저자는 해군 연구소, 유나이티드 콩 보드, 그리고 자금 지원을위한 국립 과학 재단 (National Science Foundation)의 사무실을 인정합니다.

1. 제사기 암모늄 소금과 Nafion의 변경 <ol><li> 약 강력 5퍼센트 W / V Nafion 서스펜션의 병을 흔들어. 그 Nafion를 보장 30 초간 용액에 균일하게 정지됩니다. </li><li> 유리관 (유리병 볼륨 2.5 ML에서 10 ML에 포함할 수있는)으로 지금은 다시 정지 Nafion 2 ML을 피펫. </li><li> 3 배 어금니 초과 (Nafion 폴리머에 sulfonic 산 그룹에 상대적인) 알킬 암모늄 브로마이드 소금 (적절한 질량은 표 1에 표시된)의 측정과 Nafion의 2 ML이 들어있는 유리병에 이것을 추가합니다. </li><li> 10-15분위한 1,500 RPM의 소용돌이 병을. </li><li> 약 측정 플라스틱 무게 트레이에 점성 용액을 부어. 3인치 X 3, 무게 트레이에 약병에서 잔여 솔루션을 전송하는 피펫을 사용합니다. </li><li> 용매는 무게 트레이의 하단 (FI에 노란색 / 갈색, 투명 필름을두고, 무게는 배 밖으로 증발하도록 허용gure 2). 용매 증발의 비율은 증발하여 모든 용매에 대한 더 이상의 6 시간 소요 있도록해야합니다. 용매 증발 너무 빠른 경우, 흰색, 피각질의 재료는 폴리머의 micellar 구조가 파괴되었음을 나타내는 투명 필름 대신에 형성되며,이 절차를 다시 시작해야합니다. 용매 증발이 너무 느린 경우에는 증발의 너무 느린 일반적으로 볼품없는, 오렌지 젤로 연결하고 절차를 다시 시작해야하기 때문에, 드 가습기가 필요할 수 있습니다. 37 ° C. - 용매 증발에 대한 일반적인 온도 범위는 20입니다 건조를위한 실제 조건 방에 상대 습도와 온도의 함수지만, 그것은 중요 겔 형성하도록하기 위해서 건조 마이셀 구조를 유지할 느리게되고,하지만 너무 느려니다. </li><li> 채우기 18M Ωcm 드 이온화된 물 (물 10-20 ML), 커버와 함께 보트를 저울질하고, 초과 알킬 암모늄 브로마이드 소금과 HBR를 제거하는 12~24시간 동안 약물 사용하실 수 있습니다. </li><li&gt;는 물속으로 (혹은 피펫) 붓고 트레이 때마다 채우기 위해 충분한 DI 워터로 3 회 씻어. 이 단계 동안 폴리머 필름 중 하나를 잃게하지 않도록주의하십시오. </li><li> 폴리머는이 시점에서 완전히 건조까지 밝혀낸 앉을 수있는 무게 트레이를 허용, 폴리머는 분명하고 다소 부서지기 쉬운 플라스틱 필름이어야합니다. 공기가 매우 습기가있다면 다시, 드 가습기는 적시에 증발을 완료하는 데 필요할 수 있습니다. </li><li> 주걱을 사용하여 조심스럽게 무게 트레이에서 말린 필름을 제거하고 깨끗한 유리관에 그 파일을 전송합니다. </li><li> 에탄올 2 ML 그리고 3 세라믹 혼합 비즈, 4 시간 동안 와동을 추가 또는 폴리머 필름이 완전히 다시 정지 때까지. </li></ol> 2. 활동 Assays위한 TBAB-수정된 Nafion에 효소의 고정 <ol><li> 건조 효소의 경우 1.5 ML의 microcentrifuge 튜브로 효소 중 1-10 밀리그램을 무게, 그리고 1-10 MG / ML 효소 솔루션을 만드는 데 100 밀리미터 산도 7 인산염 완충액 1 ML을 추가합니다. 정R 용액에 효소는 단백질의 양을 결정하는 bicinchoninic 산성 (BCA) 검정 13 사용하고 1-10 밀리그램 / ML로 단백질 농도를 가지고 100 MM 인산 버퍼의 적절한 금액을 추가합니다. 1-10 MG / ML은 일반적으로 1-50 nanomoles / ML에 해당합니다. </li><li> 1 MG / ML 효소 용액 120 μL, 10 초 동안 60 알킬 암모늄 - 수정된 Nafion 솔루션 μL, 그리고 소용돌이를 추가합니다. (이 혼합물은 복제 다수에 확장할 수 있습니다. 2시 1분에서 효소 - 투 - 폴리머 용액 비율을 유지.) </li><li> 피펫 60 세 별도 1cm 2 cuvettes의 하단에 효소 / 폴리머 솔루션의 μL, 그리고 하룻밤 건조하실 수 있습니다. </li></ol> 3. 고정화 NAD 의존 탈수소 효소의 분석 <ol><li> cuvette에 50 밀리미터 나트륨 pyrophosphate (산도 8.8) 1.3 ML, 15 MM NAD (신선한)의 1.5 ML, 그리고 0.1 ML 물을 추가합니다. </li><li> UV / VIS 분광 광도계 (예 : ThermoScientific Evolut에 cuvette를 배치이온 260 바이오 및 써모 Spectronic Genesys 20) 340 nm의 파장의 설정. </li><li> 분광계를 제로, 그리고 0.1 ML의 에탄올을 추가합니다. 부드럽게 5 번 아래로 솔루션을 pipetting 및하여 시약을 섞는다. 빈 경우, 별도의 물을 0.1 ML 대신 에탄올의 0.1 ML을 사용합니다. </li><li> 시약이 후 cuvette 20 분에 추가된 후 5 분 340 nm의 흡광도에서를 기록합니다. 활동 계산에 사용할 수 경사면을 얻기 위해 두 개의 데이터 포인트를 잡아. </li></ol> 4. 고정화 PQQ 종속 Dehydrogenases의 분석 <ol><li> cuvette로 인산 나트륨 1.5 ML (산도 7.3), 600 μm의 PMS 200 μL를 추가합니다. </li><li> 600 nm의 파장의 설정 UV / VIS 분광 광도계의 cuvette를 삽입 후 분광계를 제로. </li><li> 700 μL DCIP의 100 μL와 관심 기판 (에탄올, 아세트 알데히드, 글리세린, 포도당, 또는 glyceraldehyde) 200 μL를 추가하고 부드럽게 solut를 pipetting하여 시약을 섞어이온 위 아래로 5 번. 빈 들어, 200 물의 μL 대신 관심 기판을 사용합니다. </li><li> 시약이 후 cuvette 20 분에 추가된 후 5 분 600 nm의 흡광도에서를 기록합니다. </li></ol> 5. 