<em>In Situ</em> Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Pacific Northwest National Laboratory

&#x5236;&#x9020;&#x5FAE;/nanoreactors &#x529F;&#x80FD;&#x7684;&#x751F;&#x7269;&#x5927;&#x5206;&#x5B50;&#x5728;&#x751F;&#x7269;&#x8FC7;&#x7A0B;&#x4E2D;&#xFF0C;&#x53D1;&#x751F;&#x5728; nanospace &#x5185;&#x7684;&#x9176;&#x50AC;&#x5316;&#x53CD;&#x5E94;&#x52A8;&#x529B;&#x5B66;&#x8BC4;&#x4EF7;&#x7684;&#x5174;&#x8DA3;&#x6B63;&#x5728;&#x8FC5;&#x901F;&#x589E;&#x52A0;&#x3002;&#x7528;&#x4E00;&#x79CD;&#x7B80;&#x5355;&#x7684;&#x53CD;&#x80F6;&#x675F;&#x5FAE;&#x4E73;&#x65B9;&#x6CD5;&#xFF0C;&#x8FA3;&#x6839;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x9176; (HRP) &#x6A21;&#x578B;&#x751F;&#x7269;&#x5206;&#x5B50;&#xFF0C;&#x6B64;&#x5904;&#x5DE7;&#x5367;&#x75C5;&#x5728;&#x4E8C;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x9897;&#x7C92; &#xFF08;HRP@SiO(2)) &#x548C;&#x5176; biocatalytical &#x884C;&#x4E3A;&#x8BE6;&#x7EC6;&#x5730;&#x8FDB;&#x884C;&#x4E86;&#x8C03;&#x67E5;&#x3002;&#x5149;&#x8C31;&#x6D4B;&#x91CF;&#x663E;&#x793A;&#x5305;&#x5D4C;&#x7684; HRP &#x5206;&#x5B50;&#x4FDD;&#x7559;&#x5176;&#x672C;&#x673A;&#x7ED3;&#x6784;&#x548C;&#x8FC7;&#x9AD8;&#x9176;&#x6D3B;&#x6027;&#x5BF9; 3&#xFF0C;3&#39;&#xFF0C;5&#xFF0C;5&#39;-tetramethylbenzidine &#xFF08;&#x5929;&#x9A6C;&#x80A1;&#x4EFD;&#xFF09; &#x4E0E;&#x7C73;&#x51EF;&#x5229;&#x65AF;&#x5E38;&#x6570; &#xFF08;3.02&times;10(-5)molL(-1) K(m))&#x3002;&#x5305;&#x5D4C;&#x8FA3;&#x6839;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x9176;&#x663E;&#x793A;&#x826F;&#x597D;&#x7684;&#x76F4;&#x63A5;&#x7535;&#x5B50;&#x8F6C;&#x79FB;&#x884C;&#x4E3A;&#x548C;&#x51CF;&#x5C11;&#x7684; H(2)O(2)&#xFF0C;&#x5176;&#x4E2D;&#x53EF;&#x4F7F;&#x7528;&#x786B;&#x5807;&#x548C;&#x90BB;&#x82EF;&#x4E8C;&#x80FA; &#xFF08;o-PD&#xFF09; &#x4F5C;&#x4E3A;&#x7535;&#x5B50;&#x8C03;&#x89E3;&#x5458;&#x52A0;&#x5F3A;&#x5BF9;&#x654F;&#x611F;&#x7684;&#x7535;&#x50AC;&#x5316;&#x53CD;&#x5E94;&#x3002;&#x5F53;&#x4F7F;&#x7528;&#x786B;&#x5807;&#x4F5C;&#x4E3A;&#x8C03;&#x89E3;&#x4EBA;&#xFF0C;&#x7535;&#x89E3;&#x6DB2;&#x4E2D;&#x7684;&#x57FA;&#x8D28;&#x548C; HRP &#x5C40;&#x9650;&#x5728;&#x4ECB;&#x5B54;&#x96A7;&#x9053;&#x901A;&#x8FC7;&#x4E8C;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x5FAE;&#x7403;&#x4E4B;&#x95F4;&#x7684;&#x5927;&#x89C4;&#x6A21;&#x8FD0;&#x8F93;&#x662F; o PD &#x653E;&#x7F13;&#x4E2D;&#x8FA3;&#x6839;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x9176;&#x5728;&#x5BC6;&#x95ED;&#x7684; nanospace &#x4E2D;&#x7684;&#x8840;&#x7EA2;&#x7D20;&#x548C;&#x7535;&#x6781;&#x4E4B;&#x95F4;&#x7684;&#x7535;&#x5B50;&#x8F6C;&#x79FB;&#xFF0C;&#x5BFC;&#x81F4;&#x8C03;&#x89E3;&#x65F6;&#x76F8;&#x6BD4; o PD &#x4E0E;&#x786B;&#x5807;&#x5230; H(2)O(2) &#x4F4E;&#x654F;&#x611F;&#x5EA6;&#x8F83;&#x6162;&#x3002;

