Os autores agradecem o Escritório de Pesquisa Naval, United Soybean Board, e Fundação Nacional de Ciência para o financiamento.

1. Modificação do Nafion com sais de amónio quaternário <ol><li> Agitar uma garrafa de suspensão Nafion 5% w / v vigorosamente durante aprox. 30 segundos para assegurar que Nafion é suspenso uniformemente na solução. </li><li> Pipetar fora 2 mL da Nafion agora re-suspenso em um frasco de vidro (volume frasco pode conter de 2,5 mL a 10 mL). </li><li> Medir um excesso de 3 vezes molar (em relação aos grupos de ácido sulfónico no polímero Nafion) do sal de amónio de alquilo brometo de (massas adequadas são mostrados na Tabela 1) e adicionar este ao frasco contendo o 2 mL de Nafion. </li><li> Vortex do frasco a 1500 rpm por 10-15 minutos. </li><li> Despeje a solução viscosa em uma bandeja de plástico pesando que mede aprox. 3 x 3 polegadas, e usar uma pipeta para transferir qualquer solução residual a partir do frasco para a bandeja de pesagem. </li><li> Permitir que os solventes para evaporar para fora do barco pesar, deixando um amarelo / castanho película transparente, sobre a parte inferior da bandeja de pesagem (Figura 2). A taxa de evaporação de solvente deve ser tal que é necessário mais do que 6 horas para todo o solvente se evapore. Se o solvente evapora muito rápido, um material branco duro vai formar, em vez de um filme transparente, indicando que a estrutura micelar do polímero foi destruído, e você deve re-iniciar o procedimento. Se a evaporação de solventes, é muito lento, um de-umidificador pode ser necessário, porque muito lento de evaporação normalmente leva a um gel, brega laranja e você deve re-iniciar o procedimento. Gamas de temperatura típicas para a evaporação do solvente são de 20 - 37 ° C. As condições reais de secagem são uma função da humidade relativa e à temperatura da sala, mas é importante que a secagem ser lento para manter a estrutura micelar, mas não demasiado lenta para permitir a formação do gel. </li><li> Encher o barco com pesar 18M Ωcm água deionizada (10-20 mL de água), tampe e deixe de molho por 12-24 horas para remover o excesso de sais de amônio alquílicos de brometo e HBr. </li><li&gt; Verter (ou pipeta) para fora da água e enxaguar 3 vezes com água DI suficiente para encher o tabuleiro de cada vez. Tenha cuidado para não perder nenhum do filme de polímero durante esta etapa. </li><li> Permitir que a bandeja de pesagem para sentar descoberto até que o polímero é completamente seca Neste ponto, o polímero deve ser uma película transparente e um pouco quebradiço plástico. Novamente, se o ar é muito húmido, um de-humidificador pode ser necessário para completar a evaporação de uma forma atempada. </li><li> Usando uma espátula, cuidadosamente remover o filme seco a partir da bandeja de pesagem e transferi-lo para um frasco de vidro limpo. </li><li> Adicionar 2 mL de etanol e 3 cerâmicos grânulos de mistura, e vórtice durante 4 horas ou até que a película de polímero é completamente re-suspenso. </li></ol> 2. Imobilização de enzimas em TBAB-Modified Nafion para ensaios de atividade <ol><li> Para uma enzima seca, pesam-se 1-10 mg da enzima em um tubo de microcentrífuga de 1,5 ml, e adicionar 1 mL de tampão de fosfato 100 mM pH 7 para criar um 1-10 mg / mL de solução enzimática. For uma enzima que está em solução, usar um ácido bicinconínico (BCA) ensaio de 13 para determinar a quantidade de proteína, e adicionar uma quantidade apropriada