KR20220115411A - A method for detecting il-2 and soulble il-2 receptor alpha using a hydrogel and method for diagnosis of cancer using the same - Google Patents

Abstract

The present disclosure relates to a biosensor chip, a detection method using the same, a cancer diagnosis composition including the same, and a cancer diagnosis kit, wherein the biosensor chip includes: interleukin-2 (IL-2); a diswellable hydrogel containing a receptor alpha, beta, gamma or a combination thereof; and a transducer.

Description

Method for detecting IL-2 and soluble IL-2 receptor alpha using hydrogel and method for diagnosing cancer

Diswelling-capable hydrogels containing Interleukin-2 (IL-2) receptors alpha, beta, gamma, or combinations thereof; and a transducer; to a biosensor chip comprising the same, a detection method using the same, a composition for diagnosis of cancer including the same, and a diagnostic kit for cancer.

Interleukin-2 (IL-2) is one of a class of cytokine signaling molecules in the immune system. IL-2 has been identified as a key regulatory molecule in T-cell proliferation and natural killer cell activity, and thus is expressed as a T-cell stimulatory factor. IL-2 has an immuno-modulatory action mediated by three different cell surface receptors. These three receptors are the alpha, beta and gamma receptors, respectively. Receptor alpha binds specifically to IL-2 and is not involved in signal transduction, but its presence may enhance receptor affinity and play a role in the regulation of the lymphoid compartment. On the other hand, receptor beta is also part of the IL-15 receptor, and receptor gamma is also shared with the cytokines IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 and its receptors form a stable complex by binding to each other three-dimensionally, as shown in FIG. 1 . Receptors alpha and beta alone can bind IL-2, whereas receptor gamma alone does not (Mathias Rickert et al., J. Mol. Biol. (2004) 339, 1115-1128; Deborah J. Stauber et al., PNAS. February 21, 2006. vol.103. no.8, 2788-2793; Xinquan Wang et al., SCIENCE VOL 310 18 NOVEMBER 2005, 1159-1163; DAVID G. MYSZKA et al., Protein Sci. 1996 Dec; 5(12): 2468-2478; Yasuhiro Minami et al., Annu. Rev, Immunol. 1993, 11:24-67; and Onur Boyman et al., Nature Reviews Immunology volume 12, pages 180-190 (2012)).

On the other hand, IL-2 receptor alpha also exists in a water-soluble form. It is known that the levels of these IL-2 receptor alpha and IL-2 in the blood of cancer patients are increased. They exist either individually or as a complex of IL-2 and soluble IL-2 receptor alpha. It has been reported that increased levels of IL-2, soluble IL-2 receptor alpha, and complexes thereof are found in the serum of patients with various cancers, tumors, malignancies, benign tumors, carcinomas, sarcomas, neoplasms, etc. come. Specifically, as a result of analyzing the serum of patients with the following diseases, increased levels of IL-2 and soluble IL-2 receptor alpha were confirmed compared to the serum of normal persons: chronic myeloproliferative disease (cMPD); Examples include agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera (PV) and essential thrombocythemia (ET); haematological malignancies; non-hematologic malignancies; malignant melanoma and carcinoma of the kidney, head and neck, esophagus and lung; lymphocyte proliferative disorders; leukemias such as acute myelocytic leukemia, adult T-cell leukemia/lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Acute lymphoblastic leukemia; Lymphomas such as Anaplastic large-cell lymphoma, Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas (B cell), Hodgkin's lymphoma, peripheral T-cell lymphomas, multiple myeloma; Malignant solid tumors, such as malignant melanoma, soft tissue sarcomas, head and neck cancer, nasopharyngeal carcinoma, breast cancer, lung carcinoma, esophageal cancer, pancreatic cancer, stomach cancer, renal cell cancer , Hepatocellular carcinoma, ovarian cancer, colorectal cancer; Childhood neoplasia, such as acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Wilms' tumor, Sarcomas of bone and soft tissues, and hemophagocytic -histiocytic syndrome (Katerina E. Panteli et al., British Journal of Haematology, 130, 2005, 709-715; E. Bien et al., Biomarkers, February 2008; 13(1): 1-26; JUN MURAKAMI et al. , MOLECULAR AND CLINICAL ONCOLOGY 11: 474-482, 2019; Masahiko Shibata et al., Oncology 1999; 56:54-58; and Liang Wang et al., Ann Hematol (2017) 96:2079-2088). Therefore, IL-2, IL-2 receptor alpha, and their complexes can be used in the diagnosis of these various cancers.

Currently used as a cancer diagnostic kit is an enzyme-linked immunosorbent assay (ELISA). ELISA is a method for quantitatively measuring the strength and amount of an antigen-antibody reaction by measuring the activity of an enzyme by labeling an antibody or antigen with an enzyme. Most simple forms of ELISA work as follows: an antibody is attached to a substrate, and an antigen is attached to the antibody on the surface through an antigen-antibody reaction from the sample to be tested. Another secondary antibody matching the antigen can then be applied over the surface to bind the antigen. This secondary antibody is linked to the enzyme, and the unbound secondary antibody is then removed. Finally, when a substrate containing the substrate of the enzyme is added and binding occurs, it causes a detectable signal, mostly a color change. It is also possible to detect IL-2 and soluble IL-2 receptor alpha through this ELISA technique. However, in the case of detection through such an ELISA, it takes about 5 hours and takes a very long time, and the antibody does not bind to IL-2 or water-soluble IL-2 receptor alpha, but binds to other places, for example, the surface of the substrate. Undesirable, non-specific binding may occur, which reduces the accuracy of the detection method somewhat. In addition, since binding through an antigen-antibody reaction is detected, there is a problem in that even an antigen that is inactive can be detected even if there is only a fragment of the antigen binding site. In addition, since IL-2 is easily degraded in the body and has a short half-life, it may lead to a decrease in detection performance when a long detection time is required as in ELISA. In addition, since IL-2 is small in size, it is difficult to use conventional detection methods. Furthermore, detection through ELISA requires separate tests for each of IL-2 and soluble IL-2 receptor alpha, and a method for testing both these substances at once does not yet exist. Moreover, although IL-2 is not stable alone, it is relatively stable when complexed with IL-2 receptor alpha, and accurate detection is difficult because ELISA does not detect such a complex.

Accordingly, there is a need for a method for detecting IL-2, soluble IL-2 receptor alpha, and their complexes through a single test within a short time.

On the other hand, the above-mentioned background technology is possessed or acquired by the inventor in the process of deriving the disclosure of the present application, and cannot necessarily be said to be a known technology disclosed to the general public prior to the present application.

The present disclosure provides a method for detecting at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha in a short time, IL-2 receptor alpha, beta, gamma or a diswellable hydrogel containing a combination thereof; and a transducer; provides a biosensor chip comprising a. In addition, a detection method using the same, a composition for diagnosing cancer, and a cancer diagnosis kit are provided.

In a first aspect for achieving the above object, the present disclosure provides a diswellable hydrogel containing interleukin (IL)-2 receptor alpha, beta, gamma or a combination thereof; and a transducer; provides a biosensor chip comprising a.