&#x8FA3;&#x6839;&#x8FC7;&#x6C27;&#x5316;&#x7269;&#x9176;&#x7684;&#x4E8C;&#x6C27;&#x5316;&#x7845;&#x7EB3;&#x7C73;&#x5FAE;&#x7403;&#x7684;&#x751F;&#x7269;&#x6D3B;&#x6027;&#x3002;

Interest in the fabrication of micro/nanoreactors for evaluation of the function of biomolecules in biological processes, enzymatic reaction kinetics occurring inside the nanospace is rapidly increasing. With a simple reverse-micelle microemulsion method, horseradish peroxidase (HRP), a model biomolecule, was herein skillfully confined in silica nanoshells (HRP@SiO(2)) and its biocatalytical behaviors were investigated in detail. Spectroscopic measurements showed that the entrapped HRP molecules retained their native structure and had high enzymatic activity toward 3,3',5,5'-tetramethylbenzidine (TMB) with Michaelis constant (K(m)) of 3.02Ã—10(-5)molL(-1). The entrapped HRP displayed a good direct electron transfer behavior and sensitive electrocatalytic response toward the reduction of H(2)O(2), which could be enhanced using thionine and o-phenylenediamine (o-PD) as electron mediators. When using thionine as mediator, the mass transport between the substrates in electrolyte and HRP confined in silica nanospheres through the mesoporous tunnels was slower than that of o-PD, which slowed down the electron transfer between heme in HRP in the confined nanospace and the electrode, and resulted in low sensitivity to H(2)O(2) with thionine as mediator when compared to o-PD.

Bioactivity of horseradish peroxidase entrapped in silica nanospheres.

Int&eacute;r&ecirc;t dans la fabrication de micro/nanor&eacute;acteurs pour l&#39;&eacute;valuation de la fonction des biomol&eacute;cules dans les processus biologiques, cin&eacute;tique de la r&eacute;action enzymatique se produisant &agrave; l&#39;int&eacute;rieur de la nanospace augmente rapidement. Avec une m&eacute;thode simple micro&eacute;mulsion inverse-micelle, peroxydase de raifort (HRP), une biomol&eacute;cule mod&egrave;le, ici habilement plac&eacute;e dans nanocapsules de silice (HRP@SiO(2)) et ses biocatalytical les comportements ont &eacute;t&eacute; &eacute;tudi&eacute;s en d&eacute;tail. Mesures spectroscopiques ont montr&eacute; que les mol&eacute;cules HRP pi&eacute;g&eacute;s conservent leur structure native et avaient une activit&eacute; enzymatique &eacute;lev&eacute;e vers le 3, 3&#39;, 5, 5&#39;-t&eacute;tram&eacute;thylbenzidine (TMB) avec la constante de Michaelis (K(m)) de 3.02&times;10(-5)molL(-1). La HRP pi&eacute;g&eacute; affiche un comportement de transfert bon &eacute;lectronique directe et r&eacute;ponse &eacute;lectrocatalytique sensible vers la r&eacute;duction des H(2)O(2), qui pourrait &ecirc;tre am&eacute;lior&eacute;e en utilisant comme m&eacute;diateurs des &eacute;lectrons thionine et o-ph&eacute;nyl&egrave;nediamine (o-PD). Lorsque vous utilisez la thionine comme m&eacute;diateur, le transport de masse entre les substrats dans l&#39;&eacute;lectrolyte et HRP, confin&eacute;s dans des nanosph&egrave;res de silice dans les tunnels m&eacute;soporeux a &eacute;t&eacute; plus lent que celle d&#39;o-PD, qui a ralenti le transfert d&#39;&eacute;lectrons entre l&#39;h&egrave;me en HRP dans le clos nanospace et l&#39;&eacute;lectrode et a donn&eacute; lieu &agrave; une faible sensibilit&eacute; au H(2)O(2) avec la thionine comme m&eacute;diateur par rapport &agrave; o-PD.

