JP4565205B2

JP4565205B2 - Sample analysis element

Info

Publication number: JP4565205B2
Application number: JP2006528954A
Authority: JP
Inventors: 俊文大久保; 善一吉田; 信幸寺田
Original assignee: TAMA-TLO, LTD.
Current assignee: TAMA-TLO, LTD.
Priority date: 2004-07-01
Filing date: 2005-06-30
Publication date: 2010-10-20
Anticipated expiration: 2025-06-30
Also published as: WO2006004176A1; JPWO2006004176A1

Description

本発明は、血液などの検体サンプルに含まれる細胞の密度、形状、老化・劣化あるいは異常の有無を高精度かつ簡易にかつ微量で、計測および判定可能とする超小型分析素子に関するものである。 The present invention relates to a microanalytical element capable of measuring and determining the density, shape, aging / deterioration, or presence / absence of cells contained in a specimen sample such as blood with high accuracy and in a simple manner with a small amount.

予防医学への関心の高まりやＳＡＲＳ、ＡＩＤＳなどの重大感染症拡大に対する迅速な対応の観点から、血液・体液等の検体を簡易にしかも必要な生理情報を精密に分析できる機器への要請が高まっている。かつては検体のもつ生理情報の基礎となる赤血球や白血球などの血球機能計測は、顕微鏡下における目視検査により行われており、測定の迅速性はもとより測定精度が測定者の個人差により影響を受け、精度の高い客観評価が困難などの課題があった。
近年マイクロプロセッサの導入により血液の希釈や溶血処理などが自動化されるとともに、血球一つ一つを緩衝液で囲んだまま微細ノズルより高速噴射させ、血球サイズに絞り込んだアルゴンレーザビームによりその前方、後方散乱光を光検出素子で高感度に検出・カウントすることで、高精度かつ短時間に検体の生理的検査が可能なシステム（フローサイトメトリー）が開発されている（例えば、特表平１１−５１３４８６号公報や、浜松ホトニクス株式会社編集委員会、「光電子増倍管 −その基礎と応用−」、Ｃａｔ．Ｎｏ．ＴＯＴＨ９００１Ｊ０２、（１９９８）、ｐｐ．２６０−２６１等を参照）。
従来用いられているフローサイトメトリーの基本システムの一例を示した図４を参照しながら説明する。まず、事前に希釈・蛍光処理をされた検体６４を、検体導入路５２から液滴噴射ノズル５４に導入する。検体は保護緩衝液導入路５３から別途ノズル予備室に導入された保護緩衝液に包まれた状態で、ノズル５４背後に設置された液滴発生用超音波素子５１により超音波振動が与えられる。ノズル５４より当初水柱状に噴射された検体６４は、その直後に個々の血球を含んだ微細液滴となって列状に飛散する。ノズル噴射直後の保護緩衝液に囲まれた水柱状部分に、レーザ光源５６から発射したアルゴンイオンレーザをビームエキスパンダ５８及び検体照射用集光レンズ５７を介して照射する。このときレーザは血球サイズに収束されている。透過した光は集光レンズ５９にて集光し受光素子（フォトダイオード）６０で検出される。この光情報から血球密度（血球数カウント）や血球の体積情報を得ることができる。また、後方散乱光（蛍光）を散乱光集光レンズ６１及び６２にて集光し光電子増倍管６３で高感度検出することにより血球に取り込まれた蛍光分子数をカウントして血球の生理学的な情報を採取できる。血球飛散列の下流に位置する電極５５は、上記レーザの散乱光より得られた個々の血球の情報に基づき、帯電された血球６４をクーロン力により選別するために電界を発生させるもので、その飛翔軌跡を制御し、所望の血球サンプルを検体から分離することができる。
これらのシステムにより血球のシース粒状化とレーザ光学系による高速・高感度・高精度な検体分析・分離が可能となったが、その一方でレーザ発振器などの光源、レーザ光分析光学系、液滴飛散系などはその機器の規模が大きく、現状でも制御系を含めたその全システムはほぼ家庭用冷蔵庫１台分程度の体積空間を占める。近年目指す予防医学の基本の一つは、「いつでも、どこでも、誰でも、簡易かつ正確に」検体情報を取得できることにあり、これに対応した検体分析システムの超小型化、低消費エネルギー化、ユビキタス化が今後一層求められる。従ってこれら検体の生理情報検出に関わる基本原理は踏襲しながらも、ユビキタスまたは個々人に対するウェアラブル化に適応した光学系、機構系を超小型に凝縮した検体分析素子の開発が渇望される。
本発明の上記及び他の特徴及び利点は、添付の図面とともに考慮することにより、下記の記載からより明らかになるであろう。From the standpoint of increasing interest in preventive medicine and the rapid response to the spread of serious infectious diseases such as SARS and AIDS, there is an increasing demand for instruments that can easily analyze specimens such as blood and body fluids and accurately analyze the necessary physiological information. ing. In the past, blood cell functions such as red blood cells and white blood cells, which are the basis of physiological information of specimens, are measured by visual inspection under a microscope. In addition to the speed of measurement, the measurement accuracy is affected by individual differences of the measurer. There were problems such as difficulty in objective evaluation with high accuracy.
In recent years, with the introduction of microprocessors, blood dilution and hemolysis are automated, and each blood cell is jetted at a high speed from a fine nozzle while surrounded by a buffer solution, and an argon laser beam narrowed down to the size of the blood cell, A system (flow cytometry) has been developed that can detect and count backscattered light with high sensitivity by a photodetection element and can perform a physiological examination of a specimen with high accuracy and in a short time (for example, JP 11 No. 51486, Hamamatsu Photonics Co., Ltd. Editorial Committee, “Photomultiplier Tubes-Fundamentals and Applications”, Cat. No. TOTH9001J02, (1998), pp. 260-261, etc.).
A description will be given with reference to FIG. 4 showing an example of a conventional flow cytometry basic system. First, the specimen 64 that has been diluted and fluorescent in advance is introduced from the specimen introduction path 52 to the droplet ejection nozzle 54. The sample is ultrasonically vibrated by the droplet generating ultrasonic element 51 installed behind the nozzle 54 in a state of being wrapped in a protective buffer solution separately introduced into the nozzle preliminary chamber from the protective buffer solution introduction path 53. The specimen 64 initially ejected from the nozzle 54 in the form of a water column becomes a fine droplet containing individual blood cells immediately after that and scatters in a row. An argon ion laser emitted from a laser light source 56 is irradiated through a beam expander 58 and a specimen irradiating condensing lens 57 onto a water columnar portion surrounded by a protective buffer immediately after nozzle injection. At this time, the laser is focused on the blood cell size. The transmitted light is condensed by the condenser lens 59 and detected by the light receiving element (photodiode) 60. From this light information, blood cell density (blood cell count) and blood cell volume information can be obtained. Further, the backscattered light (fluorescence) is collected by the scattered light collecting lenses 61 and 62 and detected with high sensitivity by the photomultiplier tube 63, thereby counting the number of fluorescent molecules taken into the blood cell and the physiological of the blood cell. Information can be collected. The electrode 55 located downstream of the blood cell scattering row generates an electric field for sorting the charged blood cells 64 by Coulomb force based on the information of the individual blood cells obtained from the scattered light of the laser. By controlling the flight trajectory, a desired blood cell sample can be separated from the specimen.
These systems enable blood cell sheath granulation and high-speed, high-sensitivity, and high-precision specimen analysis and separation using a laser optical system. On the other hand, light sources such as laser oscillators, laser light analysis optical systems, and droplets The size of the equipment such as the scattering system is large, and the entire system including the control system still occupies a volume space of about one household refrigerator. One of the fundamentals of preventive medicine aimed at in recent years is to be able to acquire sample information "anytime, anywhere, anyone easily and accurately". The sample analysis system corresponding to this is ultra-compact, low energy consumption, ubiquitous In the future, there will be more demands. Therefore, while following the basic principle relating to the detection of physiological information of these specimens, there is a craving for the development of a specimen analysis element in which an optical system and a mechanism system adapted to wearability for ubiquitous or individuals are condensed in a very small size.
The above and other features and advantages of the present invention will become more apparent from the following description when considered in conjunction with the accompanying drawings.