고정화 포도당 산화 효소의 분석 <ol><li> cuvette에 0.2 M P-hydroxybenzoic 산, 0.02 % (W / V) 나트륨 azide, 128 U 퍼옥시데이즈, 4 aminoantipyrine 0.3 MM, 1 M 인산 칼륨, 50 MM 포도당을 포함하는 솔루션의 2.0 ML을 추가합니다. 5 번 아래로 pipetting 및하여 솔루션을 섞는다. </li><li> 510 nm의 파장의에 VIS / UV 분광 광도계 세트에 cuvette를 놓습니다. </li><li> 시약은시 20 분 cuvette하고 다시에 추가된 후 5 분 510 nm의 흡광도에서를 기록합니다. </li></ol> 6. 대표 결과 수정된 Nafion 중합체의 micellar 구조는 원래 소금 / 고분자 공동 casted 필름도 FAS 건조에 의해 중단될 수 있습니다티. 그림 2 제대로 투명하게, 밝은 갈색 필름으로 인한 건조되었습니다 소금 / 폴리머 혼합물을 보여줍니다. 마르면 너무 빨리 건조 과정 micellar 구조를 파괴할 수있다는 사실에 의한 고분자의 불투명, 백색 박편을 초래할 수있는 영화. 수정된 Nafion 고분자와 효소가 혼합 및 cuvette의 하단에 공동 캐스팅되고 나면 효소 활동 assays는 폴리머 필름 내에서 효소의 안정성을 평가하는 데 사용할 수 있습니다. 테이블 두 탈수소 효소의 2-4 쇼 분석 결과 그리고 포도당 산화 효소는 각각 다양한 바뀌었 Nafion 필름으로 고정. 수정된 Nafion의 폴리머가 실제로 특정 효소 (superactivity 불리는)의 활동을 향상시킬 수 있다고 보여주는, 버퍼 용액에 효소 수와 고정화되는 효소의 높은 활동을합니다. 다른 효소는 고분자에 그들을 고정했을 때 그들의 구체적인 활동을 감소 전송 제한이 있습니다 (그의 기판) 상당히 큰 macromolecules 있습니다 즉 cellulases 및 amylases. <table border="1"><tbody><tr><td> 사용 제사기 암모늄 염 </td><td> 3 배 초과 </td></tr><tr><td> T3A (tetrapropylammonium 브로마이드) </td><td> 32.37 MG / ML </td></tr><tr><td> TBAB (tetrabutylammonium 브로마이드) </td><td> 39.19 MG / ML </td></tr><tr><td> TPAB (tetrapentylammonium 브로마이드) </td><td> 46.​​01 MG / ML </td></tr><tr><td> TEHA (triethylhexylammonium 브로마이드) </td><td> 32.37 MG / ML </td></tr><tr><td> TMHA (trimethylhexylammonium 브로마이드) </td><td> 27.25 MG / ML </td></tr><tr><td> TMOA (trimethyloctylammonium 브로마이드) </td><td> 30.66 MG / ML </td></tr><tr><td> TMDA (trimethyldecylammonium 브로마이드) </td><td> 34.07 MG / ML </td></tr><tr><td> TMDDA (trimethyldodecylammonium 브로마이드) </td><td> 37.48 MG / ML </td></tr><tr><td> TMTDA (trimethyltetradecylammonium 브로마이드) </td><tD&gt; 40.89 밀리그램 / ML </td></tr><tr><td> TMHDA (trimethylhexadecylammonium 브로마이드) </td><td> 44.31 MG / ML </td></tr><tr><td> TMODA (trimethyloctadecylammonium 브로마이드) </td><td> 47.71 MG / ML </td></tr></tbody></table> 표 1. tetra-알킬 암모늄의 염분의 금액은 Nafion 폴리머 변형에 사용할. <table border="1"><tbody><tr><td> Nafion의 종류 </td><td> 효소 활동 (U / G) </td></tr><tr><td> 버퍼 (없음 폴리머) </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> 표 2 선택한 수정된 Nafion의 폴리머 (참고 : 고정화 활동이 효소의 초기 구체적인 활동의 기능입니다)에 고정화 NAD 의존 포도당 탈수소 효소 활동.. <table border="1"><tbody><tr><td> Nafion의 종류 </td><td> 효소 활동 (MU / g) </td></tr><tr><td> 버퍼 (없음 폴리머) </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> 표 3 PQQ 종속 포도당 탈수소 효소 활동 선택한 수정된 Nafion의 폴리머 (참고 : 고정화 활동이 효소의 초기 구체적인 활동의 기능입니다)에 고정화.. <table border="1"><tbody><tr><td> Nafion의 종류 </td><td> 효소 활동 (U / G) </td></tr><tr><td> 버퍼 (없음 폴리머) </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> . 표 4 대표 포도당 산화 효소 구체적인 활동 선택한 수정된 Nafion의 폴리머 (고정화 활동이 효소의 초기 구체적인 활동의 기능이다 메모)에 고정. <img alt="그림 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> 그림 1. Nafion 폴리머 및 효소 고정의 후속 사용에 TBAB의 정관의 도식. <img alt="그림 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> 그림 2. Nafion과 TBAB의 초기 공동 캐스트 필름의 광학 사진. 수확량에게 보를 덮고있는 투명한 밝은 갈색 필름을 건조 속도를무게 트레이의 ttom.

