LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...

The Hebrew University of Jerusalem

Yeshiva University

&#x6297;&#x51BB;&#x86CB;&#x767D; &#xFF08;&#x517B;&#x8001;&#x91D1;&#xFF09; &#x662F;&#x63A7;&#x5236;&#x51B0;&#x6676;&#x4F53;&#x751F;&#x957F;&#x7684;&#x51B0;-&#x7ED3;&#x5408;&#x86CB;&#x767D;&#x7684;&#x4E00;&#x4E2A;&#x5B50;&#x96C6;&#x3002;&#x5B83;&#x4EEC;&#x53EF;&#x80FD;&#x4F1A;&#x5BF9;&#x51B7;&#x51BB;&#x4FDD;&#x5B58;&#x7684;&#x7EC6;&#x80DE;&#x3001; &#x7EC4;&#x7EC7;&#x548C;&#x5668;&#x5B98;&#xFF0C;&#x5BF9;&#x751F;&#x4EA7;&#x548C;&#x8D2E;&#x5B58;&#x98DF;&#x7269;&#x548C;&#x4FDD;&#x62A4;&#x519C;&#x4F5C;&#x7269;&#x7684;&#x971C;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x517B;&#x8001;&#x57FA;&#x91D1;&#x7BA1;&#x7406;&#x59D4;&#x5458;&#x4F1A;&#x7684;&#x884C;&#x52A8;&#x7684;&#x8BE6;&#x7EC6;&#x7684;&#x673A;&#x5236;&#x4ECD;&#x4E0D;&#x6E05;&#x695A;&#x3002;&#x5177;&#x4F53;&#x800C;&#x8A00;&#xFF0C;&#x6709;&#x4E89;&#x8BAE;&#x7684;&#x517B;&#x8001;&#x57FA;&#x91D1;&#x7BA1;&#x7406;&#x59D4;&#x5458;&#x4F1A;&#x7ED1;&#x5B9A;&#x5230;&#x6676;&#x4F53;&#x8868;&#x9762;&#x7684;&#x53EF;&#x9006;&#x6027;&#x3002;&#x901A;&#x8FC7;&#x5B9E;&#x9A8C;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x517B;&#x8001;&#x57FA;&#x91D1;&#x7BA1;&#x7406;&#x59D4;&#x5458;&#x4F1A;&#x5BF9;&#x5176;&#x6D53;&#x5EA6;&#x6EB6;&#x6DB2;&#x4E2D;&#x7684;&#x6D3B;&#x52A8;&#x4F9D;&#x8D56;&#x4F3C;&#x4E4E;&#x8868;&#x660E;&#x7ED1;&#x5B9A;&#x662F;&#x53EF;&#x9006;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x901A;&#x8FC7;&#x4E00;&#x7CFB;&#x5217;&#x7684;&#x5B9E;&#x9A8C;&#x4E2D;&#x6E29;&#x5EA6;&#x63A7;&#x5236;&#x7684;&#x5FAE;&#x6D41;&#x63A7;&#x8BBE;&#x5907;&#xFF0C;&#x90A3;&#x91CC;&#x53EF;&#x4EE5;&#x4EA4;&#x6362;&#x5468;&#x56F4;&#x51B0;&#x6676;&#x4F53;&#x7684;&#x5A92;&#x4ECB;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x52A8;&#x9EC4;&#x7C89;&#x866B;&#x6CD5;&#x65B0;&#x793E;&#x51B0;&#x6676;&#x4F53;&#x7684;&#x7ED1;&#x5B9A;&#x662F;&#x51E0;&#x4E4E;&#x4E0D;&#x53EF;&#x9006;&#x8F6C;&#x548C;&#x8868;&#x9762;&#x7ED1;&#x5B9A;&#x517B;&#x8001;&#x57FA;&#x91D1;&#x7BA1;&#x7406;&#x59D4;&#x5458;&#x4F1A;&#x662F;&#x4E0D;&#x8DB3;&#x4EE5;&#x6291;&#x5236;&#x51B0;&#x6676;&#x4F53;&#x751F;&#x957F;&#x751A;&#x81F3;&#x5728;&#x517B;&#x8001;&#x57FA;&#x91D1;&#x7BA1;&#x7406;&#x59D4;&#x5458;&#x4F1A;&#x7684;&#x8D2B;&#x5316;&#x7684;&#x89E3;&#x51B3;&#x65B9;&#x6848;&#x3002;&#x8FD9;&#x4E9B;&#x8C03;&#x67E5;&#x7ED3;&#x679C;&#x6392;&#x9664;&#x6025;&#x6027;&#x5F1B;&#x7F13;&#x6027;&#x9EBB;&#x75F9;&#x6D3B;&#x52A8;&#x4F9D;&#x6258;&#x7684;&#x672A;&#x7ED1;&#x5B9A;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x5206;&#x5B50;&#x5B58;&#x5728;&#x7684;&#x7406;&#x8BBA;&#x3002;