Bioactivit&eacute; de la peroxydase de raifort, pris au pi&egrave;ge dans des nanosph&egrave;res de silice.

Interesse an der Herstellung von Mikro-/Nanoreactors f&uuml;r die Bewertung der Funktion von Biomolek&uuml;len in biologischen Prozessen, enzymatischen Reaktionskinetik Innern der Nanospace steigt rapide. Mit einer einfachen R&uuml;ckseite-Mizellen Mikroemulsion-Methode war Meerrettichperoxidase konjugiert (HRP), ein Modell von Biomolek&uuml;len, hierin gekonnt beschr&auml;nkt in Kiesels&auml;ure Nanoshells (HRP@SiO(2)) und seine biokatalytischen, die Verhaltensweisen im Detail untersucht worden sind. Spektroskopische Messungen zeigten, dass die eingeschlossenen HRP-Molek&uuml;le ihre native Struktur beibehalten und hohen enzymatischen Aktivit&auml;t in Richtung 3, 3&#39;, 5, 5&#39;-Tetramethylbenzidin (TMB) mit Michaelis-Konstante (K(m)) der 3.02&times;10(-5)molL(-1) hatte. Die eingeschlossene HRP angezeigt eine gute direkte Electron Transfer Verhalten und sensible elektrokatalytische Antwort auf die Verringerung der H(2)O(2), die mit Thionine und o-Phenylendiamin (o-PD) als Elektron Mediatoren erweitert werden konnte. Bei der Verwendung von Thionine als Vermittler war der Masse Transport zwischen der Substrate im Elektrolyt und HRP beschr&auml;nkt in Kiesels&auml;ure TPOT durch die Tunnel Mesopor&ouml;se langsamer als die der o-PD, die verlangsamt den Elektronentransfer zwischen H&auml;m in HRP in den engen Nanospace und der Elektrode, und f&uuml;hrte zu geringe Empfindlichkeit gegen&uuml;ber H(2)O(2) mit Thionine als Vermittler gegen&uuml;ber o-PD.

Bioaktivit&auml;t der Meerrettichperoxidase konjugiert in Kiesels&auml;ure TPOT gefangen.

Sta rapidamente aumentando l&#39;interesse per la fabbricazione di micro/nanoreattori per la valutazione della funzione di biomolecole in processi biologici, cinetica di reazione enzimatica che si verificano all&#39;interno del nanospace. Con un metodo semplice reverse-micella microemulsione, perossidasi di rafano (HRP), una modello biomolecola, fu confinato qui abilmente in silice nanoshells (HRP@SiO(2)) e sua biocatalytical comportamenti sono stati studiati in dettaglio. Le misure spettroscopiche hanno mostrato che le molecole HRP intrappolate mantenuto la loro struttura nativa e avevano un&#39;alta attivit&agrave; enzimatica verso 3, 3&#39;, 5, 5&#39;-tetrametilbenzidina (TMB) con costante di Michaelis (K(m)) di 3.02&times;10(-5)molL(-1). L&#39;HRP intrappolata visualizzato un buon elettrone diretto trasferimento comportamento e risposta electrocatalytic sensibili verso la riduzione della H(2)O(2), che potrebbe essere migliorata utilizzando tionina e o-fenilendiammina (o-PD) come mediatori di elettroni. Quando si utilizza tionina come mediatore, il trasporto di massa tra i substrati nell&#39;elettrolita e HRP confinati in silice nanosfere attraverso i tunnel mesoporosa era pi&ugrave; lento di quello di o-PD, che ha rallentato il trasferimento di elettroni tra l&#39;elettrodo ed EME in HRP nella nanospace confinati e portato a bassa sensibilit&agrave; a H(2)O(2) con tionina come mediatore rispetto all&#39;o-PD.

Bioattivit&agrave; della perossidasi di rafano intrappolate in nanosfere di silice.