de tampão fosfato 100 mM para trazer a concentração de proteína para 1-10 mg / mL. ML mg / 1-10 geralmente corresponde a 1-50 nanomoles / mL. </li><li> Para 120 uL de 1 mg / mL solução de enzima, adicionar 60 uL da solução de Nafion alquilo-amónio modificada, e vórtice durante 10 segundos. (Esta mistura pode ser aumentado para um grande número de repetições. Mantenha a razão de solução de enzima a-polímero em 2:1.) </li><li> Pipetar 60 da solução de enzima / polímero para o fundo de 3 separadas 1 cm 2 cuvetes, e permitir que a secar durante a noite. </li></ol> 3. Ensaio da enzima desidrogenase NAD-dependente Imobilizado <ol><li> Para a cuvete, adicionar 1,3 mL de 50 mM de pirofosfato de sódio (pH 8,8), 1,5 mL de 15 mM NAD (recentemente preparado), e 0,1 mL de água. </li><li> Coloque a cuvete num espectrofotómetro UV / Vis (Evolut ThermoScientific ieion 260 Bio e Thermo Spectronic Genesys 20) ajustado para um comprimento de onda de 340 nm. </li><li> Zerar o espectrômetro, e depois adicionar 0,1 ml de etanol. Misturar os reagentes suavemente pipetando a solução cima e para baixo 5 vezes. Para um espaço em branco, usar 0,1 mL de água adicional, em vez do 0,1 mL de etanol. </li><li> Ler a absorvância a 340 nm a 5 minutos após os reagentes foram adicionados à cuvete e 20 minutos depois. Traçar os dois pontos de dados para obter uma inclinação que pode ser usado para os cálculos da actividade. </li></ol> 4. Ensaio de Imobilizados PQQ-dependentes Desidrogenases <ol><li> Para a cuvete, adicionar 1,5 mL de fosfato de sódio (pH 7,3) e 200 uL de 600 PMS uM. </li><li> Coloque a cuvete num espectrofotómetro UV / Vis definido para um comprimento de onda de 600 nm e, em seguida, zero, o espectrómetro. </li><li> Adicionar 100 uL de 700 uL DCIP e 200 uL do substrato de interesse (etanol, acetaldeído, glicerol, glucose, gliceraldeído ou), e misturar os reagentes suavemente pipetando a solutião cima e para baixo 5 vezes. Para um espaço em branco, usar 200 uL de água em vez do substrato de interesse. </li><li> Ler a absorvância a 600 nm a 5 minutos após os reagentes foram adicionados à cuvete e 20 minutos depois. </li></ol> 5. Ensaio de glucose oxidase imobilizada <ol><li> Para a cuvete, adicionar 2,0 mL de uma solução contendo 0,2 M de ácido p-hidroxibenzóico, azida de sódio 0,02% (w / v), 128 U de peroxidase, 0,3 mM de 4-aminoantipirina, 1 M de fosfato de potássio, e 50 mM de glucose. Misturar a solução pipetando para cima e para baixo 5 vezes. </li><li> Coloque a cuvete de um conjunto espectrofotómetro UV / Vis para um comprimento de onda de 510 nm. </li><li> Ler a absorvância a 510 nm a 5 minutos após os reagentes foram adicionados à cuvete e, novamente, em 20 minutos depois. </li></ol> 6. Os resultados representativos A estrutura micelar do polímero Nafion modificada pode ser interrompido por secagem do sal original / polímero co-fundido filme muito fast A Figura 2 mostra uma mistura de sal / polímero que tenha sido seca correctamente resultando em um filme transparente castanho, luz. Um filme que seca muito rápido pode resultar em opacos, flocos brancos de polímero devido ao facto de o processo de secagem pode destruir a estrutura micelar. Uma vez que o Nafion modificado polímero e enzima foram misturados e co-moldada sobre o fundo de uma cuvete, ensaios de actividade enzimática pode ser usado para avaliar a estabilidade da enzima dentro do filme de polímero. Tabelas 2-4 mostram os resultados do ensaio de duas enzimas desidrogenase e glicose oxidase imobilizada em vários filmes de Nafion modificados, respectivamente. Note-se a maior actividade das enzimas que são imobilizadas vs as enzimas em solução tampão, mostrando que os polímeros Nafion modificadas podem efectivamente aumentar a actividade de certas enzimas (chamado superactivity). Outras enzimas têm limitações de transporte que diminuem a sua actividade específica quando imobilizá-los no polímero (celulases e amilases ou seja, cujos substratos são macromoléculas bastante grandes). <table border="1"><tbody><tr><td> Sal de amônio quaternário usado </td><td> 3 vezes superior </td></tr><tr><td> T3A (brometo de tetrapropilamónio) </td><td> 32,37 mg / ml </td></tr><tr><td> TBAB (brometo de tetrabutilamónio) </td><td> 39,19 mg / ml </td></tr><tr><td> TPAB (brometo de tetrapentylammonium) </td><td> 46,01 mg / ml </td></tr><tr><td> TEHA (brometo de triethylhexylammonium) </td><td> 32,37 mg / ml </td></tr><tr><td> AHMT (brometo de trimethylhexylammonium) </td><td> 27,25 mg / ml </td></tr><tr><td> TMOA (brometo de trimethyloctylammonium) </td><td> 30,66 mg / ml </td></tr><tr><td> TMDA (brometo de trimethyldecylammonium) </td><td> 34,07 mg / ml </td></tr><tr><td> TMDDA (brometo de trimethyldodecylammonium) </td><td> 37,48 mg / ml </td></tr><tr><td> TMTDA (brometo de trimethyltetradecylammonium) </td><td&gt; 40,89 mg / ml </td></tr><tr><td> TMHDA (brometo de trimethylhexadecylammonium) </td><td> 44,31 mg / ml </td></tr><tr><td> TMODA (brometo de trimethyloctadecylammonium) </td><td> 47,71 mg / ml </td></tr></tbody></table> Tabela 1. Quantidades de sais de tetra-alquil amónio a utilizar para Nafion modificação de polímeros. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> A actividade enzimática (U / g) </td></tr><tr><td> Tampão (sem polímero) </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> AHMT </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> Tabela 2 NAD-dependente actividade de desidrogenase de glucose imobilizada em seleccionados polímeros modificados Nafion (nota: actividade imobilizada é uma função da actividade específica inicial da enzima).. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> A actividade enzimática (mU / g) </td></tr><tr><td> Tampão (sem polímero) </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> Tabela 3 PQQ-dependente actividade de desidrogenase de glucose imobilizada em seleccionados polímeros modificados Nafion (nota: actividade imobilizada é uma função da actividade específica inicial da enzima).. <table border="1"><tbody><tr><td> Tipo de Nafion </td><td> A actividade enzimática (U / g) </td></tr><tr><td> Tampão (sem polímero) </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> AHMT </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> . Tabela 4 representativas actividade de oxidase de glicose específica imobilizado na seleccionados polímeros modificados Nafion (nota: actividade imobilizada é uma função da actividade específica inicial da enzima). <img alt="A Figura 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> Figura 1. Esquemática da incorporação TBAB em Nafion polímero e subsequente utilização na imobilização de enzimas. <img alt="A Figura 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> Figura 2. Fotografia óptica de um primeiro co-cast filmes de Nafion e TBAB. Retardar a secagem produz um transparente, filme castanho claro cobrindo o bottom da bandeja de pesagem.

