Complex and method for capturing sialic acid

Abstract

The invention provides a complex and a method for capturing sialic acid. The complex is capable of capturing and concentrating the sialic acid. A complex (1) according to the present invention is characterized by being represented by formula (1). Ba-L-Mp (1) (In formula (1), Ba represents a boronic acid group having a bonding constant Ka with sialic acid of 500 M <-1 > or more, Mp represents magnetic particles having strong magnetism, L represents a linking group that bonds the boronic acid group to the magnetic particles, and Mp represents a linking group that bonds the magnetic particles to the boronic acid group. The complex represented by formula (1) is in a state in which the boric acid group and the magnetic particles are chemically bonded via the linking group.

Description

Technical Field

[0001] This invention relates to a composite containing borate groups and magnetic particles, and a method for capturing sialic acid using said composite. Background Technology

[0002] Due to the heterogeneity of cancerous tissue, the types of cancer suffered by each patient are extremely diverse. In the case of breast cancer, treatment methods are extremely complex, varying depending on whether hormone therapy is available or not, or whether molecularly targeted therapy is available or not. Furthermore, cancer cells unresponsive to hormone therapy or molecularly targeted therapy have low survival rates and poor prognoses due to high metastatic potential. Cancer-related deaths are primarily caused by metastasis, thus necessitating early diagnosis of cancerous tissue, especially metastatic cancer.

[0003] In recent years, liquid biopsy has been implemented as a method for cancer screening. This method detects cancer cells or cancer-derived components that flow into bodily fluids, primarily blood. For example, exosomes secreted by cancer cells contain cancer-specific molecules that are unique to cancer cells. These cancer-specific molecules are captured and detected in the collected bodily fluids. The cancer-specific molecules differ for each type of cancer. Biomarkers are selected for each cancer type; for example, epidermal growth factor receptor variant III (EGFRvIII) is selected for glioblastoma, and developmental regulatory endothelial cell locus-1 (Del-1) is selected for breast cancer. Detection is performed using ELISA (Enzyme-Linked Immunosorbent Assay), which primarily utilizes antigen-antibody reactions (see Non-Patent Literature 1).

[0004] However, while liquid biopsy is helpful in detecting advanced cancers, it cannot be applied to detect cancer cells flowing into bodily fluids or early-stage cancers where cancerous components are present in trace amounts. Furthermore, because it involves specific detection using different biomarkers for each cancer type, it cannot provide a universal cancer screening method regardless of the cancer type.

[0005] If early-stage cancers can be included in the screening program, and a universal cancer screening system can be implemented regardless of the type of cancer, it will greatly facilitate cancer screening and significantly improve patient prognosis.

[0006] Furthermore, there have been reports of a correlation between sialylation on glycans on the cell surface and metastasis rate (see Non-Patent Literature 2). That is, sialic acid is overexpressed on the surface of metastatic cancer cells.

[0007] The effectiveness of using this property to detect sialic acid in cancer screening was also reported. Prostate-specific antigen (PSA) is used for cancer screening because it shows high values ​​in prostate cancer; however, because it also shows high values ​​in benign prostatic hyperplasia and prostatitis, it has the problem of not being able to distinguish between cancer and other cancer markers. According to this report, in 60 cases where all PSA tests were positive and it was impossible to determine whether the patient had cancer or not, 59 cases were successfully and accurately identified as either prostate cancer or non-cancer cases (see Non-Patent Literature 3). That is, sialic acid detection is reportedly effective as a cancer screening method.

[0008] Regarding the detection of sialic acid, boric acid, which selectively bonds with sialic acid, has been reported (see Non-Patent Literature 4). That is, boric acid selectively interacts with sialic acid in various sugars present in organisms.

[0009] Furthermore, while the aminophenylboronic acid used in this report has a low bonding affinity with sialic acid, subsequent studies have reported boric acids with strong bonding affinity to sialic acid (see Non-Patent Literature 5). For example, boric acid with a pyridine ring skeleton (5-boropyridinecarboxylic acid, 5-BPA) with a bonding affinity exceeding 1,000 M has been reported. -1 High bonding constant (K) a ).

[0010] Furthermore, as an achievement of the inventor (Yukichi Horiguchi), a cancer detection method taking into account the properties of sialic acid is also reported (see Non-Patent Document 6). In this report, it is reported that when surface-modified gold nanoparticles with aminophenylboronic acid are applied to the surface of metastatic cancer cells, the gold nanoparticles aggregate in the sialic acid expressed on the cancer cell surface, thus producing surface-enhanced Raman scattering different from that of normal cells. Additionally, as another achievement of the inventor (Yukichi Horiguchi), the bonding state between 5-boropyridinecarboxylic acid and sialic acid immobilized on a gold film is also reported by surface plasmon resonance (SPR) (see Non-Patent Document 7).

[0011] However, past reports involving sialic acid testing have been difficult to apply in practical cancer screening.

[0012] That is, the biological fluids used for cancer detection contain only trace amounts of sialic acid, especially in the case of early-stage cancer, where the amount of sialic acid in the biological fluids becomes extremely small, thus posing a problem of not being able to accurately detect sialic acid and thus metastatic cancer.

