JP2017529999A - 生物高分子を分離するための吸着剤 - Google Patents
生物高分子を分離するための吸着剤 Download PDFInfo
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- JP2017529999A JP2017529999A JP2017513396A JP2017513396A JP2017529999A JP 2017529999 A JP2017529999 A JP 2017529999A JP 2017513396 A JP2017513396 A JP 2017513396A JP 2017513396 A JP2017513396 A JP 2017513396A JP 2017529999 A JP2017529999 A JP 2017529999A
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- Prior art keywords
- silica
- adsorbent
- silanized
- methacrylate
- dna
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- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
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Abstract
Description
タンパク質の物理吸着は、基板と分子の間の静電相互作用および疎水性相互作用に関連する多くの因子に影響される複雑なプロセスである。改質表面へのタンパク質の吸着は、表面の濡れ性に対し全体的な依存性を呈すことが示される。タンパク質は疎水性表面(高接触角)に選択的に吸着する。リゾチーム(14.3kDa、pl11.4)などの小タンパク質は、疎水性表面に対し弱い吸着を示す。ウシ血清アルブミン(66.5kDa、pl4.7)などのより大きいタンパク質は、親水性表面(低接触角)により吸着する。
マイカおよび二酸化ケイ素などのSi−O機能性を示す表面へのDNAの吸着がよく知られている。適切なアルキルシランを用いたこのような表面の処置は、基板を改質して疎水性表面終端を生成する。このような処置は、DNAとの表面相互作用を低減し、それによりDNA付着に対する識別を促すことが知られている。
DNA吸着を排除しながら大きいおよび小さいタンパク質を吸着するための優れたバランスの表面を見つけるため、当社はある範囲のモノマーユニットとその組み合わせについて、その表面濡れ性特性の関数として性能特性を評価した。ここで、窒素ドープSi/SiO2ウェハをきれいにし、各モノマー系で改質し、サンプルの範囲に亘り接触角を測った。表1に、研究した16個の異なるポリマー系の結果をまとめる。
タンパク質の物理吸着は、基板と分子の間の静電相互作用および疎水性相互作用に関連する多くの因子に影響を受ける、複雑なプロセスである。水素結合、ファンデルワース力、π−πスタッキング、配位、およびモノマーユニットの適切な機能化を介した分散力などの、非共有結合性相互作用の選択的制御が、生物学的種の所与の基板への吸着/反発の選択的指向に関与し得る。
ポリマーを用いたシリカの改質は、典型的には、所望の物質で細孔を充填させ、これは本質的には細孔内でポリマーブラシを伸ばす。上記のように、流体力学的半径は、分析物が入り、細孔により保持されることが可能であるような、適切なサイズとするべきである。この場合、当社はこれをアクティブ表面積と定義した。しかしながら、当社は、システムのサイズ排除メカニズムの説明のために、アクティブ表面積は、より正確には、孔径および粒子間の平均介在間隔と記載されることを認める。
上記のように、クロマトグラフィーカラムで述べた任意の粒度は、実際には、自動sub-sieve定寸技法を用いて製造業者により実質的に得られる粒度分布の平均値である。実際には、粒子の広範な分布はカラム内の不均一性につながり、これは、分析分子がカラムを通過する際に通る可変経路長の増加を招き得る。したがって、均一性はテスト間の変動を低減させる鍵である。可変粒度の影響は、均一なサイズ分布のサンプルと、高度な不均一性を有するサンプルの、qPCRを用いて解析されるDNA抽出能力を比較すれば明らかである。表5は、>55%のサイズ分布を有する物質(サンプルA)と、サイズ分布が12.5%変動する物質(サンプルB)について、qPCRを用いて解析したDNA抽出能力を示す。