&#x5FAE;&#x6D41;&#x63A7;&#x5B9E;&#x9A8C;&#x63ED;&#x793A;&#x7ED1;&#x5B9A;&#x51B0;&#x6676;&#x4F53;&#x7684;&#x6297;&#x51BB;&#x86CB;&#x767D;&#x8DB3;&#x4EE5;&#x9632;&#x6B62;&#x5176;&#x7ECF;&#x6D4E;&#x589E;&#x957F;&#x3002;

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules.

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.

Prot&eacute;ines antigel (AFP) sont un sous-ensemble des prot&eacute;ines liant la glace qui contr&ocirc;lent la croissance de cristaux de glace. Ils ont le potentiel pour la cryoconservation des cellules, tissus et organes, ainsi que pour la production et le stockage de nourriture et de la protection des cultures contre le gel. Toutefois, les modalit&eacute;s d&#39;action des Pag est encore incertaine. Plus pr&eacute;cis&eacute;ment, il y a controverse au sujet de la r&eacute;versibilit&eacute; de la liaison de l&#39;AFP &agrave; surfaces cristallines. La d&eacute;pendance observ&eacute;e exp&eacute;rimentalement de l&#39;activit&eacute; des Pag sur leur concentration dans la solution semble indiquer que la liaison est r&eacute;versible. Ici, par une s&eacute;rie d&#39;exp&eacute;riences dans des dispositifs microfluidiques &agrave; temp&eacute;rature contr&ocirc;l&eacute;e, o&ugrave; le milieu entourant les cristaux de glace peut &ecirc;tre &eacute;chang&eacute;, nous montrons que la liaison d&#39;hyperactif Tenebrio molitor AFP de cristaux de glace est pratiquement irr&eacute;versible et que surface-bondissent AFPs suffisent &agrave; inhiber la croissance de cristaux de glace m&ecirc;me dans des appauvri des Pag des solutions. Ces constatations excluent les th&eacute;ories de l&#39;activit&eacute; d&#39;AFP en s&#39;appuyant sur la pr&eacute;sence de mol&eacute;cules prot&eacute;iques non li&eacute;.

Des exp&eacute;riences de microfluidique r&eacute;v&egrave;lent que les prot&eacute;ines antigel li&eacute;es aux cristaux de glace suffisant pour emp&ecirc;cher leur croissance.

Frostschutzmittel Proteine (AFPs) sind eine Teilmenge der Eis-bindenen Proteine, die Eis-Kristallwachstum zu steuern. Sie haben Potenzial f&uuml;r die Kryokonservierung von Zellen, Geweben und Organen sowie f&uuml;r Produktion und Lagerung von Lebensmitteln und Schutz der Pflanzen vor Frost. Der genaue Wirkmechanismus von AFPs ist jedoch noch unklar. Insbesondere gibt es Kontroverse &uuml;ber Reversibilit&auml;t der Bindung des AFPs Kristall Oberfl&auml;chen. Die experimentell beobachtete Abh&auml;ngigkeit der Aktivit&auml;t des AFPs auf ihre Konzentration in der L&ouml;sung wird angezeigt, um anzugeben, dass die Bindung reversibel ist. Durch eine Reihe von Experimenten im temperaturgef&uuml;hrten Microfluidic Vorrichtungen, wo das Medium umgebenden Eiskristalle ausgetauscht werden k&ouml;nnen, zeigen wir hier, dass die Bindung des hyperaktiven Tenebrio Molitor AFP Eis Kristalle praktisch irreversibel ist und Oberfl&auml;che gebundene AFPs ausreichen, Eis Kristallwachstum auch in L&ouml;sungen AFPs ausgelaugt zu hemmen. Diese Ergebnisse ausschlie&szlig;en, Theorien der AFP-Aktivit&auml;t, die unter Berufung auf das Vorhandensein von ungebundenen Proteinmolek&uuml;le.

