JP2022069994A - Magnetic particle, immunity inspection particle, and inspection reagent - Google Patents
Magnetic particle, immunity inspection particle, and inspection reagent Download PDFInfo
- Publication number
- JP2022069994A JP2022069994A JP2020178983A JP2020178983A JP2022069994A JP 2022069994 A JP2022069994 A JP 2022069994A JP 2020178983 A JP2020178983 A JP 2020178983A JP 2020178983 A JP2020178983 A JP 2020178983A JP 2022069994 A JP2022069994 A JP 2022069994A
- Authority
- JP
- Japan
- Prior art keywords
- particles
- magnetic
- core
- magnetic particle
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002245 particle Substances 0.000 title claims abstract description 308
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 244
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 8
- 238000007689 inspection Methods 0.000 title description 22
- 230000036039 immunity Effects 0.000 title 1
- 230000005484 gravity Effects 0.000 claims abstract description 98
- 239000007771 core particle Substances 0.000 claims abstract description 83
- 239000011347 resin Substances 0.000 claims abstract description 78
- 229920005989 resin Polymers 0.000 claims abstract description 78
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000003446 ligand Substances 0.000 claims abstract description 25
- 125000000524 functional group Chemical group 0.000 claims abstract description 12
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 9
- 239000000696 magnetic material Substances 0.000 claims description 65
- 230000001900 immune effect Effects 0.000 claims description 47
- 238000012360 testing method Methods 0.000 claims description 42
- 238000003018 immunoassay Methods 0.000 claims description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 15
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 15
- 230000005415 magnetization Effects 0.000 claims description 11
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 10
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 9
- 239000002270 dispersing agent Substances 0.000 claims description 7
- -1 succinimidyl group Chemical group 0.000 claims description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 6
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 3
- 125000003055 glycidyl group Chemical group C(C1CO1)* 0.000 claims description 3
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 claims description 3
- YXMISKNUHHOXFT-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) prop-2-enoate Chemical compound C=CC(=O)ON1C(=O)CCC1=O YXMISKNUHHOXFT-UHFFFAOYSA-N 0.000 claims description 2
- 229920002845 Poly(methacrylic acid) Polymers 0.000 claims description 2
- 229910001566 austenite Inorganic materials 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 229910052742 iron Inorganic materials 0.000 claims description 2
- 229920002401 polyacrylamide Polymers 0.000 claims description 2
- 229910000859 α-Fe Inorganic materials 0.000 claims description 2
- 229910000480 nickel oxide Inorganic materials 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 56
- 238000001514 detection method Methods 0.000 abstract description 36
- 230000008878 coupling Effects 0.000 abstract 1
- 238000010168 coupling process Methods 0.000 abstract 1
- 238000005859 coupling reaction Methods 0.000 abstract 1
- 239000010410 layer Substances 0.000 description 155
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 80
- 239000006185 dispersion Substances 0.000 description 63
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 60
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 52
- 239000000427 antigen Substances 0.000 description 49
- 108091007433 antigens Proteins 0.000 description 49
- 102000036639 antigens Human genes 0.000 description 49
- 239000000203 mixture Substances 0.000 description 44
- 239000011258 core-shell material Substances 0.000 description 43
- 229910052757 nitrogen Inorganic materials 0.000 description 40
- 230000005587 bubbling Effects 0.000 description 30
- 230000035945 sensitivity Effects 0.000 description 26
- 238000000034 method Methods 0.000 description 23
- 238000006116 polymerization reaction Methods 0.000 description 22
- 230000015572 biosynthetic process Effects 0.000 description 21
- 239000002344 surface layer Substances 0.000 description 21
- 230000003287 optical effect Effects 0.000 description 19
- 239000004793 Polystyrene Substances 0.000 description 14
- 229920002223 polystyrene Polymers 0.000 description 14
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 12
- 239000007995 HEPES buffer Substances 0.000 description 12
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 239000004570 mortar (masonry) Substances 0.000 description 12
- 229910001172 neodymium magnet Inorganic materials 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- XDLMVUHYZWKMMD-UHFFFAOYSA-N 3-trimethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CO[Si](OC)(OC)CCCOC(=O)C(C)=C XDLMVUHYZWKMMD-UHFFFAOYSA-N 0.000 description 10
- 239000006087 Silane Coupling Agent Substances 0.000 description 10
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 10
- 238000009396 hybridization Methods 0.000 description 10
- 229920002454 poly(glycidyl methacrylate) polymer Polymers 0.000 description 10
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 10
- 230000004580 weight loss Effects 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000012790 confirmation Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 239000010419 fine particle Substances 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 229910044991 metal oxide Inorganic materials 0.000 description 5
- 150000004706 metal oxides Chemical class 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 239000007987 MES buffer Substances 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000002612 dispersion medium Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 2
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229910052809 inorganic oxide Inorganic materials 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- AXTADRUCVAUCRS-UHFFFAOYSA-N 1-(2-hydroxyethyl)pyrrole-2,5-dione Chemical compound OCCN1C(=O)C=CC1=O AXTADRUCVAUCRS-UHFFFAOYSA-N 0.000 description 1
- KTZVZZJJVJQZHV-UHFFFAOYSA-N 1-chloro-4-ethenylbenzene Chemical compound ClC1=CC=C(C=C)C=C1 KTZVZZJJVJQZHV-UHFFFAOYSA-N 0.000 description 1
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000006173 Good's buffer Substances 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010079855 Peptide Aptamers Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000254154 Sitophilus zeamais Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- XYLMUPLGERFSHI-UHFFFAOYSA-N alpha-Methylstyrene Chemical compound CC(=C)C1=CC=CC=C1 XYLMUPLGERFSHI-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- TVFLFCXZLGULPU-UHFFFAOYSA-N imidazol-1-yl prop-2-enoate Chemical compound C=CC(=O)On1ccnc1 TVFLFCXZLGULPU-UHFFFAOYSA-N 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 238000009702 powder compression Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001141 propulsive effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000003440 styrenes Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
本発明は、磁性粒子、免疫検査用の粒子、検査試薬に関する。 The present invention relates to magnetic particles, particles for immunoassay, and test reagents.
試料に含まれる抗原や抗体などの測定対象物質を検出する検査システムにおいて、磁性粒子が使用されることがある。具体的には、試料から測定対象物質（例えば、抗原）を検出するために、抗原と特異的に結合する抗体を結合した磁性粒子、及び抗原と特異的に結合する抗体を固定したセンサーを用いる検査システムがある。試料に抗原が存在すると、抗原抗体反応により、抗体を固定したセンサーに、抗原を介して磁性粒子が結合する。このような検査システムとして、光導波路型の検査システムが挙げられる。光導波路型の検査システムでは、センサーに入射した光に対する出射光量の変化により抗原の有無を判定するものである。このような抗原の検出方法においては、検出までの時間をより短時間にすること、すなわち検出速度が速いことが求められる。 Magnetic particles may be used in inspection systems that detect substances to be measured such as antigens and antibodies contained in a sample. Specifically, in order to detect a substance to be measured (for example, an antigen) from a sample, a magnetic particle having an antibody specifically bound to the antigen and a sensor having an antibody specifically bound to the antigen fixed are used. There is an inspection system. When an antigen is present in the sample, the magnetic particles bind to the sensor on which the antibody is immobilized via the antigen by the antigen-antibody reaction. Examples of such an inspection system include an optical waveguide type inspection system. In the optical waveguide type inspection system, the presence or absence of an antigen is determined by the change in the amount of emitted light with respect to the light incident on the sensor. In such an antigen detection method, it is required that the time until detection is shorter, that is, the detection speed is high.
一方、試料に含まれる測定対象物質の検出に用いられる従来の磁性粒子として以下のものが知られている。特許文献１では、ポリマー粒子の表面に磁性体微粒子を吸着させた診断薬用粒子が開示されている（特許文献１）。
On the other hand, the following are known as conventional magnetic particles used for detecting a substance to be measured contained in a sample.
特許文献２では、無機酸化物又はポリマーを含有するコア粒子の表面に、マグネタイト粒子を含有するシェル層を有する磁性体内包粒子が開示されている（特許文献２）。特許文献３では、酸化鉄などの超常磁性金属酸化物粒子を含有するシリカ粒子の表面上にシリカ層が形成された磁性シリカ粒子が開示されている。
本発明者らが検討したところ、上記従来の磁性粒子は、光導波路型の検査システムにおいて、検出速度が遅いという課題や測定対象物質の有無によるセンサーからの出射光量の変化が小さい、という課題があることを見出した。 As examined by the present inventors, the above-mentioned conventional magnetic particles have a problem that the detection speed is slow and a problem that the change in the amount of light emitted from the sensor is small depending on the presence or absence of the substance to be measured in the optical waveguide type inspection system. I found that there is.
したがって、本発明の目的は、測定対象物質を検出する際に、検出速度が速く、測定対象物質の有無によるセンサーからの出射光量の変化が大きい磁性粒子を提供することである。 Therefore, an object of the present invention is to provide magnetic particles having a high detection speed and a large change in the amount of light emitted from the sensor depending on the presence or absence of the substance to be measured when the substance to be measured is detected.
本発明に係る磁性粒子は、シリカを含むコア粒子と、前記コア粒子の表面のシェル層とを有する磁性粒子であって、前記シェル層は、前記コア粒子に近い方から順に、磁性体粒子を含む磁性体層と、リガンドを結合できる官能基を含む樹脂層と、を有し、前記磁性粒子の体積平均粒径が０．６μｍ以上３．０μｍ以下であり、前記磁性粒子の比重が１．８ｇ／ｃｍ３以上５．０ｇ／ｃｍ３以下である。 The magnetic particles according to the present invention are magnetic particles having core particles containing silica and a shell layer on the surface of the core particles, and the shell layer contains magnetic particles in order from the one closest to the core particles. It has a magnetic material layer containing the magnetic material layer and a resin layer containing a functional group capable of binding a ligand, and the volume average particle size of the magnetic particles is 0.6 μm or more and 3.0 μm or less, and the specific gravity of the magnetic particles is 1. 8 g / cm 3 or more and 5.0 g / cm 3 or less.
本発明によれば、測定対象物質を検出する際に、検出速度が速く、測定対象物質の有無による出射光量の変化が大きい磁性粒子を提供することができる。 According to the present invention, it is possible to provide magnetic particles having a high detection speed and a large change in the amount of emitted light depending on the presence or absence of the substance to be measured when the substance to be measured is detected.
以下、本発明の実施の形態について、詳細に述べる。以下で示す各種の物性値は、特に断りのない限り、２５℃における値である。 Hereinafter, embodiments of the present invention will be described in detail. The various physical property values shown below are values at 25 ° C. unless otherwise specified.
