JP2017530371A - 電気化学的バイオセンサ及びアルブミンとその複合体の検出方法 - Google Patents
電気化学的バイオセンサ及びアルブミンとその複合体の検出方法 Download PDFInfo
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- JP2017530371A JP2017530371A JP2017532224A JP2017532224A JP2017530371A JP 2017530371 A JP2017530371 A JP 2017530371A JP 2017532224 A JP2017532224 A JP 2017532224A JP 2017532224 A JP2017532224 A JP 2017532224A JP 2017530371 A JP2017530371 A JP 2017530371A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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Abstract
Description
図6a、b、c、d、e及びfに示すこれらの実施形態は、血液サンプル中の総アルブミン及びグリコアルブミン成分を除くアルブミンを測定するために用いられる。
MB+2e−+H+→LMB
LMB+2Fe+++→MB+2Fe++
14.1gのNaCl、2.8gのKCl、17.3gの尿素、19mlのアンモニア水(25%)、0.60gのCaCl2及び0.43gのMgSO4を0.02モル/LのHClに溶解して合成尿を調製する。HCl及びアンモニア水を使って、合成尿の最終pHを6.04に調節する。蒸留水中に水酸化ナトリウムが0.1〜1Nの溶液20mlに、1〜6mgのヘミンを溶解させる。溶液を蒸留水でさらに希釈する。ヘミン溶液の最終容積は40mlであり、pHは11.5である。容積20μLのヘミン溶液を受容体として用いて、尿中アルブミンを検出する。3mgのヒトアルブミンを10mlの合成尿溶液に溶解し、微量アルブミン溶液を調製する。この原液を適切に希釈することにより、様々な濃度の微量アルブミン溶液を調製する。濃度の異なる微量アルブミン溶液を一定量の受容体(例えば20μL)と予混合し、試験用に最終容量300μLとする。
y=−1.96x+209
上記の式において、「y」は酸化還元電流値を表し、「x」は検体濃度を表す。
試料容積300μLの合成尿を1〜5μgのヘミン受容体を有する電極に載せ、電位窓を0V〜−1.4Vに指定してCH Instruments社のelectrochemical workstationを使ったサイクリックボルタモグラムから、還元ピーク電流値を得る。還元ピーク電流の値は200μAである。この電流値を表1に当てはめると、対応する尿中アルブミン濃度5mg/Lが得られる。
14.1gのNaCl、2.8gのKCl、17.3gの尿素、19mlのアンモニア水(25%)、0.60gのCaCl2及び0.43gのMgSO4を0.02モル/LのHClに溶解して合成尿を調製する。HCl及びアンモニア水を使って、合成尿の最終pHを6.04に調節する。5〜25mgのCuCl2を50mlの人工尿溶液に溶解する。容積20μLのこの溶液を受容体として用いて、尿中アルブミンを検出する。2mgのヒトアルブミンを10mlの合成尿溶液に溶解し、微量アルブミン溶液を調製する。この微量アルブミン溶液を20μLの微量の受容体と既知濃度で予混合し、最終容量220μLとする。
y=−0.0103x+4.363
上記の式において、「y」は酸化還元電流値を表し、「x」は検体濃度を表す。
試料容積220μLの合成尿を2〜6μgのCuCl2受容体を有する電極に載せ、電位窓を0.6V〜−0.4Vに指定してCH Instruments社のelectrochemical workstationを使ったサイクリックボルタモグラムから、酸化還元ピーク電流値を得る。還元ピーク電流の値は4.17μAである。この電流値を表2に当てはめると、対応する尿中アルブミン濃度18.2mg/Lが得られる。
14.1gのNaCl、2.8gのKCl、17.3gの尿素、19mlのアンモニア水(25%)、0.60gのCaCl2及び0.43gのMgSO4を0.02モル/LのHClに溶解して合成尿を調製する。HCl及びアンモニア水を使って、合成尿の最終pHを6.04に調節する。MBを脱イオン水に溶解させる。10〜45mgのMBを10mlの脱イオン水に溶解させる。1〜10mgのヘミンを40mlの合成尿に溶解させる。容積9μL(ヘミン5μL+MB4μL)のこの溶液を受容体として用いて、尿中アルブミンを検出する。3mgのヒトアルブミンを10mlの合成尿溶液に溶解し、微量アルブミン溶液を調製する。この微量アルブミン溶液を9μLの微量の受容体と既知濃度で予混合し、最終容量300μLとする。
y=−1.288x+189