In one embodiment, the receptor contained in the hydrogel binds to at least one of IL-2, IL-2 receptor alpha, and a complex of IL-2 and IL-2 receptor alpha. In one aspect, the hydrogel comprises two or more of receptors alpha, beta, and gamma. In one embodiment, the hydrogel comprises IL-2 receptor beta and gamma. In one embodiment, the hydrogel comprises IL-2 receptors alpha, beta and gamma. In one embodiment, the hydrogel comprises a copolymer consisting of a main monomer and a comonomer. In one embodiment, the main monomer is N-isopropylacrylamide, N-acryloylglycinamide (N-acryloylglycinamide), hydroxypropylcellulose (hydroxypropylcellulose), vinylcaprolactam (vinylcaprolactame), N-vinylpyrrolidone (N-vinyl pyrrolidone), 2-hydroxyethyl methacrylate, ethylene glycol; amino acids such as aspartic acid, glutamic acid, and L-lysine; selected from the group consisting of caprolactone, and vinyl methyl ether,

The comonomer is allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc) is selected from

In one embodiment, the hydrogel further comprises a crosslinking agent. In one embodiment, the crosslinking agent is, N,N'-methylene-bis-diacrylamide (MBA), polyethylene glycol (PEG) dihydroxyl (PEG dihydrocyl), PEG diamine (PEG diamine), PEG dioxyamine (PEG) dioxyamine), PEG dichloride, PEG dibromide, PEG diazide, PEG dithiol, PEG dialdehyde, PEG diepoxide (PEG) diepoxide), PEG diacrylate, PEG dimethacrylate, PEG diacetic acid, PEG disuccinic acid, PEG disuccimidylcarboxymethyl ester (PEG discuccinimidyl carboxy methyl ester), poly(ε-caprolactone)diacrylate], poly(ε-caprolactone)dimethacrylate [poly(ε-caprolactone)dimethacrylate], poly Lactide diacrylate, polylactide dimethacrylate, poly(lactide-co-glycolide) diacrylate [poly(lactide-co-glycolide)diacrylate], poly(lactide) tide-co-glycolide)dimethacrylate [poly(lactide-co-glycolide)dimethacrylate], poly(ε-caprolactone-b-ethylene glycol-b- ε-caprolactone)diacrylate [poly(ε-) caprolactone-b-ethylene glycol-b-ε-caprolactone)diacrylate], poly(ε-caprolactone-b-ethylene glycol-b-ε-caprolactone)dimethacrylate [poly(ε-caprolactone-b-ethylene glycol) -b-ε-caprolacton e)dimethacrylate], poly(lactide-b-ethylene glycol-b-lactide)diacrylate [poly(lactide-b-ethylene glycol-b-lactide)diacrylate], poly(lactide-b-ethylene glycol- b-lactide)dimethacrylate [poly(lactide-b-ethylene glycol-b-lactide)dimethacrylate], poly[(lactide-co-glycolide)-b-ethylene glycol-b-(lactide-co) -glycolide)]diacrylate {poly[(lactide-co-clycolide)-b-ethylene glycol-b-(lactide-co-glycolide)] diacrylate}, poly[(lactide-co-glycolide)-b -ethylene glycol-b-(lactide-co-glycolide)]dimethacrylate {poly[(lactide-co-clycolide)-b-ethylene glycol-b-(lactide-co-glycolide)] dimethacrylate}, poly (ε-caprolactone-co-lactide)-diacrylate [poly(ε-caprolactone-co-lactide)-diacrylate], poly(ε-caprolactone-co-lactide)-dimethacrylate [poly( ε-caprolactone-co-lactide)-dimethacrylate], poly(ε-caprolactone-co-glycolide)-diacrylate [poly(ε-caprolactone-co-glycolide)-diacrylate], poly(ε-caprolactone- co-glycolide)-dimethacrylate [poly(ε-caprolactone-co-glycolide)-dimethacrylate], poly[(caprolactone-co-lactide)-b-ethylene glycol-b-(caprolactone-co- lactide)]diacrylate {poly[(caprolactone-co-lactide)-b-ethylene glycol-b-(caprolactone-co-lactide)]diacrylate}, poly[(caprolactone-co-lactide)-b- Ethylene glycol-b-(caprolactone-co-lactide)]dimethacrylate {poly[(caprolactone-co-) lactide)-b-ethylene glycol-b-(caprolactone-co-lactide)]dimethacrylate}, poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide)] diacrylate {poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide)]diacrylate}, poly[(caprolactone-co-glycolide)-b-ethylene glycol-b -(caprolactone-co-glycolide)]dimethacrylate {poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide)]dimethacrylate} and combinations thereof is selected from In one embodiment, the hydrogel includes a copolymer consisting of 50 to 97.9% by weight of the main monomer, 2 to 40% by weight of the comonomer, and 0.1 to 10% by weight of a crosslinking agent. In one embodiment, the hydrogel is, poly(N-isopropyl acrylamide-co-allylamine) [poly(N-isopropylacrylamide-co-allylamine): poly(NIPAM-co-AA)], poly(N-iso Propylacrylamide-co-2-(dimethylamino)ethyl methacrylate)[poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl methacrylate): poly(NIPAM-co-DMAEMA)], poly(N- Isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate), poly(NIPAM-co-DMAEA)], poly(N- Isopropylacrylamide-co-acrylic acid) [poly(N-isopropylacrylamide-co-acrylic acid): poly(NIPAM-co-AAc)], poly(N-isopropylacrylamide-co-polyethylene glycol-acrylic acid) [poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid): poly(NIPAM-co-PEG-AAc)], and poly(N-isopropylacrylamide-co-methacrylic acid) [poly(N-isopropylacrylamide-co) -methacrylic acid): poly(NIPAM-co-MAAc)]. In one aspect, the hydrogel comprises a copolymer prepared using N-isopropylacrylamide as a main monomer, acrylic acid as a comonomer, and MBA (N, N'-methylene-bis-acrylamide) as a crosslinking agent. .

In one embodiment, when the receptors contained in the hydrogel bind to IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, the hydrogel is diswelled. In an aspect, a change in optical properties of the biosensor chip occurs due to the deswelling. In one aspect, the optical property is refractive index. In one aspect, the change in the optical property is detected through surface plasmon resonance.

In one embodiment, the biosensor chip detects at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha within 30 minutes.

In one aspect, the transducer includes a metal thin film. In one embodiment, the metal thin film is surface-modified with cysteamine. In one embodiment, the metal thin film is a gold thin film.

A second aspect is a method for detecting at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha,

providing the biosensor chip;

isolating serum from blood; and

It provides a detection method comprising the step of flowing the separated serum on the biosensor chip.

In one aspect, the detection method includes measuring a change in an optical property of a sensor chip.

A third aspect provides a composition for diagnosing cancer including the biosensor chip.

In one embodiment, the cancer is lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, head and neck cancer, bone marrow cancer, biliary tract cancer, chronic myeloid cancer Chronic myeloproliferative disease (cMPD), agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera (PV), essential thrombocythemia ET), haematological malignancies, non-hematologic malignancies, malignant melanomas and carcinomas of the kidney, head and neck, esophagus, nasopharynx, liver and lung, lymphoproliferative disorders, leukemia, acute myeloid leukemia Acute myelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, lymphoma, anaplastic large-cell lymphoma ), Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas, Hodgkin's lymphoma, Peripheral T-cell lymphomas ), multiple myeloma, malignant solid tumor, malignant melanoma, soft tissue sarcomas, renal cell carcinoma, childhood neoplasia, Wilms' tumor, bone tissue sarcoma ( sarcomas of bone tissues), or hemophagocytic-tissues It's a syndrome.

A fourth aspect provides a diagnostic kit for cancer, including a biosensor chip.