&#x30DE;&#x30A4;&#x30AF;&#x30ED;/nanoreactors &#x751F;&#x4F53;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3001;&#x30CA;&#x30CE;&#x7A7A;&#x9593;&#x5185;&#x3092;&#x767A;&#x751F;&#x3059;&#x308B;&#x9175;&#x7D20;&#x53CD;&#x5FDC;&#x901F;&#x5EA6;&#x8AD6;&#x306E;&#x6A5F;&#x80FD;&#x8A55;&#x4FA1;&#x306E;&#x305F;&#x3081;&#x306E;&#x4F5C;&#x88FD;&#x3078;&#x306E;&#x95A2;&#x5FC3;&#x306F;&#x6025;&#x901F;&#x306B;&#x5897;&#x52A0;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5358;&#x7D14;&#x306A;&#x9006;&#x30DF;&#x30BB;&#x30EB; &#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30A8;&#x30DE;&#x30EB; &#x30B8;&#x30E7;&#x30F3;&#x306E;&#x65B9;&#x6CD5;&#x3067;&#x306F;&#x3001;&#x897F;&#x6D0B;&#x30EF;&#x30B5;&#x30D3;&#x30DA;&#x30EB;&#x30AA;&#x30AD;&#x30B7;&#x30C0;&#x30FC;&#x30BC; (HRP) &#x30E2;&#x30C7;&#x30EB; biomolecule &#x3053;&#x3053;&#x5DE7;&#x307F;&#x306B;&#x30B7;&#x30EA;&#x30AB; nanoshells &#xFF08;HRP@SiO(2)) &#x3068;&#x305D;&#x306E; biocatalytical &#x306E;&#x6319;&#x52D5;&#x306E;&#x8A73;&#x7D30;&#x306B;&#x8ABF;&#x3079;&#x305F;&#x3002; &#x9589;&#x3058;&#x8FBC;&#x3081;&#x3060;&#x3063;&#x305F;&#x5206;&#x5149;&#x6E2C;&#x5B9A;&#x3092;&#x793A;&#x3057;&#x305F;&#x6355;&#x6349;&#x306E; HRP &#x5206;&#x5B50;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x69CB;&#x9020;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x3001;3, 3&#39;, 5, 5&#39; &#x306B;&#x5411;&#x304B;&#x3063;&#x3066;&#x9AD8;&#x9175;&#x7D20;&#x6D3B;&#x6027;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x305F;-&#x30C6;&#x30C8;&#x30E9;&#x30E1;&#x30C1;&#x30EB;&#x30D9;&#x30F3;&#x30B8;&#x30B8;&#x30F3; &#xFF08;TMB&#xFF09; &#x30DF;&#x30AB;&#x30A8;&#x30EA;&#x30B9;&#x5B9A;&#x6570; (3.02&times;10(-5)molL(-1) &#x306E; K(m))&#x3002;&#x6355;&#x6349; HRP &#x826F;&#x3044;&#x76F4;&#x63A5;&#x96FB;&#x5B50;&#x79FB;&#x52D5;&#x6319;&#x52D5;&#x3068;&#x654F;&#x611F;&#x306A;&#x96FB;&#x6C17;&#x89E6;&#x5A92;&#x5FDC;&#x7B54;&#x30C1;&#x30AA;&#x30CB;&#x30F3;&#x3068; o-&#x30D5;&#x30A7;&#x30CB;&#x30EC;&#x30F3;&#x30B8;&#x30A2;&#x30DF;&#x30F3; (o PD) &#x96FB;&#x5B50;&#x30E1;&#x30C7;&#x30A3;&#x30A8;&#x30FC;&#x30BF;&#x30FC;&#x3068;&#x3057;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x62E1;&#x5F35;&#x3067;&#x304D;&#x308B;&#x30AB;&#x30BF;&#x30E9;&#x30FC;&#x30BC;&#x306E;&#x524A;&#x6E1B;&#x306B;&#x5411;&#x3051;&#x305F;&#x8868;&#x793A;&#x3002;&#x30C1;&#x30AA;&#x30CB;&#x30F3; &#x30E1;&#x30C7;&#x30A3;&#x30A8;&#x30FC;&#x30BF;&#x30FC;&#x3068;&#x3057;&#x3066;&#x7528;&#x3044;&#x305F;&#x3068;&#x304D;, &#x96FB;&#x89E3;&#x6DB2;&#x3067;&#x57FA;&#x677F;&#x3068;&#x30B7;&#x30EA;&#x30AB; &#x30CA;&#x30CE;&#x30B9;&#x30D5;&#x30A7;&#x30A2; &#x30E1;&#x30BD;&#x30DD;&#x30FC;&#x30E9;&#x30B9; &#x30C8;&#x30F3;&#x30CD;&#x30EB;&#x306B;&#x9589;&#x3058;&#x8FBC;&#x3081; HRP &#x9593;&#x8CEA;&#x91CF;&#x8F38;&#x9001; o PD &#x30CA;&#x30CE;&#x5236;&#x9650;&#x7A7A;&#x9593; HRP &#x306B;&#x304A;&#x3051;&#x308B;&#x30D8;&#x30E0;&#x3068;&#x3001;&#x96FB;&#x6975;&#x9593;&#x306E;&#x96FB;&#x5B50;&#x79FB;&#x52D5;&#x30C0;&#x30A6;&#x30F3;&#x920D;&#x5316;&#x3068;&#x30AB;&#x30BF;&#x30E9;&#x30FC;&#x30BC; &#x30C1;&#x30AA;&#x30CB;&#x30F3; o-PD &#x3092;&#x6BD4;&#x8F03;&#x3057;&#x305F;&#x5834;&#x5408;&#x306E;&#x4EF2;&#x4ECB;&#x5F79;&#x3068;&#x3057;&#x3066;&#x4F4E;&#x611F;&#x5EA6;&#x306E;&#x7D50;&#x679C;&#x306E;&#x305D;&#x308C;&#x3088;&#x308A;&#x3082;&#x9045;&#x304B;&#x3063;&#x305F;&#x3002;

