JP2020159871A - Magnetic field measuring device and magnetic field measuring method - Google Patents
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Abstract
Description
本発明は、磁気的免疫検査により被測定物を検出するための磁界測定装置及び磁気測定方法に関し、具体的には、液体中において、被測定物質と結合している磁性物質（磁気マーカ）に由来する磁界を測定する磁界測定装置及び磁界測定方法に関する。 The present invention relates to a magnetic field measuring device and a magnetic measuring method for detecting an object to be measured by a magnetic immunological test, and specifically, to a magnetic substance (magnetic marker) bound to the substance to be measured in a liquid. The present invention relates to a magnetic field measuring device for measuring a derived magnetic field and a magnetic field measuring method.
疾患由来のタンパク質や病原菌などの生体物質を検出する免疫検査が医療診断において用いられている。免疫検査は、被測定物質である抗原と抗体が特異的に結合する抗原抗体反応が利用され、この抗体をマーカと呼ばれる物質で標識させ、抗原と結合している抗体のマーカからの信号を検出することで、抗原の量を測定することが可能となる。 Immune tests that detect biological substances such as disease-derived proteins and pathogens are used in medical diagnosis. In the immunological test, an antigen-antibody reaction in which an antigen and an antibody to be measured specifically bind to each other is used, and this antibody is labeled with a substance called a marker to detect a signal from the marker of the antibody bound to the antigen. By doing so, it becomes possible to measure the amount of antigen.
免疫検査の一つとして、被測定物質との結合能力が既知である抗体に蛍光酵素などの光学マーカを付加して標識し、被測定物質との結合の程度を光学的に検出する光学的免疫検査が行われている。ここで、多くの光学的免疫検査では、被測定物質と結合した光学マーカと結合しなかった光学マーカとを分離するための洗浄除去する工程が必要であり、検査工程が複雑で時間を要するという側面がある。 As one of the immunological tests, an antibody having a known binding ability to a substance to be measured is labeled with an optical marker such as a fluorescent enzyme to optically detect the degree of binding to the substance to be measured. Inspection is being carried out. Here, in many optical immunological tests, a step of washing and removing is required to separate the optical marker bound to the substance to be measured and the optical marker not bound to the substance to be measured, which is complicated and time-consuming. There are sides.
一方、光学的免疫検査とは異なり、磁気的手法によって被測定物質の検出を行う技術が磁気的免疫検査として知られている（特許文献１、２）。磁気的免疫検査は、磁性粒子と磁気センサを用いて抗原抗体反応を検出する手法であって、抗体に磁性粒子（以下、磁気マーカと称する）を付加して標識させ、被測定物質である抗原との結合程度を磁気マーカからの磁気信号を磁気センサを用いて検出する。具体的には、被測定物質と、磁気マーカが付加された抗体とを溶液中で結合させた試料を作製し、当該試料に外部から直流磁界を印加し、磁気マーカを磁化させる。直流磁界の印加を遮断した後、被測定物質と結合した磁気マーカ付加抗体（以下、結合マーカと称する）は、被測定物質と結合していない磁気マーカ付加抗体（未結合マーカ）より体積が大きくなり、ブラウン回転運動が遅いため、ブラウン緩和時間が比較的遅い。これにより、結合マーカは残留磁気を有する時間が長い。 On the other hand, unlike the optical immunoassay, a technique for detecting a substance to be measured by a magnetic method is known as a magnetic immunoassay (Patent Documents 1 and 2). Magnetic immunoassay is a method of detecting an antigen-antibody reaction using magnetic particles and a magnetic sensor. Magnetic particles (hereinafter referred to as magnetic markers) are added to the antibody to label the antibody, and the antigen to be measured is used. The degree of coupling with the magnetic marker is detected by using a magnetic sensor. Specifically, a sample in which a substance to be measured and an antibody to which a magnetic marker is added is bound in a solution is prepared, and a DC magnetic field is applied to the sample from the outside to magnetize the magnetic marker. The magnetic marker-added antibody (hereinafter referred to as a binding marker) bound to the substance to be measured after blocking the application of the DC magnetic field has a larger volume than the magnetic marker-added antibody (unbound marker) not bound to the substance to be measured. As a result, the brown rotation movement is slow, so the brown relaxation time is relatively slow. This allows the coupling marker to have residual magnetism for a long time.
一方、被測定物質と結合しなかった磁気マーカ付き抗体（未結合マーカ）も溶液中に存在する。未結合マーカは、単体で存在するために粒径が小さく、ブラウン回転運動が早くなる。従って、未結合マーカ抗体は磁気モーメントの方向がランダムとなりやすく、ブラウン緩和時間が早く、未結合マーカは残留磁気を有する時間が短い。これにより、結合マーカと未結合マーカのブラウン時間の差を利用することで、結合マーカのみの磁気信号を選択に検出することができる。 On the other hand, an antibody with a magnetic marker (unbound marker) that did not bind to the substance to be measured is also present in the solution. Since the unbound marker exists alone, the particle size is small and the brown rotational movement is fast. Therefore, the unbound marker antibody tends to have a random direction of magnetic moment, the brown relaxation time is short, and the unbound marker has a short residual magnetism. As a result, the magnetic signal of only the coupled marker can be selectively detected by utilizing the difference in brown time between the coupled marker and the unbound marker.
このように、磁気的免疫検査は、磁気マーカのブラウン緩和特性の違いを利用することで、磁気マーカ付加抗体を洗浄除去する工程を行うことなく、被測定物質との結合の程度を測定することができる。 In this way, the magnetic immunoassay measures the degree of binding to the substance to be measured by utilizing the difference in brown relaxation characteristics of the magnetic marker without performing the step of washing and removing the magnetic marker-added antibody. Can be done.
特許文献１−５は、磁気センサとしてＳＱＵＩＤ（Superconducting Quantum Interference Device；超伝導量子干渉素子）を使用して磁気マーカのブラウン緩和に基づく磁気信号を検出する構成について開示する。 Patent Document 1-5 discloses a configuration in which a SQUID (Superconducting Quantum Interference Device) is used as a magnetic sensor to detect a magnetic signal based on brown relaxation of a magnetic marker.
また、特許文献６は、磁気抵抗効果素子（ＭＲセンサ）を用いて、磁気マーカのブラウン緩和特性を交流磁化率の差として測定する磁界計測装置について開示する。すなわち、より体積が大きい結合マーカは、より体積が小さい未結合マーカよりも高周波の交流磁界に対する追従性が低く、交流磁化率は、周波数とブラウン緩和時間に依存する。このことから、交流磁化率を磁気抵抗効果素子（ＭＲセンサ）を用いて測定することによって、結合マーカの量を測定することができる。 Further, Patent Document 6 discloses a magnetic field measuring device that measures a brown relaxation characteristic of a magnetic marker as a difference in AC magnetic susceptibility by using a magnetoresistive sensor (MR sensor). That is, a larger volume coupling marker has lower followability to a high frequency alternating magnetic field than a smaller volume uncoupled marker, and the AC magnetic susceptibility depends on frequency and brown relaxation time. From this, the amount of coupling markers can be measured by measuring the AC magnetic susceptibility using a magnetoresistive element (MR sensor).
さらに、特許文献７は、磁界検出方向に指向性を有する薄膜磁気センサ（磁気抵抗センサ、磁気インピーダンスセンサ）を用いて、検査対象物内における磁性異物の有無を検出する磁性異物検査装置について開示する。 Further, Patent Document 7 discloses a magnetic foreign matter inspection device that detects the presence or absence of magnetic foreign matter in an inspection object by using a thin film magnetic sensor (magnetic resistance sensor, magnetic impedance sensor) having directionality in the magnetic field detection direction. ..
また、特許文献８は、本願発明者が提案した手法であり、磁気ビーズ（磁性ナノ粒子）の磁界をスイッチさせることによるブラウン緩和を利用した磁界測定装置について開示する。 Further, Patent Document 8 is a method proposed by the inventor of the present application, and discloses a magnetic field measuring device utilizing brown relaxation by switching the magnetic field of magnetic beads (magnetic nanoparticles).