In one embodiment, the cancer is lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, head and neck cancer, bone marrow cancer, biliary tract cancer, chronic myeloid cancer Chronic myeloproliferative disease (cMPD), agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera (PV), essential thrombocythemia ET), haematological malignancies, non-hematologic malignancies, malignant melanomas and carcinomas of the kidney, head and neck, esophagus, nasopharynx, liver and lung, lymphoproliferative disorders, leukemia, acute myeloid leukemia Acute myelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, lymphoma, anaplastic large-cell lymphoma ), Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas, Hodgkin's lymphoma, Peripheral T-cell lymphomas ), multiple myeloma, malignant solid tumor, malignant melanoma, soft tissue sarcomas, renal cell carcinoma, childhood neoplasia, Wilms' tumor, bone tissue sarcoma ( sarcomas of bone tissues), or hemophagocytic-tissues It's a syndrome.

The biosensor chip according to the present disclosure can quantitatively measure the concentration of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, which are markers of cancer diagnosis within a short time. Therefore, it provides a very rapid cancer diagnosis method, and it can be used very usefully because it can be detected with only a very small amount of blood.

1 is a diagram showing the binding of IL-2 and its receptors alpha, beta, and gamma.
2 illustrates an aspect of a biosensor chip according to the present disclosure.
Figure 3 (a) shows the maximum reaction value according to the concentration of the hydrogel of Examples 2 to 4, respectively, (b), (c) and (d) of Figure 3 are the hydrogels of Examples 2 to 4, respectively. It shows the reaction signal according to time of the gel.
Figure 4 (a) shows the reaction signal according to the concentration when the cancer patient serum is flowed to the hydrogel of Example 4, (b) is the cancer patient serum flowed to the hydrogel of Example 4 In this case, the reaction signal according to time is shown.
Figure 5 (a) shows the maximum reaction value according to the concentration of the hydrogel of Examples 1, 4 and 5, respectively, Figure 5 (b), (c) and (d) are Examples 1, 4, respectively And 5 shows the reaction signal according to the time of the hydrogel.
6 is a comparison of the maximum response values of the biochip of the hydrogel of Example 4 and the biochip of the pure gel as a control according to the concentrations of IL-2 and IL-2Rα.
7 shows the reaction signals confirmed when the normal serum and cancer patient serum were respectively flowed to the hydrogel of Example 4. At this time, it is corrected by subtracting the maximum reaction value confirmed in the hydrogel of the pure gel from the maximum reaction value confirmed in the hydrogel of Example 4.
FIG. 8 shows the concentration of IL-2Rα in the serum of normal people and cancer patients using the ELISA kit, which is a conventional diagnostic method.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.

Terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

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

In addition, in the description with reference to the accompanying drawings, the same components are assigned the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In the description of the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

In addition, in describing the components of the embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms. When it is described that a component is “connected”, “coupled” or “connected” to another component, the component may be directly connected or connected to the other component, but between each component another component It will be understood that may also be "connected", "coupled" or "connected".

Components included in one embodiment and components having a common function will be described using the same names in other embodiments. Unless otherwise stated, descriptions described in one embodiment may be applied to other embodiments as well, and detailed descriptions within the overlapping range will be omitted.

According to a first aspect of the present disclosure, there is provided a diswellable hydrogel containing IL-2 receptor alpha, beta, gamma or a combination thereof; and a transducer; is provided.

As described above, ELISA, which is a conventional method for detecting IL-2 and IL-2 receptor alpha, is difficult to detect rapidly, requires individual detection, and is difficult to detect complexes thereof, resulting in poor accuracy. Accordingly, the present disclosure provides a detection method using several receptors of IL-2. Since the majority of methods for detecting marker substances and methods for diagnosing cancer use antigen-antibody reactions, a method for detecting and diagnosing IL-2 through binding of IL-2 to its receptor was first developed by the present inventors. Usually, in the case of binding to a receptor, it is difficult to use in detection and diagnostic methods because of its low affinity, but in the case of IL-2 and its receptor, the affinity is high, and in particular, several receptors (ie, IL-2 receptor alpha and beta, or beta and gamma), preferably all three receptors, when IL-2 binds to form a very stable complex, so it has a high affinity. Therefore, in the present disclosure, based on the high affinity between such IL-2 and its receptors, it is used for detection and diagnostic methods. In addition, for accurate and sensitive detection of such binding, hydrogels with special properties are used in the present disclosure.

"Hydrogel" according to the present disclosure refers to a three-dimensional network formed by crosslinking a hydrophilic polymer chain as known per se in the art, for example, a Korean Patent Registration and hydrogels disclosed in No. 10-1754774. The hydrogel consists of at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt% water.

The term "diswellable" according to the present disclosure means that the physical shape of the hydrogel is changed, and the opposite phenomenon of swelling, i.e., deswelling, is possible, and the hydrogel according to the present disclosure has its softness. Due to this, it refers to a property that can spontaneously dewell when the receptors bound therein are multiple-coupled with the target material. Specifically, when multiple receptors and one target material in the hydrogel are combined with one another, the degree of crosslinking of the hydrogel increases due to the binding, which causes the hydrogel network to be located more densely and the structure inside the hydrogel is modified together. will become At this time, as the water constituting the hydrogel is drained due to the deformation of the structure, a diswelling phenomenon in which the volume of the hydrogel is reduced may occur. That is, a three-dimensional network is formed in the hydrogel upon binding of receptors (that is, a kind of cross-link is formed), and the more bonds there are, the more networking points (that is, because the density of cross-links increases). volume will be greatly reduced.

A hydrogel according to the present disclosure has an appropriate softness and/or (visco)elastic modulus to be diswell-able. The hydrogel according to the present disclosure is soft, that is, high in ductility, and for this purpose, in one embodiment, the elastic modulus of the hydrogel particles at 25° C. may be in the range of 0.01 Pa to 100 Pa ( For further discussion of ductility, see the following literature, which is incorporated herein by reference in its entirety: Mattias Karg et al., Langmuir 2019, 35, 6231-6255).

The hydrogel according to the present disclosure may be in the form of particles. The biosensor chip according to the present disclosure may include a plurality of hydrogel particles. The diameter of the hydrogel particles is from about 50 nm to about 3,000 nm, for example from about 100 nm to about 2500 nm, or from about 300 nm to about 2000 nm, preferably at least about 440 nm, more preferably about 540 nm. at least about 700 nm, and even more preferably at least about 700 nm, for example, in the range of about 700 nm to about 1300 nm.

The diameter of the hydrogel can be measured by a conventional method known to those skilled in the art, for example, dynamic light scattering (light correlation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS) and medium-angle laser light scattering (MALLS)), Light obscuration methods (such as the Coulter analysis method), or other methods (such as rheology, and light or electron microscopy) can be used.

The surface of the hydrogel particles according to the present disclosure may be modified so that a receptor may be bound to the surface thereof. In one example, the particle surface can be modified using standard binding methods including maleimide/thiol and EDC/NHS bonding. More specifically, the linkage is a carbodiimide cross-linking, a Schiff base cross-linking, an azlactone cross-linking, a carbonyl diimidazole (CDI) cross-linking, iodoacetyl ) crosslinking, hydrazide crosslinking, Mannich crosslinking, and maleimide crosslinking. Other useful binding methods for hydrogel particles are described in Hermanson et al., (2013) Bioconjugate Techniques: Academic Press, which is incorporated herein by reference in its entirety.

In the biosensor chip according to the present disclosure, IL-2 receptor alpha, beta, gamma or a combination thereof is a target substance, that is, IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha. It is possible to specifically bind to at least one of In one embodiment, the hydrogel comprises IL-2 receptor beta and gamma, wherein the receptors are capable of binding to both IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha. In one embodiment, the hydrogel comprises IL-2 receptors alpha, beta and gamma, wherein the receptors are capable of binding IL-2.