&#x751F;&#x7269;&#x6D3B;&#x6027;&#x30B7;&#x30EA;&#x30AB; &#x30CA;&#x30CE;&#x30B9;&#x30D5;&#x30A7;&#x30A2;&#x306B;&#x9665;&#x308C;&#x305F;&#x897F;&#x6D0B;&#x308F;&#x3055;&#x3073;&#x30DA;&#x30EB;&#x30AA;&#x30AD;&#x30B7;&#x30C0;&#x30FC;&#x30BC;&#x306E;&#x3002;

&#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; nanospace &#xC548;&#xC5D0; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xD6A8;&#xC18C; &#xBC18;&#xC751; &#xC18D;&#xB3C4; &#xB860;&#xC5D0;&#xC11C; biomolecules &#xAE30;&#xB2A5; &#xD3C9;&#xAC00; &#xC704;&#xD55C; &#xB9C8;&#xC774;&#xD06C;&#xB85C;/nanoreactors&#xC758; &#xC81C;&#xC870;&#xC5D0; &#xB300; &#xD55C; &#xAD00;&#xC2EC; &#xC99D;&#xAC00;. &#xAC04;&#xB2E8;&#xD55C; &#xB9AC;&#xBC84;&#xC2A4; micelle &#xC544;&#xC0AC;&#xCE74; &#xBA54;&#xC11C;&#xB4DC;&#xB85C; &#xC591; &#xACE0;&#xCD94;&#xB0C9;&#xC774; &#xACFC;&#xC0B0;&#xD654; &#xD6A8;&#xC18C; (HRP) &#xBAA8;&#xB378; biomolecule &#xC5EC;&#xAE30; &#xB2A5;&#xC219; &#xD558; &#xAC8C; &#xC2E4;&#xB9AC; &#xCE74; nanoshells (HRP@SiO(2)) &#xBC0F; &#xADF8;&#xAC83;&#xC758; biocatalytical &#xB3D9;&#xC791;&#xC744; &#xC790;&#xC138;&#xD558;&#xAC8C;&#xC5D0;&#xC11C; &#xC870;&#xC0AC; &#xD588;&#xB2E4;&#xC5D0; &#xAD6D;&#xD55C; &#xB418;&#xC5C8;&#xB2E4;. &#xBD84; &#xAD11; &#xCE21;&#xC815; &#xBCF4;&#xC600;&#xB2E4; &#xBD09;&#xB41C; HRP &#xBD84;&#xC790;&#xC758; &#xAE30;&#xBCF8; &#xAD6C;&#xC870;&#xB97C; &#xC720;&#xC9C0; &#xD558; &#xACE0; 3, 3&#39;, 5, 5&#39;-tetramethylbenzidine (TMB) Michaelis &#xC0C1;&#xC218; (3.02&times;10(-5)molL(-1) k(m))&#xC640;&#xB97C; &#xD5A5;&#xD574; &#xB192;&#xC740; &#xD6A8;&#xC18C; &#xD65C;&#xB3D9;&#xC744; &#xD588;&#xB2E4;. &#xBD09;&#xB41C; HRP &#xC88B;&#xC740; &#xC9C1;&#xC811; &#xC804;&#xC790; &#xC804;&#xC1A1; &#xB3D9;&#xC791; &#xBC0F; &#xC804;&#xC790; &#xC911;&#xC7AC;&#xC790;&#xB85C; thionine &#xBC0F; o-phenylenediamine (o-PD)&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD5A5;&#xC0C1; &#xB420; &#xC218; &#xC788;&#xB294; H(2)O(2)&#xC758; &#xAC10;&#xC18C; &#xBC29;&#xD5A5;&#xC73C;&#xB85C; &#xBBFC;&#xAC10;&#xD55C; electrocatalytic &#xC751;&#xB2F5;&#xC5D0; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. &#xC911;&#xC7AC;&#xC790;&#xB85C; &#xC11C; thionine&#xB97C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xACBD;&#xC6B0; &#xC0AC;&#xC774; &#xC804;&#xD574;&#xC9C8;&#xC5D0; &#xAE30;&#xD310; &#xBC0F; mesoporous &#xD130;&#xB110;&#xC744; &#xD1B5;&#xD574; &#xC2E4;&#xB9AC; &#xCE74; &#xB098;&#xB178;&#xC5D0; &#xAD6D;&#xD55C; &#xD558;&#xB294; HRP &#xB300;&#xB7C9; &#xC804;&#xC1A1; &#xBC00;&#xD3D0; &#xB41C; nanospace&#xC5D0;&#xC11C; HRP&#xC758; heme&#xACFC;&#xB294; &#xC804;&#xADF9; &#xAC04;&#xC758; &#xC804;&#xC790; &#xC804;&#xC1A1; &#xC18D;&#xB3C4;&#xAC00; &#xB290;&#xB824; &#xACE0; &#xADC0;&#xCC29;&#xB418; &#xC5C8; &#xB2E4; &#xC624; PD&#xC5D0; &#xBE44;&#xD574; &#xC911;&#xC7AC;&#xC790;&#xB85C; &#xC11C; thionine&#xC640; H(2)O(2) &#xB0AE;&#xC740; &#xAC10;&#xB3C4; &#xC624; PD &#xBCF4;&#xB2E4; &#xB290;&#xB9B0; &#xD588;&#xB2E4;.

&#xC2E4;&#xB9AC; &#xCE74; &#xB098;&#xB178;&#xC5D0; &#xC18D;&#xC740; &#xC591; &#xACE0;&#xCD94;&#xB0C9;&#xC774; &#xACFC;&#xC0B0;&#xD654; &#xD6A8;&#xC18C;&#xC758; bioactivity&#xC785;&#xB2C8;&#xB2E4;.

Inter&eacute;s en la fabricaci&oacute;n de micro/nanoreactors para la evaluaci&oacute;n de la funci&oacute;n de biomol&eacute;culas en procesos biol&oacute;gicos, cin&eacute;tica de la reacci&oacute;n enzim&aacute;tica que ocurre dentro de la nanospace est&aacute; aumentando r&aacute;pidamente. Con un m&eacute;todo simple microemulsi&oacute;n inversa-micela, peroxidasa de r&aacute;bano (HRP), un modelo de biomol&eacute;culas, aqu&iacute; h&aacute;bilmente se limit&oacute; en s&iacute;lice nanoc&aacute;scaras (HRP@SiO(2)) y su biocatalytical comportamientos fueron investigados en detalle. Mediciones espectrosc&oacute;picas demostraron que las mol&eacute;culas HRP atrapadas conservan su estructura nativa y ten&iacute;an alta actividad enzim&aacute;tica hacia 3, 3&#39;, 5, 5&#39;-tetrametilbencidina (TMB) con la constante de Michaelis (K(m)) de 3.02&times;10(-5)molL(-1). El HRP atrapado muestra un comportamiento de transferencia electr&oacute;nica directa buena y sensible electrocatal&iacute;tica respuesta hacia la reducci&oacute;n de h, que podr&iacute;a mejorarse utilizando Tionina y fenilenediamina (o-PD) como mediadores del electr&oacute;n. Cuando utilice Tionina como mediador, el transporte masivo entre los sustratos en electrolitos y HRP confinado en nanoesferas de silicona a trav&eacute;s de los t&uacute;neles mesoporosos fue m&aacute;s lento que el de o-PD, que retras&oacute; la transferencia de electrones entre el electrodo y heme en HRP en el nanospace confinado y dio lugar a la baja sensibilidad a h con Tionina como mediador en comparaci&oacute;n con o-PD.

