Die Autoren danken dem Office of Naval Research, United Soybean Board und der National Science Foundation für finanzielle Unterstützung.

1. Modifizierung von Nafion mit quartären Ammoniumsalzen <ol><li> Schütteln eine Flasche von 5% w / v Nafion Suspension kräftig für ca.. 30 Sekunden bis die Nafion gewährleistet ist, gleichmäßig in der Lösung suspendiert. </li><li> Pipette aus 2 ml der nun erneut suspendiert Nafion in einen Glaskolben (Fläschchen Volumen konnte von 2,5 ml bis 10 ml enthalten). </li><li> Messen einer 3-fachen molaren Überschuss (bezogen auf die Sulfonsäuregruppen der Nafion-Polymer) des Alkylammonium Bromidsalz (gegebenenfalls Massen sind in Tabelle 1 gezeigt) und addieren, um das Fläschchen mit dem 2 ml Nafion. </li><li> Vortex das Fläschchen bei 1500 rpm für 10-15 Minuten. </li><li> Gießen Sie die viskose Lösung in einen Kunststoff Waagschale, die ca. misst. 3 x 3 cm, und verwenden Sie eine Pipette, um das restliche Lösung aus der Durchstechflasche zu übertragen auf die Waagschale. </li><li> Lassen Sie die Lösungsmittel zu verdampfen aus dem Gewicht Boot, wobei ein gelb / braun, transparenten Films auf dem Boden der Waagschale (FiBild 2). Die Geschwindigkeit der Verdampfung des Lösungsmittels sollte so sein, dass es mehr als 6 Stunden für alle Lösungsmittel verdampfen erfolgt. Wenn das Lösungsmittel verdunstet zu schnell, wird ein weißes, knusprigem Material anstelle Form eines transparenten Films anzeigt, dass der mizellaren Struktur des Polymers zerstört worden ist, und Sie müssen neu starten Sie den Vorgang. Wenn Lösungsmittelverdampfung zu langsam ist, kann eine de-Luftbefeuchter erforderlich sein, weil zu langsam, der Verdunstung führt typischerweise zu einer klebrigen, orange Gel und Sie müssen neu starten Sie den Vorgang. Typische Temperaturbereiche für Lösungsmittelverdampfung sind 20 bis 37 ° C. Die tatsächlichen Bedingungen für das Trocknen eine Funktion der relativen Feuchte und Temperatur in dem Raum, aber es ist wichtig, dass das Trocknen wobei langsam Mizellenstruktur beizubehalten, aber nicht zu langsam, um die Gelbildung zu ermöglichen. </li><li> Füllen Sie das Boot wiegt mit 18m Qcm de-ionisiertem Wasser (10-20 ml Wasser), Abdeckung, und lassen Sie sie für 12-24 Stunden einweichen, um überschüssige Alkylammonium Bromidsalze und HBr zu entfernen. </li><li&gt; Gießen (oder Pipette) aus dem Wasser und spülen Sie 3-mal mit genug DI-Wasser zu füllen Sie das Fach jeder Zeit. Schreiben darauf, keine der Polymerfolie während dieses Schritts zu verlieren. </li><li> Lassen Sie die Waagschale auf die unbedeckten sitzen, bis das Polymer vollständig trocken ist An dieser Stelle sollte das Polymer eine klare und etwas spröde Kunststoff-Folie sein. Auch wenn die Luft sehr feucht ist, kann eine de-Luftbefeuchter erforderlich sein, die Verdunstung in einer fristgerechten Weise zu vervollständigen. </li><li> Mit einem Spatel vorsichtig die getrocknete Film aus der Waagschale und übertragen sie in ein sauberes Glasgefäß. </li><li> Geben Sie 2 ml Ethanol und 3 Keramik-Misch-Perlen und Wirbel für 4 Stunden oder bis der Polymerfilm vollständig resuspendiert. </li></ol> 2. Immobilisierung von Enzymen in TBAB-Modified Nafion für Aktivitätstests <ol><li> Für einen trockenen Enzym, abwiegen 1-10 mg des Enzyms in ein 1,5 ml Mikrozentrifugenröhrchen, und fügen Sie 1 ml 100 mM Phosphatpuffer pH 7, eine 1-10 mg / ml Enzymlösung zu schaffen. For ein Enzym, das in Lösung ist, mit einem Bicinchoninsäure (BCA)-Probe 13, um die Menge des Proteins zu bestimmen, und geben eine entsprechende Menge von 100 mM Phosphatpuffer, um die Proteinkonzentration auf 1-10 mg / ml zu bringen. 