monolayer-contact-doping-silicon-surfaces-nanowires-using

Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures1. MLCD results in the formation of highly controlled, ultra ...

The Hebrew University of Jerusalem

&#x529F;&#x80FD;&#x6027;&#x56FA;&#x5B9A;&#x5316;&#x548C;&#x6A2A;&#x5411;&#x7EC4;&#x7EC7;&#x6210;&#x5FAE;&#x578B;&#x548C; nanopatterns &#x86CB;&#x767D;&#x662F;&#x5C0F;&#x578B;&#x5316;&#x751F;&#x7269;&#x548C;&#x751F;&#x7269;&#x6280;&#x672F;&#x8BBE;&#x5907;&#x7684;&#x91CD;&#x8981;&#x524D;&#x63D0;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x4E00;&#x79CD;&#x9AD8;&#x6548;&#x7684;&#x7AD9;&#x70B9;&#x7279;&#x5B9A;&#x86CB;&#x767D;&#x8D28;&#x56FA;&#x5B9A;&#x5316;&#x57FA;&#x4E8E;&#x8F6C;&#x79FB;&#x9176; phosphopantetheinyl (PPT) &#x4ECE;&#x8F85;&#x9176; A (CoA)-&#x5B98;&#x80FD;&#x73BB;&#x7483;&#x578B;&#x8868;&#x9762;&#x5230;&#x7279;&#x5B9A;&#x7684;&#x80BD;&#x6807;&#x8BB0;&#x3002;&#x6211;&#x4EEC;&#x5236;&#x8BA2;&#x4E86;&#x4E00;&#x79CD;&#x81EA;&#x4E0B;&#x800C;&#x4E0A;&#x7684;&#x8868;&#x9762;&#x6539;&#x6027;&#x65B9;&#x6CD5;&#x8026;&#x5408; CoA &#x5BC6;&#x96C6;&#x5206;&#x5B50;&#x805A; (&#x4E59;&#x4E8C;&#x9187;) &#x805A;&#x5408;&#x7269;&#x5237;&#x3002;&#x4E3A; PPTase Sfp &#x878D;&#x5408;&#x5230;&#x4E0D;&#x540C;&#x7684;&#x76EE;&#x6807;&#x591A;&#x80BD;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x7AD9;&#x70B9;&#x7279;&#x5B9A;&#x9176;&#x7684;&#x56FA;&#x5B9A;&#x5316;&#x8BC1;&#x5B9E;&#x65E0;&#x6807;&#x7B7E;&#x7684;&#x5B9E;&#x65F6;&#x68C0;&#x6D4B;&#x3002;&#x5B9A;&#x91CF;&#x86CB;&#x767D;&#x8D28;-&#x86CB;&#x767D;&#x8D28;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x5B9E;&#x9A8C;&#x8BC1;&#x5B9E;&#x5927;&#x5927;&#x8D85;&#x8FC7; 50%&#x7684;&#x56FA;&#x5B9A;&#x5316;&#x86CB;&#x767D;&#x8D28;&#x5B8C;&#x5168;&#x6D3B;&#x52A8;&#x3002;&#x8BE5;&#x65B9;&#x6CD5;&#x5DF2;&#x6210;&#x529F;&#x5E94;&#x7528;&#x4E0E;&#x4E0D;&#x540C;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x57FA;&#x4E8E; PPT &#x7684;&#x56FA;&#x5B9A;&#x5316;&#x7684;&#x4E0D;&#x540C;&#x56FA;&#x5B9A;&#x5316;&#x6548;&#x7387;&#x53D1;&#x73B0;&#x4E0D;&#x540C;&#x80BD;&#x6807;&#x8BB0;&#x88AB;&#x878D;&#x5408;&#x5230; N-&#x548C; C-&#x603B;&#x7AD9;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x3002;&#x6839;&#x636E;&#x8FD9;&#x79CD;&#x56FA;&#x5B9A;&#x5316;&#x65B9;&#x6CD5;&#xFF0C;&#x6211;&#x4EEC;&#x5EFA;&#x7ACB;&#x4E86;&#x86CB;&#x767D;&#x8D28;&#x5230;&#x529F;&#x80FD;&#x7684;&#x4E8C;&#x8FDB;&#x5236;&#x5FAE;&#x89C2;&#x7ED3;&#x6784;&#x7684;&#x5149;&#x523B;&#x56FE;&#x6848;&#x3002;

&#x529F;&#x80FD;&#x6027;&#x56FA;&#x5B9A;&#x5316;&#x548C;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x9176;&#x6CD5;&#x8F6C;&#x79FB;&#x53CD;&#x5E94;&#x7684;&#x9635;&#x5217;&#x3002;

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Functional immobilization and patterning of proteins by an enzymatic transfer reaction.