図１は、本発明の好ましい一実施態様（第一実施態様）を説明する斜視図である。
図２は、本発明の別の好ましい一実施態様（第二実施態様）を説明する斜視図である。
図３は、本発明の別の好ましい一実施態様（第三実施態様）の概要を説明する斜視図である。
図４は、従来用いられているフローサイトメトリーの基本システムの一例を示す説明図である。FIG. 1 is a perspective view for explaining a preferred embodiment (first embodiment) of the present invention.
FIG. 2 is a perspective view for explaining another preferred embodiment (second embodiment) of the present invention.
FIG. 3 is a perspective view for explaining the outline of another preferred embodiment (third embodiment) of the present invention.
FIG. 4 is an explanatory diagram showing an example of a conventional flow cytometry basic system.

本発明によれば、以下の手段が提供される：
（１）マイクロ流路をその内部に形成された透明部材と、該透明部材に埋め込まれ該透明部材に形成された該マイクロ流路を隔ててその端面同士が対向して配置され、しかも該透明部材と屈折率を異にする一対の導光棒状部材と、測定部からの散乱光を集光する該透明部材に埋め込まれた散乱光集光部とから構成されることを特徴とする検体分析素子。
（２）前記の一対の導光棒状部材が一本の光ファイバからなり、前記の透明部材に形成されたマイクロ流路が該光ファイバに直交し、該光ファイバのコアを貫通して形成されていることを特徴とする（１）項に記載の検体分析素子。
（３）前記透明部材にレーザ光源及び／又は受光素子が装着されていることを特徴とする（１）又は（２）に記載の検体分析素子。
（４）前記透明部材にピンホール又はスリット部が配置されていることを特徴とする（１）〜（３）のいずれか１項に記載の検体分析素子。
（５）前記透明部材の周囲が金属膜で覆われていることを特徴とする（１）〜（４）のいずれか１項に記載の検体分析素子。
（６）前記の一対の導光棒状部材が複数組設けられていることを特徴とする（１）〜（５）のいずれか１項記載の検体分析素子。
（７）前記透明部材に複数の流路が形成され、その個々の流路ごとに、該透明部材に埋め込まれ該透明部材に形成された該流路を隔ててその端面同士が対向して配置され、しかも該透明部材と屈折率を異にする一対の導光棒状部材の組が１組又は複数組配置されていることを特徴とする（１）〜（６）のいずれか１項に記載の検体分析素子。According to the present invention, the following means are provided:
(1) A transparent member having a microchannel formed therein and an end surface facing each other across the microchannel embedded in the transparent member and formed in the transparent member, and the transparent channel Sample analysis comprising a pair of light guide rod-like members having different refractive indexes from those of the member, and a scattered light condensing part embedded in the transparent member for condensing scattered light from the measurement part element.
(2) The pair of light guide rod-shaped members are made of a single optical fiber, and the micro flow path formed in the transparent member is formed perpendicularly to the optical fiber and through the core of the optical fiber. The specimen analysis element according to item (1), wherein
(3) The sample analysis element according to (1) or (2), wherein a laser light source and / or a light receiving element is attached to the transparent member.
(4) The sample analysis element according to any one of (1) to (3), wherein a pinhole or a slit portion is disposed in the transparent member.
(5) The specimen analysis element according to any one of (1) to (4), wherein the periphery of the transparent member is covered with a metal film.
(6) The sample analysis element according to any one of (1) to (5), wherein a plurality of pairs of the pair of light guide rod members are provided.
(7) A plurality of flow paths are formed in the transparent member, and the end faces of the individual flow paths are arranged so as to face each other with the flow paths formed in the transparent member embedded in the transparent member. In addition, one set or a plurality of sets of a pair of light guide rod-shaped members having a refractive index different from that of the transparent member are arranged. Any one of (1) to (6) Sample analysis element.