本願発明者は、特許文献８により提案した磁界測定にかかる手法について、さらに鋭意研究・開発を進め、今般、より高感度な磁気的免疫検査が可能となる改良された磁界測定装置及び磁界測定方法を開発するに至った。 The inventor of the present application has further diligently researched and developed the method related to magnetic field measurement proposed in Patent Document 8, and has now improved a magnetic field measuring device and a magnetic field measuring method that enable more sensitive magnetic immunoassay. Came to develop.
本発明の目的は、比較的簡易な構成により高感度に磁気的免疫検査を実行することができる磁界測定装置及び磁界測定方法を提供することにある。 An object of the present invention is to provide a magnetic field measuring device and a magnetic field measuring method capable of performing a magnetic immunoassay with high sensitivity by a relatively simple configuration.
上記目的を達成するための本発明の磁界測定装置は、磁気的免疫検査により被測定物を検出するための磁界測定装置であって、磁性物質と該磁性物質と結合可能な被測定物とを含む試料を収容する容器を所定の移動周期で繰り返し同一移動させる移動機構と、容器の移動周期に同期して移動毎に磁界方向が反転して切り替わる磁界を、移動している容器に収容される試料に印加する磁界発生部と、磁界発生部からの磁界の影響を実質的に受けない程度に離間した位置に配置され、移動している容器に収容される試料から放出される磁界に対応する信号を検出する磁界センサとを備え、磁界発生部は発振器と該発振器に接続するコイルと該コイルを貫くヨークとを有して構成され、ヨークは錐体に形成されることを特徴とする。上記の移動形態は直線移動あるいは回転移動でもよい。 The magnetic field measuring device of the present invention for achieving the above object is a magnetic field measuring device for detecting an object to be measured by a magnetic immunological test, and a magnetic substance and an object to be measured that can be bonded to the magnetic substance are separated from each other. The moving container accommodates a moving mechanism that repeatedly moves the container containing the sample to be contained in the same movement at a predetermined movement cycle, and a magnetic field that reverses and switches the magnetic field direction each time it moves in synchronization with the movement cycle of the container. The magnetic field generator applied to the sample is located at a position separated from the magnetic field generator so as not to be affected by the magnetic field from the magnetic field generator, and corresponds to the magnetic field emitted from the sample housed in the moving container. A magnetic field sensor for detecting a signal is provided, and a magnetic field generating unit is configured to include an oscillator, a coil connected to the oscillator, and a yoke penetrating the coil, and the yoke is formed in a cone. The above movement mode may be linear movement or rotational movement.
本発明の磁界測定装置は、上記において、さらに、磁界発生部は、2n+1（ｎは0以上の整数）周期目の移動に対して、磁界強度B2n+1＝B2n+dB（B0＝0mT、dB＝所定の磁界強度増加分）の正又は負の磁界を印加し、2n+2周期目の移動に対して、磁界強度B2n+2＝B2n+1＝B2n+dBであって2n+1周期目の移動に印加した磁界と反対方向の磁界を印加することを特徴とする。また、移動機構は、一周期ごとに容器を所定時間停止させ、磁界発生部は、容器の停止中に、容器に収容される試料に磁界を所定時間印加することを特徴とする。 In the magnetic field measuring device of the present invention, in the above, the magnetic field generating unit further has a magnetic field strength B 2n + 1 = B 2n + dB (B) with respect to the movement of the 2n + 1 (n is an integer of 0 or more) period. Apply a positive or negative magnetic field ( 0 = 0 mT, dB = increase in magnetic field strength), and for the movement in the 2n + 2nd cycle, the magnetic field strength B 2n + 2 = B 2n + 1 = B 2n + dB It is characterized in that a magnetic field in the direction opposite to the magnetic field applied to the movement in the 2n + 1 cycle is applied. Further, the moving mechanism is characterized in that the container is stopped for a predetermined time every cycle, and the magnetic field generating unit applies a magnetic field to the sample contained in the container for a predetermined time while the container is stopped.
本発明の磁界測定装置は、上記において、さらに、同一の磁界強度を印加する隣接する２回の周回で検出される信号の積分値に基づいて被測定物の量を判定する演算処理部とを備えることを特徴とする。 In the above, the magnetic field measuring device of the present invention further includes an arithmetic processing unit that determines the amount of the object to be measured based on the integrated value of the signals detected in two adjacent orbits to which the same magnetic field strength is applied. It is characterized by being prepared.
本発明の磁界測定方法は、磁気的免疫検査により被測定物を検出するための磁界測定方法であって、磁性物質と該磁性物質と結合可能な被測定物とを含む試料を直流磁界により着磁させる着磁工程と、試料を収容する容器を複数回移動させる移動工程と、容器の移動周期に同期して、2n+1（ｎは0以上の整数）周期目の移動に対して、磁界強度B2n+1＝B2n+dB（B0＝0mT、dB＝所定の磁界強度増加分）の正又は負の磁界を容器に収容される試料に印加し、2n+2周期目の移動に対して、磁界強度B2n+2＝B2n+1＝B2n+dBであって2n+1周期目の移動に印加した磁界と反対方向の磁界を容器に収容される試料印加する磁界印加工程と、移動している容器に収容される試料から放出される磁界に対応する信号を検出する検出工程とを備えることを特徴とする。 The magnetic field measurement method of the present invention is a magnetic field measurement method for detecting an object to be measured by a magnetic immunological test, and a sample containing a magnetic substance and an object to be measured that can be bonded to the magnetic substance is applied by a DC magnetic field. A magnetic field for the movement of the 2n + 1 (n is an integer of 0 or more) cycle in synchronization with the magnetizing step of magnetizing, the moving step of moving the container containing the sample multiple times, and the moving cycle of the container. A positive or negative magnetic field of intensity B 2n + 1 = B 2n + dB (B 0 = 0 mT, dB = predetermined increase in magnetic field strength) is applied to the sample contained in the container to move in the 2n + 2nd cycle. On the other hand, the magnetic field strength is B 2n + 2 = B 2n + 1 = B 2n + dB, and the magnetic field applied in the direction opposite to the magnetic field applied to the movement in the 2n + 1 cycle is applied to the sample housed in the container. It is characterized by including a detection step of detecting a signal corresponding to a magnetic field emitted from a sample housed in a moving container.
本発明の磁界測定方法は、上記において、さらに、移動工程において、一周期ごとに容器を所定時間停止させ、磁界印加工程において、容器の停止中に、容器に収容される試料に磁界を所定時間印加することを特徴とする。また、同一の磁界強度を印加する隣接する２回の周回で検出される信号の積分値に基づいて被測定物の量を判定する判定工程とを備えることを特徴とする。 In the magnetic field measurement method of the present invention, the container is stopped for a predetermined time at each cycle in the moving step, and the magnetic field is applied to the sample contained in the container for a predetermined time while the container is stopped in the magnetic field application step. It is characterized by applying. Further, it is characterized by including a determination step of determining the amount of the object to be measured based on the integrated value of the signals detected in two adjacent orbits to which the same magnetic field strength is applied.
本発明の磁界測定装置及び磁界測定方法によれば、ブラウン緩和特性を利用して、より高感度な磁気的免疫検査を実行することができる。高感度な磁界測定装置を比較的簡易、小型且つ低コストで構成可能となる。 According to the magnetic field measuring device and the magnetic field measuring method of the present invention, it is possible to perform a more sensitive magnetic immunoassay by utilizing the brown relaxation characteristic. A highly sensitive magnetic field measuring device can be configured relatively simply, compactly, and at low cost.
以下、図面を参照して本発明の実施の形態について説明する。しかしながら、かかる実施の形態例が、本発明の技術的範囲を限定するものではない。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, such embodiments do not limit the technical scope of the invention.
図１は、本発明の実施の形態における磁界測定装置の概略構成例を示す図である。図１（ａ）は、磁界測定装置の全体構成を示し、図１（ｂ）は、図１（ａ）の点線囲み部分Pにおいて、後述するように、永久磁石３８が取り外され、励磁コイル３４に囲まれるヨーク３６の先端部が容器１２の底部に近接して配置される状態を示す。 FIG. 1 is a diagram showing a schematic configuration example of a magnetic field measuring device according to an embodiment of the present invention. FIG. 1 (a) shows the overall configuration of the magnetic field measuring device, and FIG. 1 (b) shows the permanent magnet 38 removed and the exciting coil 34 in the dotted line-enclosed portion P of FIG. 1 (a), as will be described later. Indicates a state in which the tip end portion of the yoke 36 surrounded by is arranged close to the bottom portion of the container 12.