Immobilisation fonctionnelle et organisation lat&eacute;rale des prot&eacute;ines en micro - et nanopatterns est une condition importante pour la miniaturisation des appareils biotechnologiques et bioanalytiques. Nous rapportons ici une approche pour l&#39;immobilisation des prot&eacute;ines sp&eacute;cifiques au site efficace bas&eacute; sur phosphopantetheinyl enzymatiques de transfert (PPT) du coenzyme A (CoA)-fonctionnalis&eacute;s de surfaces de verre de type aux balises peptidiques sp&eacute;cifiques. Nous avons con&ccedil;u une approche bottom-up modification de surface de couplage CoA dens&eacute;ment un pinceau de polym&egrave;re mol&eacute;culaire de poly (&eacute;thyl&egrave;ne glycol). In site immobilisation enzymatique des prot&eacute;ines fondues &agrave; peptides cibles diff&eacute;rentes pour la PPTase Sfp a &eacute;t&eacute; confirm&eacute;e par la d&eacute;tection d&#39;&eacute;tiquette libres en temps r&eacute;el. Exp&eacute;riences interaction prot&eacute;ine-prot&eacute;ine quantitative a confirm&eacute; que sensiblement plus de 50 % de la prot&eacute;ine immobilis&eacute;e &eacute;tait pleinement active. La m&eacute;thode a &eacute;t&eacute; appliqu&eacute;e avec succ&egrave;s avec diff&eacute;rentes prot&eacute;ines. Efficacit&eacute;s diff&eacute;rentes immobilisation d&#39;immobilisation ax&eacute;e sur le PPT ont cependant &eacute;taient observ&eacute;es pour les balises de peptide diff&eacute;rent &eacute;tant fusionn&eacute;es pour les N - et C-terminus de prot&eacute;ines. Sur la base de cette m&eacute;thode d&#39;immobilisation, nous avons &eacute;tabli photolithographique structuration des prot&eacute;ines dans les microstructures binaires fonctionnelles.

Immobilisation fonctionnelle et la structuration des prot&eacute;ines par une r&eacute;action de transfert enzymatique.

Funktionelle Immobilisation und seitlichen Organisation von Proteinen in Mikro- und Nanopatterns ist eine wichtige Voraussetzung f&uuml;r Moor&#39;schen Bioanalytische und biotechnologischer Ger&auml;te. Hier berichten wir ein Konzept f&uuml;r effiziente standortspezifische Protein Immobilisierung auf enzymatische Phosphopantetheinyl Transfer (PPT) von Coenzym A (CoA) Basis-funktionalisierten Oberfl&auml;chen der Glas-Typ spezifischen Peptid-Tags. Wir entwickelten einen Oberfl&auml;chenmodifizierung Bottom-up-Ansatz zur Kopplung CoA dicht auf molekulare Poly (Ethylene Glycol) Polymer Pinsel. Etikett-freie Echtzeiterkennung best&auml;tigte standortspezifische enzymatische Immobilisierung von Proteinen, die auf unterschiedliche Zielgruppen Peptide f&uuml;r die PPTase Sfp verschmolzen. Quantitative Protein-Protein Interaktion Experimente best&auml;tigt, dass deutlich mehr als 50 % der das immobilisierte Protein voll aktiv war. Die Methode wurde erfolgreich mit verschiedenen Proteine angewendet. Jedoch wurden verschiedene Immobilisierung Wirkungsgrade von PPT-basierte Immobilisierung f&uuml;r verschiedene Peptid-Tags wird abgesichert, die N - und C-Termini von Proteinen beobachtet. Auf der Grundlage dieser Methode Immobilisierung gr&uuml;ndeten wir fotolithografische Musterung von Proteinen in funktionale bin&auml;re Mikrostrukturen.

Funktionelle Immobilisation und Musterung der Proteine durch eine enzymatische Transfer-Reaktion.