記載する各実施形態の基本的な役割を完全に示すため、吸着剤を記載するパラメータに従って生成した。各物質の生物学的性能が、効率的なDNA抽出における、平均介在間隔、適切なポリマー選択、孔径、およびアクティブ表面積の具体的基準を示す。
シリカ粒子
15μm表面積80〜120m2/g
ファイバの表面積=2πrL
Oe=5/ファイバ数〜800/ファイバ数
上記のやり方で生成したベンジルメタクリレートナノファイバ(0.2g)を5mLのシリンジに入れた。手動の圧力を用い、ファイバを2分間圧縮して計画的な4×10−3の経路長を提供した。サンプルを次に40℃で30分間加熱し、続いてゆっくりと冷却した。活性化緩衝液(700μL)をシステムに揚水してから80μLの溶解物を加えた。流体をシステムに押し通し、溶出液を解析用に保持した。図7はこのやり方で調製したシリンジの典型的な性能を示す。
シリカの平均介在間隔(15μm、300Å)を、均一な充填を仮定して6.95μmと計算した。平均介在間隔はまた表面積の関数であり、2つのパラメータの比である。モノマーユニットを固定し、(上記のように)表面積を定義することにより、新しい構造を適切な寸法で形成して血液溶解物からDNAを抽出した。表6に示すように、約6μm未満または約23μm超の平均介在間隔を有する例では、許容可能な解析レベルでDNAを精製することはできない。
粒子分布/細孔半径
粒度および細孔半径の広範な分布はカラム内の不均一性に、ひいては分析分子がカラム内で通る可変経路長の増大につながるだろう。DNA抽出効率はこのような場合ひどく損なわれ、しばしば高いタンパク質濃度をまねく。
アクティブ表面積は、DNA抽出効率において重要な役割を果たす。上記のように、当社は、少なくとも一部の実施形態における最適な範囲は、0.1m2〜150m2であると理解する。少なくとも一部の実施形態では、具体的な範囲にそれぞれ該当する、記載のパラメータの個々の組み合わせは、血液溶解物からDNAを抽出する吸着剤の機能を決定する役割を果たし得る。
多孔質シリコンウェハの合成
p−Si(100)ウェハを形成した多孔質シリコン層の定電流陽極酸化。これを48%のHF水溶液とエタノール溶液の1:1v/v溶液を含むセルを用いて行った。電気化学セルはPTFEから構成され、これは円形断面を有した。シリコンウェハを、テフロン(登録商標)被膜バイトン(登録商標)O−リングを用いて基板に封着した。対向電極をループ状に巻き取ったタングステンワイヤで構成した。これは均一な電流分布を提供した。高電流密度(500mAcm2で5分)を用いて多孔質シリコンの層を生成した。図11は多孔質ベアシリコン(左)シラン改質多孔質シリコン(中)、およびベンジルメタクリレートで改質した多孔質シリコンを示し、図12は多孔質ベアシリコンのAFM解析を示す。
300Åシリカ(2.20g)を200℃の真空下で3時間、続いて室温のハウス真空(house vacuum)下で一晩乾燥させた。50mLの丸底フラスコとスターラーを一晩乾燥機で乾燥させた。
300Å、15μm(シラン化シリカ)を用いた重合
水中(4.8mL)のステアリン酸ナトリウム(67mg)の撹拌溶液に、シリカ、モノマー、およびペルオキソ二硫酸カリウム(2.4mg)を加えた。重合を95℃で4時間行った。混合物を室温まで冷却し濾過した。結果として生じたシリカをDMF(5×5mL)、エタノール(5×5mL)、および水(5×5mL)で洗浄してから室温で30分間風乾させた。シリカを最終的に真空で3時間乾燥させながら室温を慎重に80℃まで上げ、水で飽和させた際にトラップを変えた。
300Åシリカ(2.20g)を200℃の真空下で3時間、続いて室温のハウス真空下で一晩乾燥させた。50mLの丸底フラスコとスターラーを一晩乾燥機で乾燥させた。
300Åシリカ(1.20g)を200℃の真空下で3時間、続いて室温のハウス真空下で一晩乾燥させた。50mLの丸底フラスコとスターラーを一晩乾燥機で乾燥させた。
300Åシリカ(1.20g)を200℃の真空下で3時間、続いて室温のハウス真空下で一晩乾燥させた。50mLの丸底フラスコとスターラーを一晩乾燥機で乾燥させた。