また、本実施形態における磁性粒子が試料から検出する測定対象物質としては、後述するリガンドが挙げられるが、以下ではリガンドとして、抗原を例に挙げて説明する。 Further, examples of the substance to be measured to be detected by the magnetic particles in the present embodiment from the sample include a ligand described later, but below, an antigen will be described as an example as the ligand.
（本実施形態に係る磁性粒子の構成の概要）
本実施形態に係る磁性粒子１００は、シリカを含むコア粒子１０１と、コア粒子１０１の表面のシェル層１０２とを有し、シェル層１０２は、コア粒子１０１に近い方から順に、磁性体粒子を含む磁性体層１０３と、樹脂層１０４と、を有する。樹脂層１０４はリガンドを結合できる官能基（不図示）を含む。そして、磁性粒子１００の体積平均粒径が０．６μｍ以上３．０μｍ以下であり、磁性粒子１００の比重が１．８ｇ／ｃｍ３以上５．０ｇ／ｃｍ３以下である。このような構成により、光導波路型の検査システムにおいて、抗原等の測定対象物質を検出する際に、検出速度が速い。また、測定対象物質の有無による出射光量の変化が大きいため、感度が高い。以下、その理由について説明する。
(Outline of composition of magnetic particles according to this embodiment)
The
（光導波路型の検査システムにおいて、磁性粒子を用いて抗原を検出する方法）
光導波路型の検査システムでは、検体等の試料から抗原を検出する際に、抗原と特異的に結合する抗体を結合した磁性粒子と、抗原と特異的に結合する抗体を固定したセンサーを用いる。センサーを重力方向の下側に配置し、センサーの近傍に磁力を発生させる電磁石などを配置する。電磁石を作動させると、重力と磁力の作用で、センサー近傍に磁性粒子を引き寄せることができる。そして、抗原抗体反応を利用することで、センサー近傍に引き寄せられた磁性粒子は、抗原を介して、センサー面に設けられた抗体と結合する。さらに、重力方向の上側に電磁石を配置することで、センサー面に設けられた抗体と結合していない磁性粒子を重力と反対方向に引き上げることができる。これにより、センサーに結合する磁性粒子と結合していない磁性粒子とを分離することができる。
(Method of detecting antigen using magnetic particles in an optical waveguide type inspection system)
In the optical waveguide type inspection system, when an antigen is detected from a sample such as a sample, a magnetic particle having an antibody that specifically binds to the antigen and a sensor having an antibody that specifically binds to the antigen are fixed. The sensor is placed below the direction of gravity, and an electromagnet that generates magnetic force is placed near the sensor. When the electromagnet is activated, magnetic particles can be attracted to the vicinity of the sensor by the action of gravity and magnetic force. Then, by utilizing the antigen-antibody reaction, the magnetic particles attracted to the vicinity of the sensor bind to the antibody provided on the sensor surface via the antigen. Further, by arranging the electromagnet on the upper side in the direction of gravity, the magnetic particles not bound to the antibody provided on the sensor surface can be pulled up in the direction opposite to the direction of gravity. This makes it possible to separate the magnetic particles that are bound to the sensor from the magnetic particles that are not bound to the sensor.
光源からセンサーに光を入射させたときの、出射光量の変化を測定することで、抗原抗体反応を介してセンサー面に結合した磁性粒子の有無、濃度、量などを測定することができる。すなわち、センサー面に結合した磁性粒子が存在する（多い）場合は、存在しない（少ない）場合に比べて、出射光量が小さい。したがって、磁性粒子がセンサーに結合する前に検出される出射光量からの減衰量に基づいて、試料中の抗原などの測定対象物質の有無、濃度、量などを測定することができる。 By measuring the change in the amount of emitted light when light is incident on the sensor from the light source, it is possible to measure the presence / absence, concentration, amount, etc. of magnetic particles bound to the sensor surface via the antigen-antibody reaction. That is, when the magnetic particles bonded to the sensor surface are present (more), the amount of emitted light is smaller than when they are not present (less). Therefore, the presence / absence, concentration, amount, and the like of a substance to be measured such as an antigen in the sample can be measured based on the amount of attenuation from the amount of emitted light detected before the magnetic particles are bound to the sensor.
このような方法で測定対象物質を検出する際、磁性粒子への測定対象物質以外のタンパク質の非特異的な吸着を低減させることや、センサー面に抗原がない場合での吸着を低減させることで、検査エラーを減らすことが重要である。そのために、磁性粒子の表面に樹脂層が形成される。樹脂層は親水的な特性を持つものが好ましく、また、樹脂層には、抗体等のリガンドを結合できる官能基が含まれる。 When detecting a substance to be measured by such a method, by reducing non-specific adsorption of proteins other than the substance to be measured on magnetic particles, or by reducing adsorption when there is no antigen on the sensor surface. , It is important to reduce inspection errors. Therefore, a resin layer is formed on the surface of the magnetic particles. The resin layer preferably has hydrophilic properties, and the resin layer contains a functional group capable of binding a ligand such as an antibody.
（光導波路型の検査システムにおいて、磁性粒子に求められる性能）
従来の磁性粒子は、磁性粒子にかかる重力と磁力が十分ではないため、センサー近傍に磁性粒子を引き寄せるために時間を要してしまい、その結果として検出速度が遅くなっていた。
(Performance required for magnetic particles in an optical waveguide type inspection system)
In the conventional magnetic particles, since the gravity and the magnetic force applied to the magnetic particles are not sufficient, it takes time to attract the magnetic particles to the vicinity of the sensor, and as a result, the detection speed is slowed down.
上記特許文献１に記載の磁性粒子は、ポリマー粒子の表面に磁性体微粒子を吸着させた構成である。本発明者らが検討したところ、特許文献１に開示されている磁性粒子の比重は、１．２～１．３ｇ／ｃｍ３程度である。そのため磁性粒子を重力方向に移動させる推進力が弱く、センサー近傍に磁性粒子を引き寄せるために時間を要してしまい、検出速度が十分に得られない。
The magnetic particles described in
上記特許文献２に記載の磁性粒子は、無機酸化物又はポリマーを含有するコア粒子の表面に、マグネタイト粒子を含有するシェル層を有する構成である。しかし、特許文献２の磁性粒子の体積平均粒径は１０～５００ｎｍであり、小さい。そのため、重力と反対方向に磁場を印加した際に、抗原抗体反応を介さずにセンサー面付近に存在する磁性粒子が移動せず残存してしまい、正確な測定が難しい。
The magnetic particles described in
特許文献３に記載の磁性粒子は、酸化鉄などの超常磁性金属酸化物粒子を含有するシリカ粒子の表面上にシリカ層が形成された構成である。したがって、磁性体である超常磁性金属酸化物粒子は、シリカ粒子中に分散して存在する。本発明者らが検討した結果、光導波路型の検査システムで特許文献３の磁性粒子を用いる場合に課題があることを見出した。すなわち、光導波路型の検査システムでは、センサー面に結合した磁性粒子により出射光量が変化するが、特にセンサー界面から１００～１５０ｎｍ程度の距離にあるごく近い屈折率の高い磁性体の存在が出射光量の変化に大きく影響する。したがって、シリカ粒子中に磁性体が分散された構造では、センサー面近くの磁性体の密度が低くなる。その結果としてコア粒子の表面のシェル層に磁性体が存在する本実施形態の構造と比較して、磁性粒子１個あたりの光量変化が小さくなり、測定の感度が低くなる。また、磁性体を分散させる構成であるため、個々の磁性粒子が含有する磁性体の量がばらつきやすい。本発明者らは、光導波路型の検査システムにおいてセンサーからの出射光量の減衰量も粒子毎で大きくばらつき、検査制度が低下する、という課題があることも見出した。 The magnetic particles described in Patent Document 3 have a configuration in which a silica layer is formed on the surface of silica particles containing superparamagnetic metal oxide particles such as iron oxide. Therefore, the superparamagnetic metal oxide particles, which are magnetic materials, are dispersed in the silica particles. As a result of the study by the present inventors, it has been found that there is a problem when the magnetic particles of Patent Document 3 are used in the optical waveguide type inspection system. That is, in the optical waveguide type inspection system, the amount of emitted light changes depending on the magnetic particles bonded to the sensor surface, but the amount of emitted light is particularly due to the presence of a magnetic material having a very close high refractive index at a distance of about 100 to 150 nm from the sensor interface. It greatly affects the change of. Therefore, in the structure in which the magnetic material is dispersed in the silica particles, the density of the magnetic material near the sensor surface becomes low. As a result, the change in the amount of light per magnetic particle is small and the measurement sensitivity is low as compared with the structure of the present embodiment in which the magnetic material is present in the shell layer on the surface of the core particles. Further, since the structure is such that the magnetic material is dispersed, the amount of the magnetic material contained in each magnetic particle tends to vary. The present inventors have also found that in an optical waveguide type inspection system, the amount of attenuation of the amount of light emitted from the sensor also varies greatly from particle to particle, and there is a problem that the inspection system is lowered.
（本実施形態に係る磁性粒子の構成の詳細）
上記従来の磁性粒子の課題を踏まえ、本発明者らは、抗原抗体反応をさせる際には磁性粒子を重力方向に、抗原抗体反応をさせた後には、重力と反対方向に素早く移動させる磁性粒子の設計を検討した。その結果、本発明者らは磁性粒子の比重と、粒径（体積平均粒径）を適切な範囲にすることが重要であると考えた。
(Details of the composition of the magnetic particles according to this embodiment)
Based on the above-mentioned problems of the conventional magnetic particles, the present inventors quickly move the magnetic particles in the direction of gravity when the antigen-antibody reaction is carried out, and in the direction opposite to the gravity after the antigen-antibody reaction. I considered the design of. As a result, the present inventors considered that it is important to keep the specific gravity of the magnetic particles and the particle size (volume average particle size) in an appropriate range.
粒径は抗原の検出感度に影響するパラメータで、粒形が小さい粒子を用いると粒子の比表面積を大きくすることができるため、抗体を多く結合でき、結果的に抗原を検出する確率が上がる。しかし、粒径が小さすぎる場合、粒子の比表面積を大きくできるが、センサーから遠ざかる方向へ、すなわち重力と反対方向の移動が困難になり、結果として正確な抗原検出ができなくなることが分かった。 The particle size is a parameter that affects the detection sensitivity of the antigen, and if particles with a small grain shape are used, the specific surface area of the particles can be increased, so that a large amount of antibody can be bound, and as a result, the probability of detecting the antigen increases. However, it has been found that if the particle size is too small, the specific surface area of the particles can be increased, but it becomes difficult to move in the direction away from the sensor, that is, in the direction opposite to gravity, and as a result, accurate antigen detection cannot be performed.