In one embodiment, the hydrogel comprises at least two of IL-2 receptors alpha, beta and gamma. In this case, since the hydrogel contains two or more different types of receptors, when binding to the target substance IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, it is not a single bond but multiple It is possible to form bonds, and it is possible to induce more stable complex formation and optical changes due to diswelling of the hydrogel. For example, when the hydrogel contains IL-2 receptor beta and gamma, multiple bonds may be formed because one target material forms a bond with receptor gamma as well as receptor beta. As such, when the hydrogel contains two or more different receptor types to form multiple bonds with the target material, an effect of increasing the degree of crosslinking in the hydrogel is induced and changes in optical properties through diswelling of the hydrogel may occur. can Therefore, when one receptor is included, that is, a more stable complex is formed than when a single bond is formed, and it can also cause diswelling of the hydrogel, that is, a greater physical change of the hydrogel, thereby showing better detection performance. can

As the hydrogel is diswelled upon binding between the receptor and the target material, the hydrogel undergoes a change in physical properties. At this time, physical properties are usually mechanical properties such as strength, hardness, and elongation of materials, electromagnetic properties such as electrical conductivity, resistivity, transmittance, and refractive index, thermal properties such as thermal conductivity, coefficient of thermal expansion, specific heat, melting point, temperature such as boiling point It is a generic term for character. In one aspect, the change in the physical property is a change in the optical property, for example, a change in refractive index. In another embodiment, the change in the physical property is a change in volume.

The transducer of the biosensor chip according to the present disclosure serves to transmit a displacement signal corresponding to a change in the physical properties of the hydrogel to the analysis equipment.

The biosensor chip may be mounted on an analysis device (not shown) such as a biosensor, and may further include a physical interface (wired or wireless) for transmitting a displacement signal to the analysis device. Here, the analysis equipment is an equipment that visually/auditively/tactilely provides analysis results related to multiple binding of a target protein and a binding-mediated substrate to the user. The analysis equipment may be, for example, a Surface Plasmon Resonance (SPR) equipment from Reichert®. Also, depending on the implementation, the analysis equipment may be implemented as a mobile device. The biosensor chip is designed to be detachable from various analysis equipment and may be implemented in a replaceable manner.

The transducer outputs, as a displacement signal, a change in the physical properties of the hydrogel caused by the binding between the target material and the receptor administered on the biosensor chip. According to one implementation, the transducer may further include a physical interface unit (not shown) for transmitting the displacement signal to an external analysis equipment. According to another implementation, the transducer may further include a signal processing unit (not shown) for processing the displacement signal and a physical interface unit for transmitting the processing result to an external device.

When the physical property that changes due to diswelling of the hydrogel is the refractive index, the transducer analyzes the displacement signal, which is either an optical signal or an electrical signal, corresponding to the change in the refractive index of the hydrogel caused by the binding between the target material and the receptor. It may be a waveguide outputting as . The waveguide may be a Surface Plasmon Resonance (SPR) Waveguide, a Ring Resonator Waveguide, a Long-Period Fiber Grating Waveguide, a Grating Coupler, or a grating waveguide. (Grated Waveguide) may be any one.

When the physical property that changes due to deswelling of the hydrogel is volume, the transducer is a piezo element that outputs a displacement signal corresponding to the volume change of the hydrogel caused by the binding between the target material and the receptor to the analysis equipment may include. In this case, a Quartz Crystal Microbalance (QCM) equipment may be used as the analysis equipment.

Depending on how much or how concentrated the binding between the target material and the receptor occurs, the change in the physical properties of the hydrogel becomes greater. Using this relationship, a lookup table (not shown) generated by comparing the amount of binding and the change value of the physical properties of the hydrogel can be used to detect the binding between the target protein and the receptor and measure the amount of binding. can The lookup table may be recorded in a memory connected to an external analysis device such as a biosensor connected to the biosensor chip through a physical interface.

When the change in the physical property due to the diswelling of the hydrogel is a change in refractive index, the transducer transmits a displacement signal corresponding to the change in the refractive index of the hydrogel to the analysis equipment. In this case, the transducer may be a waveguide. Due to the change in the refractive index of the hydrogel, the effective refractive index of at least a part of the waveguide, which is a transducer, is changed. An input optical signal input to the biosensor chip through a light source of an analysis device (not shown) has a resonance frequency shifted due to a change in effective refractive index generated in the transducer, and an output optical signal whose resonance frequency is shifted or A corresponding electrical signal (displacement signal) is transmitted to the detector of the analysis instrument. The analysis equipment may measure the amount of binding between the target protein and the receptor by analyzing the displacement signal.

As another example, the physical property may be a refractive index, and the transducer transmits a displacement signal corresponding to a change in the refractive index of the hydrogel to the analysis equipment. In this case, the transducer may be a gold thin film. Surface Plasmon Resonance (SPR) occurs in the gold thin film, which is a transducer, due to a change in the refractive index of the hydrogel. In more detail, an input signal inputted to the biochip 200 from an analysis device (not shown) may change a progression path due to surface plasmon resonance generated in the gold thin film, or a leak signal may occur on the gold thin film. have. In this way, a displacement signal (output optical signal or an electrical signal corresponding thereto) such as a signal whose path is changed or a leak signal is transmitted to the detector of the analysis equipment.

As another example, the physical property may be volume, and the transducer includes a piezo element capable of transmitting an electrical signal (displacement signal) according to a change in the volume of the hydrogel to the analysis equipment. The analysis equipment may measure the amount of binding between the target protein and the receptor corresponding to the volume change of the hydrogel by analyzing the displacement signal.

According to one side, the hydrogel may include a copolymer consisting of a main monomer and a comonomer. According to one embodiment, the hydrogel may be one in which a main monomer and a comonomer are polymerized with a crosslinking agent. Preferably, monomers capable of forming hydrogels sensitive to heat, ionic strength, or pH may be used. According to one side, the main monomer may be selected from the group consisting of N-isopropyl acrylamide, poly(N-acryloylglycinamide), hydroxypropylcellulose, poly(vinylcaprolactame), and polyvinyl methyl ether. The comonomer is from the group consisting of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc) can be selected from

According to one side, the hydrogel may include 55 to 98% of the main monomer, 2 to 40% of the comonomer, and 0.1 to 5% of the crosslinking agent. According to one side, when the content of the main monomer is less than 55%, the reactivity may be reduced, and if it is more than 98%, the ability to detect multiple binding of the target protein may be reduced. According to another aspect, when the content of the comonomer is less than 2% or more than 40%, the ability to detect multiple binding of the target protein may be reduced. In addition, when the content of the crosslinking agent is less than 0.1%, it may be difficult to form a hydrogel, and when it is more than 5%, the ability to detect multiple binding of the target protein may be reduced.