Mikrofluidische Experimente zeigen, dass Frostschutzmittel Proteine gebunden Kristalle Eis gen&uuml;gen, um ihr Wachstum zu verhindern.

Proteine antigelo (AFPs) sono un sottoinsieme di ghiaccio-binding proteins che controllano la crescita del cristallo di ghiaccio. Essi hanno il potenziale per la crioconservazione di cellule, tessuti e organi, nonch&eacute; per la produzione e conservazione degli alimenti e la protezione delle colture dal gelo. Tuttavia, il meccanismo dettagliato dell&#39;azione dell&#39;AFPs &egrave; ancora poco chiaro. In particolare, non c&#39;&egrave; polemica per quanto riguarda la reversibilit&agrave; dell&#39;associazione dell&#39;AFPs per piani in cristallo. La dipendenza sperimentalmente osservata dell&#39;attivit&agrave; dell&#39;AFPs sulla loro concentrazione in soluzione sembra indicare che l&#39;associazione &egrave; reversibile. Qui, da una serie di esperimenti in dispositivi microfluidici temperatura controllata, dove il medium che circondano i cristalli di ghiaccio pu&ograve; essere scambiato, ci mostrano che l&#39;associazione di iperattivo Tenebrio molitor AFP di cristalli di ghiaccio &egrave; praticamente irreversibile e che associato a superficie AFPs sono sufficienti per inibire la crescita del cristallo di ghiaccio anche in soluzioni impoverito dell&#39;AFPs. Questi risultati escludono le teorie di attivit&agrave; AFP basandosi sulla presenza di molecole proteiche non associato.

Microfluidica esperimenti rivelano che le proteine antigelo associate a cristalli di ghiaccio &egrave; sufficiente a prevenire la loro crescita.

&#x4E0D;&#x51CD;&#x5264;&#x86CB;&#x767D;&#x8CEA; (AFPs) &#x306F;&#x3001;&#x6C37;&#x306E;&#x7D50;&#x6676;&#x6210;&#x9577;&#x5236;&#x5FA1;&#x6C37;&#x7D50;&#x5408;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x3067;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306F;&#x751F;&#x7523;&#x3068;&#x98DF;&#x54C1;&#x306E;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B8;&#x3068;&#x971C;&#x304B;&#x3089;&#x4F5C;&#x7269;&#x306E;&#x4FDD;&#x8B77;&#x306E;&#x305F;&#x3081;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x7D30;&#x80DE;&#x3001;&#x7D44;&#x7E54;&#x3001;&#x81D3;&#x5668;&#x306E;&#x51CD;&#x7D50;&#x4FDD;&#x5B58;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;AFPs &#x306E;&#x4F5C;&#x7528;&#x306E;&#x8A73;&#x7D30;&#x306A;&#x6A5F;&#x69CB;&#x306F;&#x307E;&#x3060;&#x4E0D;&#x660E;&#x3067;&#x3059;&#x3002;&#x5177;&#x4F53;&#x7684;&#x306B;&#x306F;&#x3001;&#x7D50;&#x6676;&#x8868;&#x9762;&#x3068; AFPs &#x306E;&#x7D50;&#x5408;&#x306E;&#x53EF;&#x9006;&#x6027;&#x306B;&#x95A2;&#x3059;&#x308B;&#x8AD6;&#x4E89;&#x3067;&#x3059;&#x3002;&#x6D3B;&#x52D5; AFPs &#x306E;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x306E;&#x6FC3;&#x5EA6;&#x306B;&#x3064;&#x3044;&#x3066;&#x306E;&#x5B9F;&#x9A13;&#x7684;&#x89B3;&#x6E2C;&#x306E;&#x4F9D;&#x5B58;&#x6027;&#x306F;&#x3001;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x304C;&#x53EF;&#x9006;&#x7684;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x304C;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x4E00;&#x9023;&#x306E;&#x5B9F;&#x9A13;&#x3067;&#x6C37;&#x7D50;&#x6676;&#x5468;&#x56F2;&#x306E;&#x5A92;&#x4F53;&#x3092;&#x4EA4;&#x63DB;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x6E29;&#x5EA6;&#x5236;&#x5FA1;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x3067;&#x306F;&#x975E;&#x5E38;&#x306B;&#x6D3B;&#x767A;&#x306A; Tenebrio &#x30E2;&#x30EA;&#x30BF;&#x30FC;&#x6C37;&#x306E;&#x7D50;&#x6676;&#x306B; AFP &#x306E;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x306F;&#x3001;&#x5B9F;&#x8CEA;&#x7684;&#x306B;&#x5143;&#x306B;&#x623B;&#x305B;&#x308B;&#x72B6;&#x614B;&#x3067;&#x306F;&#x306A;&#x3044;&#x3068;&#x8868;&#x9762;&#x7D50;&#x5408; AFPs &#x304C; AFPs &#x306E;&#x67AF;&#x6E07;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3082;&#x6C37;&#x7D50;&#x6676;&#x6210;&#x9577;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x306E;&#x306B;&#x5341;&#x5206;&#x3067;&#x3042;&#x308B;. &#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x8ABF;&#x67FB;&#x7D50;&#x679C;&#x306E;&#x975E;&#x9023;&#x7D50;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5206;&#x5B50;&#x306E;&#x5B58;&#x5728;&#x306B;&#x4F9D;&#x5B58;&#x3059;&#x308B; afp &#x901A;&#x4FE1;&#x6D3B;&#x52D5;&#x306E;&#x7406;&#x8AD6;&#x3092;&#x6392;&#x9664;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x5B9F;&#x9A13;&#x4E0D;&#x51CD;&#x5264;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x6C37;&#x306E;&#x7D50;&#x6676;&#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x3066;&#x5F7C;&#x3089;&#x306E;&#x6210;&#x9577;&#x3092;&#x9632;&#x3050;&#x305F;&#x3081;&#x306B;&#x5341;&#x5206;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;