Immobilizzazione funzionale e organizzazione laterale delle proteine in micro - e nanopatterns &egrave; un presupposto importante per la miniaturizzazione bioanalytical e dispositivi biotecnologici. Qui riportiamo un approccio per immobilizzazione della proteina site specific efficiente basato su trasferimento enzimatico phosphopantetheinyl (PPT) dal coenzima A (CoA)-funzionalizzato le superfici di vetro-tipo di tag specifico peptide. Abbiamo ideato un approccio bottom-up di modificazione superficiale per l&#39;accoppiamento CoA densamente per un pennello di polimero molecolare poli (etilen glicole). Site-specific immobilizzazione enzimatica delle proteine fuse di peptidi diversi target per la PPTase Sfp &egrave; stato confermato dal rilevamento in tempo reale senza etichetta. Gli esperimenti di interazione della proteina-proteina quantitativa ha confermato che significativamente pi&ugrave; del 50% della proteina immobilizzata &egrave; pienamente attivo. Il metodo &egrave; stato applicato con successo con diverse proteine. Tuttavia, efficienze diverse immobilizzazione di immobilizzazione PPT-based sono state osservate per tag diversi peptidi fuse insieme a N - e C-termini delle proteine. Sulla base di questo metodo di immobilizzazione, abbiamo stabilito fotolitografica patterning di proteine in microstrutture binari funzionale.

Immobilizzazione funzionale e creare una serie di proteine da una reazione enzimatica di trasferimento.

&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x56FA;&#x5B9A;&#x5316;&#x3068;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5916;&#x5074;&#x306E;&#x7D44;&#x7E54;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x304A;&#x3088;&#x3073; nanopatterns &#x306B;&#x306F;&#x3001;&#x30D0;&#x30A4;&#x30AA;&#x5206;&#x6790;&#x3068;&#x30D0;&#x30A4;&#x30AA; &#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x306E;&#x5C0F;&#x578B;&#x5316;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x524D;&#x63D0;&#x6761;&#x4EF6;&#x3067;&#x3059;&#x3002;&#x52B9;&#x7387;&#x7684;&#x306A;&#x90E8;&#x4F4D;&#x7279;&#x7570;&#x7684;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x56FA;&#x5B9A;&#x5316;&#x9175;&#x7D20; phosphopantetheinyl &#x306E;&#x8EE2;&#x9001; &#xFF08;PPT&#xFF09; &#x304B;&#x3089;&#x30B3;&#x30A8;&#x30F3;&#x30B6;&#x30A4;&#x30E0; &#xFF08;CoA&#xFF09; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x5831;&#x544A;-&#x5B98;&#x80FD;&#x57FA;&#x5316;&#x30AC;&#x30E9;&#x30B9;&#x578B;&#x8868;&#x9762;&#x306B;&#x7279;&#x7570;&#x7684;&#x30DA;&#x30D7;&#x30C1;&#x30C9; &#x30BF;&#x30B0;&#x3002;CoA &#x306F;&#x5BC6;&#x96C6;&#x5206;&#x5B50;&#x30DD;&#x30EA; &#xFF08;&#x30A8;&#x30C1;&#x30EC;&#x30F3;&#x30B0; &#x30EA;&#x30B3;&#x30FC;&#x30EB;&#xFF09; &#x30DD;&#x30EA;&#x30DE;&#x30FC; &#x30D6;&#x30E9;&#x30B7;&#x306B;&#x7D50;&#x5408;&#x8868;&#x9762;&#x6539;&#x8CEA;&#x30DC;&#x30C8;&#x30E0;&#x30A2;&#x30C3;&#x30D7; &#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x8003;&#x6848;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B5;&#x30A4;&#x30C8;&#x56FA;&#x6709;&#x306E;&#x9175;&#x7D20;&#x306E;&#x56FA;&#x5B9A;&#x5316;&#x306B;&#x7570;&#x306A;&#x308B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30DA;&#x30D7;&#x30C1;&#x30C3;&#x30C9; PPTase Sfp &#x306E;&#x6EB6;&#x3051;&#x308B;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x306E;&#x975E;&#x6A19;&#x8B58;&#x691C;&#x51FA;&#x306B;&#x3088;&#x3063;&#x3066;&#x78BA;&#x8A8D;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x5B9A;&#x91CF;&#x7684;&#x86CB;&#x767D;&#x8CEA;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x5B9F;&#x9A13;&#x5927;&#x5E45;&#x306B;&#x56FA;&#x5B9A;&#x3055;&#x308C;&#x305F;&#x86CB;&#x767D;&#x8CEA;&#x306E; 50 &#xFF05; &#x4EE5;&#x4E0A;&#x304C;&#x5B8C;&#x5168;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x3067;&#x3042;&#x3063;&#x305F;&#x3053;&#x3068;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x306F;&#x7570;&#x306A;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x6B63;&#x3057;&#x304F;&#x9069;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x7570;&#x306A;&#x308B;&#x56FA;&#x5B9A;&#x52B9;&#x7387;&#x56FA;&#x5B9A;&#x5316; PPT &#x30D9;&#x30FC;&#x30B9;&#x306F;&#x3001;N- &#x304A;&#x3088;&#x3073; C-&#x30C6;&#x30EB;&#x30DF;&#x30CB;&#x306B;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x878D;&#x5408;&#x3055;&#x308C;&#x3066;&#x7570;&#x306A;&#x308B;&#x30DA;&#x30D7;&#x30C1;&#x30C9; &#x30BF;&#x30B0;&#x306E;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x56FA;&#x5B9A;&#x5316;&#x6CD5;&#x306B;&#x57FA;&#x3065;&#x304F;&#x86CB;&#x767D;&#x8CEA;&#x6A5F;&#x80FD;&#x30D0;&#x30A4;&#x30CA;&#x30EA;&#x5FAE;&#x7D30;&#x69CB;&#x9020;&#x306B;&#x30D5;&#x30A9;&#x30C8;&#x30EA;&#x30BD;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3092;&#x8A2D;&#x7ACB;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x56FA;&#x5B9A;&#x5316;&#x3068;&#x9175;&#x7D20;&#x79FB;&#x52D5;&#x53CD;&#x5FDC;&#x306B;&#x3088;&#x308B;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3002;