According to one side, the hydrogel is, poly(N-isopropylacrylamide-co-allylamine) [poly(N-isopropylacrylamide-co-allylamine): poly(NIPAM-co-AA)], poly(N-iso Propyl acrylamide-co-2-(dimethylamino)ethyl methacrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl methacrylate): poly(NIPAM-coDMAEMA)], poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate)[poly(N-isopropylacrylamide-co-2-(dimethylamino)ethyl acrylate): poly(NIPAM-co-DMAEA)], poly(N-isopropyl acrylic Amide-co-acrylic acid) [poly(N-isopropyl acrylamide-co-acrylic acid): poly(NIPAM-co-AAc)], poly(N-isopropyl acrylamide-co-polyethylene glycol-acrylic acid) [poly(N -isopropyl acrylamide-co-polyethylene glycol-acrylic acid): poly(NIPAM-co-PEG-AAc)], poly(N-isopropyl acrylamide-co-methacrylic acid)[poly(N-isopropylacrylamide-co-methacrylic acid) ): poly(NIPAM-co-MAAc)], N-isopropyl acrylamide, poly(Nacryloylglycinamide [poly(Nacryloylglycinamide)], hydroxypropylcellulose, poly(vinylcarbrolactam) (vinylcaprolactame)), and may include at least one selected from the group consisting of polyvinyl methyl ether.

Preferably, the main monomer may be N-isopropyl acrylamide (temperature-sensitive hydrogel), and the comonomer may be acrylic acid (pH-sensitive hydrogel). In this case, the crosslinking agent may be MBA (N,N'-methylene-bis-acrylamide).

According to one side, the hydrogel may further include an initiator as an element for starting a polymerization reaction, and ammonium persulfate (APS) may be used as the initiator.

According to one side, the hydrogel may include 55 to 98% of N-isopropyl acrylamide, 2 to 40% of acrylic acid, and 0.1 to 5% of a BIS crosslinking agent. In particular, when acrylic acid is used as the comonomer, it is preferable to include at least 2% or more of the materials constituting the hydrogel.

The hydrogel according to an embodiment is not limited to the examples described herein, since it is possible to control the sensitivity to binding by adjusting the ratio between the components. According to one side, as the specific gravity of acrylic acid is high and the specific gravity of BIS is low, the reactivity (degree of diswelling) of the hydrogel to the multiple binding between the target protein and the receptor may increase.

The transducer surface of the biochip according to one side may have a modified surface in order to increase the binding force with the hydrogel. In one aspect, the transducer is a thin metal film, for example, a thin film of gold, the surface of which may be surface-modified with cysteamine.

The receptors formed in the hydrogel are carbodiimide cross-linking, Schiff base cross-linking, Azlactone cross-linking, carbonyl diimidazole (CDI) cross-linking, iodo It may be formed in the hydrogel by acetyl (Iodoacetyl) cross-linking, hydrazide (Hydrazide) cross-linking, Mannich (Mannich) cross-linking, or maleimide (maleimide) cross-linking. A receptor may be formed on the surface of the hydrogel by using a carboxylic acid functional group (COOH) present on the surface of the hydrogel according to an embodiment and an NH2+ group present in the protein.

According to one side, for carbodiimide crosslinking, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) , DCC (dicyclohexyl carbodiimide; dicyclohexyl carbodiimide), NaCNBH3 (sodiumcyanoborohydride; sodium cyanoborohydride), Azlactone, CDI (Carbonyl diimidazole; carbonyl diimidazole), iodoacetyl, hydra At least one crosslinking agent selected from the group consisting of zaide, diaminodipropylamine (DADPA; diaminodipropylamine), and N-hydroxysuccinimide esters (N-hydroxysuccinimide esters) may be used. By controlling the crosslinking agent, the binding properties between the receptor and the hydrogel can be controlled.

In one aspect, the biosensor chip of the present disclosure reacts at least one of a target material, IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, within about 30 minutes, for about 20 minutes. Within about 10 minutes, about 9 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute can be detected within Therefore, it provides an advantage that detection is possible within a very short time compared to ELISA, which is widely used as a conventional detection method. In addition, the biosensor chip according to the present disclosure has the advantage of being able to detect a target material even with a very small amount of sample. In one aspect, when the sample being tested is serum, detection of a target substance with only a trace amount of serum of about 10 mL or less, about 1 mL or less, about 0.5 mL or less, about 0.1 mL or less, about 10 μL or less, or about 1 μL of serum This is possible.

According to one aspect, the present disclosure provides a method for detecting at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, comprising:

providing the biosensor chip;

isolating serum from blood; and

It provides a detection method comprising the step of flowing the separated serum on the biosensor chip.

As described above, the biochip according to the present disclosure is capable of specifically detecting at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha in serum. Therefore, it is possible to accurately measure the presence and concentration of a target substance using this.

According to one aspect, the present disclosure provides a composition for diagnosis of cancer including the biosensor chip.

According to another aspect, the present disclosure provides a cancer diagnosis kit including the biosensor chip.

Here, "cancer" includes all cancers, and includes lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, two Examples include, but are not limited to, cervical cancer, bone marrow cancer, and biliary tract cancer. In addition, the cancer may further include a cancer that increases the concentration of at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha in the patient's blood. For example, the cancer is chronic myeloproliferative disease (cMPD), such as agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera ; PV) and essential thrombocythemia (ET); haematological malignancies; non-hematologic malignancies; malignant melanoma and carcinoma of the kidney, head and neck, esophagus and lung; lymphocyte proliferative disorders; leukemias such as acute myelocytic leukemia, adult T-cell leukemia/lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Acute lymphoblastic leukemia; Lymphomas such as Anaplastic large-cell lymphoma, Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas (B cell), Hodgkin's lymphoma, peripheral T-cell lymphomas, multiple myeloma; Malignant solid tumors, such as malignant melanoma, soft tissue sarcomas, head and neck cancer, nasopharyngeal carcinoma, breast cancer, lung carcinoma, esophageal cancer, pancreatic cancer, stomach cancer, renal cell cancer , Hepatocellular carcinoma, ovarian cancer, colorectal cancer; Childhood neoplasia, such as acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Wilms' tumor, Sarcomas of bone and soft tissues, and hemophagocytic -Including histological syndrome.

The term “diagnosis” according to the present disclosure refers to determining a disease name, and may include the disease name, disease state, stage, etiology, presence or absence of complications, prognosis, and recurrence of breast cancer.

It is understood that the term “about” refers to a range of numbers that one of ordinary skill in the art would consider equivalent to the recited value in terms of achieving the same function or result.

All numerical ranges set forth throughout this specification include their upper and lower values, as well as all narrower numerical ranges falling therein, and all such narrower numerical ranges are considered to be expressly and specifically set forth herein.

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (vol/vol) %.

material used

N-isopropylacrylamide (NIPAm), acrylic acid (AAc), N,N'-methylenebisacrylamide (N,N'-methylenebisacrylamide; BIS), ammonium persulfate (APS), poly Oxyethylenesorbitan monooleate (Tween 80), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC) and N -Hydroxysuccinimide (N-hydroxysuccinimide; NHS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-2, IL-2Rα IL-2Rβ and IL-2Rγ proteins were purchased from Acrobiosystems (Newark, DE, USA). Normal human serum (Off-the-Clot) was purchased from Zen-bio (Research Triangle, NC, USA). The water used throughout this example was distilled, deionized to 18.2 MΩ cm (aquaMAX-Ultra 370 water purification system, Young In, Anyang, Korea), and filtered through a 0.22 μm filter to remove particulate matter. 0.1M MES buffer (pH 5.5) was purchased from Bio-solution (Seoul, KOREA). 0.9% sodium chloride buffer was purchased from Thermo Fisher Scientific. Phosphate buffered saline (PBS, 1X) without calcium or magnesium was purchased from WELGENE (Gyeongsan, Korea).

production example

1. Method for producing hydrogel nanoparticles (nanogel)

Hydrogels have been described in previously published papers (Kim, J.; Park, Y.; Brown, A. C.; Lyon, L. A. Direct Observation of Ligand-Induced Receptor Dimerization with a Bioresponsive Hydrogel. RSC Adv. 2014, 4, 65173-65175). With reference to the content of aqueous free-radical precipitation polymerization (precipitation polymerization) was prepared as follows.