&#xBD80;&#xB3D9; &#xC561; &#xB2E8;&#xBC31;&#xC9C8; (AFPs) &#xC5BC;&#xC74C; &#xACB0;&#xC815; &#xC131;&#xC7A5; &#xC81C;&#xC5B4; &#xC5BC;&#xC74C; &#xBC14;&#xC778;&#xB529; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD558;&#xC704; &#xC9D1;&#xD569;&#xC785;&#xB2C8;&#xB2E4;. &#xADF8;&#xB4E4;&#xC740; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC138;&#xD3EC;, &#xC870;&#xC9C1; &#xBC0F; &#xC7A5;&#xAE30;&#xC758; cryopreservation&#xC5D0; &#xB300; &#xD55C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC0DD;&#xC0B0; &#xBC0F; &#xC2DD;&#xD488;&#xC758; &#xC800;&#xC7A5; &#xBC0F; &#xB18D;&#xC791;&#xBB3C; &#xC11C; &#xB9AC; &#xB85C;&#xBD80;&#xD130; &#xBCF4;&#xD638; &#xD574;&#xC57C;&#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; AFPs &#xD589;&#xB3D9;&#xC758; &#xC790;&#xC138;&#xD55C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xC544;&#xC9C1; &#xBD88;&#xBD84;&#xBA85;. &#xD2B9;&#xD788;, &#xD06C;&#xB9AC;&#xC2A4;&#xD0C8; &#xD45C;&#xBA74; Afps&#xC758; &#xBC14;&#xC778;&#xB529;&#xC758; &#xBC18;&#xC804; &#xAE30;&#xB2A5;&#xC5D0; &#xAD00;&#xD55C; &#xB17C;&#xB780;&#xC774;&#xC785;&#xB2C8;&#xB2E4;. &#xC2E4;&#xD5D8;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xCC30;&#xB41C; &#xD65C;&#xB3D9; Afps&#xC758; &#xC194;&#xB8E8;&#xC158;&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; &#xB18D;&#xB3C4;&#xC5D0; &#xC758;&#xC874; &#xBC14;&#xC778;&#xB529;&#xC774; &#xB418;&#xB3CC;&#xB9B4; &#xC218; &#xC784;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB824;&#xBA74; &#xB098;&#xD0C0;&#xB0A9;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xBBF8;&#xC138; &#xC628;&#xB3C4; &#xC81C;&#xC5B4; &#xC7A5;&#xCE58;, &#xC5BC;&#xC74C; &#xACB0;&#xC815;&#xC744; &#xB458;&#xB7EC;&#xC2FC; &#xB9E4;&#xCCB4;&#xB97C; &#xAD50;&#xD658;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC5B4;&#xB514;&#xC5D0; &#xC2E4;&#xD5D8;&#xC5D0; &#xC758;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xD65C;&#xB3D9;&#xC801;&#xC778; Tenebrio molitor AFP &#xD06C;&#xB9AC;&#xC2A4;&#xD0C8; &#xC5BC;&#xC74C;&#xC758; &#xBC14;&#xC778;&#xB529; &#xC2E4;&#xC9C8;&#xC801;&#xC73C;&#xB85C; &#xB418;&#xB3CC;&#xB9B4; &#xC218; &#xACE0; &#xD45C;&#xBA74; &#xBC14;&#xC778;&#xB529;&#xB41C; AFPs &#xC194;&#xB8E8;&#xC158; Afps&#xC758; &#xACE0;&#xAC08;&#xC5D0; &#xC5BC;&#xC74C; &#xACB0;&#xC815; &#xC131;&#xC7A5; &#xC5B5;&#xC81C; &#xD558;&#xAE30;&#xC5D0; &#xCDA9;&#xBD84; &#xB429;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC5B8;&#xBC14;&#xC6B4;&#xB4DC; &#xB2E8;&#xBC31;&#xC9C8; &#xBD84;&#xC790;&#xC758; &#xC874;&#xC7AC;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xB294; afp &#xD1B5;&#xC2E0; &#xD65C;&#xB3D9;&#xC758; &#xC774;&#xB860;&#xC744; &#xBC30;&#xC81C;.