1-10 mg / ml entspricht typischerweise bis 1-50 Nanomol / ml. </li><li> Um 120 ul der 1 mg / ml Enzymlösung mit 60 uL des Alkylammonium-modifizierte Nafion-Lösung, und Vortex für 10 Sekunden. (Dieses Gemisch kann für eine große Anzahl von Wiederholungen skaliert werden. Halten des Enzym-zu-Polymer-Lösung Verhältnis von 2:1.) </li><li> Pipette 60 pl des Enzyms / Polymer-Lösung in den Boden 3 separate 1 cm 2 Küvetten, und über Nacht trocknen lassen. </li></ol> 3. Assay von immobilisierter NAD-abhängigen Dehydrogenase-Enzym <ol><li> Um die Küvette, fügen 1,3 ml von 50 mM Natriumpyrophosphat (pH 8,8), 1,5 ml 15 mM NAD (frisch hergestellt) und 0,1 ml Wasser. </li><li> Legen Sie die Küvette in einem UV / VIS Spektralphotometer (dh ThermoScientific EvolutIon 260 Bio-und Thermo Spectronic Genesys 20) zu einer Wellenlänge von 340 nm eingestellt. </li><li> Null des Spektrometers, und dann 0,1 ml Ethanol. Mischen Sie die Reagenzien vorsichtig Pipettieren der Lösung auf und ab 5 mal. Für ein Leerzeichen mit 0,1 ml von zusätzlichem Wasser anstelle der 0,1 ml Ethanol. </li><li> Optische Dichte bei 340 nm nach 5 Minuten, nachdem die Reagenzien in die Küvette und 20 Minuten nach zugegeben. Zeichnen Sie die zwei Datenpunkte, um eine Steigung, die für die Tätigkeit Berechnungen verwendet werden können bekommen. </li></ol> 4. Assay von PQQ-abhängige Dehydrogenasen <ol><li> Um die Küvette, fügen Sie 1,5 ml Natriumphosphat (pH 7,3) und 200 ul von 600 um PMS. </li><li> Legen Sie die Küvette in einem UV / VIS Spektralphotometer, einstellbar auf eine Wellenlänge von 600 nm und dann auf Null das Spektrometer. </li><li> Fügen Sie 100 ul 700 ul und 200 ul DCIP des Substrats von Interesse (Ethanol, Acetaldehyd, Glycerin, Glucose, oder Glycerinaldehyd), und mischen Sie die Reagenzien durch vorsichtiges Pipettieren der solutIonen auf und ab 5 mal. Für einen leeren, verwenden Sie 200 ul Wasser anstelle des Substrats von Interesse. </li><li> Optische Dichte bei 600 nm nach 5 Minuten, nachdem die Reagenzien in die Küvette und 20 Minuten nach zugegeben. </li></ol> 5. Assay von immobilisierten Glucose-Oxidase <ol><li> Um die Küvette, 2,0 mL einer Lösung, die 0,2 M p-Hydroxybenzoesäure, 0,02% (w / v) Natriumazid, 128 U Peroxidase, 0,3 mM 4-Aminoantipyrin, 1 M Kaliumphosphat, und 50 mM Glucose. Mischen Sie die Lösung durch Auf-und Abpipettieren 5 mal. </li><li> Legen Sie die Küvette in einem UV / VIS Spektralphotometer einstellbar auf eine Wellenlänge von 510 nm auf. </li><li> Optische Dichte bei 510 nm nach 5 Minuten, nachdem die Reagenzien in die Küvette und wieder 20 Minuten nach zugegeben. </li></ol> 6. Repräsentative Ergebnisse Die micellare Struktur des modifizierten Nafion Polymer kann durch Trocknen des ursprünglichen Salz / Polymer-Co-gegossenen Film zu fas gestörtt. 2 zeigt ein Salz / Polymer-Mischung, die getrocknet worden ist vollständig um einen transparenten, hellbraun Film. Ein Film, der zu schnell trocknet in opaken weißen Flocken von Polymeren aufgrund der Tatsache, dass die Trocknung der micellaren Struktur zu zerstören führen. Sobald das modifizierte Nafion Polymer und Enzym gemischt worden und auf dem Boden einer Küvette zusammen gegossen, können enzymatische Aktivitäts-Assays verwendet, um die Stabilität des Enzyms in der Polymerfolie zu beurteilen. Tabellen 2-4 zeigen Testergebnisse von zwei Dehydrogenaseenzyme und Glucose-Oxidase in verschiedenen modifizierten Nafion Filme immobilisiert sind. Man beachte die höhere Aktivität der Enzyme, die gegen die Enzyme in Pufferlösung immobilisiert sind, die zeigen, dass modifizierte Polymere Nafion kann tatsächlich erhöhen die Aktivität bestimmter Enzyme (so genannte Überaktivität). Andere Enzyme haben Transport Beschränkungen auf, die spezifische Aktivität zu verringern, wenn sie bewegungsunfähig im Polymer (dh Cellulasen und Amylasen, deren Substrate sind recht groß Makromolekülen). <table border="1"><tbody><tr><td> Quaternäres Ammoniumsalz verwendet </td><td> 3-fachen Überschuss </td></tr><tr><td> T3A (Tetrapropylammoniumbromid) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (Tetrabutylammoniumbromid) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (Tetrapentylammoniumbromid) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (triethylhexylammonium Bromid) </td><td> 32,37 mg / ml </td></tr><tr><td> TMHA (trimethylhexylammonium Bromid) </td><td> 27,25 mg / ml </td></tr><tr><td> TMOA (trimethyloctylammonium Bromid) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (trimethyldecylammonium Bromid) </td><td> 34,07 mg / ml </td></tr><tr><td> TMDDA (Trimethyldodecylammonium Bromid) </td><td> 37,48 mg / ml </td></tr><tr><td> TMTDA (Trimethyltetradecylammoniumsalze Bromid) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (trimethylhexadecylammonium Bromid) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (trimethyloctadecylammonium Bromid) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tabelle 1. Mengen von Tetraalkylammonium-Salze, die zum Nafion Polymer benutzt werden können. <table border="1"><tbody><tr><td> Art der Nafion </td><td> Enzymaktivität (U / g) </td></tr><tr><td> Buffer (kein Polymer) </td><td> 16,63 ± 8,11 </td></tr><tr><td> Nafion (un-mod.) </td><td> 9,25 ± 2,21 </td></tr><tr><td> TMTDA </td><td> 3,23 ± 2,92 </td></tr><tr><td> TBAB </td><td> 3,93 ± 3,33 </td></tr><tr><td> TMDDA </td><td> 4,19 ± 1,04 </td></tr><tr><td> TMOA </td><td> 3,51 ± 1,11 </td></tr><tr><td> TMDA </td><td> 8,00 ± 4,53 </td></tr><tr><td> TMHA </td><td> 1,68 ± 1,39 </td></tr><tr><td> TMHDA </td><td> 4,83 ± 0,99 </td></tr><tr><td> TMODA </td><td> 10,45 ± 3,20 </td></tr></tbody></table> Tabelle 2 NAD-abhängige Glucose-Dehydrogenase-Aktivität in ausgewählten modifizierten Polymeren Nafion (Anmerkung: immobilisierte Aktivität ist eine Funktion der anfänglichen spezifischen Aktivität des Enzyms) immobilisiert.. <table border="1"><tbody><tr><td> Art der Nafion </td><td> Die Enzymaktivität (mU / g) </td></tr><tr><td> Buffer (kein Polymer) </td><td> 7,18 ± 0,51 </td></tr><tr><td> Nafion (un-mod.) </td><td> 70,1 ± 0,5 </td></tr><tr><td> TMTDA </td><td> 133 ± 6 </td></tr><tr><td> TBAB </td><td> 244 ± 4 </td></tr><tr><td> TMDDA </td><td> 221 ± 6 </td></tr><tr><td> TMOA </td><td> 1,78 ± 0,63 </td></tr><tr><td> TMDA </td><td> 206 ±5 </td></tr><tr><td> TEHA </td><td> 40,1 ± 50,6 </td></tr><tr><td> TMHDA </td><td> 0 </td></tr><tr><td> TMODA </td><td> 1,45 ± 0,06 </td></tr></tbody></table> Tabelle 3 PQQ-abhängige Glucose-Dehydrogenase-Aktivität in ausgewählten modifizierten Polymeren Nafion (Anmerkung: immobilisierte Aktivität ist eine Funktion der anfänglichen spezifischen Aktivität des Enzyms) immobilisiert.. <table border="1"><tbody><tr><td> Art der Nafion </td><td> Enzymaktivität (U / g) </td></tr><tr><td> Buffer (kein Polymer) </td><td> 103,61 ± 3,15 </td></tr><tr><td> Nafion (un-mod.) </td><td> 19,93 ± 10,10 </td></tr><tr><td> TMTDA </td><td> 247,25 ± 12,49 </td></tr><tr><td> TBAB </td><td> 152,27 ± 5,29 </td></tr><tr><td> TMDDA </td><td> 262,05 ± 6,26 </td></tr><tr><td> TMOA </td><td> 129,18 ± 2,31 </td></tr&gt; <tr><td> TMDA </td><td> 141,23 ± 1,97 </td></tr><tr><td> TMHA </td><td> 131,75 ± 2,89 </td></tr><tr><td> TMHDA </td><td> 132,50 ± 1,18 </td></tr><tr><td> TMODA </td><td> 136,50 ± 0,96 </td></tr></tbody></table> . Tabelle 4 Repräsentative Glucoseoxidase spezifische Aktivität in ausgewählten modifizierten Polymeren Nafion (: immobilisierte Aktivität ist eine Funktion der anfänglichen spezifischen Aktivität des Enzyms beachten) immobilisiert. <img alt="1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Abbildung 1. Schematische Darstellung der TBAB Einbau in Nafion-Polymer und die anschließende Verwendung in Enzymimmobilisierung. <img alt="2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Abbildung 2. Optische Foto von anfänglichen Co-Gießfolien von Nafion und TBAB. Langsam trocknend liefert eine transparente, hellbraune Fettfilm über das bottom der Waagschale.