A solution mixture with a total surfactant concentration of 0.5 mM was prepared by dissolving NIPAm and BIS in deionized water, followed by addition of Tween 80. At this time, the total reaction volume of the solution mixture was fixed at 100 mL. In addition, thereafter, the total monomer concentration was 100 mM, and the NIPAm, AAc and BIS contents relative to the total monomer concentration were adjusted to be 88 wt%, 10 wt%, and 2 wt%, respectively, to prepare a reaction solution of NIPAm and BIS.

The monomer solution was filtered with a 0.22 μm syringe-driven filter, which was then transferred to a 100 mL three-neck round bottom flask with a reflux condenser, thermometer and magnetic stirring bar. After equilibration to 70° C., the reaction mixture was purged with Ar while stirring at 275 rpm. After 1 hour, a solution of AAc was pipetted and the Ar purging was maintained for 10 minutes prior to initiation of polymerization. Polymerization was initiated by syringe injection of a 0.2 mL aliquot of 1 mM APS into the reaction vessel. The reaction was allowed to continue for 6 h at 70° C. under continuous Ar purging with stirring. After cooling, the resulting mixture was purified by dynamic dialysis (Spectra/Por Tube-A-Lyzer, MWCO 100 kD, Repligen, Boston, MA, USA) using pure water for 24 h, and at the 12 h mark, exchanged water.

2. Protein Conjugation of Hydrogels

(1) Example 1: Preparation of IL-2Rα-conjugated hydrogel

IL-2Rα protein (ILA-H5251), IL-2Rβ protein (ILB-H5253) and IL-2Rγ (ILG-H5256) proteins were all purchased from Acrobiosystems (Newark, DE, USA), respectively. Protein powder was dissolved in tertiary distilled water to prepare a protein solution with a concentration of 6 uM, respectively.

The freeze-dried 700 nm-sized hydrogel was dissolved in 0.1M MES (2-(N-morpholino)ethanesulfonic acid; 2-(N-Morpholino)ethanesulfonic acid) buffer (pH 5.5) to obtain 10 mg/ml 40ul of the solution was obtained. Then, it was incubated at room temperature for 1 hour. 10ul EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (20 in MES) in 40 ul of hydrogel solution (10 mg/ml) ug/ml concentration) and 10 ul NHS (N-hydroxysuccinimide) (40 ug/ml concentration in MES) were added and incubated at room temperature for 5 minutes. After that, 10 ul each of IL-2Rα protein (6 uM) and PBS were added, and incubated at room temperature for 1 hour. Thereafter, the solution was centrifuged at 8,000 rpm at a temperature of 4° C. for 5 minutes, the supernatant was removed, and the pellet was released in 80 ul of PBS. This process was repeated 4 times. The finished sample was refrigerated and used within 24 hours.

(2) Example 2: Preparation of IL-2Rβ-conjugated hydrogel

All were prepared in the same manner as in Example 1, except that IL-2Rβ protein (6 uM) was used instead of IL-2Rα protein (6 uM).

(3) Example 3: Preparation of IL-2Rγ-conjugated hydrogel

All were prepared in the same manner as in Example 1, except that IL-2Rγ protein (6 uM) was used instead of IL-2Rα protein (6 uM).

(4) Example 4: Preparation of IL-2Rβγ-conjugated hydrogel

The same procedure as in Example 1 was used except that 10 ul of IL-2Rα protein (6 uM) and 10 ul of IL-2Rβ protein (6 uM) and 10 ul of IL-2Rγ protein (6 uM) were used instead of 10 ul of PBS. manufactured through

(5) Example 5: Preparation of IL-2Rαβγ-conjugated hydrogel

The freeze-dried 700 nm-sized hydrogel was dissolved in 0.1M MES buffer (pH 5.5) to obtain 30ul of a solution having a concentration of 13.3 mg/ml. Then, it was incubated at room temperature for 1 hour. 10 ul EDC (20 ug/ml concentration in MES) and 10 ul NHS (40 ug/ml concentration in MES) were added to 30 ul of hydrogel solution (13.3 mg/ml) and incubated at room temperature for 5 minutes. Thereafter, 10 ul each of IL-2Rα protein (6 uM), IL-2Rβ protein (6 uM), and IL-2Rγ protein (6 uM) were added, and incubated at room temperature for 1 hour. After this, the solution was centrifuged at 8,000 rpm at a temperature of 4 °C for 5 minutes, the supernatant was removed, and the pellet was dissolved in 80 ul of PBS. This process was repeated 4 times. The finished sample was refrigerated and used within 24 hours.

In summary, the final concentrations of each component in the prepared solutions were as follows: hydrogel - 5 mg/ml; EDC - 2.5 ug/ml; NHS - 5 ug/ml; Each IL-2R protein - 0.8 uM.

3. Fabrication of the sensor chip

A bare gold substrate to be used as a transducer of the sensor chip was prepared by purchasing SIA Kit AU (cat.no BR-1004-05) from GE Healthcare.

In order to provide an amine group capable of binding to the hydrogel to the sensor chip, cysteamine was purchased from Sigma (cat. No M6500) and dissolved in water to a concentration of 10 mM to prepare a solution.

A cysteamine solution was flowed on the surface of the gold substrate at a rate of 2 ul/min for 300 seconds. Thereafter, the substrate was washed by flowing tertiary distilled water at a rate of 5 ul/min for 60 seconds.

The hydrogel conjugated with the receptor obtained in step 2 above was diluted 1/5 in 0.1M MES buffer (pH 5.5). This was flowed to the substrate at a rate of 2 ul/min for 60 seconds. The same flowing process was repeated 5 times in order to evenly coat the hydrogel on the gold substrate.

This process is briefly shown in FIG. 2 .

4. SPR Analysis of Sensor Chips

Solutions in which IL-2, IL-2Rα, normal human serum and cancer patient serum were dissolved/diluted in DPBS were prepared, respectively. By dissolving the powder of IL-2 and IL-2Rα, 100 μl of solutions having concentrations of 50, 100, 150, 200, and 250 nM were prepared, respectively. was prepared. In addition, in the case of cancer patient serum, 10 ul of normal serum was diluted 100-fold or 1000-fold, and then IL-2 and IL-2Rα were added to prepare a cancer patient imitation serum. At this time, the concentrations of the added IL-2 and IL-2Rα were prepared in various ways to be 2, 20, 50, 100, 150, 200, and 300 nM, respectively.

The test solution was flowed to the sensor chip prepared in step 3 at a rate of 2 ul/min, respectively, for 60 seconds. Thereafter, the proteins bound to the receptors in the hydrogel were allowed to dissociate for 120 seconds.

A biacore T200 was used to measure the SPR signal.

The measured signal was analyzed as follows:

Biacore evaluation program version 2.0 was used for signal fitting. The signals of the first hydrogel to which the protein was not bound and the second hydrogel to which the receptor protein was bound were compared, and the signals were compared with the difference in the response max at which the reaction signal was the highest. The results are shown in FIGS. 3 to 8 .

3 (a) shows the maximum reaction value according to the concentration of the hydrogel of Examples 2 to 4, respectively, and (b), (c) and (d) of FIGS. 3 (b), (c) and (d) are the hydrogels of Examples 2 to 4, respectively. It shows the reaction signal according to time of the gel. In particular, (a) of FIG. 3 shows a pure gel (neat gel), that is, not modified with a receptor, at the maximum reaction value measured when IL-2 and IL-2Rα were simultaneously flowed to the hydrogels of Examples 2 to 4 It is a graph obtained by subtracting the maximum reaction value measured when IL-2 and IL-2Rα were simultaneously flowed from the first hydrogel and corrected.

In the case of the hydrogel of Example 2 (including β receptor) and the hydrogel of Example 3 (including γ receptor), when IL-2 and IL-2Rα (marker substances), which are known to increase in blood iron in cancer patients, are flowed. It was confirmed that only a single bond between the receptor substance and the marker substance occurred or a relatively low signal was detected because no binding occurred. On the other hand, in the case of the hydrogel of Example 4 (including β receptor and γ receptor) in which multiple binding between the marker substance and the receptor substance occurs, it can be seen that the signal increases as the concentration increases. Therefore, it was confirmed that the hydrogel of Example 4 can be used as a hydrogel useful for cancer diagnosis. On the other hand, the diagnostic method using the hydrogel of the present disclosure has the advantage of being able to simultaneously target and detect IL-2 and IL-2Ra. In addition, unlike the conventional diagnostic method, the diagnostic method of the present disclosure may measure the Kd value.

4 is a view illustrating the use of the diagnostic chip of the present disclosure directly verified with IL-2 and IL-2Rα as a cancer diagnosis kit in Example 4 using the cancer patient simulating serum prepared in step 4 above. The reaction signal of the hydrogel was measured. Figure 4 (a) shows the reaction signal according to the concentration when the cancer patient serum is flowed to the hydrogel of Example 4, (b) is the cancer patient serum flowed to the hydrogel of Example 4 In this case, the reaction signal according to time is shown.

Referring to FIG. 4 , it was confirmed that the signal increased as the concentration of the marker substance in the serum of cancer patients increased. That is, it can be confirmed that the detection of IL-2 and IL-2Rα is possible among numerous proteins in the blood. In addition, it was also confirmed that cancer diagnosis is possible in a very short time because it shows the maximum response value within a very short time.

Figure 5 (a) shows the maximum reaction value according to the concentration of the hydrogel of Examples 1, 4 and 5, respectively, Figure 5 (b), (c) and (d) are Examples 1, 4, respectively And 5 shows the reaction signal according to the time of the hydrogel. In particular, FIG. 5 (a) shows the maximum reaction value measured when IL-2 and IL-2Rα were simultaneously flowed to the hydrogels of Examples 1, 4 and 5, in a pure gel, that is, the first reaction that was not modified with the receptor. It is a graph obtained by subtracting the maximum reaction value measured when IL-2 and IL-2Rα were simultaneously flowed from the hydrogel and corrected.

Referring to FIG. 5 , it was confirmed that both the hydrogels of Examples 4 and 5 bind well to the marker substances IL-2 and IL-2Rα. In particular, although it is known that the binding ability of the receptor αβγ complex to IL-2 is higher than that of the βγ complex to IL-2, it was confirmed that a similar signal appeared. Therefore, it was confirmed that both hydrogels can be utilized for cancer diagnosis. However, it was confirmed that there was a difference in the Kd value (the lower the binding force, the higher) between the hydrogels of Examples 4 and 5.

6 is a comparison of the maximum response values of the biochip of the hydrogel of Example 4 and the biochip of the pure gel as a control according to the concentrations of IL-2 and IL-2Rα.

From the results of FIG. 6 , a weak signal due to binding to a non-specific protein is detected in the pure gel as a control, but a much higher signal is confirmed in the hydrogel of Example 4. That is, it can be confirmed that the high signal identified in the hydrogel of the present disclosure is a signal due to a specific reaction with the marker proteins. Therefore, it can be effectively used for cancer diagnosis.

7 shows the reaction signals confirmed when the normal serum and cancer patient serum were respectively flowed to the hydrogel of Example 4. At this time, it is corrected by subtracting the maximum reaction value confirmed in the hydrogel of the pure gel from the maximum reaction value confirmed in the hydrogel of Example 4.

From FIG. 7, it was confirmed that a significantly higher response signal was exhibited when the serum of a cancer patient was flowed, compared to when the serum of a normal person was flowed.

Meanwhile, the same experiment was performed using an ELISA kit, which is a conventional diagnostic method. Measurements were made using Invitrogen's Human sIL-2R Instant ELISA Kit (cat.no BMS212INST), and the experiment was carried out according to the kit protocol. Unlike the test kit, 100ul (protocol-140ul) of tertiary distilled water was added, Each 50ul (protocol-10ul) of the patient's serum was put in the experiment. 8 shows the results thereof.

Comparing the results of FIG. 8 with the results of FIG. 7, the hydrogel of the present disclosure shows cancer diagnosis performance similar to that of the ELISA kit, which is a conventional diagnostic method, and it is possible to diagnose cancer conveniently within a much shorter time than the ELISA kit. that can be checked

As described above, although the embodiments have been described with reference to the limited drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques are performed in an order different from the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (26)