磁界測定装置は、試料１０を収容する容器１２を回転軸を中心に周回させる回転機構２０と、容器１２に収容される試料１０に磁界方向を切替可能に磁界を印加する磁界発生部３０と、磁界発生部３０からの磁界の影響を実質的に受けない程度に離間した位置に配置され且つ回転している容器１２に収容される試料１０から放出される磁界を検出するための磁界センサ４０とを備えて構成される。 The magnetic field measuring device includes a rotating mechanism 20 that orbits the container 12 containing the sample 10 around a rotation axis, and a magnetic field generating unit 30 that applies a magnetic field to the sample 10 housed in the container 12 so that the magnetic field direction can be switched. A magnetic field sensor 40 for detecting a magnetic field emitted from a sample 10 housed in a rotating container 12 arranged at a position separated so as to be substantially unaffected by the magnetic field from the magnetic field generating unit 30. Is configured with.
回転機構２０は、台２２に取り付けられたモータ内蔵の回転軸２４と、回転軸２４から半径方向に延びて取り付けられるアーム部２６とを有し、アーム部２６の先端部に容器１２が保持される。モータにより回転軸を回転させることで、アーム部２６に保持される容器１２は、回転軸２４を中心に周回する。回転機構２０は、回転軸とアームの構成に限られず、回転軸を中心に周回する円盤プレートを有する構成であってもよい。また、試料（サンプル）１０を収容する容器１２を移動させる機構は、回転機構に限らず、例えば、往復直線運動など別の移動形態を採用してもよい。容器１２には、磁性物質（磁気ビーズとも称する）とその磁性物質と結合可能な被測定物の混合液である試料（サンプル）１０が収容される。 The rotating mechanism 20 has a rotating shaft 24 with a built-in motor attached to the base 22, and an arm portion 26 attached extending radially from the rotating shaft 24, and the container 12 is held at the tip of the arm portion 26. To. By rotating the rotating shaft by the motor, the container 12 held by the arm portion 26 rotates around the rotating shaft 24. The rotation mechanism 20 is not limited to the configuration of the rotation shaft and the arm, and may have a configuration having a disk plate that revolves around the rotation shaft. Further, the mechanism for moving the container 12 containing the sample (sample) 10 is not limited to the rotation mechanism, and another movement form such as a reciprocating linear motion may be adopted. The container 12 contains a sample (sample) 10 which is a mixed solution of a magnetic substance (also referred to as magnetic beads) and an object to be measured that can be bonded to the magnetic substance.
磁界発生部３０は、発振器３２と、その制御により磁界を発生する励磁コイル３４と、その中心軸と同心に配置されるヨーク３６とを有して構成され、発振器３２により、励磁コイル３４の発生する磁界方向は切替可能であり、容器１２は励磁コイル３４の直上を周回し、容器１２の周回ごとにその磁界方向がスイッチングされる。すなわち、容器１２の回転回数において、容器１２に収容される試料１０に印加される磁界方向は、奇数回と偶数回で逆方向となる。加えて試料１０を磁化するために永久磁石３８を置く。 The magnetic field generation unit 30 includes an oscillator 32, an exciting coil 34 that generates a magnetic field under its control, and a yoke 36 that is arranged concentrically with its central axis. The oscillator 32 generates the exciting coil 34. The direction of the magnetic field to be applied is switchable, and the container 12 orbits directly above the exciting coil 34, and the magnetic field direction is switched every time the container 12 orbits. That is, in the number of rotations of the container 12, the direction of the magnetic field applied to the sample 10 housed in the container 12 is an odd number and an even number, which are opposite directions. In addition, a permanent magnet 38 is placed to magnetize the sample 10.
ヨーク３６は、透磁率の高い例えばNiFeを材料とする磁性体であり、その一方の先端が細く鋭い尖鋭形状に形成され、例えば、錐体形状に形成される。錐体形状のヨーク３６の頂部は尖鋭形状の先端部分であり、ヨーク３６は、その尖鋭頂部が容器１２の底面に僅かな隙間をあけて位置するように励磁コイル３４の空洞部分に配置され、好ましくは、ヨーク３６の頂部が励磁コイル３４から突出して、容器１２の底面に近接して面する。 The yoke 36 is a magnetic material made of, for example, NiFe, which has a high magnetic permeability, and one of the yokes 36 is formed into a thin and sharp pointed shape, for example, a cone shape. The top of the cone-shaped yoke 36 is a pointed tip, and the yoke 36 is placed in the cavity of the exciting coil 34 so that the pointed top is located on the bottom surface of the container 12 with a slight gap. Preferably, the top of the yoke 36 projects from the exciting coil 34 and faces the bottom surface of the container 12.
ヨーク３６の尖った先端部分を容器１２の底面に向けることで、励磁コイル３４の発生する磁束をより狭い範囲に集束し、容器１２内の試料１０をより小さい塊として凝集させ、センサ感度を向上させることができる。 By directing the pointed tip portion of the yoke 36 toward the bottom surface of the container 12, the magnetic flux generated by the exciting coil 34 is focused in a narrower range, and the sample 10 in the container 12 is aggregated as a smaller mass to improve the sensor sensitivity. Can be made to.
磁界センサ４０は、磁気インピーダンス効果を利用して磁界を検出する磁気インピーダンスセンサ（ＭＩセンサ）である。磁気インピーダンス効果は、アモルファス合金ワイヤなどの高透磁率合金磁性体に高周波電流を通電すると、周回方向の透磁率が外部磁界の印加により大幅に変化することに起因して表皮深さが変化することにより、インピーダンスが変化する現象であり、磁気センサの小型化、高感度化、低消費電力化が可能なセンサである。磁界センサ４０は、装置の小型化や高感度化の面からＭＩセンサを採用することが好ましいが、それに限らず、例えば磁気抵抗センサ（ＭＲセンサ）などの磁界を検出する機能を有する別のセンサであってもよい。 The magnetic field sensor 40 is a magnetic impedance sensor (MI sensor) that detects a magnetic field by utilizing the magnetic impedance effect. The magnetic impedance effect is that when a high-frequency current is applied to a magnetic material with a high magnetic permeability such as an amorphous alloy wire, the magnetic permeability in the circumferential direction changes significantly due to the application of an external magnetic field, and the skin depth changes. This is a phenomenon in which the impedance changes due to this, and it is a sensor that can reduce the size, sensitivity, and power consumption of the magnetic sensor. As the magnetic field sensor 40, it is preferable to adopt an MI sensor from the viewpoint of miniaturization and high sensitivity of the device, but the present invention is not limited to this, and another sensor having a function of detecting a magnetic field such as a magnetic resistance sensor (MR sensor) is used. It may be.
信号処理部５０は、磁界センサ４０からの出力信号（センサ電圧値）は演算処理する手段であり、アナログ信号の出力信号をデジタル信号に変換し、所定の演算処理装置でデジタル信号を演算処理し、後述の演算処理及び判定処理を実行する。信号処理部５０は、汎用のコンピュータ装置や特定のデジタル演算回路により実現される。 The signal processing unit 50 is a means for arithmetically processing the output signal (sensor voltage value) from the magnetic field sensor 40, converts the output signal of the analog signal into a digital signal, and arithmetically processes the digital signal with a predetermined arithmetic processing device. , The arithmetic processing and the determination processing described later are executed. The signal processing unit 50 is realized by a general-purpose computer device or a specific digital arithmetic circuit.