[0013] In order to include early-stage cancers and achieve universal cancer screening regardless of cancer type, it is necessary to develop technologies that can capture and concentrate sialic acid contained in biological fluids to improve detection performance.

[0014] Background Technology Documents

[0015] Non-patent literature

[0016] Non-patent literature 1: Oleo Science, Vol. 21, No. 2 (2021) 63

[0017] Non-patent literature 2: G. Yogeeswaran and P. Salk, Science, 1981, 212, 1514-1516.

[0018] Non-patent literature 3: Y. Haga, M. Uemura, S. Baba, K. Inamura, K. Takeuchi, N. Nomura, K. Ueda, Anal. Chem. 2019, 91, 2247.

[0019] Non-patent literature 4: H. Otsuka, E. Uchimura, H. Koshino, T. Okano, K. Kataoka, J. Am. Chem. Soc. 2003, 125, 3493.

[0020] Non-patent literature 5: A. Matsumoto, A. J. Stephenson-Brown, T. Khan, T. Miyazawa, H. Cabral, K. Kataoka, and Y. Miyahara, Chemical Science 8, 6165 (2017).

[0021] Non-patent literature 6: B. Shashni, Y. Horiguchi, K. Kurose, H. Furusho and Y. Nagasaki, Biomaterials, 2017, 134, 143-153.

[0022] Non-patent literature 7: Y. Horiguchi, K. Barthelmes, Y. Miyahara, A. Matsumoto Chemistry Letters, 2021, 50, 1467-1469. Summary of the Invention

[0023] [The problem the invention aims to solve]

[0024] The objective of this invention is to solve the various problems mentioned above in the prior art and to provide a complex capable of capturing and concentrating sialic acid, as well as a method for capturing said sialic acid.

[0025] [Technical means to solve the problem]

[0026] As a means of solving the aforementioned problem, it is shown below. That is,

[0027] <1> A composite, characterized by being represented by the following formula (1).

[0028] B a -LM p (1)

[0029] In equation (1), B a The bonding constant K with sialic acid is represented. a 500M -1 The above borate groups, M p The term (1) represents a magnetic particle with strong magnetism, and L represents a linking group that bonds the borate group to the magnetic particle. The composite represented by the formula (1) is in a state where the borate group and the magnetic particle are chemically bonded via the linking group.

[0030] <2> According to the composite described in <1>, the magnetic particles are spherical particles with a diameter of 0.1 μm to 10 μm.

[0031] <3> According to the complex described in <1> or <2>, wherein the borate group is composed of the following chemical structural formula (B a 1) and (B) a 2) Any one of the representations.

[0032] [Chemistry 1]

[0033]

[0034] Among them, the chemical structural formula (B) a In formula 1), any one of R1 to R4 represents a divalent linker that forms an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms. Furthermore, the chemical structural formula (B) a In 2), any one of R5 to R7 represents a divalent linker that forms any one of an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms.

[0035] <4> The composite according to any one of <1> to <3>, wherein the parent material forming the linking group contains: an amino group modified on the surface of the magnetic particles; a thiol compound derivative bonded to a borate group; and a linking core component having an amide bond bonded to the amino amide and a maleimide group bonded to the thiol group of the thiol compound derivative.

[0036] <5> According to the composite described in <4>, the connecting core component is an heterodifunctional compound having N-hydroxysuccinimide ester group and maleimide group.

[0037] <6> The composite according to any one of <1> to <5>, wherein the linking group has an intermediate portion that modifies the surface properties of the magnetic particles.

[0038] <7> According to the composite described in <6>, the intermediate portion is composed of a polymer chain of a homopolymer or copolymer of monomers selected from the group consisting of ethylene glycol, 2-methoxy acrylate, 2-methacryloyloxyethyl phosphocholine and 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate.

[0039] <8> The complex described in <7> has a mass-average molecular weight of 2,000 to 10,000 for the polymer chains.

[0040] <9> A method for capturing sialic acid, characterized by comprising: a bonding step, adjusting the pH of a liquid sample containing a complex described in any one of <1> to <8> and a biological fluid containing sialic acid to less than 6, so that the sialic acid bonds with the complex; a recovery step, using a magnet to separate and recover the complex after the bonding step from the liquid sample; and a dissociation step, dissociating the sialic acid from the complex after the recovery step in a concentrated solution with a pH adjusted to above 7.

[0041] <10> According to the sialic acid capture method described in <9>, the bonding step, the recovery step and the dissociation step are carried out again using the complex that has undergone the bonding step, the recovery step and the dissociation step for the same or different biological fluids.

[0042] [The effects of the invention]

[0043] According to the present invention, the various problems mentioned above in the prior art can be solved, and a complex capable of capturing and concentrating sialic acid and a method for capturing said sialic acid can be provided. Attached Figure Description

[0044] Figure 1 It is a schematic diagram representing the complex.

[0045] Figure 2 This is an explanatory diagram showing the outline of the bonding process.

[0046] Figure 3 This is an explanatory diagram illustrating the dissociation process.