<ol>
	<li>Calabrese-Barton, S., Gallaway, J., Atanassov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+biofuel+cells+for+implantable+and+microscale+devices+Chem.">Enzymatic biofuel cells for implantable and microscale devices Chem.</a> Rev. 104, 4867-4886 (2004).</li><li>Cracknell, J. A., Vincent, K. A., Armstrong, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+as+Working+or+Inspirational+Electrocatalysts+for+Fuel+Cells+and+Electrolysis.">Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis.</a> Chem. Rev. 108, 2439-2461 (2008).</li><li>Minteer, S. D., Liaw, B. Y., Cooney, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-Based+Biofuel+Cells.">Enzyme-Based Biofuel Cells.</a> Curr. Opin. Biotechnol. 18, 228-234 (2007).</li><li>Callahan, J. W., Kosicki, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Lipid+Micelles+on+Mitochondrial+Malate+Dehydrogenase.">The Effect of Lipid Micelles on Mitochondrial Malate Dehydrogenase.</a> Canadian Journal of Biochemistry. 45, 839-851 (1967).</li><li>Martinek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+the+Membrane+Environment+of+Enzymes:+Superactivity+of+Laccase+Entrapped+into+Surfactant+Reversed+Micelles+in+Organic+Solvents.">Modeling of the Membrane Environment of Enzymes: Superactivity of Laccase Entrapped into Surfactant Reversed Micelles in Organic Solvents.</a> Biokhimiya. 53, 1013-1016 (1988).</li><li>Moore, C. M., Akers, N. L., Hill, A. D., Johnson, Z. C., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+Environment+for+Immobilized+Dehydrogenase+Enzymes+by+Modifying+Nafion+with+Tetraalkylammonium+Bromides.">Improving the Environment for Immobilized Dehydrogenase Enzymes by Modifying Nafion with Tetraalkylammonium Bromides.</a> Biomacromolecules. 5, 1241-1247 (2004).</li><li>Schrenk, M. J., Villigram, R. E., Torrence, N. J., Brancato, S. J., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Mixture+Casting+Nafion+with+Quaternary+Ammonium+Bromide+Salts+on+the+Ion-Exchange+Capacity+and+Mass+Transport+in+the+Membranes.">Effects of Mixture Casting Nafion with Quaternary Ammonium Bromide Salts on the Ion-Exchange Capacity and Mass Transport in the Membranes.</a> J. Membr. Sci. 205, 3-10 (2002).</li><li>Akers, N. L., Moore, C. M., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Alcohol/O2+Biofuel+Cells+Using+Salt-Extracted+Tetrabutylammonium+Bromide/Nafion+Membranes+to+Immobilize+Dehydrogenase+Enzymes.">Development of Alcohol/O2 Biofuel Cells Using Salt-Extracted Tetrabutylammonium Bromide/Nafion Membranes to Immobilize Dehydrogenase Enzymes.</a> Electrochim. Acta. 50, 2521-2525 (2005).</li><li>Sokic-Lazic, D., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citric+Acid+Cycle+Biomimic+on+a+Carbon+Electrode.">Citric Acid Cycle Biomimic on a Carbon Electrode.</a> Biosens. Bioelectron. 24, 939-944 (2008).</li><li>Arechederra, R. L., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+Oxidation+of+Glycerol+in+an+Enzymatic+Biofuel+Cell.">Complete Oxidation of Glycerol in an Enzymatic Biofuel Cell.</a> Fuel Cells. 9, 63-69 (2009).</li><li>Germain, M., Arechederra, R. L., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroaromatic+Actuation+of+Mitochondrial+Bioelectrocatalysis+for+Self-Powered+Explosive+Sensors.">Nitroaromatic Actuation of Mitochondrial Bioelectrocatalysis for Self-Powered Explosive Sensors.</a> J. Am. Chem. Soc. 130, 15272-15273 (2008).</li><li>Addo, P. K., Arechederra, R. L., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Enzyme+Cascades+for+Methanol/Air+Biofuel+Cells+Based+On+NAD+-Dependent+Enzymes.">Evaluating Enzyme Cascades for Methanol/Air Biofuel Cells Based On NAD+-Dependent Enzymes.</a> Electroanalysis. 22, 807-812 (2010).</li><li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150, 76-85 (1985).</li></ol>