試料容積300μLの合成尿を、MBが5〜15μgとヘミンが0.1〜1μgであるMB−ヘミン受容体を有する電極に載せ、電位窓を0.6V〜−0.4Vに指定してCH Instruments社のelectrochemical workstationを使ったサイクリックボルタモグラムから、酸化還元ピーク電流値を得る。還元ピーク電流の値は187.9μAである。この電流値を表3に当てはめると、対応する尿中アルブミン濃度5mg/Lが得られる。
14.1gのNaCl、2.8gのKCl、17.3gの尿素、19mlのアンモニア水(25%)、0.60gのCaCl2及び0.43gのMgSO4を0.02モル/LのHClに溶解して合成尿を調製する。HCl及びアンモニア水を使って、合成尿の最終pHを6.04に調節する。1〜10mgのMBを10mlの合成尿に、5〜35mgのCuCl2を30mlの合成尿に溶解させ(例えば、CuCl240μL+MB30μL)、尿中アルブミンを検出する。2mgのヒトアルブミンを10mlの合成尿溶液に溶解し、微量アルブミン溶液を調製する。この微量アルブミン溶液を70μLの少量の受容体と既知濃度で予混合し、最終容量220μLとする。
y=0.1098x+0.6903
試料容積220μLの合成尿を、MBが10〜50μgとCuCl2が5〜55μgであるMB−CuCl2受容体を有する電極に載せ、電位窓を0V〜−0.5Vに指定してCH Instruments社のelectrochemical workstationを使ったサイクリックボルタモグラムから、酸化還元ピーク電流値を得る。還元ピーク電流の値は30.8μAである。この電流値を表4に当てはめると、対応する尿中アルブミン濃度9.09mg/Lが得られる。
アルブミン濃度が34.02g /Lのヒト血漿を原液として採取する。血漿を希釈して、アルブミン濃度を5g/lから32.09g/lにする。CuCl2受容体のストック溶液を70〜150g/L濃度で塩水を使って調製し、20μLの少量を希釈血漿溶液と予混合し、総量300μLを電極表面に載せて、還元ピーク電流値を測定する。
y=−19.28x+716.8
試料容積300μLの合成尿を、CuCl2が1〜10μgであるCuCl2受容体を有する電極に載せ、電位窓を1V〜−0.6Vに指定したCH Instruments社のelectrochemical workstationを使ったサイクリックボルタモグラムから、酸化還元ピーク電流値を得る。還元ピーク電流の値は676.8μAである。この電流値を表5に当てはめると、対応する尿中アルブミン濃度5g/Lが得られる。
ヘミン(Sigma Aldrich社)をアルカリ溶液に公知の方法で溶解してメトヘムアルブミンを調製する。この溶液を5mlの2%ヒトアルブミンと混合し、1NのHClでpHを7.4に調節する。 1〜7mgのMBをpH7で10mlのPBSに溶解する。濃度の異なるメトヘムアルブミン溶液をMB溶液に添加し、還元ピーク電流値を得る。還元ピーク電流値は、走査速度0.1V/秒、−0.2Vから−0.5Vまでの様々な電位窓でCH Instruments社のelectrochemical workstationを使用したものである。メトヘムアルブミン量をその対応する還元電流値(μA)と共に記録し、表6に示すような表にまとめる。表6は、以下に示す線形フィット方程式から作成することができる。
y=1.006x+41.19
101 基板
102a、102b、102c、102d、102e、102f 導電路
103a、103b、103c、103d、103e、103f 電極
104 薄膜
105 アルブミン結合性かつ電気化学的に活性の受容体
106 パッシベーション層
107 第2の薄膜
108 化学物質層
200 装置ホルダ
201 ハウジング
202 USBプラグ
203 装置挿入口
204 生体試料
300 ポイントオブケア用バイオセンサ
301 ハウジング
302 マイクロUSB
303 マイクロSDカード
304 表示部材
305 装置挿入口
306 データベース部材
307 デジタル制御部(マイクロコントローラ)
308 電源装置
309 信号調整・装置検出装置
310 湿度センサ
311 温度センサ
Claims (21)
- (i)基板に配置された少なくとも一対の導電路と、
(ii)前記導電路に接続された少なくとも1つの二電極部材と、
(iii)アルブミン結合性かつ電気化学的に活性の受容体と、を備え、前記受容体は前記少なくとも1つの二電極部材及びバイオ検体を含む生体試料と化学的に接触することを特徴とする、
生体試料を採取及び保持するための電気化学的に活性の装置。 - 前記基板がポリマ又は紙である、請求項1に記載の装置。
- 1つの三電極部材が前記基板に配置される、請求項1に記載の装置。
- 複数の二電極部材が前記基板に配置される、請求項1に記載の装置。
- 複数の三電極部材が前記基板に配置される、請求項1に記載の装置。
- 前記電極部材はパターン電極である、請求項1に記載の装置。
- 薄膜が前記電極部材に配置される、請求項1に記載の装置。
