forming a lattice structure contribute to securing a large specific surface area of ​​the nanocomposite.

[0197]

[0198] In addition, Fig. 10 is the XRD measurement result for the nanocomposite prepared according to Example 1, and Figs. 11 and 12 are the results of analyzing the nanocomposite prepared according to Example 1 through energy-dispersive X-ray spectroscopy.

[0199] First, referring to Figure 10, the distinct peaks observed in the XRD pattern indicate that the nickel oxide has high crystallinity and is uniformly formed.

[0200] In particular, it was confirmed that peaks corresponding to the (200) and (220) crystal planes appeared strongly, which indicates that the nanosheet structure of the nickel oxide is well maintained.

[0201] In addition, referring to FIGS. 11 and 12, gold (Au), nickel (Ni), and oxygen (O) components were clearly detected in the nanocomposite prepared according to Example 1, and impurity signals were hardly detected.

[0202] Through this, it was confirmed that a nanocomposite composed of nickel oxide and gold nanoparticles modified with digitonin was formed in a high-purity state on a gold-deposited silicon wafer.

[0203] The EDS mapping results also visually confirmed the uniform distribution of each component—gold (Au), nickel (Ni), and oxygen (O)—demonstrating that the nanocomposite surface was uniformly formed.

[0204]

[0205] Meanwhile, Figure 13 shows the results of the CV (cyclic voltammetry) analysis in a potassium ferricyanide-ferrocyanide solution for each sample. Here, for the CV analysis, a gold electrode sample (bare Au), a nickel oxide nanosheet sample (NiO), a nanocomposite sample composed of nickel oxide nanosheets and gold nanoparticles (NiO + GNP), and a nanocomposite sample composed of nickel oxide nanosheets and gold nanoparticles modified with digitonin (NiO + DGNP) were used.

[0206] To confirm the formation of nanocomposites formed via the drop casting method, sample-specific CVs were performed at a scan rate of 50 mV in a 5 mM potassium ferricyanide-ferrocyanide (K3[Fe(CN)6] / K4[Fe(CN)6]) solution from 10.2 V to 0.6 V. This solution plays an important role in electron transfer between the material formed on the electrode surface and the solution in electrochemical measurements.

[0207] Referring to Fig. 13, it was confirmed that a gold electrode sample with nothing formed on it (bare Au) exhibits high electrical conductivity and stable redox reaction characteristics.

[0208] In addition, it was confirmed that the nickel oxide nanosheet sample (NiO) has lower conductivity compared to the gold electrode sample (bare Au), resulting in a decrease in redox current.

[0209] In addition, it was confirmed that in a nanocomposite sample (NiO + GNP) composed of nickel oxide nanosheets and gold nanoparticles, the current density increased significantly due to the electrocatalytic effect of the gold nanoparticles.

[0210] On the other hand, in a nanocomposite sample (NiO + DGNP) composed of nickel oxide nanosheets and gold nanoparticles modified with digitonin, it was confirmed that the current density decreased due to the action of digitonin, an electrochemically inactive substance.

[0211] Through this, it was confirmed that gold nanoparticles modified with digitonin were well dispersed on nickel oxide nanosheets.

[0212] Accordingly, a nanocomposite sample (NiO + DGNP) composed of nickel oxide nanosheets and gold nanoparticles modified with digitonin can realize a composite function optimized for cholesterol sensing.

[0213] Here, nickel oxide nanosheets can strengthen the bonding force between the gold-deposited substrate and the gold nanoparticles and provide a large specific surface area, thereby increasing the reaction area of ​​the sensor, improving electrochemical stability, and increasing electron transfer efficiency.

[0214] In addition, gold nanoparticles can effectively bind with digitonin to form a cholesterol-sensing substance, promote electron transfer with excellent electrical conductivity, and provide a high surface area to volume ratio.

[0215] Furthermore, digitonin has the ability to specifically bind to cholesterol and binds to gold nanoparticles, inducing a change in the size of the gold nanoparticles according to cholesterol concentration.

[0216]

[0217] Cholesterol sensing

[0218] Figure 14 is a CV graph showing the change in cholesterol concentration of the nanocomposite prepared according to Example 1.

[0219] CV was performed according to cholesterol concentrations of 0 mM, 0.01 mM, and 1 mM. This concentration range was adopted as a value demonstrating the ability of the in vitro diagnostic biosensor for healthcare according to one embodiment of the present invention to detect cholesterol levels in the body.

[0220] In actual clinical application, the performance of the sensor can be evaluated in the range of 0 mM to 10 mM, which can include salivary cholesterol concentrations (0.12 ± 0.064 mM). The concentration range used in this experiment was determined to be sufficient to demonstrate the basic detection capability and responsiveness of the sensor.