図２は、磁界センサ４０の概略的な配置例を示す図である。磁界センサ４０は、容器１２の回転移動方向に直交する方向に並列に配置される２つのセンサ素子４０ａ、４０ｂを有し、差動センサとして動作する。後述するように、差動センサの構成として、２つのセンサ素子の一方素子の直上に容器１２を通過させ、他方の素子の上には容器１２を通過させないようにすることで、バックグラウンドノイズを相殺し、高感度化を図ることができる。また、センサ素子４０ａ、４０ｂにバイアス磁界を印加するバイアス用磁石４２がセンサ素子４０ａ、４０ｂに近接して配置され、容器１２の回転移動方向を向いたバイアス磁界を印加する。なお、ブラウン緩和を正確に観測するために、このバイアス磁界からの漏れ磁界はできるだけ抑えることが好ましく、バイアス磁界による試料１０に含まれる磁気ビーズの磁化の影響を無視できる程度に小さくする。回転している容器とバイアス用磁石４２との間には、磁気シールド４４が配置される。磁気シールド４４は、軟磁性体で形成され、回転している容器１２がバイアス用磁石４２に接近する位置に配置され、バイアス用磁石４２からの磁界を遮断する。 FIG. 2 is a diagram showing a schematic arrangement example of the magnetic field sensor 40. The magnetic field sensor 40 has two sensor elements 40a and 40b arranged in parallel in a direction orthogonal to the rotational movement direction of the container 12, and operates as a differential sensor. As will be described later, as a configuration of the differential sensor, background noise is generated by passing the container 12 directly above one element of the two sensor elements and preventing the container 12 from passing over the other element. It can be offset and the sensitivity can be increased. Further, a bias magnet 42 that applies a bias magnetic field to the sensor elements 40a and 40b is arranged close to the sensor elements 40a and 40b, and a bias magnetic field that faces the rotational movement direction of the container 12 is applied. In order to accurately observe the brown relaxation, it is preferable to suppress the leakage magnetic field from the bias magnetic field as much as possible, and the influence of the magnetization of the magnetic beads contained in the sample 10 due to the bias magnetic field is made small enough to be ignored. A magnetic shield 44 is arranged between the rotating container and the bias magnet 42. The magnetic shield 44 is made of a soft magnetic material, and the rotating container 12 is arranged at a position close to the bias magnet 42 to block the magnetic field from the bias magnet 42.
図３は、本発明の実施の形態における磁界測定装置による磁界測定方法の処理手順を示す図である。また、図４は、本発明の実施の形態に磁界測定装置の概略模式図であり、図１と同一の構成を示す。 FIG. 3 is a diagram showing a processing procedure of the magnetic field measuring method by the magnetic field measuring device according to the embodiment of the present invention. Further, FIG. 4 is a schematic schematic view of the magnetic field measuring device according to the embodiment of the present invention, and shows the same configuration as that of FIG.
容器１２に試料１０を入れて撹拌（超音波洗浄約１５秒＋振動攪拌約３０秒）し、回転機構２０のアーム部２６の所定位置にセットする（Ｓ１００）。試料１０は、磁性物質である磁気ビーズ（磁性ナノ粒子）とそれに結合可能な被測定物質との混合液である。被測定物質は、検出対象の細菌や微生物であり、被測定物質の数（想定される最大数）よりも多い磁気ビーズが投入されるよう調整される。好ましくは、被測定物質と結合しない未結合の残留磁気ビーズを少なくするように調整することで高感度化が図られる。後述では、被測定物質として大腸菌を用いた場合を例示する。実験に用いる場合のモデル細菌として、ポリマービーズを利用することもできる。 The sample 10 is placed in the container 12 and stirred (ultrasonic cleaning for about 15 seconds + vibration stirring for about 30 seconds), and set at a predetermined position on the arm portion 26 of the rotating mechanism 20 (S100). Sample 10 is a mixed solution of magnetic beads (magnetic nanoparticles) which are magnetic substances and a substance to be measured which can be bonded to the magnetic beads (magnetic nanoparticles). The substance to be measured is a bacterium or a microorganism to be detected, and the number of magnetic beads to be measured is adjusted to be larger than the number of substances to be measured (the maximum number assumed). Preferably, the sensitivity is increased by adjusting so as to reduce the number of unbound residual magnetic beads that do not bind to the substance to be measured. In the following, the case where Escherichia coli is used as the substance to be measured will be illustrated. Polymer beads can also be used as a model bacterium when used in experiments.
容器１２の初期位置は、磁界発生部３０のコイル３４の直上位置である。撹拌は、測定直前に行うことが好ましい。また、容器１２の底部厚さは0.3mm±0.05mm程度が好ましい。磁界センサ４０との距離を近づけられ高感度検出を可能とするが、容器１２の強度維持のために一定の厚さが必要である。 The initial position of the container 12 is a position directly above the coil 34 of the magnetic field generating unit 30. Stirring is preferably performed immediately before the measurement. The bottom thickness of the container 12 is preferably about 0.3 mm ± 0.05 mm. The distance from the magnetic field sensor 40 can be shortened to enable high-sensitivity detection, but a certain thickness is required to maintain the strength of the container 12.
試料１０の回転前に、永久磁石（例えばNdFeB磁石)３８を容器１２に近接配置し、例えば約10分間着磁し、試料１０を容器１２の底部に集める（Ｓ１０２）。永久磁石３８は、励磁コイル３４と容器１２の底との間隙に例えば手動で挿入される。永久磁石３８による着磁により、容器１２内の試料１０をセンサ素子４０ａ、４０ｂの一方素子寸法と同程度の面積に凝集させて集め、回転の際に一方素子の真上を通過させるようにする。 Prior to rotation of the sample 10, a permanent magnet (eg, NdFeB magnet) 38 is placed close to the container 12, magnetized for, for example, about 10 minutes, and the sample 10 is collected at the bottom of the container 12 (S102). The permanent magnet 38 is, for example, manually inserted into the gap between the exciting coil 34 and the bottom of the container 12. By magnetizing with the permanent magnet 38, the sample 10 in the container 12 is aggregated and collected in an area similar to the size of one of the sensor elements 40a and 40b, and is passed directly above the one element during rotation. ..
永久磁石３８による着磁後、永久磁石３８は容器１２の近傍から取り除かれ、続いて、さらに、発振器３２により励磁コイル３４に通電し、磁界を発生させ、試料１０に磁界を印加する（Ｓ１０４）。励磁コイル３４による磁界方向は、永久磁石３８による磁界方向と同一とする。永久磁石３８の配置及び除去は手動又は機械的な構成のいずれにより行われてもよい。一例として、励磁コイル３４による印加磁界の磁界強度（又は磁束密度）は約78mT、印加時間は約5分程度である。印加される磁界方向は、永久磁石による着磁の磁界方向と同一であり、例えばプラス方向である。発生された磁界は、ヨーク３６の尖鋭先端形状により一点に集束され、容器１２内の試料１０は、図１（ｂ）に模式的に示されるように小さな塊として凝集される。 After magnetization by the permanent magnet 38, the permanent magnet 38 is removed from the vicinity of the container 12, and then the exciting coil 34 is energized by the oscillator 32 to generate a magnetic field, and the magnetic field is applied to the sample 10 (S104). .. The magnetic field direction of the exciting coil 34 is the same as the magnetic field direction of the permanent magnet 38. The placement and removal of the permanent magnets 38 may be done either manually or in a mechanical configuration. As an example, the magnetic field strength (or magnetic flux density) of the magnetic field applied by the exciting coil 34 is about 78 mT, and the application time is about 5 minutes. The direction of the applied magnetic field is the same as the direction of the magnetic field magnetized by the permanent magnet, for example, the positive direction. The generated magnetic field is focused at one point by the sharp tip shape of the yoke 36, and the sample 10 in the container 12 is aggregated as a small mass as schematically shown in FIG. 1 (b).
このように、回転開始前においては、永久磁石３８による着磁と励磁コイル３４による着磁を行うことで、被測定物質と結合している磁気ビーズを含む試料１０を容器１２の底部へ集め、さらに、尖鋭形状のヨーク３６により磁界を集束して、できるだけ小さな塊として凝集させ、センサ素子面に対して十分に小さな塊に凝集された試料１０が磁界センサ近傍を通過するようにすることで、磁界センサ４０の検出感度が高まり、ＳＮ比が向上する。 In this way, before the start of rotation, by magnetizing with the permanent magnet 38 and magnetizing with the exciting coil 34, the sample 10 containing the magnetic beads bonded to the substance to be measured is collected at the bottom of the container 12. Further, the magnetic field is focused by the sharp yoke 36 and aggregated as a small mass as much as possible so that the sample 10 aggregated into a sufficiently small mass with respect to the sensor element surface passes near the magnetic field sensor. The detection sensitivity of the magnetic field sensor 40 is increased, and the SN ratio is improved.