本発明者らは、鋭意検討を重ねた結果、検体中の血球数や形状、光の透過・吸収率などの生理学的な情報を検出・分析する素子において、検体を流すマイクロメートルオーダーの幅の流路（本明細書において「マイクロ流路」という。）をその内部に形成した透明部材をベースに、ベース材に比して屈折率の異なる部材を流路を隔ててその端面どうしが対向した構成とすることにより、流路を流れる血球にレーザ光などの光を前記の端面（横方向）から確実に照射し、かつ光の前方散乱、後方散乱、後方反射などの光学情報を分離して精密に検出・測定可能な光学系をチップ状素子に集約することができ、しかも安価に製造できることを見出した。本発明はこのような知見に基づきなされるに至ったものである。
以下、本発明の好ましい一実施態様について、添付の図面に基づいて詳細に説明をする。なお、各図の説明において同一の要素には同一の符号を付す。
図１は、本発明の好ましい一実施態様（第一実施態様）を説明する斜視図である。
本発明の検体素子は透明部材１からなり、その一面には検体用流路２として溝が形成されている。透明部材１には、例えばガラスなどを用いることもできるが、血液との適合性や製造コストの観点から紫外線硬化型樹脂が好ましく用いられる。本発明では検体５として主に赤血球、白血球を対象とすることができる。流路２の大きさはマイクロメートルオーダーの極微細な幅であり、検体に応じて適宜の幅に定めることができる。検体が赤血球の場合、赤血球は直径８μｍ程度、厚み１μｍ程度であるため、これが集団ではなく個々に通過できるように、導光棒状部材３及び４の端面３ｂ及び４ｃ間を横切る狭小部分は、外径が７〜８μｍ程度から最大１２〜１３μｍ程度とすることが好ましく、約１０μｍ程度がより好ましい。流路２の形成方法は特に限定されず任意の方法を用いることができるが、例えば光硬化性樹脂を用いた光造形法などが好ましい。なお、図示していないが、透明部材１の流路２が形成されている面には透明部材１と同材質の部材を透明カバー（プレート）として接合して溝にふたをすることにより、流路２を管状とする（図１では透明カバーを接合する前の状態を示す）。接合方法としては、熱その他による圧着などの任意の方法を用いることができる。
また、透明部材１内には流路２を挟んで端面同士が対向するように導光棒状部材３及び４が配置されている。導光棒状部材３及び４には透明部材１と屈折率の異なる部材が用いられる。これにより、導光棒状部材３及び４がコア部、透明部材１がクラッド部として光導波路が形成され、導光棒状部材３及び４を光が伝搬することができる。導光棒状部材３及び４として光ファイバを用いてもよい。導光棒状部材３及び４の形状及び外径は特に限定されないが、例えば外径３〜４μｍ以下のシングルモード特性をもつものや、外径１０μｍ以上のマルチモード特性をもつものを用いることができ、またこれらを複数用いてもよい。流路２を挟んだ導光棒状部材３及び４の端面同士（３ｂと４ｃ）の対向位置は、導光棒状部材３及び４が一直線になるように配置してもよいが、導光棒状部材３及び４（コア部）の伝搬光モードや血球の光学特性との関係で、血球の流れの方向等にずらして配置してもよい。
本発明の検体分析素子には、導光棒状部材３の一端３ａに光を導入するようにレーザ光源６が配置され、また、導光棒状部材４の末端４ｄから放出された光を受光するように受光素子９が配置される。受光素子９は、図１に示したように光検出側の導光棒状部材４の端部４ｄに対向するように透明部材１に装着されていてもよい。また、受光素子９は図２に示したように透明部材１から分離して配置してもよく、その場合は光検出側の光導波路の端部と受光素子９との間に集光レンズ８を配置することが好ましい。
レーザ光源６は特に限定されず、例えばガスレーザや半導体レーザ等を用いることができる。受光素子９は特に限定されず、例えばフォトダイオード等を用いることができる。
また、透明部材１内には散乱光集光部３１が配置されている。散乱光集光部３１は、透明部材１の一部にシリンドリカル凸レンズ様の切り欠きを設けることで形成できる。散乱光集光部３１は、導光棒状部材３及び４の端部３ｂ及び４ｃ間の流路部分（測定部２ａ）で散乱した前方又は後方散乱光を受光し、受光素子１０へと散乱光を導く役割を果たす。この散乱光集光部３１により導光棒状部材３ｂ及び４ｃ間を血球５が通過する際における散乱光を集光レンズと同等の機能によって受光素子１０に効率よく導くことができ、素子自体をシンプルな構成とし、しかも、透明部材１の外部に集光レンズを配置する必要がないので集光レンズの設置角度に依存する測定誤差を生じにくくすることができる。受光素子１０は前記受光素子９と同様に特に限定されず、例えばフォトダイオード等を用いることができる。特に、後方散乱光の強度に関しては検体（血球）の屈折率との関係からも極めて微弱な場合があるが、受光素子として例えばアバランシェフォトダイオードを用いることで、極めて高い受光感度で散乱光（光変調）を採取することができる。
また、検体照射用の集光レンズ７と導光棒状部材３の端部３ａとの間にビームスプリッタ３２を配置し、検体からの反射光を受光素子３３にて測定することもできる。これにより、レーザ入力強度のふらつきを少なくすることができる。
本発明の検体分析素子の使用方法について説明する。最初に、バックグランドを測定するために、流路２にシース液（保護緩衝液、または希釈液と血漿の混合液）を満たした状態で測定を行う。まず、レーザ発振器６から集光レンズ７を介して導光棒状部材３の端部３ａにレーザ光を導入する。導入する光の波長は適宜切り替えることができ、光導波路が複数ある場合には光導波路ごとに波長を変えることもできる。導光棒状部材３を伝搬した光は流路２（測定部２ａ）にて発散するが対向側の導光棒状部材４に再度導入され、導光棒状部材４の末端４ｄから出光して、受光素子９により検出される。流路２（測定部２ａ）で散乱した後方散乱光は散乱光集光部３１により集光され受光素子１０により検出される。なお、図示していないが、コア伝搬光の進行方向斜め前方に散乱光集光部及び受光素子を配置することで、前方光散乱光を受光素子により検出することができる。
次に、流路２に検体５を流して同様に測定を行う。なお、測定に際して、検体中の血球に蛍光処理をしてもよい。検体５は流路２内を個々に独立した状態で流れる。検体５が導光棒状部材３ｂ及び４ｃ間の測定部２ａを通過すると、レーザ光が前方、後方に散乱されると同時に検体５に一部吸収される。受光素子９により検体５によるレーザ光の吸収がモニタされる一方、受光素子１０により後方散乱光がモニタされる。また、検体５からの反射光は、集光レンズ７と導光棒状部材３の端部３ａとの間に配置されたビームスプリッタ３２を介して受光素子３３により検出される。測定値をバックグランド値と比較することで検体の量や大きさ、形状の異常などを正確に測定することができる。
図２は、本発明の別の好ましい一実施態様（第二実施態様）を説明する斜視図である。
図２の検定分析素子は、光導波路として光ファイバを用いたものである。透明部材１内にコア部２２及びクラッド部２３からなる光ファイバが設けられており、コア部２２を貫通するように検体通過穴（マイクロ流路）２１が設けられている。本実施態様は、本来良好な光の導波路である光ファイバそのものを用いることで、光ファイバのコアを横断した穴を形成するだけで光ファイバと直行するマイクロ流路を形成することができ、光造形、光リソグラフィなどに伴うマスクが不要で、また作製過程においてコア端の対向精度などに配慮する必要もなく、比較的簡易に血液分析素子の主要部を構成できる利点がある。
本実施態様の検体分析素子は、まず光ファイバを透明液体状樹脂で覆い固め、加工用レーザを用いて該光ファイバに直交し、光ファイバのコア部分２２を貫通するように穴２１を開けることにより作製することができる。穴の寸法は特に限定されないが、検体中の血球が個々に通過できる程度の寸法が好適である。
本実施態様に用いることができる光ファイバとしては特に限定されず、コア径が３〜４μｍ程度のシングルモード光ファイバや、コア径が１０μｍ程度以上のマルチモード光ファイバを用いることができる。また、図２には一本の光ファイバとこのコア部分を貫通する１個の穴（流路）の例のみを示したが、配列するファイバ数やその種別、導入レーザ光の波長等は特に限定されない。ファイバの種別や波長を変えることで、血球に対する照射空間分解能を変えたり、吸収・散乱に関する波長依存などの情報を得ることができる。
光ファイバの他端からレーザ光を導入すると穴２１（通常はシース液で満たされている。）部分により伝搬損失が生じ、反対側に配置された受光素子９にてその光パワーが損失分だけ減少した状態で検出される。シース液中の血球が光ファイバコア２２の対向部分を通過することで、同様にレーザ光の吸収、前方、後方への散乱が発生し、受光素子にて感知されるレーザ光パワーがこれに応じて変化する。これの低周期信号成分フィルタにより分離・検出・処理することにより単位時間あたりの流通血球数がカウントできる。また血球が通過していない時の受光パワーと比較することにより血球による光の吸収が評価できる。また、図示していないが、図１と同様に散乱光集光部および受光素子が設けられ、前方または後方散乱される光パワーを検出できる。
なお、本実施態様の他の構成や作用、効果は、第一実施態様とほぼ同様である。
なお、上記の第一実施態様または第二実施態様において、レーザ光源は透明部材１とは個別に配置し、集光レンズを含めた形で位置や光軸調整をしてはじめて検出素子としての機能を実現できるようにしているが、レーザ光源自体やこれのヒートシンクとなる金属部材ごと透明部材１に全体または部分的に埋め込むことも可能であり、また、光導波路への光の導入も、第一実施態様における散乱光集光部のような切り欠き部を応用したり、微小ボールレンズを透明部材内に設けたりすることで集光レンズを代替できる。これにより、光照射系、流路、集光レンズ、光源などの個々の光学要素を個別に光軸合わせやアセンブルする手間を省くことができ、信頼性や安定性、さらに耐衝撃性の高い検体分析素子を実現できる。
また、透明部材の導光棒状部材４の末端部４ｄ近傍において、特定の方向のみの散乱光を通過させるスリットまたはピンホール様の遮光部材を埋め込むことで、他の部分で散乱・反射伝搬して受光素子に混入する有害光（背景光、外乱光）を遮断し、検出の精度・確度を上げることもできる。また、上記透明部材１の周囲を遮光金属膜で覆うことで、同様に不要な周囲光を遮断し、精度・確度の高い検体の光学情報を検出できる。
図３は、本発明の別の好ましい一実施態様（第三実施態様）の概要を説明する斜視図である。図３では透明部材１中の流路２並びに導光棒状部材３及び４に注目して示しているが、レーザ光源や受光素子、散乱光集光部などの他の構成要素については第一実施態様と同様である。
図３に示すように、透明部材１に複数組の流路２及び導光棒状部材３及び４を設けてもよい。図３では、流路２が平行に配置され、１流路につき１組の導光棒状部材３及び４が配置されている。図示していないが、導光棒状部材３及び４は透明部材１の端面まで伸びている。各導光棒状部材は、光導波路内を伝搬する光情報に他の流路の影響が紛れ込むのを防止するため、他の流路と物理的に交差しないように三次元的に配置されている。すなわち図３では、透明部材１に埋め込まれた各導光棒状部材３及び４は流路２の下を通り、測定する流路２についてのみ該流路２を隔ててその端面同士が対向して配置されている。なお、１流路につき複数の光導波路が配置されてもよいが、その場合も各光導波路は他の流路と物理的に交差しないように三次元的に配置される。また、図３において、各流路２の両端には検体ため４１が設けられている。
なお、図３では透明部材に形成された１層の構成を示したが、これを重ねて積層構造としてもよい。
本実施態様によれば、多数形成された流路に同一の検体を流し、同一波長の光をそれぞれの光導波路に導入してそれらの応答を観測することにより、血球に関する応答のばらつきを短時間に評価できる。また、光導波路ごとに波長や偏光方向を変えた光を導入することにより、それぞれ血球に対する波長（吸収、散乱性）や偏光依存性などの特性も短時間に評価できる。さらに、検体をフィルタなどによって分離し、流路ごとにその成分を分けて流して個々の光応答を観測することにより、同時に検体の多数の成分の特性を知ることも可能となる。一方、液体成分のみを流す流路と、液体分に血球が浮遊した成分を流す流路を別にし、採取した光応答信号の差分を取ることで、微弱な信号中から血球の応答を高精度に分離検出することもできる。
本実施態様の他の構成や作用、効果は、第一実施態様または第二実施態様とほぼ同様である。
また、各実施態様において、光導波路及び流路がいずれも直線状に配置された構成について説明したが、これらは曲率をもって形成・配置されてもよい。
本発明の検体分析素子は、数十μｍ〜約１ｃｍという超小型素子であり、容易に持ち運ぶことができ、いつでも、どこでも、簡易かつ正確に検体分析が可能である。