[0221] Referring to Figure 14, it can be seen that the current density tends to decrease as the cholesterol concentration increases.

[0222] This is because as cholesterol binds to digitonin-modified gold nanoparticles (DGNP), the size of the gold nanoparticles decreases, and consequently, the specific surface area of ​​the nanocomposite is reduced, leading to a decrease in current density.

[0223] Through this, it can be confirmed that the nanocomposite prepared according to Example 1 responds to changes in cholesterol concentration.

[0224] In addition, Figure 15 is a graph showing the change in current density according to the cholesterol concentration of the nanocomposite prepared according to Example 1.

[0225] Referring to Fig. 15, the relationship between cholesterol concentration and current density can be observed in the linear graph, and the correlation coefficient (R 2 With a value of 0.944, it shows that the relationship between the measured value and the actual value is consistent. This result indicates a linear relationship between cholesterol concentration and current density, serving as an important indicator of the sensor's accuracy and reliability.

[0226]

[0227] Although the present invention has been described in detail using preferred embodiments, the scope of the invention is not limited to specific embodiments and should be interpreted by the appended claims. Furthermore, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the invention.

Claims

1. Entry; An electrode layer provided on the above-mentioned substrate; and A sensor layer provided on the electrode layer and composed of a nanocomposite; comprising, The above nanocomposite is, Metal oxide nanosheets forming a lattice structure; and An in vitro diagnostic biosensor for healthcare comprising metal nanoparticles dispersed on the metal oxide nanosheet and modified to respond to a target biomaterial.

2. In Paragraph 1, An in vitro diagnostic biosensor for healthcare in which, when the target biomaterial is cholesterol, the metal nanoparticles are modified into digitonin.

3. In Paragraph 2, The above-mentioned digitonin induces a change in the size of the metal nanoparticles according to the concentration of the above-mentioned cholesterol, an in vitro diagnostic biosensor for healthcare.

4. In Paragraph 3, The above metal nanoparticles are gold (Au) nanoparticles, and the in vitro diagnostic biosensor for healthcare is composed of gold (Au) nanoparticles.

5. In Paragraph 4, An in vitro diagnostic biosensor for healthcare, wherein the digitonin is immobilized on the surface of the gold (Au) nanoparticles by sulfuric acid (H2SO4) which promotes an esterification reaction between the carboxyl group (-COOH) on the surface of the gold (Au) nanoparticles and the hydroxyl group (-OH) of the digitonin.

6. In Paragraph 1, The above electrode layer is made of gold (Au), and The above metal oxide nanosheet is an in vitro diagnostic biosensor for healthcare, composed of nickel oxide (NiO).

7. In Paragraph 6, The above metal oxide nanosheet is an in vitro diagnostic biosensor for healthcare having X-ray diffraction peaks for (200) and (220) crystal planes.

8. A step of preparing, respectively, a substrate having an electrode layer, a metal oxide nanosheet, and metal nanoparticles modified to respond to a target biomaterial; A step of preparing a mixture by slurrying and mixing the prepared metal oxide nanosheets and the modified metal nanoparticles, respectively; and A method for manufacturing an in vitro diagnostic biosensor for healthcare, comprising the step of forming a sensor layer composed of a nanocomposite on the electrode layer from the above-manufactured mixture.

9. In Paragraph 8, When the above target biomaterial is cholesterol, the metal nanoparticles are modified into digitonin, and A method for manufacturing an in vitro diagnostic biosensor for healthcare, wherein the metal nanoparticles are composed of gold (Au) nanoparticles.

10. In Paragraph 9, The above electrode layer is made of gold (Au), and A method for manufacturing an in vitro diagnostic biosensor for healthcare, wherein the metal oxide nanosheets are made of nickel oxide (NiO).

11. In Paragraph 10, The above preparation step is, The process of synthesizing the above gold (Au) nanoparticles; A process of obtaining initially modified gold (Au) nanoparticles from the above-described synthesized gold (Au) nanoparticles; and The process includes adding digitonin to a solution containing the initially modified gold (Au) nanoparticles obtained above to obtain gold (Au) nanoparticles modified with digitonin, wherein A method for manufacturing an in vitro diagnostic biosensor for healthcare, wherein sulfuric acid (H2SO4) is further added during the process of obtaining gold (Au) nanoparticles modified with the above-mentioned digitonin.

12. In Paragraph 8, A method for manufacturing an in vitro diagnostic biosensor for healthcare, wherein the step of preparing the above mixture involves mixing the slurried metal oxide nanosheets and modified metal nanoparticles in a 1:1 ratio.

13. In Paragraph 8, A method for manufacturing an in vitro diagnostic biosensor for healthcare, wherein in the step of forming the sensor layer, a sensor layer composed of a nanocomposite is formed on the electrode layer from the prepared mixture through drop casting.

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