&#xBBF8;&#xC138; &#xC2E4;&#xD5D8; &#xACF5;&#xAC1C; &#xBD80;&#xB3D9; &#xC561; &#xB2E8;&#xBC31;&#xC9C8; &#xACB0;&#xC815;&#xC744; &#xC5BC;&#xC74C;&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xB41C; &#xADF8;&#xB4E4;&#xC758; &#xC131;&#xC7A5;&#xC744; &#xBC29;&#xC9C0; &#xD558;&#xAE30; &#xC704;&#xD574; &#xCDA9;&#xBD84; &#xD558;&#xB2E4;.

Las prote&iacute;nas anticongelantes (AFP) son un subconjunto de hielo prote&iacute;nas que controlan el crecimiento de cristales de hielo. Tienen potencial para la criopreservaci&oacute;n de c&eacute;lulas, tejidos y &oacute;rganos, as&iacute; como para la producci&oacute;n y almacenamiento de alimentos y protecci&oacute;n de cultivos de las heladas. Sin embargo, el mecanismo detallado de acci&oacute;n de AFP es todav&iacute;a incierto. Espec&iacute;ficamente, hay controversia con respecto a la reversibilidad de encuadernaci&oacute;n de AFP a las superficies de cristal. La dependencia observada experimentalmente de la actividad de la AFP en su concentraci&oacute;n en la soluci&oacute;n parece indicar que la Uni&oacute;n es reversible. Aqu&iacute;, por una serie de experimentos en dispositivos microflu&iacute;dicos temperatura controlada, donde el medio que rodea los cristales de hielo puede intercambiarse, demostramos que la Uni&oacute;n de hiperactivo Tenebrio molitor AFP a cristales de hielo es pr&aacute;cticamente irreversible y que AFP superficie determinada es suficiente para inhibir el crecimiento de cristales de hielo incluso en soluciones agotadas de AFP. Estos resultados descartan las teor&iacute;as de la actividad de la AFP bas&aacute;ndose en la presencia de mol&eacute;culas de prote&iacute;na independiente.

Microflu&iacute;dicos experimentos revelan que las prote&iacute;nas anticongelantes a cristales de hielo es suficiente para evitar su crecimiento.

The growth dynamics of DO ice in liquid HO in a microfluidic device were investigated between the melting points of DO ice (3.8 °C) and HO ice (0 °C). As the temperature was decreased at rates between 0.002 °C/s and 0.1 °C/s, the ice front advanced but retreated immediately upon cessation of cooling, regardless of the temperature. This is a consequence of the competition between diffusion of HO into the DO ice, which favors melting of the interface, and the driving force for growth supplied by cooling. Raman microscopy tracked H/D exchange across the solid HO-solid DO interface, with diffusion coefficients consistent with transport of intact HO molecules at the DO ice interface. At fixed temperatures below 3 °C, the DO ice front melted continuously, but at temperatures near 0 °C a scalloped interface morphology appeared with convex and concave sections that cycled between growth and retreat. This behavior, not observed for DO ice in contact with DO liquid or HO ice in contact with HO liquid, reflects a complex set of cooperative phenomena, including H/D exchange across the solid-liquid interface, latent heat exchange, local thermal gradients, and the Gibbs-Thomson effect on the melting points of the convex and concave features.