シリカ粒子の混合物を、相補的または同一のポリマー系で、例えば、限定されるわけではないが、ある実施形態ではスチレンおよび2-ヒドロキシエチルメチルメタクリレートモノマー系をそれぞれ用いて、2つの別箇のバッチの物質をまず改質することにより調製した。所望の比率の粒子混合物は、抽出されるサンプル型に応じて抽出特性を確定するために、組成物を質量組成の比率により調整することを可能にする。
シリコンチップ(バルクで多孔質、1cm2)を200℃の真空下で3時間、続いて室温のハウス真空下で一晩乾燥させた。50mLの丸底フラスコとスターラーを一晩乾燥機で乾燥させた。
ポリビニルアルコール(PVA)
10重量%のPVA(mw89000〜98000)溶液を、銅棒に貼ったアルミホイルに揚水した。針とコレクタの間の距離は16cmであり、20kVの電圧を印加した。ポリマー溶液の流速は0.075mL/hだった。環境条件:19%R.H.、20.3℃。
THF溶液中の37.5重量%のP(BzMA)を、銅棒に貼ったアルミホイルに揚水した。針とコレクタの間の距離は10cmであり、20kVの電圧を印加した。ポリマー溶液の流速は0.05mL/分だった。環境条件:21%R.H.、20.6℃。図15は、ポリベンジルメタクリレート(P(BzMA))の電界紡糸マットの光学像を示す。
17.5%w/wポリマー/アセトンにおいて、溶液を調整し、室温で水中に一晩放置した。あるいは、ホイル上の2cm2の正方形のファイバマットを、40℃の水中において、緩酸(1Mエタン酸)、エタノールと水の50:50の組み合わせ、メタノールと水の50:50の組み合わせで処置した。
表8に、本開示の一部の実施形態で用いることが可能ないくつかの他のモノマーを列挙する。
サンプル事前処置
ガラスの事前処置ができないプラスチック容器で生成したサンプルを除く全てのモノリスサンプル調整物について、以下の基本的な手順を辿った。0.1M水酸化ナトリウム洗浄(5分)、脱イオン水洗浄(20分)、メタノール洗浄(5分)、窒素流下での乾燥、3-(トリメトキシシリル)プロピルメタクリレート(MSMA):メタノール(1:1)の注入、容器端の封止、35℃の水槽への容器の浸漬(17時間)、メタノール洗浄(13分)、脱イオン水洗浄(13分)、窒素流下での乾燥。
事前コンディショニングの直後、容器を、ラジカル重合を介した、機能化のための重合反応混合物で充填することができた。モノマー、ラジカル開始剤、およびポロジェニック(porogenic)溶媒系からなる、キャピラリまたは他の容器に注入される反応混合物を、このように調整した。モノマー混合物は予め混ぜられ、種々の比率のスチレン(Sty)、ブチルメタクリレート(But)、および/またはベンジルメタクリレート(Bnz)を、ジビニルベンゼン(DVB)またはエチレングリコールジメタクリレート(EDMA)という架橋剤とともに含んだ。活性剤、ビニルベンゼンスルホン酸(VBSA)、および/またはアゾビスイソブチロニトリル(AIBN)を次に加えた。2つのポロジェニック溶媒系のうち1つを次に反応混合物(シクロヘキサノール、水、およびNMPまたは1-プロパン-1-オール、1,4-ブタンジオール)に加えた。モノマー混合物を超音波処理した(15分/均一化まで)。次にモノリス容器にモノマー混合物を注入し、端を封止した。残ったモノマー混合物をガラス容器に移した。容器を70℃の水槽に20時間浸漬するか、または、UV光で15分間処置した。サンプルをメタノールで洗浄した(15分)。
一例では、DNA抽出カセット(DEC、図18)−吸着剤を保持するように設計されている−を用いて、40μLの(ビーズ粉砕を用いた機械的溶解により生成された)血液溶解物または溶解緩衝液のみからDNAを精製した。上記の作業は、吸着剤で充填したDEC(表2〜5)がDNAを血液溶解物から精製可能であることを示した。図21は、連続流路DECを用いた大腸菌の抽出を示す。図は、濃度を低くした大腸菌サンプル(細胞/mL)由来のインプット溶解物質から生じたDECの100μL画分における、溶出DNAの濃度(pg/mL)を示す。
Claims (22)
- 生物高分子を分離するための吸着剤であって、ポリ(アリールメタクリレート)、ポリ(アリールアクリレート)、ポリ(ヘテロアリールメタクリレート)、ポリ(ヘテロアリールアクリレート)およびそれらのコポリマーからなる群から選択されるポリマーで少なくとも部分的に覆われた、または形成されたシラン化物質を含む、吸着剤。
- 前記シラン化物質は、シラン化シリカ粒子、シラン化シリカファイバ、およびシラン化シリカのモノリシックな膜または構造からなる群から選択される無機物質である、請求項1に記載の吸着剤。