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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>

관심의 어떠한 충돌 선언 없습니다.

설명한 절차에 tetra-알킬의 암모늄 염은 효소를 고정하고 안정화하는 데 사용할 수 micellar 폴리머를 만드는 상용 Nafion을 수정하는 데 사용됩니다. assays는 폴리머는 활동의 높은 보존과 효소의 다양한 고정하는 데 사용할 수있는 절차 쇼에 설명되어. 관심 효소가 매우 낮은 활동이 아니면 불순한 경우, 높은 집중력이 필요할 수 있습니다 10 밀리그램 / ML 이상의 농도에서 효소를 immobilizing 않는 한 부...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Nafion </td><td> 시그마 - 올드 리치 </td><td> 70,160 </td></tr><tr><td> Tetra의 alkylammonium의 브로마이드 소금 </td><td> 시그마 - 올드 리치 </td><td> N / A </td></tr><tr><td> 알코올 탈수소 효소 </td><td> 시그마 - 올드 리치 </td><td> A3263 </td></tr><tr><td> 니코틴 아데닌 dinucleotide (NAD) </td><td> Simga - 올드 리치 </td><td> N7004 </td></tr><tr><td> 나트륨 pyrophosphate </td><td> 시그마 - 올드 리치 </td><td> P8010 </td></tr><tr><td> Phenazine의 methosulfate (PMS) </td><td> 시그마 - 올드 리치 </td><td> P9625 </td></tr><tr><td> 2,6-Dichloroindophenol (DCIP) </td><td> 시그mA - 올드 리치 </td><td> D1878 </td></tr><tr><td> 포도당 산화 효소 </td><td> 시그마 - 올드 리치 </td><td> G7141 </td></tr><tr><td> 4 Hydroxybenzoic 산 </td><td> 시그마 - 올드 리치 </td><td> 240,141 </td></tr><tr><td> 나트륨 azide </td><td> 시그마 - 올드 리치 </td><td> S8032 </td></tr><tr><td> 퍼옥시데이즈 </td><td> 시그마 - 올드 리치 </td><td> P8375 </td></tr><tr><td> 4 aminoantipyrine </td><td> 시그마 - 올드 리치 </td><td> 06,800 </td></tr><tr><td> VIS / UV 분광 </td><td> 써모 </td><td> 진화 260 생물이나 Spectronic Genesys 20 </td></tr><tr><td> 와동 지니 </td><td></td><td></td></tr><tr><td> 분석 균형 </td><td></td><td></td></tr></tbody></ta경&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