また、本発明の検体分析素子は、血液や体液のみならず、尿、唾液、汗等に含有している癌細胞の検出にも応用することができ、さらに、水やアルコールなどの様々な液体中の微生物の検出等にも広く応用することができる。
本発明の検体分析素子は、検体を流すマイクロ流路をその内部に形成した透明部材をベースに、ベース材に比して屈折率の異なる部材を流路を隔ててその端面どうしが対向した構成とすることにより、流路を流れる検体、例えば血液の個々の血球にレーザ光を確実に照射して、レーザ光の前方散乱、後方散乱、後方反射などの光学情報を分離して解析し、血球の数、形状（形状の異常）などを精密に検出可能な光学系をコンパクトなチップ状素子上に集約することができ、しかも安価に製造することができる。
また、本発明の検体分析素子は、光導波路として光ファイバを用いることができ、これにより簡易で生産性の高い検体分析素子を実現できる。
また、本発明の検体分析素子は、上記透明部材にシリンドリカル（凸）レンズ状の散乱光集光部が設けられることで、透明部材の外部に集光レンズを配置することなく光検出素子に確実に散乱光を導くことができる。また、レーザ光源や、これのヒートシンク、受光素子などは微小化した物を使用できるので上記透明部材に装着・配置することにより、光照射系、流路、集光レンズ、光源などの個々の光学要素を個別に光軸合わせやアセンブルする手間を省くことができ、信頼性、安定性、及び耐衝撃性の高い検体分析素子を実現できる。
また、本発明の検体分析素子は、上記透明部材の導光棒状部材末端部近傍にピンホール又はスリット部を配置することにより、検体以外の部分で散乱・反射した検出に有害な光（背景光、外乱光）の混入を遮断して、精度・確度の高い検体の光学情報を検出できる。また、本発明の検体分析素子は、上記透明部材の周囲を遮光金属膜で覆うことで、同様に有害な周囲光を遮断して、精度・確度の高い検体の光学情報を検出できる。
また、本発明の検体分析素子は、上記透明部材に多数の流路を集積形成し、その個々の流路ごとに、ベースとなる透明部材と屈折率を異にし、その端面が流路を隔てて対向する配置となる一対の導光棒状部材の組を１組又は複数組配置することにより、同一検体に照射光波長を変えてその光学的性質を短時間に子細に判定でき、また同一波長の光を多数の検体中の血球に照射することで個々の血球特性の計測確度を高め、かつ計測結果の分布そのものを医療情報として得られるなど、多様な生理学的情報をコンパクトな素子構成をもって測定することができる。As a result of intensive studies, the inventors of the present invention have an element that detects and analyzes physiological information such as the number and shape of blood cells in a sample, light transmission / absorption rate, etc. A transparent member having a channel (referred to as “microchannel” in the present specification) formed therein is used as a base, and a member having a refractive index different from that of the base material is opposed to each other across the channel. By adopting the configuration, the blood cells flowing in the flow path are surely irradiated with light such as laser light from the end face (lateral direction), and optical information such as light forward scattering, back scattering and backward reflection is separated. It has been found that optical systems that can be precisely detected and measured can be integrated into a chip-like element and can be manufactured at low cost. The present invention has been made based on such findings.
Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. In the description of each drawing, the same elements are denoted by the same reference numerals.
FIG. 1 is a perspective view for explaining a preferred embodiment (first embodiment) of the present invention.
The sample element of the present invention comprises a transparent member 1, and a groove is formed as a sample channel 2 on one surface thereof. For the transparent member 1, for example, glass or the like can be used, but an ultraviolet curable resin is preferably used from the viewpoint of compatibility with blood and manufacturing cost. In the present invention, the specimen 5 can mainly target red blood cells and white blood cells. The size of the flow path 2 is an extremely fine width on the order of micrometers, and can be set to an appropriate width according to the specimen. When the specimen is red blood cells, the red blood cells have a diameter of about 8 μm and a thickness of about 1 μm. Therefore, a narrow portion that crosses between the end faces 3 b and 4 c of the light guide rod-shaped members 3 and 4 is outside so that they can pass individually instead of a group. The diameter is preferably about 7 to 8 μm to a maximum of about 12 to 13 μm, more preferably about 10 μm. Although the formation method of the flow path 2 is not specifically limited, Although arbitrary methods can be used, the optical modeling method etc. which used photocurable resin etc. are preferable, for example. Although not shown in the drawing, a member made of the same material as that of the transparent member 1 is joined as a transparent cover (plate) to the surface of the transparent member 1 where the flow path 2 is formed, and the groove is covered. The path 2 is tubular (shown in FIG. 1 before joining the transparent cover). As a bonding method, any method such as heat or other pressure bonding can be used.
In addition, light guide rod-like members 3 and 4 are arranged in the transparent member 1 so that the end faces face each other with the flow path 2 interposed therebetween. For the light guide rod-shaped members 3 and 4, a member having a refractive index different from that of the transparent member 1 is used. Thereby, an optical waveguide is formed with the light guide rod-shaped members 3 and 4 as the core portion and the transparent member 1 as the cladding portion, and light can propagate through the light guide rod-shaped members 3 and 4. Optical fibers may be used as the light guide rod-like members 3 and 4. The shape and outer diameter of the light guide rod-shaped members 3 and 4 are not particularly limited. For example, those having a single mode characteristic with an outer diameter of 3 to 4 μm or multimode characteristics having an outer diameter of 10 μm or more can be used. A plurality of these may be used. The opposing positions of the end faces (3b and 4c) of the light guide rod members 3 and 4 across the flow path 2 may be arranged so that the light guide rod members 3 and 4 are in a straight line. Depending on the propagation light modes of 3 and 4 (core part) and the optical characteristics of blood cells, they may be shifted in the direction of blood cell flow.
In the sample analysis element of the present invention, a laser light source 6 is disposed so as to introduce light into one end 3a of the light guide rod-shaped member 3, and light emitted from the end 4d of the light guide rod-shaped member 4 is received. The light receiving element 9 is disposed on the surface. As shown in FIG. 1, the light receiving element 9 may be attached to the transparent member 1 so as to face the end 4 d of the light guide rod-shaped member 4 on the light detection side. Further, the light receiving element 9 may be arranged separately from the transparent member 1 as shown in FIG. 2, and in this case, the condensing lens 8 is provided between the end of the optical waveguide on the light detection side and the light receiving element 9. Is preferably arranged.
The laser light source 6 is not specifically limited, For example, a gas laser, a semiconductor laser, etc. can be used. The light receiving element 9 is not particularly limited, and for example, a photodiode or the like can be used.
A scattered light condensing unit 31 is disposed in the transparent member 1. The scattered light condensing part 31 can be formed by providing a cutout like a cylindrical convex lens in a part of the transparent member 1. The scattered light condensing unit 31 receives the forward or back scattered light scattered by the flow path portion (measurement unit 2 a) between the end portions 3 b and 4 c of the light guide rod-shaped members 3 and 4, and scattered light to the light receiving element 10. To play a leading role. The scattered light condensing unit 31 can efficiently guide the scattered light when the blood cell 5 passes between the light guide rod-shaped members 3b and 4c to the light receiving element 10 by the same function as the condensing lens, and the element itself is simple. In addition, since it is not necessary to dispose the condensing lens outside the transparent member 1, it is possible to make it difficult to cause a measurement error depending on the installation angle of the condensing lens. The light receiving element 10 is not particularly limited like the light receiving element 9, and for example, a photodiode or the like can be used. In particular, the intensity of backscattered light may be very weak due to the relationship with the refractive index of the specimen (blood cell). However, by using an avalanche photodiode, for example, as the light receiving element, scattered light (light Modulation).
Further, a beam splitter 32 may be arranged between the specimen irradiating condensing lens 7 and the end portion 3 a of the light guide rod-shaped member 3, and reflected light from the specimen may be measured by the light receiving element 33. Thereby, the fluctuation of the laser input intensity can be reduced.
A method for using the sample analysis element of the present invention will be described. First, in order to measure the background, measurement is performed in a state where the flow path 2 is filled with a sheath liquid (protective buffer solution or a mixture of a diluent and plasma). First, laser light is introduced from the laser oscillator 6 to the end portion 3 a of the light guide rod-shaped member 3 through the condenser lens 7. The wavelength of light to be introduced can be switched as appropriate, and when there are a plurality of optical waveguides, the wavelength can be changed for each optical waveguide. The light propagating through the light guide rod-shaped member 3 diverges in the flow path 2 (measurement unit 2a), but is reintroduced into the light guide rod-shaped member 4 on the opposite side, and is emitted from the end 4d of the light guide rod-shaped member 4 to receive the light. Detected by element 9. The backscattered light scattered by the flow path 2 (measurement unit 2a) is collected by the scattered light collecting unit 31 and detected by the light receiving element 10. Although not shown in the drawing, the forward light scattered light can be detected by the light receiving element by arranging the scattered light condensing unit and the light receiving element obliquely forward in the traveling direction of the core propagation light.
Next, the sample 5 is made to flow through the flow path 2 and the measurement is performed in the same manner. In the measurement, the blood cells in the sample may be subjected to fluorescence treatment. The specimen 5 flows through the flow path 2 in an independent state. When the specimen 5 passes through the measuring portion 2a between the light guide rod-shaped members 3b and 4c, the laser light is scattered forward and backward and simultaneously absorbed by the specimen 5 in part. While the light receiving element 9 monitors the absorption of the laser light by the specimen 5, the light receiving element 10 monitors the backscattered light. Reflected light from the specimen 5 is detected by the light receiving element 33 via the beam splitter 32 disposed between the condenser lens 7 and the end 3 a of the light guide rod-shaped member 3. By comparing the measured value with the background value, it is possible to accurately measure the amount, size, shape abnormality, etc. of the specimen.
FIG. 2 is a perspective view for explaining another preferred embodiment (second embodiment) of the present invention.
The test analysis element of FIG. 2 uses an optical fiber as an optical waveguide. An optical fiber including a core portion 22 and a clad portion 23 is provided in the transparent member 1, and a specimen passage hole (microchannel) 21 is provided so as to penetrate the core portion 22. In this embodiment, by using the optical fiber itself, which is an originally good optical waveguide, it is possible to form a micro flow path that goes directly to the optical fiber simply by forming a hole that crosses the core of the optical fiber. There is an advantage that the main part of the blood analysis element can be configured relatively easily without the need for masks associated with stereolithography, optical lithography, etc., and without having to consider the facing accuracy of the core end in the manufacturing process.
In the sample analysis element of this embodiment, the optical fiber is first covered with a transparent liquid resin, and a hole 21 is formed so as to pass through the core portion 22 of the optical fiber perpendicular to the optical fiber using a processing laser. Can be produced. The size of the hole is not particularly limited, but a size that allows blood cells in the specimen to pass individually is preferable.
The optical fiber that can be used in this embodiment is not particularly limited, and a single mode optical fiber having a core diameter of about 3 to 4 μm or a multimode optical fiber having a core diameter of about 10 μm or more can be used. FIG. 2 shows only an example of one optical fiber and one hole (flow channel) penetrating through the core portion. The number of fibers to be arranged and their types, the wavelength of introduced laser light, etc. It is not limited. By changing the type and wavelength of the fiber, it is possible to change the spatial resolution of irradiation with respect to blood cells and to obtain information such as wavelength dependence regarding absorption and scattering.
When laser light is introduced from the other end of the optical fiber, a propagation loss occurs in the hole 21 (usually filled with the sheath liquid), and the light power of the light receiving element 9 arranged on the opposite side is equal to the loss. Detected in a reduced state. As the blood cells in the sheath liquid pass through the opposite part of the optical fiber core 22, the laser light is similarly absorbed and scattered forward and backward, and the laser light power sensed by the light receiving element corresponds to this. Change. The number of circulating blood cells per unit time can be counted by separating / detecting / processing with the low-cycle signal component filter. Further, light absorption by blood cells can be evaluated by comparing with the light receiving power when blood cells are not passing through. Although not shown, a scattered light condensing unit and a light receiving element are provided in the same manner as in FIG. 1, and the light power scattered forward or backward can be detected.
The other configurations, operations, and effects of the present embodiment are substantially the same as those of the first embodiment.
In the first embodiment or the second embodiment described above, the laser light source is disposed separately from the transparent member 1 and functions as a detection element only after the position and optical axis are adjusted including the condenser lens. However, it is possible to embed all or part of the laser light source itself and the metal member serving as the heat sink thereof in the transparent member 1 as well as introducing light into the optical waveguide. The condensing lens can be replaced by applying a notch portion such as the scattered light condensing portion in the embodiment or by providing a minute ball lens in the transparent member. This eliminates the need to individually align and assemble individual optical elements such as the light irradiation system, flow path, condenser lens, and light source, and makes the specimen highly reliable, stable, and impact resistant. An analysis element can be realized.
Also, in the vicinity of the end portion 4d of the light guide rod-like member 4 of the transparent member, a slit or a pinhole-like light shielding member that allows passage of scattered light only in a specific direction is embedded, so that it is scattered / reflected and propagated in other parts. The harmful light (background light, disturbance light) mixed in the light receiving element can be blocked to improve the detection accuracy and accuracy. Further, by covering the periphery of the transparent member 1 with a light shielding metal film, unnecessary ambient light can be similarly blocked, and optical information of the specimen with high accuracy and accuracy can be detected.
FIG. 3 is a perspective view for explaining the outline of another preferred embodiment (third embodiment) of the present invention. In FIG. 3, the flow path 2 and the light guide rod-shaped members 3 and 4 in the transparent member 1 are shown with attention, but the other components such as the laser light source, the light receiving element, and the scattered light condensing part are first implemented. This is the same as the embodiment.
As shown in FIG. 3, a plurality of sets of flow paths 2 and light guide rod-shaped members 3 and 4 may be provided on the transparent member 1. In FIG. 3, the flow path 2 is arranged in parallel, and one set of light guide rod-like members 3 and 4 is arranged for each flow path. Although not shown, the light guide rod members 3 and 4 extend to the end face of the transparent member 1. Each light guide rod-like member is three-dimensionally arranged so as not to physically intersect with other flow paths in order to prevent the influence of other flow paths from being mixed into the optical information propagating in the optical waveguide. . That is, in FIG. 3, each light guide rod-like member 3 and 4 embedded in the transparent member 1 passes under the flow channel 2, and only the flow channel 2 to be measured is opposed to the end surfaces with the flow channel 2 therebetween. Has been placed. Note that a plurality of optical waveguides may be arranged for each flow path, but in this case as well, each optical waveguide is arranged three-dimensionally so as not to physically intersect with other flow paths. In FIG. 3, samples 41 are provided at both ends of each flow path 2.
In addition, in FIG. 3, although the structure of one layer formed in the transparent member was shown, it is good also as a laminated structure by overlapping this.
According to this embodiment, the same specimen is caused to flow through a plurality of formed channels, light of the same wavelength is introduced into the respective optical waveguides, and their responses are observed. Can be evaluated. In addition, by introducing light whose wavelength and polarization direction are changed for each optical waveguide, characteristics such as wavelength (absorption and scattering) and polarization dependence on blood cells can be evaluated in a short time. Furthermore, it is possible to know the characteristics of a large number of components of the specimen at the same time by separating the specimen with a filter or the like, observing the individual optical responses by separating the components for each flow path. On the other hand, by separating the flow path for flowing only liquid components and the flow path for flowing components with blood cells floating in the liquid, and taking the difference between the collected optical response signals, the response of blood cells can be accurately detected from weak signals. It can also be detected separately.
Other configurations, operations, and effects of this embodiment are substantially the same as those of the first embodiment or the second embodiment.
In each embodiment, the configuration in which both the optical waveguide and the flow path are linearly described has been described. However, these may be formed and arranged with a curvature.
The sample analysis element of the present invention is an ultra-small element of several tens of μm to about 1 cm, can be easily carried, and can easily and accurately analyze a sample anytime and anywhere.
Moreover, the sample analysis element of the present invention can be applied to detection of cancer cells contained in not only blood and body fluids but also urine, saliva, sweat, etc., and various liquids such as water and alcohol. It can also be widely applied to the detection of microorganisms therein.
The sample analysis element of the present invention has a configuration in which the end faces face each other with a channel having a refractive index different from that of the base material with a microchannel that flows the sample inside, with a channel having a different refractive index as the base material. By irradiating a sample flowing through the flow path, for example, individual blood cells of blood with laser light, and separating and analyzing optical information such as forward scattering, back scattering, and backward reflection of the laser light, The optical system capable of accurately detecting the number, shape (abnormality of shape), etc. can be integrated on a compact chip-like element, and can be manufactured at low cost.
In addition, the sample analysis element of the present invention can use an optical fiber as an optical waveguide, thereby realizing a simple and highly productive sample analysis element.
In addition, the sample analysis element of the present invention is provided with a cylindrical (convex) lens-shaped scattered light condensing part on the transparent member, so that the light detection element can be reliably disposed without arranging a condensing lens outside the transparent member. Can be scattered light. In addition, the laser light source, its heat sink, and light receiving element can be miniaturized, so by mounting and arranging on the transparent member, individual optics such as light irradiation system, flow path, condenser lens, light source, etc. The labor for aligning and assembling the elements individually can be saved, and a specimen analysis element with high reliability, stability, and impact resistance can be realized.
In addition, the specimen analyzing element of the present invention is arranged such that a pinhole or a slit part is arranged in the vicinity of the end of the light guide rod-like member of the transparent member, so that light harmful to detection (background light) scattered and reflected by parts other than the specimen. Therefore, the optical information of the specimen can be detected with high accuracy and accuracy. Moreover, the sample analysis element of the present invention can detect optical information of a sample with high accuracy and accuracy by covering the periphery of the transparent member with a light shielding metal film to similarly block harmful ambient light.
In the sample analysis element of the present invention, a large number of flow paths are integrated and formed on the transparent member, and the refractive index of the individual flow paths is different from that of the transparent member serving as a base, and the end surfaces separate the flow paths. By arranging one or a plurality of pairs of light guide rod members that are opposed to each other, it is possible to change the irradiation light wavelength for the same specimen and to determine its optical properties in a short time, and to the same wavelength Measures various physiological information with a compact device configuration, such as irradiating blood cells in many specimens with high accuracy to measure individual blood cell characteristics and obtaining the distribution of measurement results as medical information. can do.