- 前記薄膜を、ボロン酸、フェニルボロン酸(PBA)、アミノフェニルボロン酸(APBA)と、その誘導体からなる群から選択されるボロネート親和剤で、好ましくはアミノフェニルボロン酸(APBA)で処理する、請求項7に記載の装置。
- 前記受容体は、少なくとも有機物、無機物、金属ポルフィリン物質であり、好ましくはヘミン、ヘマチン、アルカリヘマチン、塩化銅(CuCl2)、銅塩(Cu(II))、メチレンブルー、メチレンブルーとヘミン、ヘマチン、アルカリヘミン又はアルカリヘマチンとの組合せ、及び、メチレンブルーと銅塩(Cu(II))との組み合わせからなる、請求項1に記載の装置。
- 前記アルブミンは、尿中アルブミン、フィト血清アルブミン(HSA)、グリコアルブミン(GA)又はメトヘムアルブミン(MHA)である、請求項1に記載の装置。
- 前記装置は、ハウジングの内部に配置され、前記ハウジングはカートリッジ又はカセットである、請求項1に記載の装置。
- 電気化学的に活性の装置を保持するホルダであって、
(i)ハウジングの内部に配置された装置を検出し信号を調整する手段と、
(ii)前記ハウジングの一端に配置されたUSBコネクタと、前記ハウジングの他端に配置された導電ポートと、
(iii)生体試料を採取及び保持するために前記導電ポートを介して前記ハウジングに接続するように配置された電気化学的に活性の装置と、を備え、
前記装置は、少なくとも一対の導電路と、少なくとも1つの二電極部材と、アルブミン結合性かつ電気化学的に活性の受容体とが基板に配置され、前記受容体は前記少なくとも1つの二電極部材及びバイオ検体を含む前記生体試料と化学的に接触することを特徴とするホルダ。 - 生体試料中のバイオ検体濃度を測定するためのポイントオブケア用バイオセンサであって、
(i)表示部材と導電ポートとを有するハウジングと、
(ii)生体試料を採取及び保持するために前記導電ポートを介して前記ハウジングに接続するように配置された電気化学的に活性の装置であって、少なくとも一対の導電路と、少なくとも1つの二電極部材と、アルブミン結合性かつ電気化学的に活性の受容体とが基板に配置され、前記受容体は前記少なくとも1つの二電極部材及びバイオ検体を含む前記生体試料と化学的に接触する装置と、
(iii)前記ハウジングの内部に配置され、前記装置に印加される酸化還元電位から酸化還元電流を測定し、アルブミン濃度に直線的にマッチングさせることによりアルブミンバイオ検体濃度を読み取り表示するように構成されたデジタル制御部と、を備えることを特徴とするバイオセンサ。 - データベース部材は、前記デジタル制御部に接続され、相互作用する酸化還元電流と併せて生体試料中のアルブミンバイオ検体濃度の基準値を記憶する、請求項13に記載のポイントオブケア用バイオセンサ。
- (a)アルブミン結合性かつ電気化学的に活性の受容体に化学的に接触し、少なくとも1つのバイオ検体を含む少量の生体試料を載置した少なくとも1つの二電極部材に酸化還元電位を印加する工程と、
(b)前記少なくとも1つの二電極部材の対応する酸化還元電位に直線的にマッチングさせることにより生体試料中の少なくともアルブミンバイオ検体の濃度を測定する工程と、
を有する生体試料中のバイオ検体濃度を測定する方法。 - 前記生体試料は、フィトの血液及び尿である、請求項15に記載の方法。
- 前記少量は、1〜300マイクロリットル(μL)の範囲である、請求項15に記載の方法。
- 薄膜が前記少なくとも1つの二電極部材に配置される、請求項15に記載の方法。
- 前記薄膜を、ボロン酸、フェニルボロン酸(PBA)、アミノフェニルボロン酸(APBA)及びその誘導体からなる群から選択されるボロネート親和剤で、好ましくはアミノフェニルボロン酸(APBA)で処理する、請求項15に記載の方法。
- 前記受容体は、少なくとも有機物、無機物、金属ポルフィリン物質であり、好ましくはヘミン、ヘマチン、アルカリヘミン、アルカリヘマチン、塩化銅(CuCl2)、銅塩(Cu(II))、メチレンブルー、メチレンブルーとヘミンとの組合せ、及び、メチレンブルーと銅塩(Cu(II))との組み合わせからなる、請求項15に記載の方法。
- 前記アルブミンは、尿中アルブミン、フィト血清アルブミン(HSA)、グリコアルブミン(GA)又はメトヘムアルブミン(MHA)である、請求項15に記載の方法。
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WO2016038505A3 (en) | 2016-05-19 |
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US11435344B2 (en) | 2022-09-06 |
US20170241996A1 (en) | 2017-08-24 |
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WO2016038505A2 (en) | 2016-03-17 |
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