また、同じ粒径であれば、比重が大きな粒子の方が重力方向の移動速度は速くできるため、検出速度を速くすることができる。一方、比重が大きすぎると、重力と反対方向に磁性粒子を引き上げる際に時間がかかり、検出速度が遅くなる。 Further, if the particle size is the same, the particles having a larger specific gravity can move faster in the direction of gravity, so that the detection speed can be increased. On the other hand, if the specific gravity is too large, it takes time to pull up the magnetic particles in the direction opposite to gravity, and the detection speed becomes slow.
そこで、本発明者らは、光導波路型の検査システムで用いられる磁性粒子以下の３つの要件を備えることが重要であることを見出した。 Therefore, the present inventors have found that it is important to have the following three requirements for magnetic particles used in an optical waveguide type inspection system.
１つ目の要件は、コア粒子とコア粒子の表面のシェル層とを有する磁性粒子であることである。２つ目の要件は、磁性粒子の体積平均粒径が０．６μｍ以上３．０μｍ以下であることである。３つ目の要件は磁性粒子の比重が１．８ｇ／ｃｍ３以上５．０ｇ／ｃｍ３以下であることである。 The first requirement is that the magnetic particles have a core particle and a shell layer on the surface of the core particle. The second requirement is that the volume average particle size of the magnetic particles is 0.6 μm or more and 3.0 μm or less. The third requirement is that the specific gravity of the magnetic particles is 1.8 g / cm 3 or more and 5.0 g / cm 3 or less.
２つ目、３つ目の要件を実現する設計として、コア粒子がシリカを有し、シェル層がコア粒子に近い方から順に、磁性体粒子を含む磁性体層と樹脂層とを有する構成が適していることを見出した。コア粒子としてシリカを用いることで従来のような樹脂のコア粒子に比べて比重を大きくでき、かつ大きすぎない構成にできる。シェル層として磁性体層と樹脂層とを含むことで、磁性粒子全体の粒径を適切な範囲にすることができる。また、樹脂層がリガンドを結合できる官能基を有することで、磁性粒子の表面にリガンドを結合できる。 As a design that realizes the second and third requirements, a configuration in which the core particles have silica and the shell layer has a magnetic material layer containing magnetic material particles and a resin layer in order from the one closest to the core particles. I found it suitable. By using silica as the core particles, the specific gravity can be increased as compared with the conventional resin core particles, and the configuration can be made not too large. By including the magnetic material layer and the resin layer as the shell layer, the particle size of the entire magnetic particles can be set in an appropriate range. Further, since the resin layer has a functional group capable of binding the ligand, the ligand can be bound to the surface of the magnetic particles.
よって、上記の構成により、光導波路型の検査システムにおいて、抗原等の測定対象物質を検出する際に、検出速度が速い。また、測定対象物質の有無による出射光量の変化が大きいため、測定の感度が高くなる。 Therefore, with the above configuration, the detection speed is high when detecting a substance to be measured such as an antigen in an optical waveguide type inspection system. In addition, since the amount of emitted light changes greatly depending on the presence or absence of the substance to be measured, the measurement sensitivity becomes high.
以下では、本実施形態に係る磁性粒子の各構成要素について詳細を説明する。 Hereinafter, each component of the magnetic particles according to the present embodiment will be described in detail.
＜コア粒子、磁性体粒子、磁性粒子＞
本実施形態において、磁性体層の領域、すなわちコア粒子とシェル層の境界は、透過型電子顕微鏡（ＴＥＭ）で観察することができる。なぜなら、ＴＥＭは、比重の異なる成分を、コントラストを持って撮影することができるため、磁性粒子中の磁性体層の領域を同定することが可能だからである。
<Core particles, magnetic particles, magnetic particles>
In this embodiment, the region of the magnetic layer, that is, the boundary between the core particles and the shell layer can be observed with a transmission electron microscope (TEM). This is because the TEM can photograph components having different specific densities with contrast, so that it is possible to identify the region of the magnetic material layer in the magnetic particles.
本実施形態における磁性粒子の平均粒径は、乾燥粒子の場合はＴＥＭ画像で観察される２０個の粒子の長径の平均値から求めることができ、溶媒中の平均粒子サイズは動的光散乱法（ＤＬＳ）から求めることができる。コア粒子がシリカ等の無機粒子の場合は、乾燥粒子と溶媒中の粒子とで大きく変化しないため、以下の計算は、乾燥粒子を前提に見積もりを行った。したがって、本実施形態における磁性粒子の平均粒径は、体積平均粒径である。 In the case of dry particles, the average particle size of the magnetic particles in this embodiment can be obtained from the average value of the major axis of the 20 particles observed in the TEM image, and the average particle size in the solvent is a dynamic light scattering method. It can be obtained from (DLS). When the core particles are inorganic particles such as silica, there is no significant change between the dried particles and the particles in the solvent. Therefore, the following calculation was made on the premise of the dried particles. Therefore, the average particle size of the magnetic particles in this embodiment is the volume average particle size.
磁性粒子の比重は、磁性粒子の体積平均粒径、コア粒子の平均粒径、材料、磁性体層の組成、磁性体層の平均厚さから算出することができる。具体的には、磁性粒子の比重Ｄｐは、磁性粒子の体積Ｖｐ、コア粒子の比重Ｄｃ、コア粒子の体積Ｖｃ、磁性体層の比重Ｄｍ、磁性体層の体積Ｖｍから式（１）で示される。 The specific gravity of the magnetic particles can be calculated from the volume average particle size of the magnetic particles, the average particle size of the core particles, the material, the composition of the magnetic material layer, and the average thickness of the magnetic material layer. Specifically, the specific gravity Dp of the magnetic particles is represented by the formula (1) from the volume Vp of the magnetic particles, the specific gravity Dc of the core particles, the volume Vc of the core particles, the specific gravity Dm of the magnetic material layer, and the volume Vm of the magnetic material layer. Is done.
本実施形態に係る磁性粒子の体積平均粒径は０．６μｍ以上３．０μｍ以下である。体積平均粒径が０．６μｍより小さくなると、光導波路型の検査システムのように磁場により磁性粒子を移動させるような場合において、重力と反対方向に移動させることが困難になり正しい測定が難しい。また、体積平均粒径が３．０μｍを超えると、磁性粒子の単位質量あたりの表面積が小さくなるため、結合できる抗体の量が限られ、抗原抗体反応が可能な領域が小さくなり、抗原抗体反応の確率が低下してしまう。抗原の検出感度と検査時間を考えた場合、本実施形態に係る磁性粒子の体積平均粒径は、０．７μｍ以上であることが好ましく、２．７μｍ以下であることが好ましく、２．０μｍ以下であることがより好ましい。 The volume average particle size of the magnetic particles according to this embodiment is 0.6 μm or more and 3.0 μm or less. When the volume average particle size is smaller than 0.6 μm, it becomes difficult to move the magnetic particles in the direction opposite to the gravity in the case where the magnetic particles are moved by a magnetic field as in an optical waveguide type inspection system, and correct measurement is difficult. Further, when the volume average particle size exceeds 3.0 μm, the surface area per unit mass of the magnetic particles becomes small, so that the amount of antibody that can be bound is limited, the region where the antigen-antibody reaction is possible becomes small, and the antigen-antibody reaction becomes small. The probability of Considering the detection sensitivity of the antigen and the inspection time, the volume average particle size of the magnetic particles according to this embodiment is preferably 0.7 μm or more, preferably 2.7 μm or less, and 2.0 μm or less. Is more preferable.
また、本実施形態に係る磁性粒子の比重は、１．８ｇ／ｃｍ３以上５．０ｇ／ｃｍ３以下である。また、本実施形態に係る磁性粒子の比重は、３．５ｇ／ｃｍ３以下であることが好ましく、３．０ｇ／ｃｍ３以下であることが好ましく、２．５ｇ／ｃｍ３以下であることがさらに好ましく、２．１ｇ／ｃｍ３以下であることが特に好ましい。本実施形態における磁性粒子の比重は、上記式（１）より算出することができる。 The specific gravity of the magnetic particles according to this embodiment is 1.8 g / cm 3 or more and 5.0 g / cm 3 or less. Further, the specific gravity of the magnetic particles according to the present embodiment is preferably 3.5 g / cm 3 or less, preferably 3.0 g / cm 3 or less, and 2.5 g / cm 3 or less. It is more preferably 2.1 g / cm 3 or less, and particularly preferably 2.1 g / cm 3. The specific gravity of the magnetic particles in this embodiment can be calculated from the above formula (1).
（磁性体粒子）
磁性体とは、磁場の印加により磁化される材料のことである。磁性体粒子は、金属、及び金属酸化物からなる群より選択される少なくとも一種を含むことが好ましい。金属としては、鉄、マンガン、ニッケル、コバルト、クロムが挙げられる。金属酸化物としては酸化鉄、例えば、マグネタイト（Ｆｅ３Ｏ４）、γ－酸化鉄（ＩＩＩ）（γ－Ｆｅ２Ｏ３）、フェライトからなる群より選択される少なくとも一種が挙げられる。
(Magnetic particles)
A magnetic material is a material that is magnetized by the application of a magnetic field. The magnetic particles preferably contain at least one selected from the group consisting of metals and metal oxides. Examples of the metal include iron, manganese, nickel, cobalt and chromium. Examples of the metal oxide include at least one selected from the group consisting of iron oxide, for example, magnetite (Fe 3 O 4 ), γ-iron oxide (III) (γ-Fe 2 O 3 ), and ferrite.
磁性体粒子の平均粒形は、０ｎｍより大きく２０ｎｍ以下であることが好ましい。特に、平均粒径が０ｎｍより大きく２０ｎｍ以下であり、マグネタイトを含む磁性体粒子は飽和磁化が大きく、かつ、超常磁性体であるため残留磁化が小さく好ましい。例えば、磁性体粒子の飽和磁化を、１８ｅｍｕ／ｇ以上とすることができる。 The average grain size of the magnetic particles is preferably larger than 0 nm and 20 nm or less. In particular, magnetic particles containing magnetite having an average particle size of more than 0 nm and 20 nm or less are preferable because they have a large saturation magnetization and are superparamagnetic materials and therefore have a small residual magnetization. For example, the saturation magnetization of the magnetic particles can be 18 emu / g or more.
ここで、磁化とは、磁性体に外部磁場をかける際に、その磁性体が分極して磁気モーメントを持つ現象のことであり、飽和磁化とは、磁場の強さとともに増大する磁化が飽和する値のことである。また、残留磁化とは、磁性体に外部磁場をかけた後に磁場ゼロにした場合に、磁性体に残留する磁化のことである。 Here, the magnetization is a phenomenon in which the magnetic material is polarized and has a magnetic moment when an external magnetic field is applied to the magnetic material, and the saturation magnetization is that the magnetization that increases with the strength of the magnetic field is saturated. It is a value. Further, the residual magnetization is the magnetization remaining in the magnetic material when the magnetic field is set to zero after applying an external magnetic field to the magnetic material.