Bioactividad de peroxidasa de r&aacute;bano atrapado en nanoesferas de s&iacute;lice.

Investigation of the catalytic activity and stability of enzymes in confined nano/microspace provides valuable contributions to the fundamental understanding of biological reactions taking place on a mesoscopic scale within confined spaces. In this paper, macroporous silica foam (MSF) is used as a nanoreactor to co-confine glucose oxidase (GOD) and horseradish peroxidase (HRP). Then, the enzymatic cascade reactions, which act in tandem inside nanoreactors, for oxidation of glucose and 3,3',5,5'-tetramethylbenzidine (TMB) were studied. The catalytic kinetic parameters of apparent Michaelis constant (K(m)(app)) and maximum rate (V(max)) were obtained from Lineweaver-Burk plot by UV-vis spectrometry. Results showed that the catalytic activity of the co-confined enzymes is reduced compared to that of free enzymes in solution at room temperature. The stabilities of co-confined enzymes in denaturing agents, such as guanidinium chloride (GdmCl) and urea, were higher than those of free enzymes in solution. When employing a co-confined bienzyme system as a biosensor for the detection of glucose, a wider linear range of glucose was obtained for the co-confined bienzyme system than for free enzymes in solution.

Catalytic activity and stability of glucose oxidase/horseradish peroxidase co-confined in macroporous silica foam.

The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency.

Enzymatic reactivity of glucose oxidase confined in nanochannels.

On the basis of the photo-induced electron transfer (PET) from CdTe quantum dots (QDs) to cytochrome P450 2C9 (CYP2C9), a light-controlled drug metabolism system was successfully designed by using CYP2C9 functionalized-CdTe QDs as photocatalysts.

Cytochrome P450 enzyme functionalized-quantum dots as photocatalysts for drug metabolism.

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis.

Quantitative evaluation of biological reaction kinetics in confined nanospaces.

A graphene nano-cage with regulatable space for the assembly of a cytochrome P450 1A2-UDP-glucuronosyltransferase 1A10 bienzyme complex has been constructed via a click reaction, and successfully used to study drug sequential metabolism using an electrochemically-driven method.

Electrochemically driven drug metabolism via a CYP1A2-UGT1A10 bienzyme confined in a graphene nano-cage.

The development of synthetic nanopores and nanochannels that mimick ion channels in living organisms for biosensing applications has been, and still remains, a great challenge. Although the biological applications of nanopores and nanochannels have achieved considerable development as a result of nanotechnology advancements, there are few reports of a facile way to realize those applications. Herein, a nanochannel-based electrochemical platform was developed for the quantitative detection of biorelated small molecules such as potassium ions (K(+)) and adenosine triphosphate (ATP) in a facile way. For this purpose, K(+) or ATP G-quadruplex aptamers were covalently assembled onto the inner wall of porous anodic alumina (PAA) nanochannels through a Schiff reaction between -CHO groups in the aptamer and amino groups on the inner wall of the PAA nanochannels under mild reaction conditions. Conformational switching of the aptamers confined in the nanochannels occurs in the presence of the target molecules, resulting in increased steric hindrance in the nanochannels. Changes in steric hindrance in the nanochannels were monitored by the anodic current of indicator molecules transported through the nanochannels. As a result, quantitative detection of K(+) and ATP was realized with a concentration ranging from 0.005 to 1.0 mM for K(+) and 0.05 to 10.0 mM for ATP. The proposed platform displayed significant selectivity, good reproducibility, and universality. Moreover, this platform showed its potential for use in the detection of other aptamer-based analytes, which could promote its development for use in biological detection and clinical diagnosis.

Quantitative detection of potassium ions and adenosine triphosphate via a nanochannel-based electrochemical platform coupled with G-quadruplex aptamers.