[0047] Figure 4This is a diagram showing the synthetic process of a compound formed by the bonding of boric acid and thiol derivatives.

[0048] Figure 5 This diagram illustrates the preparation process of a composite formed by the combination of borate groups and magnetic particles via linking groups.

[0049] Figure 6(a) shows the results of the quantitative test for sialic acid.

[0050] Figure 6(b) is a graph showing the analytical results of the number of sialic acid molecules dissociated in the quantitative test (pH 5.0) and the comparative test (pH 7.6).

[0051] Figure 7 This is a graph showing the quantitative test results of sialic acid involved in the reuse of complexes.

[0052] Figure 8(a) is a graph showing the results of concentration test 1.

[0053] Figure 8(b) is a graph showing the results of concentration test 2. Detailed Implementation

[0054] (complex)

[0055] The composite of the present invention is represented by the following formula (1).

[0056] B a -LM p (1)

[0057] In equation (1), B a The bonding constant K with sialic acid is represented. a 500M -1 The above borate groups, M p The term (1) represents a magnetic particle with strong magnetism, and L represents a linking group that bonds the borate group to the magnetic particle. The composite represented by the formula (1) is in a state where the borate group and the magnetic particle are chemically bonded via the linking group.

[0058] The magnetic particles are configured for the purpose of recovering the sialic acid bonded to the borate group using a magnet.

[0059] In this specification, "strong magnetism" means the property that is attracted by the magnet, including ferrimagnetism.

[0060] The magnetic particles can be appropriately selected according to the purpose, without particular restrictions. Known magnetic particles such as ellipsoids and spheres formed of strongly magnetic materials can be used, with spherical particles having a particle size of 0.1 μm to 10 μm being preferred.

[0061] If the particle size is less than 0.1 μm, the attraction of the magnet may be insufficient to counteract Brownian motion in the liquid. If the particle size exceeds 10 μm, the total surface area of ​​the dispersed particle group per unit area may decrease, thus reducing the capture efficiency of the sialic acid.

[0062] The borate group is a group that selectively bonds to sialic acid from among many sugars in biological substances, and is configured for the purpose of capturing sialic acid in biological fluids.

[0063] Furthermore, in this specification, the term "sialic acid" unless otherwise stated includes, in addition to sialic acid compounds in which the amino or hydroxyl groups of neuraminic acid are substituted, glycoproteins formed by the bonding of said sialic acid compounds with functionalized sialic acid groups, and cells, extracellular vesicles, etc., containing said glycoproteins.

[0064] As for the borate group (B(OH)2R-), as long as the bonding constant K with the sialic acid is... a 500M -1 Therefore, without particular restrictions, it is permissible to choose from any form having any functional group (R-). The bonding constant K with the sialic acid is... a Less than 500M -1 Therefore, it is not easy to capture cells or free proteins containing the sialic acid.

[0065] The functional group (R-) has a bonding group with the sialic acid and a bonding group with the linking group.

[0066] There are no particular limitations on the bonding group with the sialic acid; for example, alkyl, aryl, nitrogen-containing heterocyclic groups, etc., can be used. Furthermore, these groups can also have any substituents.

[0067] Furthermore, there are no particular limitations on the bonding group that is connected to the linking group. For example, any divalent linking group that forms any one of the bonds of ester, ether, amide, carbamate and sulfur with the linking group can be cited.

[0068] It is known that the nitrogen-containing heterocyclic group undergoes hydrogen bonding with the carboxyl group of the sialic acid via the non-shared electron pair of the nitrogen atom in the ring, resulting in a high bonding constant K. a (Refer to non-patent literature 5).

[0069] Examples of nitrogen-containing heterocyclic groups include: monocyclic groups such as pyridyl and pyrimidinyl; bicyclic groups such as indolyl, indololinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, naphthidyl, pteridinyl, and purine; and condensed polycyclic groups such as acridineyl, carbazoyl, phenidyl, phenazinyl, and benzoisoquinolinyl.

[0070] If the nitrogen atom in these nitrogen-containing heterocyclic groups that is bonded to the hydrogen sialic acid is ionized to become a quaternary nitrogen cation, it will lose its non-shared electron pair and thus become tertiary.

[0071] In addition, as the nitrogen-containing heterocyclic group, the hydrogen atom at any position is replaced with a group that provides any divalent linker that can form any one of the bonds of ester, ether, amide, carbamate and sulfur bonds with the linking group (e.g., hydroxyl or amino, which provides a divalent linker such as -NH- through dehydration condensation).

[0072] As suitable borate groups, the following chemical structural formulas can be cited (B a 1) and (B) a 2) Any of the groups represented by the group.

[0073] [Chemistry 2]

[0074]

[0075] Among them, the chemical structural formula (B) a In formula 1), any one of R1 to R4 represents a divalent linker that forms an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms. Furthermore, the chemical structural formula (B) a In 2), any one of R5 to R7 represents a divalent linker that forms any one of an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms.

[0076] Among them, in the chemical structural formula (B) a In 1), 5-boropyridinecarboxylic acid, where R2 is a carboxyl group and R1, R3, and R4 are hydrogen atoms, has a high bonding constant K exceeding 1,000 with the sialic acid. a .