Dynamics and unsteady morphologies at ice interfaces driven by DO-HO exchange.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

In some cold-adapted organisms, over a dozen isoforms of antifreeze (glyco)proteins or AF(G)Ps are present. Although these isoforms are structurally similar, their ability to inhibit ice growth varies significantly, and, in some fish, passive isoforms can be much more abundant than the active ones. Laboratory experiments demonstrated more than a decade ago that mixtures of AFP isoforms can exhibit synergistic enhancement of each other's activity. The mechanism of this synergy effect has remained obscure and is addressed here. Using cold-stages, microfluidics, and fluorescence microscopy, the activity of binary mixtures of structurally distinct AF(G)Ps from different fish and plant species was measured. While several mixtures exhibited enhancement, some mixtures exhibited antagonism. These latter mixtures included AF(G)Ps that bind to the same crystal planes, thereby exhibiting competition. Fluorescence microscopy experiments with a synergistic mixture of two isoform types labeled with different dyes showed they bound to different crystal planes. These results helped develop a kinetic description of the mechanism by which AF(G)Ps achieve synergy. The requirements of an active isoform include high adsorption rates, and prism plane binding, while passive isoforms usually bind to a pyramidal plane at slower rates. For synergy to occur, an active isoform first binds to the faster growing prism plane. This binding slows the advancement of the prism plane and creates more pyramidal surfaces to which a passive isoform bind. These results, in part, explain the biological observation of isoform distribution in fish, and the physical chemistry of the synergistic crystal growth inhibition by two inhibitors.

Synergy between Antifreeze Proteins Is Driven by Complementary Ice-Binding.

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X-ray diffraction reveal a complex lamellar structure of benzamide form II needle-like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non-classical morphologies.

Crystals of Benzamide, the First Polymorphous Molecular Compound, Are Helicoidal.

Since some antifreeze proteins and glycoproteins (AF(G)Ps) cannot directly bind to all ice crystal planes, they change ice crystal morphology by minimizing the area of the crystal planes to which they cannot bind until crystal growth is halted. Previous studies found that growth along the -axis (perpendicular to the basal plane, the crystal plane to which these AF(G)Ps cannot bind) is accelerated by some AF(G)Ps, while growth of other planes is inhibited. The effects of this growth acceleration on crystal morphology and on the thermal hysteresis activity are unknown to date. Understanding these effects will elucidate the mechanism of ice growth inhibition by AF(G)Ps. Using cold stages and an infrared laser, ice growth velocities and crystal morphologies in AF(G)P solutions were measured. Three types of effects on growth velocity were found: concentration-dependent acceleration, concentration-independent acceleration, and concentration-dependent deceleration. Quantitative crystal morphology measurements in AF(G)P solutions demonstrated that the adsorption rate of the proteins to ice plays a major role in determining the morphology of the bipyramidal crystal. These results demonstrate that faster adsorption rates generate bipyramidal crystals with diminished basal surfaces at higher temperatures compared to slower adsorption rates. The acceleration of growth along the -axis generates crystals with smaller basal surfaces at higher temperatures leading to increased growth inhibition of the entire crystal.

Ice Growth Acceleration by Antifreeze Proteins Leads to Higher Thermal Hysteresis Activity.

The mechanism by which safranine O (SFO), an ice growth inhibitor, halts the growth of single crystal tetrahydrofuran (THF) clathrate hydrates was explored using microfluidics coupled with cold stages and fluorescence microscopy. THF hydrates grown in SFO solutions exhibited morphology changes and were shaped as truncated octahedrons or hexagons. Fluorescence microscopy and microfluidics demonstrated that SFO binds to the surface of THF hydrates on specific crystal planes. Cryo-TEM experiments of aqueous solutions containing millimolar concentrations of SFO exhibited the formation of bilayered lamellae with an average thickness of 4.2±0.2 nm covering several μm . Altogether, these results indicate that SFO forms supramolecular lamellae in solution, which might bind to the surface of the hydrate and inhibit further growth. As an ice and hydrate inhibitor, SFO may bind to the surface of these crystals via ordered water molecules near its amine and methyl groups, similar to some antifreeze proteins.

Ran Drori

Institute of Biochemistry,
Food Science,
and Nutrition,
The Robert H. Smith Faculty of Agriculture,
Food,
and Environment,
Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment

The Hebrew University of Jerusalem

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization