著者は、SENSOTEM と NANOTIC の研究プロジェクトの枠組みの中でベルギーのワロン地域だけでなく、国家資金の科学研究 (F.R.S.-F.N.R.S) 金融支援を認めます。

1. タンパク質サンプル調製
<ol><li>生成し、ブッ cAb Lys3 私たちの以前の研究15で報告されたハイブリッドの蛋白質を浄化します。50 mM リン酸バッファー pH 7.4 次の組成のタンパク質を格納: 塩化ナトリウム 8 g、KCl の 0.2 g、Na2HPO4 1.44 g、800 mL の蒸留水に溶解した KH2PO4 0.24 g 前に 7.4 に溶液の pH を修正L. フィルター 1 の解決策の最終的な音量を調整するタンパク質溶液を滅菌します。</li>
	<li>鶏卵白リゾチーム (リゾチーム) 原液を準備します。リン酸バッファー生理食塩水 (PBS は、2.1.1 のステップを参照) の 10 mL の市販購入したリゾチーム (40,000 単位/mg) 100 mg を溶解します。0.22 μ m のカットオフ フィルターを用いたろ過によるタンパク質溶液を滅菌します。</li>
</ol>2. バイオ センサーを試金します。
<ol><li>
ソリューションおよびバッファーの準備
	<ol>
<li>50 mM PBS を準備するには、8 g の NaCl、KCl の 0.2 g、Na2HPO4、1.44 g と 800 mL の蒸留水で KH2PO4 0.24 g 溶解します。1 l. フィルター滅菌し、4 ° C で保存する溶液の量を調整する前に N HCl または 1 N NaOH pH 1 7.4 に調整します。</li>
		<li>100 mL の PBS は、前述のように準備にカゼイン加水分解物の 3 g を溶解することにより彩度/遮断ソリューションを準備 (2.1.1 の手順を参照してください)。フィルター滅菌し、4 ° C で保存</li>
		<li>100 mL の PBS は、前述のように準備にカゼイン加水分解物の 1 g を溶解することにより結合ソリューションを準備 (2.1.1 の手順を参照してください)。フィルター滅菌し、4 ° C で保存</li>
		<li>洗浄液の準備 (0.1% トゥイーン - PBS) 100 mL の PBS は、前述のように準備の Tween 20 (100%) 100 μ L の追加によって (2.1.1 の手順を参照してください)。4 ° C でのストア</li>
		<li>電極の準備ソリューションを準備 (1% トリトン X-100-PBS) トリトン X-100 (100%) 1 mL を 100 mL の PBS は、前述のように準備に追加して (手順 2.1.1 参照)。4 ° C でのストア</li>
		<li>最終巻の蒸留水で KCl の 26 g を溶解することにより電極再生液 (3.5 M KCl) を準備 100 mL。フィルター滅菌と 4 ° C で保存</li>
		<li>蒸留水 1 l. フィルター滅菌し、4 ° C で保存の最終巻ににおける NaCl の 0.29 g を溶解することにより 5 mM の NaCl 水溶液を準備します。5 mM の NaCl 水溶液 20 mL のベンジル ペニシリンの 26.7 mg を溶解することにより検出ソリューション (4 mM ベンジル) を準備します。フィルター滅菌し、-20 ° C で保存</li>
	</ol>
</li>
	<li>
センサーの作製と再生 注: ポリアニリン被覆センサー チップを開発し、博士 p. Bogaerts、s. ユヌス博士と教授 Y. Glupczynski (カトリック大学のルーバン ・ ラ ・ ヌーブ - 朱モン Godinne) によって提供されました。これらのセンサーを合成に使用するポリアニリンの電気重合プロトコルと同様に、センサーの説明は彼らの前の仕事17で詳細に説明します。簡単に言えば、このシステムは、古典的なプリント回路基板 (PCB) の技術によって製造された 8 つの個々 のチップの再利用可能なセンサーを使用します。個々 のチップは 3 つの電極から成るスポットをラウンドします。一番上は作用電極、ポリアニリンの電気合成だった。中間の 1 つは、参照電極と下部電極が対向電極を構成します。参照とカウンター電極の両方はカーボン層の上に固体の銀/塩化銀アマルガムを使用して官能基化します。<ol>
<li>ヒントを含む電極作製の 300 μ L/ウェルの 96 ウェル プレートのウェルに浸すことによって電極の 3 洗浄を実行 (1% トリトン X-100-PBS を参照してくださいステップ 2.1.5。)。室温で穏やかな混合によって 2 分の各洗浄を実行します。</li>
		<li>含む室温で穏やかな混合で 2 分間蒸留水 300 μ L/ウェルの 96 ウェル プレートのウェルにヒントを浸すことによって電極をすすいでください。</li>
		<li>含む再生ソリューションの 300 μ L/ウェルの 96 ウェル プレートのウェルにヒントを浸すことによって電極を再生成 (3.5 M の KCl、参照 2.1.6) 4 ° C または室温で 1 h で一晩。</li>
		<li>含む生理食塩水リン酸バッファー 300 μ L/ウェルの 96 ウェル プレートのウェルにヒントを浸すことによって電極の 3 洗浄を実行 (手順 2.1.1 を参照)。室温で穏やかな混合で 2 分の各洗浄を実行します。</li>
	</ol>
</li>
	<li>
センサーに対して結合の試金
	<ol>
<li>40 μ g/mL の PBS で電極表面上に作製したリゾチームの 15 μ L のドロップを堆積することにより電極のパニ (ポリアニリン) 表面にリゾチームのコート。4 ° C または 1 時間室温で一晩インキュベートします。</li>
		<li>リン酸バッファー生理食塩水で電極の 3 つの洗浄を実行 (手順 2.1.1 参照) 含む生理食塩水リン酸バッファー 300 μ L/ウェルの 96 ウェル プレートのウェルにセンサー チップの電極部分を浸すことによって。室温で穏やかな混合によって 2 分の各洗浄を実行します。</li>
		<li>ブロッキング溶液 50 μ L のドロップを追加することで、電極を飽和状態 (ステップ 2.1.2 参照) 電極表面上に。室温で 1 時間インキュベートします。洗浄 3 回、前に記載されているステップ (ステップ 2.3.2 を参照)。</li>
		<li>20 μ g/ml ソリューション バインドにブッ cAb Lys3 ソリューションを希釈 (手順 2.1.3 参照) の電極上にこの希釈液 15 μ L の滴を適用し、。室温で 10 分間インキュベートします。抗原生物学: ナノボディで反応後は洗浄液を使用して前の手順で 3 回に記載されて洗浄 (2.1.4 の手順を参照してください)。PBS で一度電極をすすいで (2.1.1 の手順を参照してください)。</li>
		<li>検出用センサー チップを経由してデジタル ・ マルチメータの銅回路部分を差し込みます。次に、50 μ L の滴検出ソリューションを適用することによってセンサー応答を開始 (2.1.7 のステップを参照) に、正極と負極 (2.1.7 のステップを参照) に食塩 5 mM の 50 μ L の滴を適用します。室温で 30 分間インキュベートします。デジタル ・ マルチメータとコンダクタンスを監視します。 注: マルチメータと定められた博士 p. Bogaerts、s. ユヌス博士と教授 Y. Glupczynski (カトリック大学のルーバン ・ ラ ・ ヌーブ - 朱モン Godinne。この電気化学測定装置は USB ポート経由でコンピューター制御し、センサーの 8 つの異なるチップを同時に分析します。ユヌス氏と同僚の17によって作成されたソフトウェアは、時間とリファレンスとサンプルの電極間のコンダクタンスの違いの測定を表すリアルタイム プロットを作成します。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Higgins, I. J., Lowe, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+the+principles+and+applications+of+biosensors.">Introduction to the principles and applications of biosensors.</a> Philos Trans R Soc Lond B Biol Sci. 316, 3-11 (1987).</li><li>Byrne, B., Stack, E., Gilmartin, N., O'Kennedy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+sensors:+principles,+problems+and+potential+for+detection+of+pathogens+and+associated+toxins.">Antibody-based sensors: principles, problems and potential for detection of pathogens and associated toxins.</a> Sensors (Basel). 9, 4407-4445 (2009).</li><li>Huston, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+of+antibody+binding+sites:+recovery+of+specific+activity+in+an+anti-digoxin+single-chain+Fv+analogue+produced+in+Escherichia+coli.">Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.</a> Proc Natl Acad Sci U S A. 85, 5879-5883 (1988).</li><li>Mechaly, A., Zahavy, E., Fisher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+implementation+of+a+single-chain+Fv+antibody+for+specific+detection+of+Bacillus+anthracis+spores.">Development and implementation of a single-chain Fv antibody for specific detection of Bacillus anthracis spores.</a> Appl Environ Microbiol. 74, 818-822 (2008).</li><li>Hamers-Casterman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+antibodies+devoid+of+light+chains.">Naturally occurring antibodies devoid of light chains.</a> Nature. 363, 446-448 (1993).</li><li>Sheriff, S., Constantine, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+minimal+antigen-binding+fragment.">Redefining the minimal antigen-binding fragment.</a> Nat Struct Biol. 3, 733-736 (1996).</li><li>Stijlemans, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+conserved+cryptic+epitopes+of+infectious+agents+by+single+domain+antibodies.+African+trypanosomes+as+paradigm.">Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm.</a> J Biol Chem. 279, 1256-1261 (2004).</li><li>Thanongsaksrikul, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+V+H+H+that+neutralizes+the+zinc+metalloproteinase+activity+of+botulinum+neurotoxin+type+A.">A V H H that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A.</a> J Biol Chem. 285, 9657-9666 (2010).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate-specific+antigen+immunosensing+based+on+mixed+self-assembled+monolayers,+camel+antibodies+and+colloidal+gold+enhanced+sandwich+assays.">Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays.</a> Biosens. Bioelectron. 21, 483-490 (2005).</li><li>Yolken, R. H., Wee, S. B., Van Regenmortel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+beta-lactamase+in+enzyme+immunoassays+for+detection+of+microbial+antigens.">The use of beta-lactamase in enzyme immunoassays for detection of microbial antigens.</a> J Immunol Methods. 73, 109-123 (1984).</li><li>Kojima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+circularly+permutated+beta-lactamase+tethered+to+antibody+domains+by+specific+small+molecules.">Activation of circularly permutated beta-lactamase tethered to antibody domains by specific small molecules.</a> Bioconjug Chem. 22, 633-641 (2011).</li><li>Iwai, H., Kojima-Misaizu, M., Dong, J., Ueda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+Ligand-Dependent+Enzyme+by+Fusing+Circularly+Permuted+Antibody+Variable+Region+Domains.">Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains.</a> Bioconjug Chem. 27, 868-873 (2016).</li><li>Vandevenne, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacillus+licheniformis+BlaP+beta-lactamase+as+a+model+protein+scaffold+to+study+the+insertion+of+protein+fragments.">The Bacillus licheniformis BlaP beta-lactamase as a model protein scaffold to study the insertion of protein fragments.</a> Protein Sci. 16, 2260-2271 (2007).