[0077] As the bonding group with the sialic acid, when the alkyl or aryl group is selected, it is preferable to have a structure substituted with a group having a non-shared electron pair (e.g., -O-, =O, -N, hydroxyl, carbonyl, amino, etc.) that forms a hydrogen bond with the sialic acid. The high bonding constant K is achieved through the hydrogen bond between the non-shared electron pair group and the sialic acid. a .

[0078] The linking group has the function of bonding the magnetic particles to the borate group.

[0079] Therefore, the linking group is designed to have a mechanism that bonds to the magnetic particles and a mechanism that bonds to the borate group.

[0080] There are no particular limitations on the bonding mechanism with the magnetic particles; examples include bonding mechanisms based on amino groups modified on the surface of the magnetic particles. There are also no particular limitations on the compound that provides the amino group to the surface of the magnetic particles; in addition to amines, amine derivatives may also be used.

[0081] There are no particular restrictions on the bonding mechanism with the borate group. However, based on considerations of bond stability, it is preferable to use a mechanism that forms any one of the following bonds with the borate group: ester bond, ether bond, amide bond, carbamate bond, or sulfur bond.

[0082] Furthermore, considering the ease with which the magnetic particles can be provided with functional surfaces, the mechanism that bonds to the borate group via these bonds is preferably composed of a thiol-terminated compound.

[0083] Therefore, as the bonding mechanism with the borate group, a mechanism of a thiol compound derivative is preferably used, which has a functional group (e.g., amino or hydroxyl) bonded to the borate group and the thiol group.

[0084] There are no particular limitations on the parent material for forming the connecting group, which includes a connecting core component that connects the bonding mechanism with the magnetic particles and the bonding mechanism with the borate group.

[0085] As the connecting core component, for example, when the amino group modified on the surface of the magnetic particles is selected as the bonding mechanism with the magnetic particles, and the thiol compound derivative is selected as the bonding mechanism with the borate group, the amino group readily reacts with N-hydroxysuccinimide ester (NHS ester) to form a stable amide bond, thus making it easy to handle. In addition, the thiol group of the thiol compound derivative readily bonds with the maleimide group, so it can be composed of a heterodifunctional compound having the N-hydroxysuccinimide ester and the maleimide group.

[0086] Furthermore, in this specification, "the parent material for forming the linking group" refers to the linking group that is chemically bonded to the borate group and the magnetic particles, meaning the material that forms the linking group in the preliminary stage of forming the chemical bond with the borate group and the magnetic particles.

[0087] There are no particular limitations on the heterodifunctional compound used. However, considering its easy solubility in water and the formation of a strong bond, a crosslinker having both the N-hydroxysuccinimide ester and the maleimide group is preferred, namely succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (SMCC) and its derivatives.

[0088] Based on the above, the parent material for forming the connecting group is preferably configured to include: an amino group that has undergone surface modification of the magnetic particles; a thiol compound derivative bonded to the borate group; and a connecting core component having an amide bond bonded to the aminoamide and a maleimide group bonded to the thiol group of the thiol compound derivative.

[0089] Furthermore, there are no particular limitations on the linking group, but in order to suppress the non-specific adsorption of inclusions and the like on the surface of the magnetic particles, it is preferable to have an intermediate portion that modifies the surface properties of the magnetic particles.

[0090] As the intermediate portion, from the viewpoint of effectively suppressing the non-specific adsorption of the inclusions, it is preferably composed of a polymer chain of a homopolymer or copolymer of monomers selected from the group consisting of ethylene glycol, 2-methoxy acrylate, 2-methacryloyloxyethyl phosphocholine and 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate.

[0091] There is no particular limitation on the mass-average molecular weight of the polymer chain, but 2,000 to 10,000 is preferred. If the mass-average molecular weight is less than 2,000, it may be difficult to suppress the non-specific adsorption of inclusions, etc. If it exceeds 10,000, the steric hindrance of the polymer chain may sometimes lead to a decrease in the number of bonds of the borate groups to the magnetic particles per unit area via the linking groups, thereby reducing the sialic acid capture capacity.

[0092] There are no particular limitations on the method of introducing the intermediate site, and it can be introduced by known methods. However, when using the thiol compound derivative, it can be introduced in the form of an intermediate chain of a polymer compound with one end capped by a functional group (e.g., amino or hydroxyl) associated with the borate group and the other end capped by the thiol group (e.g., forming NH2-PEG-SH (where PEG means polyethylene glycol chain, the same below) as the forming master material -NH-PEG-S- etc).

[0093] Furthermore, the bonding mechanism with the magnetic particles, the bonding mechanism with the borate group, the connecting core component, and the intermediate portion described above can be formed using known forming materials and reagents. Although the connecting group has a novel structure, it can be formed by referring to known reaction procedures for each part.

[0094] (Methods for capturing sialic acid)

[0095] The sialic acid capture method of the present invention includes at least a bonding step, a recovery step, and a dissociation step.

[0096] The following explanation is based on the diagram.