本発明の検体分析素子は、超小型化、低消費エネルギー化、ユビキタス化した検体分析システムとして利用でき、血液などの検体サンプルに含まれる細胞の密度、形状、老化・劣化あるいは異常の有無を高精度かつ簡易にかつ微量で、計測および判定することができる。
本発明をその実施態様とともに説明したが、我々は特に指定しない限り我々の発明を説明のどの細部においても限定しようとするものではなく、添付の請求の範囲に示した発明の精神と範囲に反することなく幅広く解釈されるべきであると考える。The specimen analysis element of the present invention can be used as an ultra-compact, low-power-consumption, ubiquitous specimen analysis system that increases the density, shape, aging / deterioration or abnormality of cells contained in specimen samples such as blood. Measurement and determination can be performed with accuracy, simplicity, and a small amount.
While this invention has been described in conjunction with its embodiments, we do not intend to limit our invention in any detail of the description unless otherwise specified and are contrary to the spirit and scope of the invention as set forth in the appended claims. I think it should be interpreted widely.

Claims

A transparent member having a microchannel formed therein, and the end surfaces of the transparent member embedded in the transparent member and opposed to each other across the microchannel formed in the transparent member, and being refracted from the transparent member A specimen comprising a pair of light guide rod-like members having different rates and a cylindrical convex lens-like scattered light condensing portion embedded in the transparent member for condensing the scattered light from the measuring portion Analytical element.

The pair of light guide rod-shaped members are made of a single optical fiber, and the micro flow path formed in the transparent member is formed perpendicular to the optical fiber and penetrating through the core of the optical fiber. The sample analysis element according to claim 1.

The sample analysis element according to claim 1, wherein a laser light source and / or a light receiving element is attached to the transparent member.

The sample analysis element according to claim 1, wherein a pinhole or a slit portion is disposed in the transparent member.

The specimen analysis element according to claim 1, wherein a periphery of the transparent member is covered with a metal film.

The sample analysis element according to any one of claims 1 to 5, wherein a plurality of pairs of the pair of light guide rod members are provided.

A plurality of flow paths are formed in the transparent member, and each of the flow paths is embedded in the transparent member and the end faces thereof are arranged to face each other across the flow path formed in the transparent member, and The sample analysis element according to claim 1, wherein one set or a plurality of sets of a pair of light guide rod-like members having a refractive index different from that of the transparent member are arranged.