回転前における励磁コイル３４による着磁後、励磁コイル３４の通電を一旦停止し、所定時間（約10秒）ほどおき、磁界印加を止める（B＝0）。その後、励磁コイル３４直上の容器１２（その内部の試料１０）に励磁コイル３４による磁界印加を磁界強度０から段階的に増大させながら回転させていく。 After magnetization by the exciting coil 34 before rotation, the energization of the exciting coil 34 is temporarily stopped, and the magnetic field application is stopped after a predetermined time (about 10 seconds) (B = 0). After that, the container 12 (sample 10 inside the container 12) directly above the exciting coil 34 is rotated while the magnetic field applied by the exciting coil 34 is gradually increased from 0.
具体的には、試料１０を励磁コイル３４上に停止させた状態で、励磁コイル３４直上の試料１０に対して励磁コイル３４により印加する磁界を増大させ（Ｓ１０６）、磁界方向をプラス（正）方向として、その増大させた磁界を印加する（Ｓ１０８）。印加する磁界強度は、前の周回より増大させる。奇数回の回転で印加する磁界強度B2n+1（2n+1：回転回数、nの初期値は0）は、前の周回2nに印加した磁界強度B2nに所定増加分dBを増加させた値とする。所定増加分dBは例えば6mTであり、一周目の前の磁界強度B0＝0とする。したがって、一回転目の回転前に印加する磁界強度Bは、初期値B0＝0に所定増加分dBを加算した値であるので、一回転目の回転前に印加する磁界強度は6mTとなる。１回転目で印加する磁界方向はプラス方向（正方向）、マイナス方向（負方向）のどちらでもよいが、周回毎に磁界方向を反転させて交互に磁界方向を切り替える。 Specifically, with the sample 10 stopped on the exciting coil 34, the magnetic field applied by the exciting coil 34 to the sample 10 directly above the exciting coil 34 is increased (S106), and the magnetic field direction is positive (positive). As the direction, the increased magnetic field is applied (S108). The applied magnetic field strength is increased from the previous circuit. The magnetic field strength B 2n + 1 (2n + 1: number of rotations, initial value of n is 0) applied in odd-numbered rotations increased the magnetic field strength B 2n applied in the previous circuit 2n by a predetermined increase dB. Use as a value. The predetermined increase dB is, for example, 6 mT, and the magnetic field strength B 0 = 0 before the first lap is assumed. Therefore, the magnetic field strength B applied before the first rotation is a value obtained by adding a predetermined increase dB to the initial value B 0 = 0, so that the magnetic field strength applied before the first rotation is 6 mT. .. The magnetic field direction applied in the first rotation may be either a positive direction (positive direction) or a negative direction (negative direction), but the magnetic field direction is reversed for each lap and the magnetic field directions are alternately switched.
磁界強度の所定増加分dB分増大させた磁界をプラス方向に約30秒間印加した後、磁界の印加を停止し、試料１０を一回転させ、その周回中に磁界センサ４０の一方素子の直上を通過させ、磁気ビーズの漏れ磁界を測定する（Ｓ１１０）。例えば３／４周期後に磁界センサ４０の一方素子の直上を通過し、周回毎に磁気ビーズの漏れ磁界を測定する。 After applying a magnetic field increased by a predetermined increase in magnetic field strength by dB in the positive direction for about 30 seconds, the application of the magnetic field is stopped, the sample 10 is rotated once, and during its orbit, directly above one element of the magnetic field sensor 40. It is passed through and the leakage magnetic field of the magnetic beads is measured (S110). For example, after a 3/4 cycle, it passes directly above one element of the magnetic field sensor 40, and the leakage magnetic field of the magnetic beads is measured every round.
磁界センサ４０は差動センサ構成であるので、試料１０が直上を通過する一方素子と試料１０が直上を通過しない他方素子との出力の差分値を得ることで、バックグラウンドノイズが相殺された高精度な出力信号（センサ電圧値）が得られる。回転速度は例えば200 degree/s程度である。回転速度は、回転速度が速いと液相が不安定になるため、遠心力による加速度が重力加速度に対して十分小さくなる程度とする。磁界センサ４０の一方素子と試料１０の入った容器１２の底部との間隙は200μm〜300μm程度とすることが好ましい。間隙を狭くするほど少量の磁気ビーズの検出が可能となり、より少量の被測定物質（細菌）を検出することができるようになる。 Since the magnetic field sensor 40 has a differential sensor configuration, the background noise is canceled out by obtaining the difference value between the output of one element in which the sample 10 passes directly above and the other element in which the sample 10 does not pass directly above. An accurate output signal (sensor voltage value) can be obtained. The rotation speed is, for example, about 200 degree / s. The rotation speed should be such that the acceleration due to centrifugal force is sufficiently smaller than the gravitational acceleration because the liquid phase becomes unstable when the rotation speed is high. The gap between one element of the magnetic field sensor 40 and the bottom of the container 12 containing the sample 10 is preferably about 200 μm to 300 μm. The narrower the gap, the smaller the amount of magnetic beads can be detected, and the smaller the amount of the substance to be measured (bacteria) can be detected.
図５は、磁界センサ４０の一方素子上を通過する容器１２内の試料１０の位置関係を示す図である。例えば容器１２の断面径が磁界センサ４０の一方素子の幅よりも大きい場合、容器１２内において、回転前の着磁により試料１０を容器１２の底部に集める際に、容器１２の底部の左右一方側に偏らせて凝集させ、その凝集した試料１０の小さな塊は磁界センサ４０の一方素子（図５では、センサ素子４０ａ）の幅よりも十分に小さい大きさであって、一方素子の直上を通過させ、他方素子（図５では、センサ素子４０ｂ）の直上を通過させないようにする。 FIG. 5 is a diagram showing the positional relationship of the sample 10 in the container 12 passing over one element of the magnetic field sensor 40. For example, when the cross-sectional diameter of the container 12 is larger than the width of one element of the magnetic field sensor 40, when the sample 10 is collected at the bottom of the container 12 by magnetism before rotation in the container 12, one of the left and right sides of the bottom of the container 12 is collected. The agglomerated sample 10 is biased to the side and aggregated, and the small mass of the aggregated sample 10 is sufficiently smaller than the width of one element of the magnetic field sensor 40 (sensor element 40a in FIG. 5), and is directly above the one element. Allow it to pass, but not directly above the other element (sensor element 40b in FIG. 5).
前の周回でプラスの磁界方向に印加して一回転させた後、試料１０を励磁コイル３４上に停止させ、次に、前の周回と同じ磁界強度でマイナス（負）の磁界方向にて励磁コイル３４により磁界を印加する（Ｓ１１２）。すなわち、偶数回目の2n+2周期目の回転に対して、磁界強度B2n+2＝B2n+1＝B2n+dBであって、2n+1周期目の移動に印加した磁界と反対方向の磁界を印加する。よって、二回転目の回転前に印加するマイナス方向磁界は、前の周回と同じ6mTであり、印加時間は、プラス方向の印加と同様に約30秒程度である。マイナス（負）の磁界方向に30秒間磁界を印加した後、磁界の印加を停止し、試料１０を一回転させ、前の周回と同様に、その周回中に磁界センサ４０の一方素子の直上を通過させ、磁気ビーズの漏れ磁界を測定する（Ｓ１１４）。 After applying in the positive magnetic field direction in the previous circuit and making one rotation, the sample 10 is stopped on the exciting coil 34, and then excited in the negative (negative) magnetic field direction with the same magnetic field strength as the previous circuit. A magnetic field is applied by the coil 34 (S112). That is, the magnetic field strength is B 2n + 2 = B 2n + 1 = B 2n + dB for the even-numbered rotation in the 2n + 2nd cycle, which is the opposite direction to the magnetic field applied to the movement in the 2n + 1th cycle. Apply the magnetic field of. Therefore, the negative magnetic field applied before the second rotation is 6 mT, which is the same as the previous rotation, and the application time is about 30 seconds, which is the same as the positive application. After applying the magnetic field in the negative (negative) magnetic field direction for 30 seconds, the application of the magnetic field is stopped, the sample 10 is rotated once, and in the same as the previous orbit, directly above one element of the magnetic field sensor 40 during the orbit. It is passed through and the leakage magnetic field of the magnetic beads is measured (S114).
上記ステップＳ１０６乃至Ｓ１１４の処理を、回転回数が所定数（例えば50〜60回転）に達するまで繰り返される（Ｓ１１６）。すなわち、一回転目以降について、奇数回転目において、磁界強度を所定増加分だけ増大させ、プラス方向の磁界を印加してから、試料１０を回転させ、偶数回転目においては、その前のプラス方向の回転と同じ磁界強度で、磁界方向を反転させたマイナス方向の磁界を印加してから、試料１０を回転させ、周回毎に磁界センサ４０により測定を行う。 The process of steps S106 to S114 is repeated until the number of rotations reaches a predetermined number (for example, 50 to 60 rotations) (S116). That is, for the first and subsequent rotations, the magnetic field strength is increased by a predetermined increase in the odd rotation, a magnetic field in the positive direction is applied, and then the sample 10 is rotated, and in the even rotation, the previous positive direction is applied. After applying a magnetic field in the negative direction in which the magnetic field direction is reversed with the same magnetic field strength as the rotation of the sample 10, the sample 10 is rotated and the measurement is performed by the magnetic field sensor 40 for each rotation.
本発明では、周回毎に極性を反転させた磁界を試料１０に印加して回転させる。これにより、試料１０に含まれる磁気ビーズは磁界方向に回転しようとする。このとき、磁気ビーズのみ（被測定物質と結合していない未結合の磁気ビーズ）であれば、磁気ビーズの体積（または回転半径）は、被測定物質と比較して十分に小さいので緩和時間が短く、励磁コイル３４による磁界により比較的容易に磁化回転するが、被測定物質と結合している磁気ビーズは、緩和時間が比較的長く、磁化回転しにくい状態となる。 In the present invention, a magnetic field whose polarity is reversed for each round is applied to the sample 10 to rotate the sample 10. As a result, the magnetic beads contained in the sample 10 tend to rotate in the direction of the magnetic field. At this time, if only the magnetic beads (unbonded magnetic beads that are not bonded to the substance to be measured), the volume (or radius of gyration) of the magnetic beads is sufficiently smaller than that of the substance to be measured, so that the relaxation time is reduced. It is short and magnetizes and rotates relatively easily due to the magnetic field generated by the exciting coil 34, but the magnetic beads bonded to the substance to be measured have a relatively long relaxation time and are in a state where it is difficult to magnetize and rotate.
所定の回転回数の回転及び各回転ごとの測定が実施されると、得られたセンサ電圧値を信号処理部５０により演算処理する（Ｓ１１８）。 When the rotation of a predetermined number of rotations and the measurement for each rotation are performed, the obtained sensor voltage value is arithmetically processed by the signal processing unit 50 (S118).
信号処理部５０は、同じ磁界強度の磁界を印加した隣接する２回の回転（プラス方向磁界を印加した奇数回の回転とマイナス方向磁界を印加した偶数回の回転）のセンサ電圧値を用いて、以下の式１で定義される磁化回転相当量を算出する。 The signal processing unit 50 uses the sensor voltage values of two adjacent rotations in which a magnetic field having the same magnetic field strength is applied (an odd number of rotations in which a positive magnetic field is applied and an even number of rotations in which a negative magnetic field is applied). , The amount equivalent to the magnetization rotation defined by the following equation 1 is calculated.
図６は、磁界センサ４０の出力電圧の測定データを示すグラフである（被測定物質：大腸菌）。横軸は磁界センサ４０の通過位置（回転角度）、縦軸は磁界センサ４０の出力電圧を示す。容器１２が磁界センサ４０を通過する位置に応じて出力電圧はプラス電圧値とマイナス電圧値に変化する値となる。 FIG. 6 is a graph showing measurement data of the output voltage of the magnetic field sensor 40 (measured substance: Escherichia coli). The horizontal axis represents the passing position (rotation angle) of the magnetic field sensor 40, and the vertical axis represents the output voltage of the magnetic field sensor 40. The output voltage becomes a value that changes into a positive voltage value and a negative voltage value according to the position where the container 12 passes through the magnetic field sensor 40.
グラフａは、プラス方向磁界を印加した奇数回の回転におけるセンサ電圧値の例であり、グラフｂは、マイナス方向磁界を印加した偶数回の回転におけるセンサ電圧値の例であり、グラフｃは、グラフａとグラフｂのセンサ電圧値の加算値、グラフｄは、グラフａとグラフｂのセンサ電圧値の差分値を示す。 Graph a is an example of the sensor voltage value in an odd number of rotations in which a positive magnetic field is applied, graph b is an example of a sensor voltage value in an even number of rotations in which a negative magnetic field is applied, and graph c is an example of a sensor voltage value. The sum of the sensor voltage values of the graph a and the graph b, and the graph d show the difference value of the sensor voltage values of the graph a and the graph b.
グラフａでは、センサ電圧値は、０付近からマイナス側に変化し、その後マイナス電圧からプラス電圧に極性が反転し、さらにマイナス電圧に戻り０付近に収束する波形となり、グラフｂでは、その逆の波形、すなわち、センサ電圧値は０付近からプラス側に変化し、その後プラス電圧からマイナス電圧に極性が反転し、さらにプラス電圧に戻り０付近に収束する波形となる。 In graph a, the sensor voltage value changes from around 0 to the minus side, then the polarity is reversed from minus voltage to plus voltage, then returns to minus voltage and converges to near 0, and in graph b, the opposite is true. The waveform, that is, the sensor voltage value changes from the vicinity of 0 to the positive side, then the polarity is reversed from the positive voltage to the negative voltage, and then returns to the positive voltage and converges to the vicinity of 0.
図示されるように、奇数回回転におけるセンサ電圧値の０付近からマイナス側への変化点をt1o、マイナス側からプラス側への変化点t2o、プラス側からマイナス側への変化点t3o、マイナス側から０付近への変化点をt4oとし、偶数回回転におけるセンサ電圧値の０付近からプラス側への変化点をt1e、プラス側からマイナス側への変化点t2e、マイナス側からプラス側への変化点t3e、プラス側から０付近への変化点をt4eとし、各期間（角度）のセンサ電圧値の波形積分値を求め、式１を算出する。式１における積分記号
は、奇数回(odd)回転における期間（角度）t1oとt2o間における波形積分値を表す。奇数回と偶数回の波形積分値の加算値が大きいほど、奇数回と偶数回の波形の差が大きいことになり、式１は、印加する磁界をプラス方向からマイナス方向に変化させた場合の試料１０の磁化反転しにくさを表す指標となる。式１の値が比較的小さければ、被測定物質の量（数）が比較的少ないため、周回毎に極性が反転する磁場に追随して試料１０の磁化方向も反転していること示し、式１の値が比較的大きければ、被測定物質の量（数）が比較的多いため、周回ごとの磁場のスイッチングに追従できず試料１０が極性反転しにくくなっていると考えられる。すなわち奇数回と偶数回の波形の相違は、被測定物質の量（数）と相関関係を有することを示唆している。
As shown in the figure, the change point of the sensor voltage value from near 0 to the minus side in odd-numbered rotation is t1o, the change point from the minus side to the plus side is t2o, the change point from the plus side to the minus side is t3o, and the minus side. The point of change from to near 0 is t4o, the point of change of the sensor voltage value from near 0 to the plus side in even-numbered rotation is t1e, the point of change from the plus side to the minus side t2e, and the change from the minus side to the plus side. Let t3e be the point, and t4e be the point of change from the plus side to near 0, obtain the waveform integrated value of the sensor voltage value for each period (angle), and calculate Equation 1. Integral symbol in Equation 1
Represents the waveform integral value between the period (angle) t1o and t2o in odd rotations (odd). The larger the addition value of the odd-numbered and even-numbered waveform integral values, the larger the difference between the odd-numbered and even-numbered waveforms. Equation 1 shows the case where the applied magnetic field is changed from the positive direction to the negative direction. It is an index showing the difficulty of reversing the magnetization of the sample 10. If the value of Equation 1 is relatively small, the amount (number) of the substance to be measured is relatively small, and therefore, it is shown that the magnetization direction of the sample 10 is also inverted following the magnetic field whose polarity is inverted every time. If the value of 1 is relatively large, the amount (number) of the substance to be measured is relatively large, and it is considered that the sample 10 cannot easily reverse the polarity because it cannot follow the switching of the magnetic field for each circuit. That is, the difference between the odd-numbered and even-numbered waveforms suggests that there is a correlation with the amount (number) of the substance to be measured.