&#xAE30;&#xB2A5;&#xC131; immobilization&#xC640; &#xB9C8;&#xC774;&#xD06C;&#xB85C;-&#xBC0F; nanopatterns &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xCE21;&#xBA74; &#xC870;&#xC9C1; bioanalytical &#xBC0F; &#xBC14;&#xC774;&#xC624; &#xC18C;&#xC790; miniaturizing&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC804;&#xC81C; &#xC870;&#xAC74;&#xC785;&#xB2C8;&#xB2E4;. &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC0AC;&#xC774;&#xD2B8; &#xAD00;&#xB828; &#xB2E8;&#xBC31;&#xC9C8; immobilization &#xAE30;&#xBC18; &#xD6A8;&#xC18C; phosphopantetheinyl &#xC804;&#xC1A1; (PPT)&#xC5D0;&#xC11C; &#xCF54;&#xC5D4;&#xC790;&#xC784; (CoA) &#xC811;&#xADFC;, &#xC5EC;&#xAE30; &#xBCF4;&#xACE0;-&#xD2B9;&#xC815; &#xD3A9; &#xD2F0; &#xB4DC; &#xD0DC;&#xADF8;&#xC5D0; &#xC720;&#xB9AC; &#xD615; &#xD45C;&#xBA74; functionalized. &#xC6B0;&#xB9AC; &#xBD84;&#xC790; &#xD3F4; &#xB9AC; (&#xC5D0;&#xD2F8;&#xB80C; &#xAE00;&#xB9AC;&#xCF5C;) &#xD3F4;&#xB9AC;&#xBA38; &#xBE0C;&#xB7EC;&#xC2DC;&#xC5D0; Coa&#xB97C; &#xBC00;&#xB3C4;&#xAC00; &#xCEE4;&#xD50C;&#xB9C1;&#xC5D0; &#xB300; &#xD55C; &#xC0C1;&#xD5A5;&#xC2DD; &#xD45C;&#xBA74; &#xAC1C; &#xC9C8; &#xBC29;&#xBC95;&#xC744; &#xACE0;&#xC548; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. PPTase Sfp&#xC5D0; &#xB300; &#xD55C; &#xB2E4;&#xB978; &#xB300;&#xC0C1; &#xD3A9; &#xD2F0; &#xB4DC;&#xB97C; &#xC735;&#xD569; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC0AC;&#xC774;&#xD2B8;&#xBCC4; &#xD6A8;&#xC18C; immobilization &#xB77C;&#xBCA8; &#xBB34;&#xB8CC; &#xC2E4;&#xC2DC;&#xAC04; &#xAC10;&#xC9C0;&#xC5D0; &#xC758;&#xD574; &#xD655;&#xC778; &#xB410;&#xB2E4;. &#xC815;&#xB7C9;&#xC801; &#xB2E8;&#xBC31;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC2E4;&#xD5D8; &#xD06C;&#xAC8C; &#xC6C0;&#xC9C1;&#xC774;&#xC9C0; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; 50%&#xB294; &#xC644;&#xBCBD; &#xD558; &#xAC8C; &#xD65C;&#xC131;&#xC744; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xBA54;&#xC11C;&#xB4DC;&#xB294; &#xC131;&#xACF5;&#xC801;&#xC73C;&#xB85C; &#xB2E4;&#xB978; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xD568;&#xAED8; &#xC801;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, immobilization PPT &#xAE30;&#xBC18;&#xC758; &#xB2E4;&#xB978; immobilization &#xD6A8;&#xC728;&#xC131;&#xC5D0;&#xB294; N-&#xC640; C-&#xD14C;&#xB974;&#xBBF8;&#xB2C8; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC735;&#xD569; &#xB418;&#xB294; &#xB2E4;&#xB978; &#xD3A9; &#xD2F0; &#xB4DC; &#xD0DC;&#xADF8;&#xC5D0; &#xB300; &#xD55C; &#xAD00;&#xCC30; &#xB418;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. &#xC774; &#xB3D9;&#xC6D0; &#xC815;&#xC9C0; &#xBC29;&#xBC95;&#xC5D0; &#xADFC;&#xAC70; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC; &#xAE30;&#xB2A5; &#xBC14;&#xC774;&#xB108;&#xB9AC; &#xB9C8;&#xC774;&#xD06C;&#xB85C; &#xAD6C;&#xC870;&#xC5D0; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; photolithographic &#xD328;&#xD134;&#xD654; &#xC124;&#xB9BD;.