（磁性粒子の移動速度の計算）
光導波路型の検査システムのように磁性粒子に磁場を印加し移動させて抗原を検出する場合、その検出速度を向上させるためには、磁性粒子の沈降速度、つまり重力方向への移動速度を速くすることが有効である。
(Calculation of moving speed of magnetic particles)
When a magnetic field is applied to a magnetic particle to move it to detect an antigen as in an optical waveguide type inspection system, in order to improve the detection speed, the settling speed of the magnetic particle, that is, the moving speed in the direction of gravity is increased. It is effective to do.
磁性粒子に作用する力は、重力方向に磁場を印加した場合、磁場をＯＦＦにした場合、重力と反対方向に磁場を印加した場合の三つのケースがあり、それぞれ、運動方程式を示すと式（３）（４）（５）となる。 There are three cases of the force acting on the magnetic particles: when a magnetic field is applied in the direction of gravity, when the magnetic field is turned off, and when a magnetic field is applied in the direction opposite to gravity. 3) (4) (5).
（重力方向に磁場を印加した場合）
（磁場をＯＦＦにした場合）
（重力方向と反対に磁場を印加した場合）
ｆ＝３×π×η×Ｒｐ×ｖ・・・（６）
ｂ＝Ｖｐ×Ｄｆ×ｇ・・・（７）
ｍ＝Ｖｐ×Ｄｐ・・・（８）
(When a magnetic field is applied in the direction opposite to the direction of gravity)
f = 3 × π × η × Rp × v ... (6)
b = Vp × Df × g ... (7)
m = Vp × Dp ... (8)
ここで、ｖは磁性粒子の移動速度（ｃｍ／ｓ）、ｇは重力加速度（９８０．７ｃｍ／ｓ２）、Ｄｐは磁性粒子の比重（ｇ／ｃｍ３）、Ｖｐは磁性粒子の体積（ｃｍ３）、Ｄｆは磁性粒子が分散している分散媒の密度（ｇ／ｃｍ３）を表す。また、Ｒｐは平均粒径（ｃｍ）、ηは前記分散媒の粘度（Ｐａ・ｓ）を表す。ストークスの式より、磁性粒子の移動速度ｖは、磁性粒子の体積平均粒形の２乗に比例して大きくなる。平均粒径が同じであれば、比重が大きい粒子の方が移動速度は速い。また、磁力Ｆｍ（Ｎ）は、磁場と磁化の積に比例し、式（９）であらわされる。ここで、ｑは磁性粒子１個あたりの磁化（ｅｍｕ／個）、Ｈは印加磁場（Ｏｅ）である。
Ｆｍ＝ｑ×Ｈ×１０５・・・（９）
Here, v is the moving speed of the magnetic particles (cm / s), g is the gravitational acceleration (980.7 cm / s 2 ), Dp is the specific gravity of the magnetic particles (g / cm 3 ), and Vp is the volume of the magnetic particles (cm). 3 ), Df represents the density (g / cm 3 ) of the dispersion medium in which the magnetic particles are dispersed. Further, Rp represents the average particle size (cm), and η represents the viscosity (Pa · s) of the dispersion medium. From Stokes' equation, the moving speed v of the magnetic particles increases in proportion to the square of the volume average grain shape of the magnetic particles. If the average particle size is the same, the particles with a larger specific gravity have a faster moving speed. Further, the magnetic force Fm (N) is proportional to the product of the magnetic field and the magnetization, and is expressed by the equation (9). Here, q is the magnetization per magnetic particle (emu / piece), and H is the applied magnetic field (Oe).
Fm = q × H × 10 5 ... (9)
以上の関係式から分散媒中の粒子の移動速度を算出すると、重力方向に磁場を印加した場合、磁場をＯＦＦにした場合、重力と反対方向に磁場を印加した場合の三つのケースのそれぞれの移動速度は式（１０）（１１）（１２）であらわされる。 When the moving speed of the particles in the dispersion medium is calculated from the above relational expression, each of the three cases when the magnetic field is applied in the direction of gravity, when the magnetic field is turned off, and when the magnetic field is applied in the direction opposite to gravity. The moving speed is expressed by the equations (10), (11) and (12).
磁性粒子の移動速度を式（１０）（１１）（１２）を用いて算出した。ここで、磁性体層の厚さは１００ｎｍとし、磁性体粒子（比重が５．２ｇ／ｃｍ３）が樹脂（比重１．１ｇ／ｃｍ３）の中に３２％含有している構造を想定した。但し、磁性体層と樹脂層が積層された構成でも同様の算出結果が得られる。印加磁場は、下磁場も上磁場も２００Ｏｅとし、コア粒子を、シリカ粒子（比重２．０ｇ／ｃｍ３）にした場合と、ポリスチレン粒子（比重１．０ｇ／ｃｍ３）にした場合とで比較した。算出結果を表１に示す。 The moving speed of the magnetic particles was calculated using the equations (10), (11) and (12). Here, the thickness of the magnetic material layer is 100 nm, and it is assumed that the magnetic material particles (specific gravity: 5.2 g / cm 3 ) are contained in the resin (specific gravity 1.1 g / cm 3 ) at 32%. .. However, the same calculation result can be obtained even in a configuration in which a magnetic material layer and a resin layer are laminated. The applied magnetic field is 200 Oe for both the lower magnetic field and the upper magnetic field, and comparison is made between the case where the core particles are silica particles (specific gravity 2.0 g / cm 3 ) and the case where the core particles are polystyrene particles (specific gravity 1.0 g / cm 3 ). bottom. The calculation results are shown in Table 1.
（磁性体層）
本実施形態において磁性体層の形成方法について説明する。
(Magnetic material layer)
The method of forming the magnetic material layer will be described in this embodiment.
コア粒子の表面に磁性体粒子を被覆して磁性体層を形成するためには、先ずコア粒子と磁性体粒子とを混合し、コア粒子の表面に磁性体粒子を物理的に吸着させる方法がある。本実施形態で述べる物理的に吸着させる方法とは、化学反応を伴わない吸着法、結合法を指すものである。また、その他に溶液中でコア粒子に磁性体粒子を樹脂と同時に付着させる化学的被覆法もある。 In order to coat the surface of the core particles with the magnetic particles to form a magnetic layer, a method of first mixing the core particles and the magnetic particles and physically adsorbing the magnetic particles on the surface of the core particles is used. be. The method of physically adsorbing described in this embodiment refers to an adsorption method and a bonding method that do not involve a chemical reaction. In addition, there is also a chemical coating method in which magnetic particles are attached to core particles at the same time as resin in a solution.
物理的に付着させる方法でコア粒子の表面に磁性体粒子を固定するためには、物理的に強い力を外部から加えることにより複合化を実現させる方法も有効である。例えば乳鉢、自動乳鉢、ボールミル、ブレード加圧式粉体圧縮法、メカノフュージョン法のようなメカノケミカル効果を利用するもの、あるいはジェットミル、ハイブリダイザーなど高速気流中衝撃法を利用するものが挙げられる。効率よくかつ強固に複合化を実施するには物理的吸着力が強いことが望ましい。その方法としては攪拌翼付き容器中で攪拌翼の周速度が好ましくは１５ｍ／秒以上、より好ましくは３０ｍ／秒以上、さらに好ましくは４０～１５０ｍ／秒で実施することが挙げられる。また、コア粒子の表面は隙間が少なくなるように磁性体層で被覆されていることが好ましい。 In order to fix the magnetic particles on the surface of the core particles by the method of physically adhering them, it is also effective to realize the composite by applying a physically strong force from the outside. For example, those using a mechanochemical effect such as a mortar, an automatic mortar, a ball mill, a blade pressure type powder compression method, and a mechanofusion method, or a jet mill, a hybridizer, etc., which utilize a high-speed airflow impact method can be mentioned. It is desirable that the physical adsorption force is strong in order to carry out the compounding efficiently and firmly. As the method, the peripheral speed of the stirring blade is preferably 15 m / sec or more, more preferably 30 m / sec or more, and further preferably 40 to 150 m / sec in a container with a stirring blade. Further, it is preferable that the surface of the core particles is coated with a magnetic material layer so that the gaps are reduced.
（樹脂層）
上記磁性体層の上に設けられる樹脂層について説明する。以下、「（メタ）アクリレート」と記載した場合は、「アクリレート、又はメタクリレート」を表すものとする。
(Resin layer)
The resin layer provided on the magnetic material layer will be described. Hereinafter, when the term "(meth) acrylate" is used, it means "acrylate or methacrylate".
磁性体粒子の表層には、樹脂層が形成される。樹脂層は、抗体等のリガンドを結合することができる官能基を有する。抗体を結合できる官能基は、アミノ基、カルボキシ基、水酸基、メルカプト基、チオール基、グリシジル基、マレイミド基、スクシンイミジル基、及びグリシジルオキシ基で構成される群から選ばれる少なくとも一種を含むことが好ましい。 A resin layer is formed on the surface layer of the magnetic particles. The resin layer has a functional group capable of binding a ligand such as an antibody. The functional group to which the antibody can be bound preferably contains at least one selected from the group composed of an amino group, a carboxy group, a hydroxyl group, a mercapto group, a thiol group, a glycidyl group, a maleimide group, a succinimidyl group, and a glycidyloxy group. ..
樹脂層を形成する際は、（メタ）アクリル酸などのカルボキシ基を有するモノマー；（メタ）アクリルアミドなどのアミノ基を有するモノマー；グリシジル（メタ）アクリレートなどのエポキシ基を有するモノマー；Ｎ－スクシンイミジルアクリレートなどのスクシンイミジル基を有するモノマー；を用いることが好ましい。言い換えると、樹脂層は、ポリ（メタ）アクリル酸ポリマー、ポリ（メタ）アクリルアミドポリマー、ポリグリシジル（メタ）アクリレート、ポリＮ－スクシンイミジルアクリレートを含むことが好ましい。 When forming the resin layer, a monomer having a carboxy group such as (meth) acrylic acid; a monomer having an amino group such as (meth) acrylamide; a monomer having an epoxy group such as glycidyl (meth) acrylate; It is preferable to use a monomer having a succinimidyl group such as imidazole acrylate. In other words, the resin layer preferably contains a poly (meth) acrylic acid polymer, a poly (meth) acrylamide polymer, a polyglycidyl (meth) acrylate, and a poly N-succinimidyl acrylate.