Keine Interessenskonflikte erklärt.

In der beschriebenen Verfahren werden Tetraalkylammonium-Salze eingesetzt, um kommerzielle Nafion zu modifizieren, um micellaren Polymere, die verwendet zu immobilisieren und stabilisieren Enzyme können zu schaffen. Die beschriebenen Tests in dem Verfahren zeigen, dass das Polymer verwendet werden, um eine Vielzahl von Enzymen mit einem hohen Beibehaltung der Aktivität zu immobilisieren. Wenn das interessierende Enzym hat eine sehr geringe Aktivität oder unrein ist, kann eine höhere Konzentration erforderlich sein u...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Tetra Alkylammonium Bromidsalze </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> Alkohol-Dehydrogenase </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Nicotinamid-Adenin-Dinukleotid (NAD) </td><td> Simga-Aldrich </td><td> N7004 </td></tr><tr><td> Natriumpyrophosphat </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Phenazinmethosulfat (PMS) </td><td> Sigma-Aldrich </td><td> P9625 </td></tr><tr><td> 2,6-Dichlorindophenol (DCIP) </td><td> Sigma-Aldrich </td><td> D1878 </td></tr><tr><td> Glucose-Oxidase </td><td> Sigma-Aldrich </td><td> G7141 </td></tr><tr><td> 4-Hydroxybenzoesäure </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> Natriumazid </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Peroxidase </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-Aminoantipyrin </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> UV / VIS Spektralphotometer </td><td> Thermo </td><td> Evolution 260 Bio oder Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Analysenwaage </td><td></td><td></td></tr></tbody></taBLE&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Im letzten Jahrzehnt gab es eine Fülle von Anwendungen für Enzyme immobilisiert und stabilisiert einschließlich Biokatalyse, Biosensoren und Biobrennstoffzellen. 03.01 In den meisten bioelektrochemische Anwendungen werden Enzyme oder Organellen auf einer Elektrodenoberfläche mit dem Einsatz von irgendeiner Art von immobilisierten Polymermatrix. Diese Polymer-Gerüst, sollten sich die Enzyme stabil und ermöglichen die einfache Diffusion von Molekülen und Ionen in die und aus der Matrix. Die meisten Polymere für diese Art der Immobilisierung verwendet werden auf der Grundlage Polyamine oder Polyalkohole - Polymere, die die Natur der Enzyme, dass sie und kapseln Stabilisierung des Enzyms durch Wasserstoff oder ionische Bindung zu imitieren. Ein weiteres Verfahren zur Stabilisierung von Enzymen ist die Verwendung von Micellen, die hydrophoben Regionen, die einzukapseln und stabilisieren Enzyme enthalten kann. 4,5 Insbesondere hat die Minteer Gruppe eine mizellare Polymer auf im Handel erhältlich Nafion entwickelt. 6,7 Nafionselbst ist ein Polymer, das micellare für den Kanal-unterstützte Diffusion von Protonen und anderen kleinen Kationen ermöglicht, aber die Micellen und Kanäle sind extrem klein ist und das Polymer sehr sauer durch Sulfonsäure Seitenketten, was ungünstig für Enzymimmobilisierung ist. Wenn jedoch Nafion mit einem Überschuß an hydrophoben Alkyl Ammoniumsalze, wie Tetrabutylammoniumbromid (TBAB) gemischt wird, ersetzen die quaternäre Ammoniumkationen die Protonen und werden die Gegenionen zu den Sulfonatgruppen an der Polymerseitenketten (1). Dies führt zu größeren Micellen und Kanälen innerhalb des Polymers, das für die Diffusion von großen Substraten und Ionen, die notwendig für die enzymatische Funktion wie Nikotinamid-Adenin-Dinukleotid (NAD) sind zu ermöglichen. Diese modifizierte Polymer Nafion wurde verwendet, um viele verschiedene Arten von Enzymen sowie Mitochondrien für den Einsatz in Biosensoren und Biobrennstoffzellen immobilisieren. 12.08 Dieser Beitrag beschreibt ein neuartiges Verfahren für die Herstellung dieser micEllar Polymer Enzymimmobilisierung Membran, die Enzyme stabilisieren können. Die Synthese des mizellenförmigen Enzymimmobilisierung Membran, dem Verfahren zur Immobilisierung von Enzymen in der Membran und den Assays zur Untersuchung enzymatische spezifische Aktivität des immobilisierten Enzyms sind unten aufgeführt.