- 前記シラン化物質は、多孔質有機物質および多孔質有機膜からなる群から選択される有機物質である、請求項1に記載の吸着剤。
- 前記ポリマーは、アニソールメチルメタクリレート、フェニルエタノールメタクリレート、ピリジンメチルメタクリレート、およびナフタレンメチルメタクリレートからなる群から選択される反復ユニットを含む、請求項2または3に記載の吸着剤。
- 前記吸着剤は約65°〜約100°の範囲の濡れ性値を有する、請求項1〜4の何れかに記載の吸着剤。
- 前記吸着剤の表面積は約0.1m2〜約130m2の範囲である、請求項1〜5の何れかに記載の吸着剤。
- 前記シラン化無機物質には、平均孔径が約1nm〜約100nmの範囲のシラン化シリカ粒子が含まれる、請求項1〜6の何れかに記載の吸着剤。
- 前記平均孔径は約30nmである、請求項7に記載の吸着剤。
- 前記シラン化無機物質には平均直径が約200ミクロン未満のシラン化シリカ粒子が含まれる、請求項1〜8の何れかに記載の吸着剤。
- 前記平均直径は約15ミクロンである、請求項9に記載の吸着剤。
- 請求項1〜10の何れかに記載の吸着剤を含む成形品であって、微粒子状、モノリシック状、または膜状から選択される形状で、平均介在距離は約10nm超である、成形品。
- 前記平均介在距離は約12ミクロン未満である、請求項11に記載の成形品。
- 前記成形品は膜形状で、前記吸着剤は多孔質の有機または無機のマトリックスに組み込まれている、請求項12に記載の成形品。
- ポリ(ベンジルメタクリレート)で少なくとも部分的に覆われたシラン化シリカを含む吸着剤を作成する方法であって、
シリカをジメチルビニルクロロシランのトリフルオロトルエン溶液に懸濁するステップと;
前記液体を取り除き、前記シリカを新規のジメチルビニルクロロシランのトリフルオロトルエン溶液に再懸濁するステップと;
前記液体をオプションとして取り除き、前記シリカを再度、新規の5%のジメチルビニルクロロシランのトリフルオロトルエン溶液に再懸濁するステップと;
結果として生じるシラン化シリカを集め、乾燥させるステップと;
前記シラン化シリカ、ベンジルメタクリレート、およびペルオキソ二硫酸カリウムを、ステアリン酸ナトリウムの撹拌水溶液に加えるステップと;
結果として生じる、ポリ(ベンジルメタクリレート)で覆われたシラン化シリカを含む吸着剤を集め、乾燥させるステップと、を含む方法。 - 生体サンプルを溶解し、前記生体サンプルから核酸を単離するための二重処理装置あって、
サンプルインプットユニットと、
サンプル溶解ユニットと、
核酸抽出ユニットと、
核酸受付ユニットとを含み、
前記核酸抽出ユニットは、ポリ(アリールメタクリレート)、ポリ(アリールアクリレート)、ポリ(ヘテロアリールメタクリレート)、ポリ(ヘテロアリールアクリレート)およびそれらのコポリマーからなる群から選択されるポリマーで少なくとも部分的に覆われた、または形成されたシラン化物質を含み、前記シラン化物質は、前記生体サンプル由来のタンパク質および他の非核酸を保持し、かつ前記核酸は前記核酸受付ユニットに入ることができるように構成されている、二重処理装置。 - 前記サンプル溶解ユニットは、前記生体サンプルを溶解するように前記サンプル溶解ユニット内で回転するように構成されたブレードを含む、請求項15に記載の装置。
- 前記サンプル溶解ユニットはビーズを含む、請求項15に記載の装置。
- 前記装置はさらに、前記サンプル溶解ユニットのブレードの回転を制御するように構成されたスプリング搭載プランジャを含む、請求項16に記載の装置。
- 前記装置はさらに、前記生体サンプルが前記サンプル溶解ユニットおよび/または前記核酸抽出ユニットで処理される際に移動キャリヤとして機能するように構成された緩衝液を含む、請求項15に記載の装置。
- 前記核酸受付ユニットはキャップを含む、請求項15に記載の装置。
- 前記キャップは、前記装置から取り外すことが可能な、加圧封止されたキャップである、請求項20に記載の装置。
- 生物高分子を分離するための吸着剤であって、前記吸着剤は、ポリマーで少なくとも部分的に覆われた、または形成された、シラン化物質を含み、前記吸着剤の平均介在間隔は約6μm超および約23μm未満、孔径は約10nm超および約200nm未満、ならびに表面積は約0.1m2超および約150m2以下である、吸着剤。
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