<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>

Não há conflitos de interesse declarados.

No processo descrito, os sais de tetra-alquil amónio são utilizados para modificar Nafion comercial para criar polímeros micelares que podem ser usados ​​para imobilizar e estabilizar as enzimas. Os ensaios descritos no procedimento revelam que o polímero pode ser usado para imobilizar uma grande variedade de enzimas com uma elevada retenção de actividade. Se a enzima de interesse tem uma actividade muito baixa ou é impuro, uma concentração mais elevada pode ser necessária e não deve afectar o processo de...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Nafion </td><td> Sigma-Aldrich </td><td> 70160 </td></tr><tr><td> Brometo de tetra sais alquilamónio </td><td> Sigma-Aldrich </td><td> n / a </td></tr><tr><td> Álcool desidrogenase </td><td> Sigma-Aldrich </td><td> A3263 </td></tr><tr><td> Nicotinamida adenina dinucleotídeo (NAD) </td><td> Simga-Aldrich </td><td> N7004 </td></tr><tr><td> Pirofosfato de sódio </td><td> Sigma-Aldrich </td><td> P8010 </td></tr><tr><td> Fenazina metossulfato (PMS) </td><td> Sigma-Aldrich </td><td> P9625 </td></tr><tr><td> 2,6 Dichloroindophenol-(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> Ácido 4-hidroxibenzóico </td><td> Sigma-Aldrich </td><td> 240141 </td></tr><tr><td> A azida de sódio </td><td> Sigma-Aldrich </td><td> S8032 </td></tr><tr><td> Peroxidase </td><td> Sigma-Aldrich </td><td> P8375 </td></tr><tr><td> 4-aminoantipirina </td><td> Sigma-Aldrich </td><td> 06800 </td></tr><tr><td> UV / Vis Espectrofotômetro </td><td> Thermo </td><td> Evolução Bio 260 ou Spectronic Genesys 20 </td></tr><tr><td> Vortex Genie </td><td></td><td></td></tr><tr><td> Balança analítica </td><td></td><td></td></tr></tbody></table&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Durante a última década, tem havido uma riqueza de aplicação para as enzimas imobilizadas e estabilizadas incluindo biocatálise, biossensores, e células de biocombustível. 1-3 Em aplicações mais bioelectrochemical, enzimas ou organelas são imobilizados sobre uma superfície do eléctrodo com a utilização de algum tipo de polímero de matriz. Este andaime polímero deve manter as enzimas estável e permitir a difusão fácil de moléculas e iões dentro e para fora da matriz. A maioria dos polímeros utilizados para este tipo de imobilização são baseados em poliaminas ou poliálcoois - polímeros que imitam o ambiente natural das enzimas que eles encapsular e estabilizar a enzima através de hidrogénio ou ligações iónicas. Outro método para a estabilização de enzimas envolve o uso de micelas, que contêm regiões hidrofóbicas que podem encapsular e estabilizar as enzimas 4,5. Em particular, o grupo Minteer desenvolveu um polímero baseado em micelar Nafion comercialmente disponível. 6,7 Nafionem si é um polímero micelar que permite a difusão de canal-assistida de protões e de outros catiões pequenas, mas as micelas e os canais são extremamente pequeno e que o polímero é muito ácida, devido às cadeias sulfónicos lado ácido, o que é desfavorável para a imobilização de enzimas. No entanto, quando Nafion é misturado com um excesso de hidrofóbicos sais de amónio de alquilo tais como brometo de tetrabutilamónio (TBAB), os catiões de amónio quaternário substituir os protões e tornar-se os contra-iões para os grupos sulfonato sobre as cadeias laterais de polímero (Figura 1). Isto resulta em maiores micelas e canais dentro do polímero que permitem a difusão de grandes substratos e iões que são necessários para a função enzimática, tais como dinucleótido adenina nicotinamida (NAD). Este modificado Nafion polímero tem sido usado para imobilizar muitos tipos diferentes de enzimas, bem como as mitocôndrias para uso em biossensores e células de biocombustível. 8-12 Este documento descreve um novo processo para fazer este microfoneEllar polímero membrana imobilização de enzimas que podem estabilizar as enzimas. A síntese da membrana micelar enzima imobilização, o procedimento para a imobilização de enzimas dentro da membrana, e os ensaios para estudar a actividade enzimática específica da enzima imobilizada são detalhados abaixo.

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

Este artigo vai descrever o procedimento para a síntese de uma membrana Nafion hidrofobicamente modificado imobilização de enzimas e como para imobilizar proteínas e / ou enzimas no interior da membrana e testar a sua actividade específica.

Nafion-Sal modificado hidrofóbico para imobilização de enzimas e Estabilização

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 visualizações.  University of Utah. Este artigo vai descrever o procedimento para a síntese de uma membrana Nafion hidrofobicamente modificado imobilização de enzimas e como para imobilizar proteínas e / ou enzimas no interior da membrana e testar a sua actividade específica.

Vídeo: 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).

A imobilização de bactérias for Imaging por Microscopia de Força Atômica

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 ...

Bioengenharia

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: uma plataforma automatizada para Discovery Biomassa Enzima e Otimização Cocktail

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.

Camada por camada de colágeno Deposição em microfluídicos para Microtissue Estabilização

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.

Wet Chemistry and Peptide Imobilização em Polytetrafluoroethylene para Melhoria de adesão celular

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.

Targeted Plasma Membrane entrega de um hidrofóbica Carga encapsulado em um cristal líquido nanopartículas Transportador

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.

Imobilização local-dirigido da Proteína morfogenética óssea 2 para superfícies sólidas pela química do clique

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.

Fabricação de um Nafion-revestido, reduzido grafeno óxido/polianilina Chemiresistive Sensor de pH de Monitor em tempo real durante a fermentação microbiana

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.

Superfície-imobilização funcional dos Genes usando várias etapas Strand deslocamento litografia

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 para o encapsulamento de compostos hidrofóbicos e hidrofílicos em nanopartículas poliméricas

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.

Imobilização de indivíduos ao vivo Caenorhabditis elegans , usando um Chip de Microfluidic polidimetilsiloxano ultrafino com retenção de água

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