Watch this Scientific Journal Video about Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization at JoVE.com

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

In diesem Artikel wird das Verfahren zur Synthese eines hydrophob modifizierten Nafion Membran und Enzymimmobilisierung, wie Proteine ​​und / oder Enzyme in der Membran zu immobilisieren und Testen ihrer spezifischen Aktivität beschreiben.

Hydrophobe Salz-modifizierte Nafion zur Enzymimmobilisierung und Stabilisierung

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

hydrophobic-salt-modified-nafion-for-enzyme-immobilization

Research

JoVE Journal

Bioengineering

16.1K Aufrufe.  University of Utah. In diesem Artikel wird das Verfahren zur Synthese eines hydrophob modifizierten Nafion Membran und Enzymimmobilisierung, wie Proteine ​​und / oder Enzyme in der Membran zu immobilisieren und Testen ihrer spezifischen Aktivität beschreiben.

Serie: Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Bakterielle Immobilisierung für Imaging von Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Forschung

Biotechnik

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
<div id="myEventWatcherDiv">
	 </div>

GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: eine automatisierte Plattform für Biomasse Enzym-Entdeckung und Cocktail-Optimierung

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Schicht-für-Schicht der Kollagenabscheidung in Mikrofluidik-Vorrichtungen zum Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Nasschemie und Peptid Immobilisation auf Polytetrafluoroethylen für verbesserte Zellhaftung

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Gezielte Plasma Membrane Lieferung eines Hydrophobic Transport Encapsulated in einem Liquid Crystal Nanoparticle Träger

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Site-verwiesene Immobilisierung von Knochen morphogenetische Protein 2 an feste Oberflächen durch Click-Chemie

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Herstellung von Nafion-beschichtet, reduziert Graphene Oxid/Polyaniline Chemiresistive Sensor Monitor pH in Echtzeit während der mikrobiellen Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

Flash-NanoPrecipitation für die Kapselung der hydrophoben und hydrophilen Verbindungen in Polymeren Nanopartikel

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Immobilisierung von Live Caenorhabditis Elegans Einzelpersonen, die eine ultraflache Polydimethylsiloxan Mikrofluidik-Chip mit Wassereinlagerungen

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...