[0097] The method for capturing sialic acid uses the complex described in this invention. Figure 1 The complex is schematically represented in the figure. The symbol 1 in the figure represents the complex.

[0098] like Figure 2 As shown, the bonding step involves adjusting the pH of a liquid sample containing complex 1 and biological fluid containing sialic acid (such as sialic acid protein 2) to less than 6, so that the sialic acid (such as sialic acid protein 2) bonds to complex 1. Furthermore, Figure 2 This is an explanatory diagram showing the outline of the bonding steps.

[0099] The sialic acid (such as sialic acid protein 2) selectively bonds to the borate group of complex 1 based on pH conditions. The pH condition is less than 6, preferably about 4 to 5.9.

[0100] As a method for adjusting the pH of the liquid sample to less than 6, examples include adding an acidic solution such as an acetate buffer until the target pH is obtained. Furthermore, this adjustment method also includes the dispersion operation itself, such as when the pH of the sample solution obtained by dispersing complex 1 in the biological fluid is initially less than 6.

[0101] There are no particular limitations on the biological fluids mentioned, and examples include blood and urine. Blood can be used in cases where it is used to examine metastatic cancer.

[0102] The recovery step involves using a magnet to separate and recover the composite 1 from the liquid sample after the bonding step.

[0103] There are no particular limitations on the method of recycling. For example, the following methods can be used: discarding the liquid in the container by bringing a permanent magnet close to the container of the liquid sample and causing the composite 1 to adhere to the wall of the container; or discarding the liquid in the container by using an electromagnet disposed near the container to cause the composite 1 to adhere to the wall of the container.

[0104] The dissociation step is as follows: Figure 3 As shown, the step involves dissociating the sialic acid (sialic acid protein 2, etc.) from complex 1 after the recovery step in a concentrated solution with a pH adjusted to 7 or higher. Furthermore, Figure 3 This is an explanatory diagram showing a summary of the dissociation steps.

[0105] As described in the embodiments below, even after the sialic acid (such as sialic acid protein 2) temporarily forms a strong bond with the complex 1 during the bonding step, it will selectively break the bond with the borate group of the complex 1 based on the pH value condition, and dissociate from the complex 1. The pH value condition is 7 or higher, preferably around 7 to 9.

[0106] As a method to adjust the pH of the concentrated solution to 7 or higher, an example is to add an alkaline solution such as a phosphate buffer that has been adjusted to be alkaline until the target pH value is obtained.

[0107] Furthermore, the concentrated sialic acid obtained after the dissociation step can be used as a test sample for cancer screening.

[0108] Therefore, according to the sialic acid capture method, the properties of sialic acid involved in metastatic cancer can be utilized to encompass early-stage cancers and achieve universal cancer screening regardless of cancer type.

[0109] However, as Figure 2 As shown, during the bonding step, sialic acid (sialic acid protein 2, etc.) that is not bonded to complex 1 may sometimes be generated due to its presence at a location far from complex 1.

[0110] On the other hand, as described in the embodiments below, after the series of steps of the bonding step, the recycling step and the dissociation step, the complex 1 still maintains its bonding and dissociation with the sialic acid based on the pH value conditions. In the second and subsequent bonding and dissociation steps when the complex 1 is reused, the sialic acid can also be bonded and dissociated.

[0111] Therefore, for the same biological fluid, the composite 1 used can be reused after the first series of steps for the second and subsequent series of steps, which can improve convenience.

[0112] Furthermore, the biological fluids need not be the same; there are cases where different biological fluids collected from the same patient at different times or different body locations are subjected to the series of steps separately. Even in this case, the composite 1 used in the first series of steps for the biological fluids can be reused for the second and subsequent series of steps for the biological fluids, thus improving convenience.

[0113] That is, as a method for capturing sialic acid, the complex 1 that has undergone the bonding step, the recovery step and the dissociation step can be reused for the same or different biological fluids, and the bonding step, the recovery step and the dissociation step are implemented.

[0114] [Example]

[0115] (Example 1: Manufacturing of the composite)

[0116] pass Figure 4 The synthetic process shown yields a bonded compound of boric acid and a thiol derivative. Specifically, the following steps are performed.

[0117] First, 33.6 mg (0.2 mmol) of 5-boropyridinecarboxylic acid (5-BPA, manufactured by Combi-Blocks) and 12.5 mL of N,N-dimethylformamide (DMF, manufactured by Fujifilm and Hikari Pure Chemical Industries) were added to a round-bottom flask and then subjected to ultrasonic treatment to obtain a solution.

[0118] Next, 175 μL of triethylamine (manufactured by Fujifilm and Kohden Chemical Co., Ltd.) and 84 mg (0.025 mmol) of polyethylene glycol (NH2-PEG-SH, mass average molecular weight: 3,400, manufactured by Biopharma PEG Scientific) with an amino group at one end and a thiol group at the other end were added to a flask, followed by ultrasonic treatment and stirring to obtain a solution.