磁化反転相当量を表す式は、上記式１に限らず、さまざまな定義式を採用しうる。例えば、以下の式２、式３、式４を用いてもよい。 The equation expressing the magnetization reversal equivalent amount is not limited to the above equation 1, and various definition equations can be adopted. For example, the following equations 2, 3 and 4 may be used.
図７は、式１の演算結果と回転回数との関係を示すグラフである。被測定物質は大腸菌(E. Coli)とし、試料は大腸菌と磁気ビーズ（磁性ナノ粒子）の混合液である。図７（ａ）は、被測定物質を含まない（磁気ビーズのみ）試料１０の測定に基づくグラフであり、図７（ｂ）は、大腸菌を約10４（CFU/ml）含む試料１０の測定に基づくグラフであり、図７（ｃ）は、大腸菌を約10５（CFU/ml）含む試料１０の測定に基づくグラフであり、図７（ｄ）は、大腸菌を約10６（CFU/ml）含む試料１０の測定に基づくグラフである。 FIG. 7 is a graph showing the relationship between the calculation result of Equation 1 and the number of rotations. The substance to be measured is E. coli, and the sample is a mixture of Escherichia coli and magnetic beads (magnetic nanoparticles). 7 (a) is a graph based on the measurement of not including the material to be measured (magnetic beads only) sample 10, FIG. 7 (b), about 10 4 (CFU / ml) of E. coli measurement of the sample 10 including a graph based on FIG. 7 (c) is a graph based on measurement of the sample 10 containing about 10 5 (CFU / ml) E. coli, FIG. 7 (d), about 10 6 (CFU / ml E. coli ) Is a graph based on the measurement of the sample 10 containing.
図７では、各回転及び各磁界強度に対する演算結果をプロットし、その演算結果を４係数ロジスティック関数で近似した近似曲線を示す。演算結果を近似する関数は、これに限らず、他の関数を用いることもできる。図７によれば、被測定物質である大腸菌の量（数）が多くなるほど、回転回数がより早い（印加する磁界強度がより小さい）段階で式１の演算結果値が低下していく傾向があることが判明した。 FIG. 7 plots the calculation results for each rotation and each magnetic field strength, and shows an approximate curve in which the calculation results are approximated by a 4-coefficient logistic function. The function that approximates the operation result is not limited to this, and other functions may be used. According to FIG. 7, as the amount (number) of Escherichia coli, which is the substance to be measured, increases, the calculation result value of Equation 1 tends to decrease at the stage where the number of rotations is faster (the applied magnetic field strength is smaller). It turned out to be.
大腸菌の量が少ないほど、磁気ビーズがより大きな磁性体として凝集することから、回転回数が増大して磁界強度を上げていっても、磁界の極性反転に追従しにくくなることから、演算結果値が低下するタイミングは遅くなり、大腸菌の量が多いほど、磁気ビーズは凝集しにくく、早い回転回数の段階（磁界強度が低い段階）で磁界の極性反転に追従しやすくなり、回転回数を増大させて磁界強度も上がっていっても極性反転が発生しづらい状況になっていると考えられる。大腸菌の量にかかわらず、回転を繰り返す毎に、極性反転に追従しない割合が増えていき、最終的には、磁界強度を上げていっても、試料の磁化方向はランダムとなり、式１の演算結果値は０近傍に収束していくが、プラス方向磁界の回転とマイナス方向磁界の回転の２回転毎に、磁界強度を上げていくことで、被測定物質の量に応じた演算結果値が低下するタイミングの違いをより大きくすることができる。 The smaller the amount of Escherichia coli, the more the magnetic beads aggregate as a larger magnetic material, and even if the number of rotations increases and the magnetic field strength increases, it becomes difficult to follow the polarity reversal of the magnetic field. The timing of the decrease is delayed, and the larger the amount of Escherichia coli, the more difficult it is for the magnetic beads to aggregate, and the easier it is to follow the polarity reversal of the magnetic field at the stage of high rotation times (the stage of low magnetic field strength), increasing the number of rotations. It is considered that the polarity reversal is unlikely to occur even if the magnetic field strength is increased. Regardless of the amount of Escherichia coli, the proportion that does not follow the polarity reversal increases with each rotation, and finally, even if the magnetic field strength is increased, the magnetization direction of the sample becomes random, and the calculation of Equation 1 The result value converges to near 0, but by increasing the magnetic field strength every two rotations of the positive magnetic field rotation and the negative magnetic field rotation, the calculation result value according to the amount of the substance to be measured can be obtained. The difference in the timing of reduction can be made larger.
この測定結果に基づいて、式１の演算結果値が低下していくタイミングから、大腸菌の量を推定することが可能となる。例えば、式１の演算結果値の最大値の1/2の値になる回転回数又はその時の磁界強度を指標として、大腸菌の量を判定する。 Based on this measurement result, it is possible to estimate the amount of Escherichia coli from the timing when the calculation result value of Equation 1 decreases. For example, the amount of Escherichia coli is determined using the number of rotations, which is half the maximum value of the calculation result value of Equation 1, or the magnetic field strength at that time as an index.
図８は、大腸菌の量と、式１の演算結果値の最大値の1/2の値になる磁界強度との関係を示すグラフである。大腸菌の量が多くなるほど、磁界強度の値は小さくなる傾向が明らかとなった。被測定物質に対してあらかじめ図８のグラフを求めておき、被測定物質数が未知の試料の評価の際には、センサ電圧値と印加した磁界強度から図８の曲線を用いて被測定物質の量を判定することができる（図６のＳ１２０）。信号処理部５０が、測定されたセンサ電圧値から、式１の演算を行い、図８のグラフデータと比較し、被測定物の数を判定する。 FIG. 8 is a graph showing the relationship between the amount of Escherichia coli and the magnetic field strength which is 1/2 of the maximum value of the calculation result value of Equation 1. It was clarified that the value of the magnetic field strength tends to decrease as the amount of E. coli increases. The graph of FIG. 8 is obtained in advance for the substance to be measured, and when evaluating a sample whose number of substances to be measured is unknown, the substance to be measured is used from the sensor voltage value and the applied magnetic field strength using the curve of FIG. Can be determined (S120 in FIG. 6). The signal processing unit 50 performs the calculation of Equation 1 from the measured sensor voltage value, compares it with the graph data of FIG. 8, and determines the number of objects to be measured.
図９は、被測定物質をう蝕菌（Ｓ．ｍｕｔａｎｓ）とした場合における式１の演算結果と回転回数との関係を示すグラフである。試料は、う蝕菌と磁気ビーズ（磁性ナノ粒子）の混合液である。図９は、被測定物質を含まない（磁気ビーズのみ）試料１０、う蝕菌を約10４（CFU/ml）含む試料１０、う蝕菌を約10５（CFU/ml）含む試料１０、う蝕菌を約10６（CFU/ml）含む試料１０の測定に基づくグラフを示す。 FIG. 9 is a graph showing the relationship between the calculation result of Equation 1 and the number of rotations when the substance to be measured is S. mutans. The sample is a mixed solution of caries bacteria and magnetic beads (magnetic nanoparticles). Figure 9 does not include a material to be measured (magnetic beads only) sample 10, the Ushokukin about 10 4 (CFU / ml) containing the sample 10, about 10 5 (CFU / ml) of Ushokukin containing sample 10, The graph based on the measurement of the sample 10 containing about 10 6 (CFU / ml) of carious bacteria is shown.
