JP5401724B2 - 被覆磁性微粒子を用いたバイオセンシング方法及び該方法に用いるバイオセンシング装置 - Google Patents
被覆磁性微粒子を用いたバイオセンシング方法及び該方法に用いるバイオセンシング装置 Download PDFInfo
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Description
反応モデル系の調製
本実施例においては、脳性ナトリウム利尿ペプチド(BNP、Brain Natriuretic Peptide)を検出するためのサンドイッチELISAを反応モデル系とした。反応モデル系はまず、脳性ナトリウム利尿ペプチド(以下、BNPとして参照する)の抗体であるBC203(塩野義製薬株式会社)を金基板(5mm x 5 mm)上に吸着させ固相化し、併せて粒径約 200 nmのポリマー被覆磁性微粒子(以下、FGビーズとして参照する)、粒径約27nmのクエン酸被覆磁性微粒子、市販の磁性粒子であるDYNAビーズ(粒径2.8μm、DYNAL、カルボキシル基)の3種類の磁性微粒子の表面にBNPのもう一つの抗体であるKY2(塩野義製薬株式会社)を固定化することで形成した。なお、上述したそれぞれの抗体−磁性微粒子複合体については、単位磁性微粒子重量あたりに固定化される抗体量が等しくなるように調製した。
48穴プレートのウェル内に、BC203を吸着させて固相化した金基板を静置し、200μLの反応溶液(10 mM Hepes(pH7.9), 50 mM KCl, 1.0 mM EDTA, 0.1% Tween20, 0.03% skimmilk)で満たした。次に、当該ウェルにBNP(ペプチド研究所)を1ng添加し、ELISA用プレートシェイカー(NISSIN)上で5分間振とうした後、KY2−FGビーズ複合体を上記反応溶液で浸漬された金基板上に添加した。その後、48穴プレートの下方より磁石を近づけた状態で所定時間(1分、10分、100分)静置した。所定時間経過後、金基板を洗浄溶液(10mM Hepes (pH7.9), 50 mM KCl, 1.0 mM EDTA, 0.1% Tween20)で3回洗浄した。最後に、各金基板をMilli Qで洗浄した後、走査型電子顕微鏡で観察した。併せて、バックグラウンドとして、BNPを添加しない反応系についても上述したのと同様の手順で実験を行ない、さらに、比較例として、磁石を用いない反応系についても上述したのと同様の手順で実験を行なった。また、バックグラウンドおよび比較例についても反応後の金基板を走査型電子顕微鏡で観察した。走査型電子顕微鏡の観察結果から表面被覆密度(%)を算出した。なお、本実施例において表面被覆密度(%)は、下記式(1)で定義されるものであり、走査型電子顕微鏡で観察された5視野を平均して算出した。
異なる量(1〜105pg)のBNPを添加した反応系について、上述したのと同様の条件で実験を行ない、それぞれの反応系についての表面被覆密度(%)を算出した。図6は、反応系に添加したBNP量(pg)と表面被覆密度(%)との関係を示す。図6に示されるように、BNP量(pg)の増加に従って表面被覆密度(%)が増加しており、一定の濃度依存性を示した。
粒径の異なる3つの磁性微粒子について、基板に対する吸着安定性を比較検証した。上述したのと同様の条件のウェルに対し、KY2−クエン酸被覆微粒子複合体(粒径約27 nm)、KY2−FGビーズ複合体(粒径約 200 nm)、およびKY2−DYNAビーズ複合体(粒径2.8μm)をそれぞれ加え、金基板表面の垂直方向に磁力を働かせた状態で10分間反応させた後、それぞれの反応系について表面被覆密度(%)を算出した。併せて、別に調製した同様の3つの反応系について、ELISA用プレートシェイカー(NISSIN)上で振とうさせながら10分間反応させた後、それぞれの反応系について表面被覆密度(%)を算出した。下記表2は、上述した3つの複合体についての表面被覆密度(%)を示す。併せて、図7に、表2の結果を棒グラフにして示した。
本実施例においては、DNAハイブリダイゼーションを反応モデル系とした。蛍光を具備した粒径200 nm のFGビーズ上に35塩基の一本鎖DNA(つくばオリゴサービス)を固定化しDNA−蛍光FGビーズ複合体を調製した。ガラス基板 (5 mm x 5mm) 表面上にFGビーズ上に固定化した一本鎖DNAの相補鎖DNA(35塩基)を吸着させ固相化した。48穴プレートのウェル内に相補鎖DNAが固相化したガラス基板を置き、200μLの反応溶液(10 mM Hepes(pH7.9), 50 mM KCl, 1.0 mM EDTA, 0.1% Tween20)で満たした。DNA−蛍光FGビーズ複合体を反応溶液に浸漬されたガラス基板に対して異なる密度(DNA分子数/1μm四方)で添加し、48穴プレートの下方より磁石を近づけながらELISA用プレートシェイカー(NISSIN)で1分間振とうした。洗浄溶液(10mM Hepes(pH7.9), 50 mM KCl, 1.0 mM EDTA, 0.1% Tween20)で3回洗浄後、蛍光顕微鏡でガラス基板上に吸着されたDNA−蛍光FGビーズ複合体を蛍光顕微鏡により観察し、蛍光カウント値を測定した。図8は、反応系に添加したDNA−蛍光FGビーズ複合体の密度(DNA分子数/1μm四方)と蛍光カウント値との関係を示す。図8に示されるように、DNA−蛍光FGビーズ複合体の密度の増加に従って蛍光カウント値が増加しており、一定の濃度依存性を示した。
Claims (3)
- アフィニティ反応を利用したバイオセンシング方法であって、
前記アフィニティ反応におけるリガンドを平均粒径が10〜200nmの単分散性のポリマー被覆磁性微粒子であって、ポリマー層に蛍光物質が導入された微粒子に固定化し、該ポリマー被覆磁性微粒子を前記アフィニティ反応の反応場に向けて強制的に数分間磁気誘導する工程と、
前記反応場を振とうしながら洗浄する工程と、
蛍光検出器を用いてセンシングを行なう工程とを含む
方法。 - 前記反応場から遠ざかる方向に前記磁性微粒子を磁気誘導しながら洗浄する工程をさらに含む、請求項1に記載の方法。
- 前記磁性微粒子は、官能基を含む分子によって被覆された磁性微粒子である、請求項1または2に記載の方法。
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