[0119] Next, 18.2 mg (0.055 mmol) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholine hydrochloride (DMT-MM, manufactured by Fujifilm and Koujun Pharmaceutical Co., Ltd.) as a triazine condensing agent was added to a flask, and the mixture was stirred for 4 hours to obtain the reaction product of 5-BPA and NH2-PEG-SH (BPEG disulfide).

[0120] Next, a solution obtained by diluting a 5M sodium hydroxide aqueous solution (manufactured by Fujifilm and Koichi Chemical Co., Ltd.) to 100mM was subjected to dialysis (molecular weight cutoff: 3,500) to exchange the solution from the DMF and remove unreacted 5-BPA.

[0121] Next, the dialyzed solution was stirred for approximately 12 hours. Then, the same dialyzed process was performed twice more to carefully remove unreacted 5-BPA. Subsequently, the solution was dialyzed with pure water and exchanged, followed by freeze-drying to remove moisture, yielding a dry solid of BPEG disulfide.

[0122] The obtained BPEG disulfide was in a state of disulfide with a thiol terminus bonded by oxidation. Therefore, it was reduced to a thiol molecule using tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl, manufactured by Fujifilm and Koujun Pharmaceutical Co., Ltd.) as a reducing agent, to obtain a compound (BPEG-SH) of boric acid and a thiol derivative. Furthermore, this reduction treatment was performed just before bonding with magnetic particles, as described below.

[0123] pass Figure 5 The preparation process shown herein involves preparing a composite material formed by combining borate groups with magnetic particles via linking groups. Specifically, the following operations are performed.

[0124] First, 5 mg of succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (SMCC, manufactured by ProteoChem) which is the core component for connection is dissolved in 100 μL of DMF.

[0125] Next, 1μm Sera-Mag TM 1 mL of SpeedBead amino-blocking particles (manufactured by Global Life Sciences Solutions Operations UK Ltd. (Cytiva), containing azide groups) was added to a microtube. While using a magnet to attract the magnetic particles to the side of the microtube, a pipette was inserted into the microtube to remove the azide groups. The tube was then rinsed twice with pure water to obtain a dispersion of the magnetic particles in 1 mL of pure water. These magnetic particles are spherical particles with a diameter of 1 μm and an amino-modified surface.

[0126] Next, the DMF solution of SMCC was mixed with the aqueous dispersion of magnetic particles and stirred for 1 hour. The reacted magnetic particles were then washed with pure water to remove unreacted SMCC. This yielded magnetic particles with SMCC surface treatment.

[0127] Next, the previously obtained BPEG disulfide was dissolved in 1 mL of pure water at a concentration of 9.2 mg to prepare a 2.6 mM BPEG aqueous solution. In addition, 100 μL of a 200 mM aqueous solution of TCEP-HCl (Mw: 286.65, 57.4 mg / mL, manufactured by Fujifilm and Koichi Chemical Co., Ltd.) was prepared.

[0128] The TCEP (hydrochloric acid solution) and BPEG aqueous solution were mixed and reacted for 1 hour to obtain the previously described BPEG-SH (refer to...). Figure 4 ) solution.

[0129] Next, the surface-treated magnetic particles of SMCC were added to the BPEG-SH solution and reacted for 1 hour using a tubular shaker. By thioetherification of the maleimide groups of SMCC with the thiol groups of BPEG-SH, a dispersion containing the reaction product (the composite of Example 1) with boric acid groups fixed on the surface of the magnetic particles via linking groups was obtained.

[0130] (Example 2: pH-based selective binding of sialic acid)

[0131] First, as an example, fetoglobulin (manufactured by Merck (Sigma-Aldrich)) was prepared as a model of sialic acid, serving as a protein with high sialic acid density. Next, fetoglobulin was dissolved in 10 mM acetate buffer at pH 5.0 to prepare a 1 mg / mL fetoglobulin aqueous solution. 1 mL of the fetoglobulin aqueous solution and 64 μL of the dispersion of the complex from Example 1 were added to a microvolume tube and mixed, thereby binding the fetoglobulin to the complex (binding step).

[0132] Furthermore, for simplicity, fetoglobulin, a protein with high sialic acid density, will be referred to as "sialic acid" below. However, Figures 6-8, which are described below, show the results of measuring or analyzing the amount or number of fetoglobulin itself using a "sialic acid quantitative kit". To avoid the misunderstanding that this is the result of measuring or analyzing the amount or number of sialic acid that is released from fetoglobulin due to enzymes, the abbreviation "sialic acid" will be avoided in the graphs in Figures 6-8, and it will be accurately labeled as "fetoglobulin" (a protein with high sialic acid density). In addition, in the description of Figures 6-8, "sialic acid" will be indicated as "fetoglobulin".

[0133] Next, with the magnetic particles adsorbed onto the side of the microtube using a magnet, a pipette was inserted into the microtube to absorb and remove unreacted sialic acid. Then, the complex from the bonding step was washed twice with 1 mL of 10 mM acetate buffer. Subsequently, the 10 mM acetate buffer in the microtube was removed by pipetting (recovery step).

[0134] Next, 50 μL of 10 mM phosphate buffer (pH 7.6) was added to the microtube containing the residual sialic acid complex and the mixture was shaken and stirred to dissociate the complex from the sialic acid (dissociation step).