また、上記モノマー以外に、グリセロール（メタ）アクリレート、２－ヒドロキシエチル（メタ）アクリレート、メトキシエチル（メタ）アクリレート、ポリエチレングリコールモノ（メタ）アクリレートなどの親水性基を有する（メタ）アクリレート類；スチレン、ｐ－クロロスチレン、α－メチルスチレンなどのスチレン類；なども用いることができる。磁性粒子への非特異的な吸着を抑制するために、親水性基を有する（メタ）アクリレート類を用いることが好ましい。 In addition to the above monomers, (meth) acrylates having hydrophilic groups such as glycerol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, methoxyethyl (meth) acrylate, and polyethylene glycol mono (meth) acrylate; styrene. , P-chlorostyrene, α-methylstyrene and other styrenes; and the like can also be used. In order to suppress non-specific adsorption to magnetic particles, it is preferable to use (meth) acrylates having a hydrophilic group.
また、抗体を結合できる官能基は、樹脂の重合の後に付加することも可能である。例えば、（メタ）アクリル酸などのカルボキシル基を有するモノマーを重合して得られる樹脂に、メルカプトジオールを付加することでチオール基を導入できる。また、重合して得られる樹脂に、Ｎ－（２－ヒドロキシエチル）マレイミドを付加することでマレイミド基を導入することができる。 In addition, the functional group to which the antibody can be bound can be added after the polymerization of the resin. For example, a thiol group can be introduced by adding a mercaptodiol to a resin obtained by polymerizing a monomer having a carboxyl group such as (meth) acrylic acid. Further, a maleimide group can be introduced by adding N- (2-hydroxyethyl) maleimide to the resin obtained by polymerization.
＜免疫検査用の粒子＞
本発明の免疫検査用の粒子は、上述の構成の磁性粒子と、上記リガンドを結合できる官能基に結合したリガンドとを有する。
<Particles for immunoassay>
The particles for immunoassay of the present invention include magnetic particles having the above-mentioned constitution and a ligand bound to a functional group capable of binding the ligand.
＜リガンド＞
上記の説明では、リガンドの例として抗体、測定対象物質として抗原を例に説明したが、これら以外のリガンドを用いることができる。
<Ligand>
In the above description, an antibody has been described as an example of the ligand, and an antigen has been described as an example of the substance to be measured, but ligands other than these can be used.
ここで、リガンドとは、特定の標的物質が有する受容体に特異的に結合する化合物のことである。リガンドとして例えば、抗体、抗原、天然由来核酸、人工核酸、アプタマー、ペプチドアプタマー、オリゴペプチド、酵素又は補酵素などが挙げられる。リガンドが測定対象物質と結合する部位は決まっており、選択的または特異的に高い親和性を有する。リガンドと測定対象物質として例えば、抗原と抗体、酵素タンパク質とその基質、ホルモンや神経伝達物質などのシグナル物質とその受容体、核酸などが例示される。リガンドと測定対象物質は逆にしてもよい。すなわち抗体をリガンド、抗原を測定対象物質としてもよいし、抗原をリガンド、抗体を測定対象物質としてもよい。なお、本実施形態のリガンドはこれらに限定されない。 Here, the ligand is a compound that specifically binds to a receptor possessed by a specific target substance. Examples of the ligand include an antibody, an antigen, a naturally occurring nucleic acid, an artificial nucleic acid, an aptamer, a peptide aptamer, an oligopeptide, an enzyme or a coenzyme and the like. The site where the ligand binds to the substance to be measured is fixed, and has a high affinity selectively or specifically. Examples of the ligand and the substance to be measured include an antigen and an antibody, an enzyme protein and its substrate, a signal substance such as a hormone and a neurotransmitter and its receptor, and a nucleic acid. The ligand and the substance to be measured may be reversed. That is, an antibody may be used as a ligand and an antigen as a substance to be measured, or an antigen may be used as a ligand and an antibody may be used as a substance to be measured. The ligand of the present embodiment is not limited to these.
また、複数種の抗体が結合した免疫検査用の粒子を用いることで、複数種の測定対象物質を検出することが容易になる。また、測定対象物質が抗体に認識される認識部位が複数ある場合、複数の認識部位に応じた複数種の抗体を免疫検査用の粒子に結合させておくとよい。 In addition, by using particles for immunoassay to which a plurality of types of antibodies are bound, it becomes easy to detect a plurality of types of substances to be measured. In addition, when there are a plurality of recognition sites in which the substance to be measured is recognized by the antibody, it is advisable to bind a plurality of types of antibodies corresponding to the plurality of recognition sites to the particles for immunological examination.
＜検査試薬＞
本実施形態における検査試薬は、上記の免疫検査用の粒子と、免疫検査用の粒子を分散させる分散剤とを有する。検査試薬における免疫検査用の粒子の含有量は、分散剤の全質量を１００質量％として、０．００１質量％以上２０質量％以下であることが好ましく、０．０１質量％以上１０質量％以下であることがさらに好ましい。検査試薬には、分散剤やブロッキング剤などを含んでいてもよい。分散剤やブロッキング剤などは、２種以上を組み合わせてもよい。分散剤としては、リン酸緩衝液、グリシン緩衝液、グッド緩衝液、トリス緩衝液、アンモニア緩衝液などの緩衝液が挙げられる。
<Test reagent>
The test reagent in the present embodiment has the above-mentioned particles for an immunological test and a dispersant for dispersing the particles for an immunological test. The content of particles for immunoassay in the test reagent is preferably 0.001% by mass or more and 20% by mass or less, and 0.01% by mass or more and 10% by mass or less, assuming that the total mass of the dispersant is 100% by mass. Is more preferable. The test reagent may contain a dispersant, a blocking agent, or the like. Two or more kinds of dispersants, blocking agents and the like may be combined. Examples of the dispersant include buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer.
＜検出方法＞
本実施形態において、試料に含まれる測定対象物質の検出方法は以下の工程を少なくとも有する。以下ではリガンドを抗体とした例を説明する。
<Detection method>
In the present embodiment, the method for detecting the substance to be measured contained in the sample has at least the following steps. An example of using a ligand as an antibody will be described below.
（第１工程）
第１工程では、重力方向の下側に第１の抗体が固定されたセンサー容器内に、測定対象物質を含む試料と、免疫検査用の粒子と免疫検査用の粒子を分散させる分散剤を有する検査試薬とを添加する。その際に、センサー底面には光導波路が形成されていて、光導波路から出射された光量を検出している。
(First step)
In the first step, a sample containing the substance to be measured and a dispersant for dispersing the immunological test particles and the immunological test particles are provided in a sensor container in which the first antibody is immobilized on the lower side in the direction of gravity. Add the test reagent. At that time, an optical waveguide is formed on the bottom surface of the sensor to detect the amount of light emitted from the optical waveguide.
（第２工程）
第２工程では、免疫検査用の粒子が測定対象物質を介して第１の抗体に結合するように、重力方向と同じ方向に磁場を印加する。この時、磁性粒子はセンサー面に粒子が複数個連なったように針状に堆積する。
(Second step)
In the second step, a magnetic field is applied in the same direction as the direction of gravity so that the particles for immunological examination bind to the first antibody via the substance to be measured. At this time, the magnetic particles are deposited in a needle shape as if a plurality of particles are connected on the sensor surface.
（第３工程）
第３工程では、磁場をＯＦＦにして針状に連なった免疫検査用の粒子をセンサー面に沈降させる。この工程で、免疫検査用の粒子はセンサー面を覆うように配置される。試料に測定対象物が存在すれば、この工程で免疫検査用の粒子側の第２の抗体はセンサー面に設けられた第１の抗体と抗原を介して結合する。光導波路から出射される光の光量は、この段階で最も低くなる。
(Third step)
In the third step, the magnetic field is turned off and needle-shaped particles for immunological examination are settled on the sensor surface. In this step, the immunological test particles are arranged so as to cover the sensor surface. If an object to be measured is present in the sample, the second antibody on the particle side for immunoassay is bound to the first antibody provided on the sensor surface via an antigen in this step. The amount of light emitted from the optical waveguide is the lowest at this stage.
（第４工程）
第４工程では、測定対象物質を介して第１の抗体と結合していない免疫検査用の粒子が、センサー面から遠ざかるように磁場を印加する。この工程で、センサー面から離れる免疫検査用の粒子が存在すれば、導波路からの出射光量はその粒子の数に従って増加する。また、第一の工程の光量を初期値とすると、この光量と比較して検出される光量が低い場合は、抗原を介して結合している免疫検査用の粒子が存在することがわかり、その結果、試料内に測定対象物質が存在することがわかる。
(4th step)
In the fourth step, a magnetic field is applied so that the immunological test particles that are not bound to the first antibody via the substance to be measured move away from the sensor surface. In this step, if particles for immunoassay are present away from the sensor surface, the amount of light emitted from the waveguide increases with the number of particles. In addition, assuming that the amount of light in the first step is the initial value, if the amount of light detected is lower than this amount of light, it is found that particles for immunoassay that are bound via the antigen are present. As a result, it can be seen that the substance to be measured exists in the sample.
本実施形態に係る検出方法において、第１の抗体、第２の抗体は測定対象物質に結合可能であればよい。第１の抗体、第２の抗体とは同じであってもよいし、異なっていてもよい。また、第２の抗体は、異なる測定対象物質に結合するとうに複数の抗体が付与されていても良い。 In the detection method according to the present embodiment, the first antibody and the second antibody may be bound to the substance to be measured. It may be the same as or different from the first antibody and the second antibody. Further, the second antibody may be imparted with a plurality of antibodies so as to bind to different substances to be measured.
以下、実施例、及び比較例を挙げて本発明をさらに詳細に説明するが、本発明は、その要旨を超えない限り、下記の実施例によって何ら限定されるものではない。なお、成分量に関して「部」、及び「％」と記載しているものは特に断らない限り質量基準である。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded. In addition, regarding the amount of components, those described as "part" and "%" are based on mass unless otherwise specified.
＜実施例１＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社日本触媒製のシリカ粒子（平均粒子サイズ：０．５μｍ、比重：２．０ｇ／ｃｍ３）（以下シリカコア粒子）を６ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を１３ｇ得た。
<Example 1>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 6 g of silica particles manufactured by Nippon Catalyst Co., Ltd. (average particle size: 0.5 μm, specific gravity: 2.0 g / cm 3 ) (hereinafter referred to as silica core particles) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 13 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子０．５ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 0.5 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours.