図９では、各回転及び各磁界強度に対する演算結果をプロットし、その演算結果を４係数ロジスティック関数で近似した近似曲線を示す。演算結果を近似する関数は、これに限らず、他の関数を用いることもできる。図９によれば、被測定物質であるう蝕菌の量（数）が多くなるほど、回転回数がより遅い（印加する磁界強度がより大きい）段階で式１の演算結果値が低下していく傾向があることが判明した。これは図７の大腸菌の結果と異なるが、その理由として、う蝕菌は抗原抗体反応以前の段階で菌同士が凝集体を形成していることが多く、磁気ビーズとの反応後はう蝕菌の大きな凝集体の周りに磁気ビーズが結合しており、う蝕菌が増えるに従って凝集体が大きくなり、磁気ビーズの極性を反転させるためには印加する磁界強度がより大きくする必要があるためと考えられる。上記は電子顕微鏡写真でも観察された（図１０）。図１０は、う蝕菌の凝集体と磁気ビーズの電子顕微鏡写真である。また、図１１は、う蝕菌の量と、式１の演算結果値の最大値の1/2の値になる磁界強度との関係を示すグラフである。う蝕菌の量が多くなるほど、磁界強度の値は大きくなる傾向が明らかとなった。ここから大腸菌と同様、う蝕菌の量を求めることができる。 FIG. 9 plots the calculation results for each rotation and each magnetic field strength, and shows an approximate curve in which the calculation results are approximated by a 4-coefficient logistic function. The function that approximates the operation result is not limited to this, and other functions may be used. According to FIG. 9, as the amount (number) of caries bacteria to be measured increases, the calculation result value of Equation 1 decreases at the stage where the number of rotations is slower (the applied magnetic field strength is larger). It turns out that there is a tendency. This is different from the result of Escherichia coli in FIG. 7, because the caries bacteria often form agglomerates before the antigen-antibody reaction, and the caries after the reaction with the magnetic beads. Magnetic beads are bound around large aggregates of bacteria, and as the number of caries bacteria increases, the aggregates grow larger, and in order to reverse the polarity of the magnetic beads, the applied magnetic field strength needs to be greater. it is conceivable that. The above was also observed in electron micrographs (Fig. 10). FIG. 10 is an electron micrograph of caries aggregates and magnetic beads. Further, FIG. 11 is a graph showing the relationship between the amount of caries bacteria and the magnetic field strength which is 1/2 of the maximum value of the calculation result value of Equation 1. It was clarified that the value of the magnetic field strength tends to increase as the amount of caries bacteria increases. From here, as with Escherichia coli, the amount of caries bacteria can be determined.
本発明の実施の形態では、磁気ビーズ（磁性ナノ粒子）及びこれと結合可能な被測定物を含む液体を回転させ、その周回ごとに極性が反転する磁界を印加し、その磁性の変化に対応する出力信号を周回毎に検出し、その隣接周回の出力信号の差異を利用して被測定物の量を測定可能とし、より高感度な磁気的免疫検査を行うことができる。 In the embodiment of the present invention, a liquid containing magnetic beads (magnetic nanoparticles) and an object to be measured that can be bonded to the magnetic beads (magnetic nanoparticles) is rotated, and a magnetic field whose polarity is reversed every time the liquid is rotated is applied to respond to the change in magnetism. The output signal to be measured can be detected for each lap, and the amount of the object to be measured can be measured by using the difference in the output signals of the adjacent laps, so that a more sensitive magnetic immunoassay can be performed.
本発明は、上記実施の形態に限定されるものではなく、本発明の分野における通常の知識を有する者であれば想到し得る各種変形、修正を含む要旨を逸脱しない範囲の設計変更があっても、本発明に含まれることは勿論である。 The present invention is not limited to the above-described embodiment, and there are design changes within a range that does not deviate from the gist including various modifications and modifications that can be conceived by a person having ordinary knowledge in the field of the present invention. Of course, it is also included in the present invention.
１０：試料、１２：容器、２０：回転機構、２２：台、２４：回転軸、２６：アーム部、３０：磁界発生装置、３２：発振器、３４：励磁コイル、３６：ヨーク、３８：永久磁石、４０：磁界センサ、４０ａ：センサ素子、４０ｂ：センサ素子、４２：バイアス用磁石、４４：磁気シールド、５０：信号処理部 10: Sample, 12: Container, 20: Rotating mechanism, 22: Table, 24: Rotating shaft, 26: Arm, 30: Magnetic field generator, 32: Oscillator, 34: Exciting coil, 36: Yoke, 38: Permanent magnet , 40: Magnetic field sensor, 40a: Sensor element, 40b: Sensor element, 42: Bias magnet, 44: Magnetic shield, 50: Signal processing unit
Claims (9)
磁性物質と該磁性物質と結合可能な前記被測定物とを含む試料を収容する容器を所定の移動周期で繰り返し同一移動させる移動機構と、
前記容器の移動周期に同期して移動毎に磁界方向が反転して切り替わる磁界を、移動している前記容器に収容される試料に印加する磁界発生部と、
前記磁界発生部からの磁界の影響を実質的に受けない程度に離間した位置に配置され、移動している前記容器に収容される試料から放出される磁界に対応する信号を検出する磁界センサとを備え、
前記磁界発生部は発振器と該発振器に接続するコイルと該コイルを貫くヨークとを有して構成され、前記ヨークは錐体に形成されることを特徴とする磁界測定装置。 A magnetic field measuring device for detecting an object to be measured by a magnetic immunological test.
A moving mechanism that repeatedly moves the container containing the magnetic substance and the sample containing the object to be measured that can be bonded to the magnetic substance in the same movement cycle.
A magnetic field generator that applies a magnetic field that reverses and switches in the direction of the magnetic field each time it moves in synchronization with the movement cycle of the container to the sample contained in the moving container.
A magnetic field sensor that detects a signal corresponding to the magnetic field emitted from the sample contained in the moving container, which is arranged at a position separated so as not to be substantially affected by the magnetic field from the magnetic field generating portion. With
A magnetic field measuring device characterized in that the magnetic field generating unit includes an oscillator, a coil connected to the oscillator, and a yoke penetrating the coil, and the yoke is formed in a cone.
前記磁界発生部は、前記容器の停止中に、前記容器に収容される試料に磁界を所定時間印加することを特徴とする請求項２に記載の磁界測定装置。 The moving mechanism stops the container for a predetermined time every cycle.
The magnetic field measuring device according to claim 2, wherein the magnetic field generating unit applies a magnetic field to a sample housed in the container for a predetermined time while the container is stopped.
磁性物質と該磁性物質と結合可能な前記被測定物とを含む試料を直流磁界により着磁させる着磁工程と、
前記試料を収容する容器を複数回移動させる移動工程と、
前記容器の移動周期に同期して、2n+1（ｎは0以上の整数）周期目の移動に対して、磁界強度B2n+1＝B2n+dB（Ｂ0＝0mT、dB＝所定の磁界強度増加分）の正又は負の磁界を前記容器に収容される試料に印加し、2n+2周期目の移動に対して、磁界強度B2n+2＝B2n+1＝B2n+dBであって2n+1周期目の移動に印加した磁界と反対方向の磁界を前記容器に収容される試料印加する磁界印加工程と、
移動している前記容器に収容される試料から放出される磁界に対応する信号を検出する検出工程とを備えることを特徴とする磁界測定方法。 A magnetic field measurement method for detecting an object to be measured by a magnetic immunological test.
A magnetizing step of magnetizing a sample containing a magnetic substance and the object to be measured that can be bonded to the magnetic substance by a DC magnetic field.
A moving step of moving the container containing the sample a plurality of times, and
Magnetic field strength B 2n + 1 = B2n + dB (B0 = 0mT, dB = predetermined magnetic field strength) with respect to the movement of the 2n + 1 (n is an integer of 0 or more) cycle in synchronization with the movement cycle of the container. A positive or negative magnetic field (increase) is applied to the sample contained in the container, and the magnetic field strength is B 2n + 2 = B 2n + 1 = B 2n + dB with respect to the movement in the 2n + 2nd cycle. A magnetic field application step of applying a magnetic field in the direction opposite to the magnetic field applied to the movement in the 2n + 1 cycle is applied to the sample housed in the container.
A magnetic field measuring method comprising a detection step of detecting a signal corresponding to a magnetic field emitted from a sample housed in the moving container.
前記磁界印加工程において、前記容器の停止中に、前記容器に収容される試料に磁界を所定時間印加することを特徴とする請求項７に記載の磁界測定方法。 In the moving step, the container is stopped for a predetermined time every cycle.
The magnetic field measurement method according to claim 7, wherein in the magnetic field application step, a magnetic field is applied to a sample housed in the container for a predetermined time while the container is stopped.
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