[0135] Based on the above, the sialic acid capture method of Example 2 was implemented.

[0136] Next, with the magnetic particles adsorbed onto the wall of the microtube using a magnet, the dissociated sialic acid in 10 mM phosphate buffer was quantified using the Agilent AdvanceBio TotalSialic Acid Quantitation Kit (manufactured by Agilent Technologies).

[0137] The quantitative test was conducted on a total of 5 samples, with the amount of the complex dispersion set to 64 μL as previously stated, and also changed to 16 μL, 4 μL, 2 μL, and 1 μL.

[0138] In addition, for comparison purposes, an equal volume of 10 mM phosphate buffer was used instead of 10 mM acetate buffer, and a comparative experiment was conducted with the pH always set to 7.6. This comparative experiment was also conducted on a total of 5 samples, with the volume of the complex dispersion set at 64 μL, 16 μL, 4 μL, 2 μL, and 1 μL.

[0139] The quantitative test results of sialic acid (labeled as "fetoprotein" in the figure) are shown in Figure 6(a).

[0140] As shown in Figure 6(a), (1) in the quantitative test with pH 5.0 using 10 mM acetate buffer, the amount of sialic acid quantified increased with the increase of the amount of complex dispersion, indicating that sialic acid was successfully bonded to the complex of Example 1. That is, even when a complex is formed using magnetic particles and connecting groups added for the surface treatment of the magnetic particles with boric acid, the boric acid groups can be bonded to sialic acid. The capture of sialic acid by the complex of Example 1 was not confirmed in the comparative test conducted with pH 7.6, therefore it is dependent on pH conditions.

[0141] Furthermore, (2) the sialic acid that had not dissociated from the complex was not detected in the quantitative test. Therefore, it can be concluded that by implementing a dissociation step with a pH of 7.6, the sialic acid temporarily bonded to the complex of Example 1 was successfully dissociated from the complex during the bonding step with a pH of 5.0. That is, the selective bonding based on pH conditions is effective not only in the scenario of bonding sialic acid to the complex, but also in the scenario of dissociating sialic acid bonded to the complex, even when sialic acid is bonded at a high bonding constant (K). a It is bonded to the complex and can also be dissociated from the complex by controlling the subsequent pH conditions to stably obtain unbonded sialic acid.

[0142] In addition, in the quantitative test (pH 5.0) and comparative test (pH 7.6) with a sample containing 64 μL of complex dispersion as the subject, the analysis results of the number of dissociated sialic acid (labeled as "number of fetal proteins" in the figure) are shown in Figure 6(b).

[0143] As shown in Figure 6(b), the quantitative test (pH 5.0) showed a significant difference compared to the comparative test (pH 7.6), confirming the dissociation of sialic acid. This strongly supports the effectiveness of controlling pH to achieve both the bonding of sialic acid in the bonding step and the dissociation of sialic acid in the dissociation step.

[0144] (Example 3: Reusability test of the composite)

[0145] Except that in the sialic acid capture method of Example 2, the dispersion volume of the complex from Example 1 was set to only 64 μL, the bonding step, recovery step, and dissociation step were performed in the same manner. This series of steps of bonding step, recovery step, and dissociation step is referred to as step 1.

[0146] After the dissociation step, a pipette is inserted into the microvolume tube while the magnetic particles are adsorbed onto the wall using a magnet, and the dissociated sialic acid solution is recovered by pipetting. Then, a 1 mg / mL sialic acid aqueous solution (the fetoglobulin aqueous solution from Example 2, hereinafter the same) is added to the microvolume tube containing the remaining magnetic particles as fresh sialic acid. Additionally, 10 mM acetate buffer is added to set the pH to 5.0, and a bonding step is performed. Next, the recovery and dissociation steps are performed in the same manner as the sialic acid capture method in Example 2. This series of bonding, recovery, and dissociation steps is referred to as the second step, and this series of bonding, recovery, and dissociation steps is repeated three times. These repeated steps are designated as the third to fifth steps.

[0147] Based on the above, the sialic acid capture method of Example 3 was implemented.

[0148] In the sialic acid capture method of Example 3, the complex used in the first step is reused in the second to fifth steps for five bonding and dissociation operations with sialic acid.

[0149] For the sialic acid dissociation solutions collected in each of the first to fifth steps, the same quantitative test as in Example 2 was performed.

[0150] Quantitative results of sialic acid (labeled "fetoglobulin") involved in the reuse of the complex are shown in the figure. Figure 7 .

[0151] Such as Figure 7 As shown, the amount of sialic acid obtained in the fifth step remained more than 80% of that in the first step, confirming the high reusability of the complex.

[0152] In actual cancer screening scenarios, it is assumed that the amount of sialic acid contained in biological fluids is extremely small. In a single test, due to the detection limit of concentration, sialic acid may be missed due to non-reaction with the complex.

[0153] At this point, it is also considered to conduct a second and subsequent multiple tests on the same biological fluid. However, in each of the second and subsequent tests, starting from the manufacturing process of the composite described in Example 1, there is a significant difference in convenience compared to directly reusing the composite used in the first test.