続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して、樹脂（ポリグリシジルメタクリレート）で被覆された粒子が完成した。 Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the particles were thoroughly washed with pure water to complete the particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が０．７μｍであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体粒子の密度を見積もると約３２％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は２．０５ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した磁性粒子の平均粒径をＤＬＳで測定したところ、０．７７μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 0.7 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic particles was estimated to be about 32%. The specific gravity of the magnetic particles was 2.05 g / cm 3 calculated from TEM and TG. The average particle size of the magnetic particles dispersed in pure water at a concentration of 0.01% was measured by DLS and found to be 0.77 μm.
（カルボキシ基形成）
得られた磁性粒子６０ｍｇの水分散液１６０００ｍｇと、トリエチルアミン（東京化成工業製）１４５ｍｇでｐＨ１０に調整した溶液にメルカプトこはく酸５０ｍｇを溶解させ、６０℃で１５時間攪拌処理した。生成した磁性粒子を純水に分散し処理を完了した。すなわち、樹脂層にカルボキシ基を有する磁性粒子を作製した。
(Carboxy group formation)
50 mg of mercaptopodium acid was dissolved in a solution adjusted to
（免疫検査用の粒子の作製）
純水で十分に洗浄した上記磁性粒子１０ｍｇを、ＭＥＳ緩衝液に分散させ、水溶性カルボジイミド（ＷＳＣ）、及びＮ－ヒドロキシスクシンイミド（ｓｕ－ＮＨＳ）を加えて２５℃で３０分間撹拌した。その後、ネオジム磁石で捕集しながら溶液を除去しながらＭＥＳ緩衝液で洗浄し、ＭＥＳ緩衝液で再分散させて、抗体の終濃度が２ｍｇ／ｍＬとなるように抗ＣＲＰ抗体を加えた。その後、２５℃で６０分間撹拌し、ネオジム磁石で捕集しながら磁性粒子を回収し、磁性粒子をＨＥＰＥＳ緩衝液で洗浄し、抗ＣＲＰ抗体が結合した免疫検査用の粒子の分散液を得た。
(Preparation of particles for immunoassay)
10 mg of the magnetic particles sufficiently washed with pure water were dispersed in MES buffer, water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (su-NHS) were added, and the mixture was stirred at 25 ° C. for 30 minutes. Then, the solution was washed with MES buffer while collecting with a neodymium magnet, redispersed with MES buffer, and anti-CRP antibody was added so that the final concentration of the antibody was 2 mg / mL. Then, the mixture was stirred at 25 ° C. for 60 minutes, the magnetic particles were collected while being collected by a neodymium magnet, and the magnetic particles were washed with HEPES buffer to obtain a dispersion of particles for immunoassay to which an anti-CRP antibody was bound. ..
磁性粒子に抗ＣＲＰ抗体が結合していることは、抗ＣＲＰ抗体を加えたＭＥＳ緩衝液中の抗体の濃度の減少量を、タンパク質を比色定量することが可能なＢＣＡ（ビシンコニン酸）アッセイで確認した。 The binding of the anti-CRP antibody to the magnetic particles is a BCA (bicinchoninic acid) assay that can quantify the amount of decrease in the antibody concentration in the MES buffer containing the anti-CRP antibody by colorimetric determination of the protein. confirmed.
（免疫検査用の粒子の検出感度の確認）
上記の免疫検査用の粒子の分散液を用いて、検出感度の確認を行った。あらかじめ、抗ＣＲＰ抗体を結合させた光導波路型センサーを電磁石上にセットした。また、この時、光導波路には、６３５ｎｍの波長の光を入射させ、出射光を光量センサーで検出しながら感度テストを行った。
(Confirmation of detection sensitivity of particles for immunological test)
The detection sensitivity was confirmed using the above-mentioned dispersion of particles for immunological examination. In advance, an optical waveguide type sensor to which an anti-CRP antibody was bound was set on an electromagnet. At this time, light having a wavelength of 635 nm was incident on the optical waveguide, and a sensitivity test was performed while detecting the emitted light with a light amount sensor.
免疫検査用の粒子は含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた。この分散液をセンサー内に２００μＬ滴下した。電磁石で１分間重力方向に磁場を印加した後、磁場をＯＦＦして５分間静置した。次に、電磁石で３０秒間粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用粒分散液滴下直後の光量と同等の光量で、抗原がない場合は光量の低下が無いことを確認した。 Particles for immunoassay were dispersed in HEPES buffer so that the content was 0.01%. 200 μL of this dispersion was dropped into the sensor. After applying a magnetic field in the direction of gravity for 1 minute with an electromagnet, the magnetic field was turned off and allowed to stand for 5 minutes. Next, the amount of emitted light was measured after applying a magnetic field with an electromagnet in a direction in which the particles moved away from the sensor for 30 seconds. It was confirmed that the amount of emitted light was the same as the amount of light immediately after the particles dispersed droplets for immunological examination, and there was no decrease in the amount of light when there was no antigen.
次に、免疫検査用の粒子の含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で１分間、重力方向に磁場を印加した後、磁場をＯＦＦにして５分間静置した。次に、電磁石で３０秒間、粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の光量に対して３４％低下しており、抗原を検出できていることを確認した。 Next, 30 μL of CRP antigen was added to 200 μL of the solution dispersed in HEPES buffer so that the content of the particles for the immunological test was 0.01%, and the mixture was sufficiently mixed. 200 μL was dropped into the sensor. After applying a magnetic field in the direction of gravity for 1 minute with an electromagnet, the magnetic field was turned off and allowed to stand for 5 minutes. Next, the amount of light emitted after applying a magnetic field in the direction in which the particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was 34% lower than the amount of light immediately after dropping the dispersion of particles for immunoassay, confirming that the antigen could be detected.
＜実施例２＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社日本触媒製のシリカ粒子（平均粒子サイズ：１．０μｍ、比重：２．０ｇ／ｃｍ３）を１２ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を２０ｇ得た。
<Example 2>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 12 g of silica particles manufactured by Nippon Shokubai Co., Ltd. (average particle size: 1.0 μm, specific gravity: 2.0 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 20 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子１ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 1 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が１．２μｍであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３２％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．９４ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ、１．２８μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 1.2 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 32%. The specific gravity of the magnetic particles was 1.94 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was measured by DLS and found to be 1.28 μm.
（感度評価）
実施例１と同様の手順で、粒子表面にカルボキシル基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で１分間重力方向に磁場を印加した後、磁場をＯＦＦにして４分間静置した。次に、電磁石で３０秒間粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の光量に対して３０％低下しており、抗原を検出できていることを確認した。
(Sensitivity evaluation)
A carboxyl group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.01%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 1 minute with an electromagnet, the magnetic field was turned off and allowed to stand for 4 minutes. Next, the amount of emitted light was measured after applying a magnetic field with an electromagnet in a direction in which the particles moved away from the sensor for 30 seconds. The amount of emitted light was 30% lower than the amount of light immediately after dropping the dispersion of particles for immunological examination, confirming that the antigen could be detected.
＜実施例３＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社日本触媒製のシリカ粒子（平均粒子サイズ：１．３μｍ、比重：２．０ｇ／ｃｍ３）を１５ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を２２ｇ得た。
<Example 3>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 15 g of silica particles manufactured by Nippon Shokubai Co., Ltd. (average particle size: 1.3 μm, specific gravity: 2.0 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 22 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子１．３ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 1.3 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して、樹脂（ポリグリシジルメタクリレート）で被覆された粒子が完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the particles were thoroughly washed with pure water to complete the particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が１．５μｍであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３３％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．８９ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ１．５７μｍであった。
(Analysis of the produced magnetic particles)
The prepared particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 1.5 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 33%. The specific gravity of the magnetic particles was 1.89 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was measured by DLS and found to be 1.57 μm.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシル基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で１分間、重力方向に磁場を印加した後、磁場をＯＦＦして３分３０秒間静置した。次に、電磁石で３０秒間、粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の光量に対して２５％低下しており、抗原を検出できていることを確認した。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxyl group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.01%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 1 minute with an electromagnet, the magnetic field was turned off and allowed to stand for 3 minutes and 30 seconds. Next, the amount of light emitted after applying a magnetic field in the direction in which the particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was 25% lower than the amount of light immediately after dropping the dispersion of particles for immunoassay, confirming that the antigen could be detected.
＜実施例４＞
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社日本触媒製のシリカ粒子（平均粒子サイズ：２．５μｍ、比重：２ｇ／ｃｍ３）を３０ｇ秤量した。磁性体微粒子は、株式会社フェローテック製の疎水化処理された磁性体微粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を３８ｇ得た。
<Example 4>
(Formation of magnetic layer on the surface of core particles)
As core particles, 30 g of silica particles manufactured by Nippon Shokubai Co., Ltd. (average particle size: 2.5 μm, specific gravity: 2 g / cm 3 ) were weighed. As the magnetic fine particles, 10 g of the hydrophobized magnetic fine particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 38 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子２．５ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 2.5 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して、樹脂（ポリグリシジルメタクリレート）で被覆された粒子が完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the particles were thoroughly washed with pure water to complete the particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が２．７μｍであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３２％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．８４ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ２．８２μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 2.7 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 32%. The specific gravity of the magnetic particles was 1.84 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was 2.82 μm as measured by DLS.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用の粒子の含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で１分間、重力方向に磁場を印加した後、磁場をＯＦＦして３分１０秒間静置した。次に、電磁石で３０秒間、磁性粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の光量に対して２０％低下しており、抗原を検出できていることを確認した。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the particles for the immunological test is 0.01%, mix well, and then put the dispersion of the particles for the immunological test in the sensor. 200 μL was added dropwise. After applying a magnetic field in the direction of gravity for 1 minute with an electromagnet, the magnetic field was turned off and allowed to stand for 3 minutes and 10 seconds. Next, the amount of light emitted after applying a magnetic field in the direction in which the magnetic particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was 20% lower than the amount of light immediately after dropping the dispersion of particles for immunological examination, confirming that the antigen could be detected.