&#xAE30;&#xB2A5; immobilization&#xC640; &#xC804;&#xC1A1; &#xD6A8;&#xC18C; &#xBC18;&#xC751;&#xC5D0; &#xC758;&#xD574; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD328;&#xD134;&#xD654; &#xD569;&#xB2C8;&#xB2E4;.

Inmovilizaci&oacute;n funcional y organizaci&oacute;n lateral de las prote&iacute;nas en micro y nanopatrones es un requisito previo importante para miniaturizar bioanal&iacute;tica y dispositivos biotecnol&oacute;gicos. Aqu&iacute;, Divulgamos un acercamiento para inmovilizaci&oacute;n de prote&iacute;na espec&iacute;fica eficiente basado en transferencia enzim&aacute;tica phosphopantetheinyl (PPT) de coenzima A (CoA)-funcionalizados superficies de vidrio tipo para etiquetas de p&eacute;ptido espec&iacute;fico. Ideamos un enfoque bottom-up modificaci&oacute;n superficial para acoplamiento CoA densamente a un pincel de pol&iacute;mero poli molecular (etilenglicol). Site-specific inmovilizaci&oacute;n enzim&aacute;tica de las prote&iacute;nas fusionadas a p&eacute;ptidos diferentes para la PPTase Sfp fue confirmada por la detecci&oacute;n en tiempo real libre de etiqueta. Experimentos de interacci&oacute;n prote&iacute;na-prote&iacute;na cuantitativa confirman que significativamente m&aacute;s del 50% de la prote&iacute;na inmovilizado era totalmente activo. El m&eacute;todo fue aplicado con &eacute;xito con diferentes prote&iacute;nas. Sin embargo, se observaron eficiencias diferentes inmovilizaci&oacute;n de inmovilizaci&oacute;n basadas en PPT para etiquetas de diferentes p&eacute;ptidos se fundieron a la N - y C-termini de prote&iacute;nas. Sobre la base de este m&eacute;todo de inmovilizaci&oacute;n, establecimos patrones photolithographic de prote&iacute;nas en microestructuras binarios funcionales.

Inmovilizaci&oacute;n funcional y el patr&oacute;n de prote&iacute;nas por una reacci&oacute;n enzim&aacute;tica de la transferencia.

Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns.

We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Quantitative ligand binding studies by solid phase detection techniques confirmed functional integrity of a transmembrane receptor reconstituted into these PSM. Colocomotion of individual ligand-receptor complexes was detected, demonstrating the applicability for single molecule fluorescence techniques.

Reconstitution of membrane proteins into polymer-supported membranes for probing diffusion and interactions by single molecule techniques.

Micropatterned polymer-supported membranes (PSM) are established as a tool for confining the diffusion of transmembrane proteins for single molecule studies. To this end, a photochemical surface modification with hydrophobic tethers on a PEG polymer brush is implemented for capturing of lipid vesicles and subsequent fusion. Formation of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy. Free diffusion of transmembrane proteins reconstituted into micropatterned PSM is demonstrated by FRAP and by single-molecule tracking. By exploiting the confinement of diffusion within micropatterned PSM, the diffusion and interaction dynamics of individual transmembrane receptors are quantitatively resolved.

Article	Year
Functional immobilization and patterning of proteins by an enzymatic transfer reaction. Analytical chemistry\| PubMed ID: 20092261	2010
Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns. Analytical chemistry\| PubMed ID: 21186833	2011
Reconstitution of membrane proteins into polymer-supported membranes for probing diffusion and interactions by single molecule techniques. Analytical chemistry\| PubMed ID: 21838222	2011
Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 23109503	2013

Sharon Waichman

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