[0154] Furthermore, when different biological fluids collected from the same patient at different times or different body locations are examined in a unified manner, the manufacturing process of the complex described in Example 1 is applied to each collected biological fluid, which is significantly different in terms of convenience compared to directly reusing the complex used in the first examination of the biological fluid.

[0155] according to Figure 7 The high reusability of the identified composite material offers excellent convenience in practical applications.

[0156] (Example 4: Concentration test of sialic acid)

[0157] In the sialic acid capture method of Example 2, except that the sialic acid concentration of the sialic acid aqueous solution was changed from 1 mg / mL to 10 μg / mL, the volume of the sialic acid aqueous solution was changed from 1 mL to 10 mL, and the dispersion volume of the complex in Example 1 was set to only 64 μL, the bonding step, recovery step and dissociation step were performed in the same manner.

[0158] Next, after the dissociation step, the supernatant is extracted while the magnetic particles are adsorbed at the bottom of the microtube using a magnet, thereby recovering the dissociated solution of sialic acid.

[0159] The recovered sialic acid dissociation solution was subjected to the same quantitative test as in Example 2. This test was designated as Concentration Test 1 (sialic acid concentration: 10 μg / mL).

[0160] In addition, in Concentration Experiment 1, the sialic acid concentration in the sialic acid aqueous solution was changed from 10 μg / mL to 1 μg / mL, and the volume of the sialic acid aqueous solution was changed from 10 mL to 100 mL. The quantification of the sialic acid dissociation solution was performed in the same manner. This experiment is designated as Concentration Experiment 2 (sialic acid concentration: 1 μg / L).

[0161] The results of concentration test 1 are shown in Figure 8(a). The results of concentration test 2 are shown in Figure 8(b). Furthermore, in these figures, sialic acid is not referred to as sialic acid, but rather as "fetoglobulin".

[0162] As shown in Figures 8(a) and 8(b), in either of the concentration experiments 1 and 2, the concentration of sialic acid in the dissociated sialic acid solution was approximately 21 times higher than that in the initial aqueous sialic acid solution, indicating successful and significant concentration of sialic acid. Using this concentrate can significantly improve the detection performance of cancer screenings utilizing sialic acid.

[0163] [Explanation of Symbols]

[0164] 1: Complex

[0165] 2: Sialoprotein.

Claims

1. A composite, characterized in that... It is represented by the following formula (1), B a -L-M p (1) In equation (1), B a K represents the bonding constant with sialic acid. a 500M -1 The above borate groups, M p The term (1) represents a magnetic particle with strong magnetism, and L represents a linking group that bonds the boric acid group to the magnetic particle. The composite represented by the formula (1) is in a state where the boric acid group and the magnetic particle are chemically bonded through the linking group.

2. The composite according to claim 1, wherein the magnetic particles are spherical particles with a diameter of 0.1 μm to 10 μm.

3. The composite according to claim 1 or 2, wherein the borate group is formed by the following chemical structural formula (B a 1) and (B) a 2) any representation, [Chemistry 1] in, The chemical structural formula (B) a In formula 1), any one of R1 to R4 represents a divalent linker that forms an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms; furthermore, the chemical structural formula (B a In 2), any one of R5 to R7 represents a divalent linker that forms any one of an ester bond, ether bond, amide bond, carbamate bond, or sulfur bond with the linking group, and the others independently represent hydrogen atoms or alkyl groups having 1 to 2 carbon atoms.

4. The composite according to claim 1 or 2, wherein the parent material forming the linking group comprises: an amino group modified on the surface of the magnetic particles; a thiol compound derivative bonded to a borate group; and a linking core component having an amide bond bonded to the amino amide and a maleimide group bonded to the thiol group of the thiol compound derivative.

5. The composite according to claim 4, wherein the connecting core component is an isomorphous difunctional compound having N-hydroxysuccinimide ester group and maleimide group.

6. The composite according to claim 1 or 2, wherein the linking group has an intermediate portion that modifies the surface properties of the magnetic particles.

7. The composite according to claim 6, wherein the intermediate portion is composed of a polymer chain of a homopolymer or copolymer of monomers selected from the group consisting of ethylene glycol, 2-methoxyacrylate, 2-methacryloyloxyethyl phosphocholine and 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate.

8. The composite according to claim 7, wherein the mass-average molecular weight of the polymer chains is 2,000 to 10,000.

9. A method for capturing sialic acid, characterized in that... include: The bonding step involves adjusting the pH of a liquid sample containing the complex as described in claim 1 or 2 and a biological body fluid containing sialic acid to less than 6, so that the sialic acid is bonded to the complex. In the recovery step, a magnet is used to separate and recover the composite from the liquid sample after the bonding step; and The dissociation step involves dissociating the sialic acid from the complex after the recovery step in a concentrated solution with a pH adjusted to above 7.

10. The method for capturing sialic acid according to claim 9, wherein the bonding step, the recovery step and the dissociation step are performed again using the complex that has undergone the bonding step, the recovery step and the dissociation step for the same or different biological fluids.

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