＜比較例１＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、綜研化学株式会社製のポリスチレン粒子（平均粒径：０．５μｍ、比重：１．１ｇ／ｃｍ３）を３ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、ポリスチレン粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を１０ｇ得た。
<Comparative Example 1>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 3 g of polystyrene particles manufactured by Soken Chemical Co., Ltd. (average particle size: 0.5 μm, specific gravity: 1.1 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 10 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子０．７ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 0.7 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が０．７μｍであることが確認できた。また、ポリスチレンコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、ポリスチレンコア粒子の比重を１．１ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３３％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した磁性粒子の比重は１．６９ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ０．７２μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 0.7 μm. Further, on the surface layer of the polystyrene core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (including magnetic particles). The layer and the resin layer are combined to form a magnetic layer). The weight loss during heating is measured by TG, assuming that the specific gravity of the magnetic particles (magnetite) is 5.2 g / cm 3 , the specific density of the polystyrene core particles is 1.1 g / cm 3 , and the specific density of the resin is 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 33%. The specific gravity of the magnetic particles was 1.69 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was 0.72 μm as measured by DLS.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．００５％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で４分３０秒間、重力方向に磁場を印加した後、磁場をＯＦＦして７分２０間、静置した。次に、電磁石で３０秒間、磁性粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の出射光量に対して約３４％低下しており、抗原を検出できていることを確認した。ただし、シリカコア粒子の同じサイズと比較すると下磁場印加時と磁場ＯＦＦ時にシリカコアと比較して時間を要し、検出時間を長く設定する必要があった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.005%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 4 minutes and 30 seconds with an electromagnet, the magnetic field was turned off and allowed to stand for 7 minutes and 20 seconds. Next, the amount of light emitted after applying a magnetic field in the direction in which the magnetic particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was reduced by about 34% with respect to the amount of emitted light immediately after the dispersion of particles for immunological examination was dropped, confirming that the antigen could be detected. However, compared with the same size of the silica core particles, it takes more time than the silica core when the lower magnetic field is applied and when the magnetic field is turned off, and it is necessary to set the detection time longer.
＜比較例２＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社モリテックス製のポリスチレン粒子（平均粒径：１．０μｍ、比重：１．１ｇ／ｃｍ３）を７ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、ポリスチレン粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を１５ｇ得た。
<Comparative Example 2>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 7 g of polystyrene particles manufactured by Moritex Co., Ltd. (average particle size: 1.0 μm, specific gravity: 1.1 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 15 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子０．８ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 0.8 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が１．２μｍであることが確認できた。また、ポリスチレンコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、ポリスチレンコア粒子の比重を１．１ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３２％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した磁性粒子の比重は１．５１ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ１．２７μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 1.2 μm. Further, on the surface layer of the polystyrene core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (including magnetic particles). The layer and the resin layer are combined to form a magnetic layer). The weight loss during heating is measured by TG, assuming that the specific gravity of the magnetic particles (magnetite) is 5.2 g / cm 3 , the specific density of the polystyrene core particles is 1.1 g / cm 3 , and the specific density of the resin is 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 32%. The specific gravity of the magnetic particles was 1.51 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was 1.27 μm as measured by DLS.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．００５％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で２分３０秒間重力方向に磁場を印加した後、磁場をＯＦＦして７分４０秒間静置した。次に、電磁石で３０秒間粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の出射光量に対して約３０％低下しており、抗原を検出できていることを確認した。ただし、シリカコア粒子の同じサイズと比較すると下磁場印加時と磁場ＯＦＦ時にシリカコアと比較して時間を要し、検出時間を長く設定する必要があった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.005%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 2 minutes and 30 seconds with an electromagnet, the magnetic field was turned off and allowed to stand for 7 minutes and 40 seconds. Next, the amount of emitted light was measured after applying a magnetic field with an electromagnet in a direction in which the particles moved away from the sensor for 30 seconds. The amount of emitted light was reduced by about 30% with respect to the amount of emitted light immediately after the dispersion of particles for immunological examination was dropped, confirming that the antigen could be detected. However, compared with the same size of the silica core particles, it takes more time than the silica core when the lower magnetic field is applied and when the magnetic field is turned off, and it is necessary to set the detection time longer.
＜比較例３＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、綜研化学株式会社製のポリスチレン粒子（平均粒径：１．３μｍ、比重：１．１ｇ／ｃｍ３）を７ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、ポリスチレン粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を１５ｇ得た。
<Comparative Example 3>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 7 g of polystyrene particles manufactured by Soken Chemical Co., Ltd. (average particle size: 1.3 μm, specific gravity: 1.1 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 15 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子２ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 2 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が１．５μｍであることが確認できた。また、ポリスチレンコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、ポリスチレンコア粒子の比重を１．１ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性層の磁性体微粒子の密度を見積もると約３３％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．４５ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒子サイズをＤＬＳで測定したところ、平均粒子サイズは１．６２μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 1.5 μm. Further, on the surface layer of the polystyrene core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (including magnetic particles). The layer and the resin layer are combined to form a magnetic material layer). The weight loss during heating is measured by TG, assuming that the specific gravity of the magnetic particles (magnetite) is 5.2 g / cm 3 , the specific density of the polystyrene core particles is 1.1 g / cm 3 , and the specific density of the resin is 1.0 g / cm 3 . The density of the magnetic fine particles in the magnetic layer was estimated to be about 33%. The specific gravity of the magnetic particles was 1.45 g / cm3, which was calculated from TEM and TG. Moreover, when the average particle size of the particles dispersed in pure water at a concentration of 0.01% was measured by DLS, the average particle size was 1.62 μm.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．００５％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で２分間、重力方向に磁場を印加した後、磁場をＯＦＦして７分間静置した。次に、電磁石で３０秒間、粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の光量に対して約２３％低下しており、抗原を検出できていることを確認した。ただし、シリカコア粒子の同じサイズと比較すると下磁場印加時と磁場ＯＦＦ時にシリカコアと比較して時間を要し、検出時間を長く設定する必要があった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.005%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 2 minutes with an electromagnet, the magnetic field was turned off and allowed to stand for 7 minutes. Next, the amount of light emitted after applying a magnetic field in the direction in which the particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was about 23% lower than the amount of light immediately after dropping the dispersion of particles for immunological examination, confirming that the antigen could be detected. However, compared with the same size of the silica core particles, it takes more time than the silica core when the lower magnetic field is applied and when the magnetic field is turned off, and it is necessary to set the detection time longer.
＜比較例４＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、株式会社モリテックス製のポリスチレン粒子（平均粒径：２．５μｍ、比重：１．１ｇ／ｃｍ３）を１３ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、ポリスチレン粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を２０ｇ得た。
<Comparative Example 4>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 13 g of polystyrene particles manufactured by Moritex Co., Ltd. (average particle size: 2.5 μm, specific gravity: 1.1 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 20 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子３．８ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 3.8 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が２．７μｍであることが確認できた。また、ポリスチレンコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、ポリスチレンコア粒子の比重を１．１ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性体層の磁性体粒子の密度を見積もると約３１％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．３１ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ２．８５μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 2.7 μm. Further, on the surface layer of the polystyrene core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (including magnetic particles). The layer and the resin layer are combined to form a magnetic material layer). The weight loss during heating is measured by TG, assuming that the specific gravity of the magnetic particles (magnetite) is 5.2 g / cm 3 , the specific density of the polystyrene core particles is 1.1 g / cm 3 , and the specific density of the resin is 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 31%. The specific gravity of the magnetic particles was 1.31 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was 2.85 μm as measured by DLS.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．００５％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で１．５分間、重力方向に磁場を印加した後、磁場をＯＦＦして５分３０秒間、静置した。次に、電磁石で３０秒間、磁性粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の出射光量に対して約１９％低下しており、抗原を検出できていることを確認した。ただし、シリカコア粒子の同じサイズと比較すると下磁場印加時と磁場ＯＦＦ時にシリカコアと比較して時間を要し、検出時間を長く設定する必要があった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.005%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 1.5 minutes with an electromagnet, the magnetic field was turned off and allowed to stand for 5 minutes and 30 seconds. Next, the amount of light emitted after applying a magnetic field in the direction in which the magnetic particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was reduced by about 19% with respect to the amount of emitted light immediately after the dispersion of particles for immunological examination was dropped, confirming that the antigen could be detected. However, compared with the same size of the silica core particles, it takes more time than the silica core when the lower magnetic field is applied and when the magnetic field is turned off, and it is necessary to set the detection time longer.
＜比較例５＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、シリカ粒子（平均粒径：０．３μｍ、比重：２ｇ／ｃｍ３）を３ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を１３ｇ得た。
<Comparative Example 5>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 3 g of silica particles (average particle size: 0.3 μm, specific gravity: 2 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 13 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェル構造の粒子０．３ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石でコアシェル構造の粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 0.3 g of the obtained core-shell structure particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting particles having a core-shell structure with a neodymium magnet, and the mixture was sufficiently washed with pure water, and then 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆されたコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water to complete the core-shell particles coated with the resin (polyglycidyl methacrylate).
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径が０．５μｍであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性層の磁性体粒子の密度を見積もると約３３％の割合であった。また、磁性粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は２．１９ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ０．５２μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the volume average particle size was 0.5 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic particles in the magnetic layer was estimated to be about 33%. The specific gravity of the magnetic particles was 2.19 g / cm 3 , which was calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was measured by DLS and found to be 0.52 μm.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシル基を形成し、感度特性を評価した。免疫検査用の粒子は含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた。この分散液をセンサー内に２００μＬ滴下した。電磁石で２分間、重力方向に磁場を印加した後、磁場をＯＦＦして７分間静置した。次に、電磁石で３０秒間、磁性粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定したが。出射光量は、免疫検査用の粒シオン分散液を滴下した直後の出射光量から８０％低下しており、抗原がないにもかかわらず光量の低下が生じ正確な検出ができなかった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxyl group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Particles for immunoassay were dispersed in HEPES buffer so that the content was 0.01%. 200 μL of this dispersion was dropped into the sensor. After applying a magnetic field in the direction of gravity for 2 minutes with an electromagnet, the magnetic field was turned off and allowed to stand for 7 minutes. Next, the amount of light emitted after applying a magnetic field in the direction in which the magnetic particles move away from the sensor was measured with an electromagnet for 30 seconds. The amount of emitted light was 80% lower than the amount of emitted light immediately after dropping the grain Zion dispersion for immunological examination, and the amount of light was reduced even though there was no antigen, and accurate detection could not be performed.
＜比較例６＞
（磁性粒子の作製）
（コア粒子の表面への磁性体層の形成）
コア粒子として、シリカ粒子（平均粒径：４．８μｍ、比重：２ｇ／ｃｍ３）を５０ｇ秤量した。磁性体粒子は、株式会社フェローテック製の疎水化処理された磁性体粒子（ＥＭＧ１４００）を１０ｇ秤量した。これらの粒子を乳鉢で十分粉砕、混合した混合粒子を作製した。次に、この混合粒子をハイブリダイゼーションシステムＮＨＳ－０型（奈良機械製作所（株）製）を使用して、羽の回転数１６２００ｒｐｍ（回転速度１００ｍ／秒で５分間処理した。この処理により、シリカコア粒子の表層に磁性体粒子が被覆された（磁性体層が形成された）コアシェル構造の粒子を５３ｇ得た。
<Comparative Example 6>
(Making magnetic particles)
(Formation of magnetic layer on the surface of core particles)
As core particles, 50 g of silica particles (average particle size: 4.8 μm, specific gravity: 2 g / cm 3 ) were weighed. As the magnetic particles, 10 g of hydrophobized magnetic particles (EMG1400) manufactured by Ferotec Co., Ltd. was weighed. These particles were sufficiently crushed in a mortar and mixed to prepare mixed particles. Next, the mixed particles were treated using a hybridization system NHS-0 type (manufactured by Nara Kikai Seisakusho Co., Ltd.) at a rotation speed of 16200 rpm (rotation speed 100 m / sec) for 5 minutes. 53 g of particles having a core-shell structure in which the surface layer of the particles was coated with the magnetic material particles (the magnetic material layer was formed) was obtained.