Graphite-phase polymeric carbon nitride (GPPCN) has emerged as a promising metal-free material toward optoelectronics and (photo)catalysis. However, the insolubility of GPPCN remains one of the biggest impediments toward its potential applications. Herein, we report that GPPCN could be dissolved in concentrated sulfuric acid, the first feasible solvent so far, due to the synergistic protonation and intercalation. The concentration was up to 300 mg/mL, thousands of time higher than previous reported dispersions. As a result, the first successful liquid-state NMR spectra of GPPCN were obtained, which provides a more feasible method to reveal the finer structure of GPPCN. Moreover, at high concentration, a liquid crystal phase for the carbon nitride family was first observed. The successful dissolution of GPPCN and the formation of highly anisotropic mesophases would greatly pave the potential applications such as GPPCN-based nanocomposites or assembly of marcroscopic, ordered materials.

Dissolution and liquid crystals phase of 2D polymeric carbon nitride.

Surface patterns of well-defined nanostructures play important roles in fabrication of optoelectronic devices and applications in catalysis and biology. In this paper, the diporphyrin honeycomb film, composed of titanium dioxide, protoporphyrin IX, and hemin (TiO2/PPIX/Hem), was synthesized using a dewetting technique with the well-defined polystyrene (PS) monolayer as a template. The TiO2/PPIX/Hem honeycomb film exhibited a higher photoelectrochemical response than that of TiO2 or TiO2/PPIX, which implied a high photoelectric conversion efficiency and a synergistic effect between the two kinds of porphyrins. The TiO2/PPIX/Hem honeycomb film was also a good photosensitizer due to its ability to generate singlet oxygen ((1)O2) under irradiation by visible light. This led to the use of diporphyrin TiO2/PPIX/Hem honeycomb film for the photocatalytic inactivation of bacteria. In addition, the photocatalytic activities of other metal-diporphyrin-based honeycomb films, such as TiO2/MnPPIX/Hem, TiO2/CoPPIX/Hem, TiO2/NiPPIX/Hem, TiO2/CuPPIX/Hem, and TiO2/ZnPPIX/Hem, were investigated. The result demonstrated that the photoelectric properties of diporphyrin-based film could be effectively enhanced by further coupling of porphyrin with metal ions. Such enhanced performance of diporphyrin compounds opened a new way for potential applications in various photoelectrochemical devices and medical fields.

Nanostructured 2D Diporphyrin Honeycomb Film: Photoelectrochemistry, Photodegradation, and Antibacterial Activity.

Novel chemiluminescent (CL) imaging microtiter plates with high-throughput, low-cost, and simple operation for detection of four biomarkers related to Down's syndrome screening were developed and evaluated. To enhance the sensitivity of CL immunosensing, soybean peroxidase (SBP) was used instead of horseradish peroxide (HRP) as a label enzyme. The microtiter plates were fabricated by simultaneously immobilizing four capture monoclonal antibodies, anti-inhibin-A, anti-unconjugated oestriol (anti-uE3), anti-alpha-fetoprotein (anti-AFP), and beta anti-HCG (anti-β-HCG), on nitrocellulose (NC) membrane to form immunosensing microtiter wells. Under a sandwiched immunoassay, the CL signals on each sensing site of the microtiter plates were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging method for detection of four target antigens in a well at the same time. The linear response to the analyte concentration ranged from 0.1 to 40 ng/mL for inhibin-A, 0.075 to 40 ng/mL for uE3, 0.2 to 400 ng/mL for AFP, and 0.4 to 220 ng/mL for β-HCG. The proposed microtiter plates possessed high-throughput, good stability, and acceptable accuracy for detection of four antigens in clinical serum samples and demonstrated potential for practical applicability of the proposed method to Down's syndrome screening. Graphical Abstract Schematic evaluation of the microtiter plater for simultaneous detection of the four biomarkers.

Novel chemiluminescent imaging microtiter plates for high-throughput detection of multiple serum biomarkers related to Down's syndrome via soybean peroxidase as label enzyme.

Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM-3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO2 nanotubes.

Jiachao Yu

Fundamental & Computational Sciences Directorate

Pacific Northwest National Laboratory

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Jiachao Yu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Fundamental & Computational Sciences Directorate

Pacific Northwest National Laboratory

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Fundamental & Computational Sciences Directorate