지난 10 년 동안, biocatalysis, biosensors, 그리고 생물 연료 전지를 포함하여 고정화 및 안정화 효소에 대한 응용 프로그램의 풍부한이있었습니다. 가장 bioelectrochemical 애플 리케이션에서 1-3, 효소 또는 organelles은 어떤 유형의를 이용하여 전극 표면에 고정화되어 폴리머 매트릭스. 이 폴리머 비계는 효소 안정 유지하고와 모체의 밖으로 분자 및 이온의 손쉬운 보급을 위해 허용해야합니다. 고정 이러한 유형에 사용되는 대부분의 고분자는 polyamines 또는 polyalcohols을 기반으로하는 - 그들이 수소 또는 이온 결합을 통해 효소를 캡슐하고 안정화하는 효소의 자연 환경을 모방 고분자. 효소를 안정화하는 또 다른 방법은 효소를 캡슐과 안정 수있는 소수성 영역을 포함하는 micelles의 사용을 포함한다. 특히 4,5, Minteer 그룹은 상용 Nafion에 근거 micellar 폴리머를 개발하였습니다. 6,7 Nafion은자체는 양자와 기타 작은 양이온의 채널을 이용한 확산을 허용 micellar 고분자이지만, micelles 및 채널은 매우 소규모이며 폴리머 효소 고정을위한 불리 술폰산 사이드 체인으로 인해 매우 산성이다. 그러나, Nafion은 같은 tetrabutylammonium의 브로마이드 (TBAB)와 같은 소수성 알킬 암모늄 소금의 과잉과 함께 혼합되면 제사기 암모늄 양이온은 양자를 교체하고 폴리머 사이드 체인 (그림 1)에서 산염 그룹 카운터 이온이된다. 이러한 니코틴 아데닌 dinucleotide (NAD)와 같은 효소 기능을 위해 필요한 대형 기판과 이온의 확산을 허용 고분자 내에서 큰 micelles 및 채널에있는이 발생합니다. 이 수정된 Nafion 폴리머는 biosensors과 생물 연료 전지에 사용하기 위해 여러 효소의 종류뿐만 아니라, mitochondria를 고정하는 데 사용되었습니다. 8-12 본 논문이 마이크를 만들기위한 새로운 절차를 설명합니다효소의 안정화 수 ellar 폴리머 효소 고정 막. micellar 효소 고정 막, 멤브레인 내에 효소를 immobilizing 절차 및 고정화 효소의 효소 구체적인 활동을 유학 assays의 합성은 아래 자세히 설명되어 있습니다.

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

이 문서 hydrophobically 수정된 Nafion의 효소 고정 막 방법과 멤브레인 내에 단백질 및 / 또는 효소를 고정하고 구체적인 활동을 테스트하는 방법을 합성을위한 절차를 설명합니다.

효소 고정 및 안정화를위한 소수성 소금으로 바뀌었 Nafion

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 조회수.  University of Utah. 이 문서 hydrophobically 수정된 Nafion의 효소 고정 막 방법과 멤브레인 내에 단백질 및 / 또는 효소를 고정하고 구체적인 활동을 테스트하는 방법을 합성을위한 절차를 설명합니다.

비디오: 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).

원자 힘 현미경에 의해 영상에 대한 세균 고정

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 ...

연구

생체공학

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).
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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 : 바이오 매스의 효소 발견과 칵테일 최적화를위한 자동 플랫폼

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.

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

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.

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

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.

액정 나노 입자 캐리어의 소수성화물 캡슐화의 대상 플라즈마 멤브레인 배달

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.

뼈 Morphogenetic 단백질의 2 고체 표면에 화학 클릭 하 여 사이트 감독 동원 정지

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.

Nafion 코팅의 제조, 미생물 발효 하는 동안 실시간 산도 모니터에 그래 핀 산화물/폴리아닐린 Chemiresistive 센서 감소

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.

다단계 가닥 변위 리소 그래피를 사용 하 여 유전자의 기능 표면-동원 정지

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.

고분자 나노 입자에 소수 성 및 친수성 화합물의 캡슐화에 대 한 NanoPrecipitation 플래시

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.

물 유지와 울트라입니다 미세 칩을 사용 하 여 라이브 꼬마 선 충 의 동원 정지

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