（樹脂層の形成）
次に、得られたコアシェルコクゾウの粒子５ｇを秤量し２０ｍＬのメタノールに分散させた。この分散液に、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を３８μＬ追加して３時間撹拌した。次に、ネオジム磁石で磁性粒子を捕集しながらメタノールを除去し、純水で十分洗浄した後６０ｍＬの純水を追加して水分散液を得た。
(Formation of resin layer)
Next, 5 g of the obtained core shell maize weevil particles were weighed and dispersed in 20 mL of methanol. 38 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion as a silane coupling agent, and the mixture was stirred for 3 hours. Next, methanol was removed while collecting magnetic particles with a neodymium magnet, and after sufficient washing with pure water, 60 mL of pure water was added to obtain an aqueous dispersion.
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングしながら２時間撹拌した。続いて、この分散液にキシダ化学製スチレンモノマーを０．５ｍＬ添加して３０分撹拌した。この段階で、窒素バブリングから窒素フローに切り替えた。 Next, this dispersion was placed in a four-necked flask (200 mL) and stirred for 2 hours while bubbling nitrogen. Subsequently, 0.5 mL of a styrene monomer manufactured by Kishida Chemical was added to this dispersion, and the mixture was stirred for 30 minutes. At this stage, we switched from nitrogen bubbling to nitrogen flow.
次に、０．０５ｇの過硫酸カリウム（シグマアルドリッチ製）を予め窒素バブリングで脱気した純水２０ｍＬに溶解して、フラスコに添加した。次に、オイルバスを用いて、６０℃に昇温して５時間保持した。続いて、グリシジルメタクリレート（キシダ化学製）を１ｍＬ添加して、さらに５時間保持して重合を終了した。重合終了後、純水で十分に洗浄して樹脂（ポリグリシジルメタクリレート）で被覆したコアシェル粒子を完成した。 Next, 0.05 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 20 mL of pure water previously degassed by nitrogen bubbling and added to the flask. Next, using an oil bath, the temperature was raised to 60 ° C. and held for 5 hours. Subsequently, 1 mL of glycidyl methacrylate (manufactured by Kishida Chemical) was added and held for another 5 hours to complete the polymerization. After the completion of the polymerization, the core-shell particles were sufficiently washed with pure water and coated with a resin (polyglycidyl methacrylate) to complete the core-shell particles.
（作製した磁性粒子の分析）
作製した磁性粒子をＴＥＭで観察した。２０個の粒子の長径の長さを測定して平均値を算出した結果、体積平均粒径５．０μｍの粒子サイズであることが確認できた。また、シリカコア粒子の表層には、平均粒径が１０ｎｍの磁性体粒子を含む層が５０ｎｍの厚さに積層されていて、その上に樹脂層５０ｎｍが形成されていた（磁性体粒子を含む層と樹脂層を合わせて磁性体層とする）。磁性体粒子（マグネタイト）の比重を５．２ｇ／ｃｍ３、シリカコア粒子の比重を２．０ｇ／ｃｍ３、樹脂の比重を１．０ｇ／ｃｍ３として、ＴＧにより加熱時の重量減少を測定して磁性層の磁性体微粒子の密度を見積もると約３２％の割合であった。また、粒子の比重は、ＴＥＭとＴＧから算出した粒子の比重は１．７２ｇ／ｃｍ３であった。また、純水に０．０１％の濃度で分散した粒子の平均粒径をＤＬＳで測定したところ１．７９μｍであった。
(Analysis of the produced magnetic particles)
The produced magnetic particles were observed by TEM. As a result of measuring the length of the major axis of 20 particles and calculating the average value, it was confirmed that the particle size had a volume average particle diameter of 5.0 μm. Further, on the surface layer of the silica core particles, a layer containing magnetic particles having an average particle size of 10 nm was laminated to a thickness of 50 nm, and a resin layer of 50 nm was formed on the layer (layer containing magnetic particles). And the resin layer are combined to form a magnetic material layer). The weight loss during heating was measured by TG, with the specific gravity of the magnetic particles (magnetite) being 5.2 g / cm 3 , the specific gravity of the silica core particles being 2.0 g / cm 3 , and the specific density of the resin being 1.0 g / cm 3 . The density of the magnetic fine particles in the magnetic layer was estimated to be about 32%. The specific gravity of the particles was 1.72 g / cm 3 calculated from TEM and TG. The average particle size of the particles dispersed in pure water at a concentration of 0.01% was 1.79 μm as measured by DLS.
（免疫検査用の粒子の検出感度の確認）
実施例１と同様の手順で、粒子表面にカルボキシ基を形成し、感度特性を評価した。免疫検査用粒子の含有量が０．０１％となるようにＨＥＰＥＳ緩衝液に分散させた液２００μＬにＣＲＰ抗原を３０μＬ添加し十分混合した後、免疫検査用の粒子の分散液をセンサー内に２００μＬ滴下した。電磁石で４０秒間重力方向に磁場を印加した後、磁場をＯＦＦして２分５０秒間静置した。次に、電磁石で３０秒間粒子がセンサーから遠ざかる方向に磁場を印加した後出射光量を測定した。出射光量は、免疫検査用の粒子の分散液を滴下した直後の出射光量に対して約７％低下していたが、変化量が小さく正確な検出が困難であった。
(Confirmation of detection sensitivity of particles for immunological test)
A carboxy group was formed on the particle surface in the same procedure as in Example 1, and the sensitivity characteristics were evaluated. Add 30 μL of CRP antigen to 200 μL of the solution dispersed in HEPES buffer so that the content of the immunological test particles is 0.01%, mix well, and then add 200 μL of the dispersion of the immunological test particles in the sensor. Dropped. After applying a magnetic field in the direction of gravity for 40 seconds with an electromagnet, the magnetic field was turned off and allowed to stand for 2 minutes and 50 seconds. Next, the amount of emitted light was measured after applying a magnetic field with an electromagnet in a direction in which the particles moved away from the sensor for 30 seconds. The amount of emitted light was about 7% lower than the amount of emitted light immediately after dropping the dispersion of particles for immunoassay, but the amount of change was small and accurate detection was difficult.
１００ 磁性粒子
１０１ コア粒子
１０２ シェル層
１０３ 磁性体層
１０４ 樹脂層
100
Claims (14)
前記シェル層は、前記コア粒子に近い方から順に、磁性体粒子を含む磁性体層と、
リガンドを結合できる官能基を含む樹脂層と、を有し、
前記磁性粒子の体積平均粒径が０．６μｍ以上３．０μｍ以下であり、前記磁性粒子の比重が１．８ｇ／ｃｍ３以上５．０ｇ／ｃｍ３以下である磁性粒子。 A magnetic particle having a core particle containing silica and a shell layer on the surface of the core particle.
The shell layer includes a magnetic material layer containing magnetic material particles in order from the one closest to the core particles.
With a resin layer containing a functional group capable of binding a ligand,
Magnetic particles having a volume average particle size of 0.6 μm or more and 3.0 μm or less, and a specific gravity of 1.8 g / cm 3 or more and 5.0 g / cm 3 or less.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020178983A JP2022069994A (en) | 2020-10-26 | 2020-10-26 | Magnetic particle, immunity inspection particle, and inspection reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020178983A JP2022069994A (en) | 2020-10-26 | 2020-10-26 | Magnetic particle, immunity inspection particle, and inspection reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2022069994A true JP2022069994A (en) | 2022-05-12 |
Family
ID=81534193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020178983A Pending JP2022069994A (en) | 2020-10-26 | 2020-10-26 | Magnetic particle, immunity inspection particle, and inspection reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2022069994A (en) |
-
2020
- 2020-10-26 JP JP2020178983A patent/JP2022069994A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5279357B2 (en) | Composite particle, method for producing the same, dispersion, magnetic biosensing device, and magnetic biosensing method | |
| US20120061608A1 (en) | Functional particle with rough-surfaced polymer coating | |
| US11231414B2 (en) | Magnetic composite particles, method for manufacturing the same, and immunoassay particles | |
| WO2015050149A1 (en) | Fluorescence-labeled particle | |
| WO2015180110A1 (en) | Method for preparing magnetic microsphere for separation of biological protein and use thereof | |
| WO2015050150A1 (en) | Method for detecting target substance | |
| JP4935973B2 (en) | Organic polymer particles and method for producing the same | |
| WO2004042397A1 (en) | High sensitivity magnetic marker used for immune response measurement | |
| WO2019208711A1 (en) | Particle and production method therefor | |
| JP6900207B2 (en) | A method for producing a probe-binding carrier and a method for detecting or separating a target substance. | |
| JP4984025B2 (en) | Organic polymer particle, method for producing the same, and probe binding particle | |
| CN114555650A (en) | Granules and method for producing granules | |
| JP2022069994A (en) | Magnetic particle, immunity inspection particle, and inspection reagent | |
| JP2006226689A (en) | Magnetic particle for immunological examination | |
| JP2006226690A (en) | Magnetic particle for immunological examination | |
| JP2005069926A (en) | Magnetic particle for immunological inspection | |
| JP2013167528A (en) | Functional particle subjected to fluorine-containing polymer coating | |
| WO2022209952A1 (en) | Particles and method for producing same | |
| JP6289705B1 (en) | Method for producing probe binding carrier, probe binding carrier and method for detecting or separating target substance | |
| JP4803366B2 (en) | ORGANIC POLYMER PARTICLE AND METHOD FOR PRODUCING THE SAME, ORGANIC POLYMER PARTICLE FOR PROBE CONNECTION AND MANUFACTURING METHOD THEREOF | |
| JP2020183946A (en) | Particle, affinity particle, inspection reagent, and detection method | |
| JP4985916B2 (en) | Method for producing carboxyl group-containing particles | |
| WO2022259989A1 (en) | Polarized light-emitting particles for specimen inspection | |
| WO2022259946A1 (en) | Method for detecting and measuring target substance on basis of measurement of polarization anisotropy, and particles used therefor | |
| JP2021060403A (en) | Magnetic particle for immunoassay and manufacturing method therefor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7421 Effective date: 20201105 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20231023 |
|
| RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7421 Effective date: 20231213 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20240516 |