a diswellable hydrogel containing interleukin (IL)-2 receptor alpha, beta, gamma, or a combination thereof; and
transducer;
A biosensor chip comprising a.
The biosensor chip of claim 1, wherein the receptor contained in the hydrogel binds to at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha.
The biosensor chip of claim 1 , wherein the hydrogel comprises two or more of receptors alpha, beta, and gamma.
The biosensor chip of claim 1 , wherein the hydrogel comprises IL-2 receptor beta and gamma.
The biosensor chip of claim 1 , wherein the hydrogel comprises IL-2 receptors alpha, beta and gamma.
The biosensor chip according to claim 1, wherein the hydrogel comprises a copolymer consisting of a main monomer and a comonomer.
According to claim 6, wherein the main monomer is, N- isopropyl acrylamide, N- acryloylglycinamide (N-acryloylglycinamide), hydroxypropyl cellulose (hydroxypropylcellulose), vinyl caprolactam (vinylcaprolactame), N- vinyl p Rollidone (N-vinyl pyrrolidone), 2-hydroxyethyl methacrylate (2-hydrocyethyl methacrylate), ethylene glycol (ethylene glycol); amino acids such as aspartic acid, glutamic acid, and L-lysine; selected from the group consisting of caprolactone, and vinyl methyl ether,
The comonomer is allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc) the group consisting of Which will be selected from, a biosensor chip.
The biosensor chip of claim 1, wherein the hydrogel further comprises a crosslinking agent.
According to claim 8, wherein the crosslinking agent, N, N'-methylene-bis-diacrylamide (MBA), polyethylene glycol (PEG) dihydroxyl (PEG dihydrocyl), PEG diamine (PEG diamine), PEG dioxyamine (PEG dioxyamine), PEG dichloride, PEG dibromide, PEG diazide, PEG dithiol, PEG dialdehyde, PEG diepoxide (PEG diepoxide), PEG diacrylate, PEG dimethacrylate, PEG diacetic acid, PEG disuccinic acid, PEG disuccimidylcarboxy Methyl ester (PEG discuccinimidyl carboxy methyl ester), poly(ε-caprolactone)diacrylate [poly(ε-caprolactone)diacrylate], poly(ε-caprolactone)dimethacrylate [poly(ε-caprolactone)dimethacrylate] , polylactide diacrylate, polylactide dimethacrylate, poly(lactide-co-glycolide) diacrylate [poly(lactide-co-glycolide)diacrylate], poly (lactide-co-glycolide)dimethacrylate [poly(lactide-co-glycolide)dimethacrylate], poly(ε-caprolactone-b-ethylene glycol-b-ε-caprolactone)diacrylate [poly( ε-caprolactone-b-ethylene glycol-b-ε-caprolactone)diacrylate], poly(ε-caprolactone-b-ethylene glycol-b- ε-caprolactone)dimethacrylate [poly(ε-caprolactone-b- ethylene glycol-b-ε-caprolac tone)dimethacrylate], poly(lactide-b-ethylene glycol-b-lactide)diacrylate [poly(lactide-b-ethylene glycol-b-lactide)diacrylate], poly(lactide-b-ethylene glycol- b-lactide)dimethacrylate [poly(lactide-b-ethylene glycol-b-lactide)dimethacrylate], poly[(lactide-co-glycolide)-b-ethylene glycol-b-(lactide-co) -glycolide)]diacrylate {poly[(lactide-co-clycolide)-b-ethylene glycol-b-(lactide-co-glycolide)] diacrylate}, poly[(lactide-co-glycolide)-b -ethylene glycol-b-(lactide-co-glycolide)]dimethacrylate {poly[(lactide-co-clycolide)-b-ethylene glycol-b-(lactide-co-glycolide)] dimethacrylate}, poly (ε-caprolactone-co-lactide)-diacrylate [poly(ε-caprolactone-co-lactide)-diacrylate], poly(ε-caprolactone-co-lactide)-dimethacrylate [poly( ε-caprolactone-co-lactide)-dimethacrylate], poly(ε-caprolactone-co-glycolide)-diaacrylate [poly(ε-caprolactone-co-glycolide)-diacrylate], poly(ε-caprolactone- co-glycolide)-dimethacrylate [poly(ε-caprolactone-co-glycolide)-dimethacrylate], poly[(caprolactone-co-lactide)-b-ethylene glycol-b-(caprolactone-co- lactide)]diacrylate {poly[(caprolactone-co-lactide)-b-ethylene glycol-b-(caprolactone-co-lactide)]diacrylate}, poly[(caprolactone-co-lactide)-b- Ethylene glycol-b-(caprolactone-co-lactide)]dimethacrylate {poly[(caprolactone- co-lactide)-b-ethylene glycol-b-(caprolactone-co-lactide)]dimethacrylate}, poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide) )]diacrylate {poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide)]diacrylate}, poly[(caprolactone-co-glycolide)-b-ethylene glycol -b-(caprolactone-co-glycolide)]dimethacrylate {poly[(caprolactone-co-glycolide)-b-ethylene glycol-b-(caprolactone-co-glycolide)]dimethacrylate} and combinations thereof Which is selected from the group consisting of, a biosensor chip.
The biosensor chip of claim 1 , wherein the hydrogel comprises a copolymer consisting of 50 to 97.9% by weight of the main monomer, 2 to 40% by weight of the comonomer, and 0.1 to 10% by weight of the crosslinking agent.
According to claim 1, wherein the hydrogel, poly (N-isopropyl acrylamide-co-allylamine) [poly (N-isopropylacrylamide-co-allylamine): poly (NIPAM-co-AA)], poly (N -Isopropyl acrylamide-co-2-(dimethylamino)ethyl methacrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl methacrylate): poly(NIPAM-co-DMAEMA)], poly( N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate), poly(NIPAM-co-DMAEA)], poly( N-isopropylacrylamide-co-acrylic acid) [poly(N-isopropylacrylamide-co-acrylic acid): poly(NIPAM-co-AAc)], poly(N-isopropylacrylamide-co-polyethylene glycol-acrylic acid) [poly(N-isopropylacrylamide-co-polyethylene glycol-acrylic acid): poly(NIPAM-co-PEG-AAc)], and poly(N-isopropylacrylamide-co-methacrylic acid) [poly(N-isopropylacrylamide) -co-methacrylic acid): poly(NIPAM-co-MAAc)], the biosensor chip comprising at least one selected from the group consisting of.
According to claim 1, wherein the hydrogel, N- isopropyl acrylamide as a main monomer, acrylic acid as a comonomer, and MBA (N, N'-methylene-bis-acrylamide) as a crosslinking agent a copolymer prepared using a copolymer Which will include, a biosensor chip.
The method of claim 1, wherein when the receptors contained in the hydrogel bind to IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, the hydrogel is diswelled. biosensor chip.
The biosensor chip of claim 13 , wherein a change in optical properties of the biosensor chip occurs due to the deswelling.
The biosensor chip of claim 14 , wherein the optical property is a refractive index.
The biosensor chip of claim 14 , wherein the change in the optical properties is detected through surface plasmon resonance.
The biosensor chip of claim 1 , wherein the biosensor chip detects at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha within 30 minutes.
The biosensor chip of claim 1 , wherein the transducer comprises a metal thin film.
The biosensor chip of claim 18, wherein the metal thin film is surface-modified with cysteamine.
The biosensor chip of claim 18, wherein the metal thin film is a gold thin film.
A method for detecting at least one of IL-2, IL-2 receptor alpha, or a complex of IL-2 and IL-2 receptor alpha, comprising:
providing the biosensor chip according to any one of claims 1 to 19;
Separating serum from blood; and
A detection method comprising the step of flowing the separated serum onto the biosensor chip.
22. The method of claim 21, wherein the detecting method comprises measuring a change in an optical property of a sensor chip.
A composition for diagnosis of cancer comprising the biosensor chip according to any one of claims 1 to 20.
The method of claim 23, wherein the cancer is lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, head and neck cancer, bone marrow cancer, biliary tract cancer, Chronic myeloproliferative disease (cMPD), agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera (PV), true thrombocytopenia ( essential thrombocythemia (ET), haematological malignancies, non-hematologic malignancies, malignant melanomas and carcinomas of the kidney, head and neck, esophagus, nasopharynx, liver and lung, lymphoproliferative disorders, leukemia, acute Acute myelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, Acute lymphoblastic leukemia, lymphoma, Anaplastic large-cell lymphoma cell lymphoma), Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas, Hodgkin's lymphoma, Peripheral T-cell lymphoma cell lymphomas), multiple myeloma, malignant solid tumor, malignant melanoma, soft tissue sarcomas, renal cell carcinoma, childhood neoplasia, Wilms' tumor, bone tissue Sarcomas of bone tissues, or hemophagocytic-tissues constitutive syndrome.
A cancer diagnostic kit comprising the biosensor chip according to any one of claims 1 to 20.
The method of claim 25, wherein the cancer is lung cancer, esophageal cancer, thymus cancer, breast cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, bladder cancer, head and neck cancer, bone marrow cancer, biliary tract cancer, Chronic myeloproliferative disease (cMPD), agnogenic myeloid metaplasia (MMM), chronic myeloid leukemia (CML), polycythaemia vera (PV), true thrombocytopenia ( essential thrombocythemia (ET), haematological malignancies, non-hematologic malignancies, malignant melanomas and carcinomas of the kidney, head and neck, esophagus, nasopharynx, liver and lung, lymphoproliferative disorders, leukemia, acute Acute myelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, Acute lymphoblastic leukemia, lymphoma, Anaplastic large-cell lymphoma cell lymphoma), Cutaneous T-cell lymphoma, Mycosis fungoides, Non-Hodgkin's lymphomas, Hodgkin's lymphoma, Peripheral T-cell lymphoma cell lymphomas), multiple myeloma, malignant solid tumor, malignant melanoma, soft tissue sarcomas, renal cell carcinoma, childhood neoplasia, Wilms' tumor, bone tissue Sarcomas of bone tissues, or hemophagocytic The diagnostic kit, which is a direct syndrome.

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