</li><li>Vandevenne, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+easy+development+of+versatile+tools+to+study+protein/ligand+interactions.">Rapid and easy development of versatile tools to study protein/ligand interactions.</a> Protein Eng Des Sel. 21, 443-451 (2008).</li><li>Crasson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+functionalization+of+a+nanobody+using+protein+insertion+technology.">Enzymatic functionalization of a nanobody using protein insertion technology.</a> Protein Eng Des Sel. 28, 451-460 (2015).</li><li>Yunus, S., Attout, A., Vanlancker, G., Bertrand, P., Ruth, N., Galleni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+probe+electrochemically+active+material+state+in+portable+sensor+applications.">A method to probe electrochemically active material state in portable sensor applications.</a> Sensors and Actuators B: Chemical. 156, 35-42 (2011).</li><li>Bogaerts, P., Yunus, S., Massart, M., Huang, T. D., Glupczynski, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+BYG+Carba+Test,+a+New+Electrochemical+Assay+for+Rapid+Laboratory+Detection+of+Carbapenemase-Producing+Enterobacteriaceae.">Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae.</a> J Clin Microbiol. 54, 349-358 (2016).</li><li>Wang, L. P., Wang, W., Di, L., Lu, Y. N., Wang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+under+electrical+stimulation+of+neural+probe+coated+with+polyaniline.">Protein adsorption under electrical stimulation of neural probe coated with polyaniline.</a> Colloids Surf B Biointerfaces. 80, 72-78 (2010).</li><li>Piletsky, S., Piletska, E., Bossi, A., Turner, N., Turner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalization+of+porous+polypropylene+membranes+with+polyaniline+for+protein+immobilization.">Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization.</a> Biotechnol. Bioeng. 82, 86-92 (2003).</li><li>Khatkhatay, M. I., Desai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+performances+of+four+enzymes+used+in+ELISA+with+special+reference+to+beta-lactamase.">A comparison of performances of four enzymes used in ELISA with special reference to beta-lactamase.</a> J Immunoassay. 20, 151-183 (1999).</li><li>Worn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+in+vitro+stability+and+in+vivo+performance+of+anti-GCN4+intrabodies+as+cytoplasmic+inhibitors.">Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors.</a> J Biol Chem. 275, 2795-2803 (2000).</li><li>Ostermeier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+allosteric+protein+switches+by+domain+insertion.">Engineering allosteric protein switches by domain insertion.</a> Protein Eng Des Sel. 18, 359-364 (2005).</li><li>Choi, J. H., Laurent, A. H., Hilser, V. J., Ostermeier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+protein+switches+based+on+an+ensemble+model+of+allostery.">Design of protein switches based on an ensemble model of allostery.</a> Nat Commun. 6, 6968 (2015).</li><li>Collinet, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+accepted+insertions+of+proteins+within+protein+domains.">Functionally accepted insertions of proteins within protein domains.</a> J Biol Chem. 275, 17428-17433 (2000).</li><li>Betton, J. M., Jacob, J. P., Hofnung, M., Broome-Smith, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+a+bifunctional+protein+by+insertion+of+beta-lactamase+into+the+maltodextrin-binding+protein.">Creating a bifunctional protein by insertion of beta-lactamase into the maltodextrin-binding protein.</a> Nat Biotechnol. 15, 1276-1279 (1997).</li><li>Ay, J., Gotz, F., Borriss, R., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+Bacillus+hybrid+enzyme+GluXyn-1:+native-like+jellyroll+fold+preserved+after+insertion+of+autonomous+globular+domain.">Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain.</a> Proc Natl Acad Sci U S A. 95, 6613-6618 (1998).</li><li>Ruth, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccination+for+the+priming+of+neutralizing+antibodies+against+non-immunogenic+STa+enterotoxin+from+enterotoxigenic+Escherichia+coli.">DNA vaccination for the priming of neutralizing antibodies against non-immunogenic STa enterotoxin from enterotoxigenic Escherichia coli.</a> Vaccine. 23, 3618-3627 (2005).</li><li>Zervosen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cattle+serum+antibody+responses+against+TEM+beta-lactamase+and+the+nonimmunogenic+Escherichia+coli+heat-stable+enterotoxin+(STaI).">Characterization of the cattle serum antibody responses against TEM beta-lactamase and the nonimmunogenic Escherichia coli heat-stable enterotoxin (STaI).</a> FEMS Immunol Med Microbiol. 54, 319-329 (2008).</li><li>Chevigne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bifunctional+hybrid+beta-lactamases+for+epitope+mapping+and+immunoassay+development.">Use of bifunctional hybrid beta-lactamases for epitope mapping and immunoassay development.</a> J Immunol Methods. 320, 81-93 (2007).</li><li>Ke, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+an+engineered+beta-lactamase+maltose+binding+protein+fusion+protein:+insights+into+heterotropic+allosteric+regulation.">Structure of an engineered beta-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.</a> PloS One. 7, 39168 (2012).</li><li>Saeedfar, K., Heng, L. Y., Ling, T. L., Rezayi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiometric+urea+biosensor+based+on+an+immobilised+fullerene-urease+bio-conjugate.">Potentiometric urea biosensor based on an immobilised fullerene-urease bio-conjugate.</a> Sensors (Basel). 13, 16851-16866 (2013).</li><li>D'Orazio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensors+in+clinical+chemistry.">Biosensors in clinical chemistry.</a> Clin Chim Acta. 334, 41-69 (2003).</li><li>Szucs, J., Pretsch, E., Gyurcsanyi, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiometric+enzyme+immunoassay+using+miniaturized+anion-selective+electrodes+for+detection.">Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection.</a> Analyst. 134, 1601-1607 (2009).</li><li>Ding, J., Wang, X., Qin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+galvanostatic+control+of+a+polymeric+membrane+ion-selective+electrode+for+potentiometric+immunoassays.">Pulsed galvanostatic control of a polymeric membrane ion-selective electrode for potentiometric immunoassays.</a> ACS Appl Mater Interfaces. 5, 9488-9493 (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polymeric+liquid+membrane+electrode+responsive+to+3,3',5,5'-tetramethylbenzidine+oxidation+for+sensitive+peroxidase/peroxidase+mimetic-based+potentiometric+biosensing.">A polymeric liquid membrane electrode responsive to 3,3',5,5'-tetramethylbenzidine oxidation for sensitive peroxidase/peroxidase mimetic-based potentiometric biosensing.</a> Anal Chem. 86, 4416-4422 (2014).</li><li>Grieshaber, D., MacKenzie, R., Voros, J., Reimhult, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Biosensors+-+Sensor+Principles+and+Architectures.">Electrochemical Biosensors - Sensor Principles and Architectures.</a> Sensors (Basel). 8, 1400-1458 (2008).</li><li>Bakker, E., Pretsch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+potentiometry.">Nanoscale potentiometry.</a> Trends Analyt Chem. 27, 612-618 (2008).</li><li>Zhang, D., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensors+and+bioelectronics+on+smartphone+for+portable+biochemical+detection.">Biosensors and bioelectronics on smartphone for portable biochemical detection.</a> Biosens Bioelectron. 75, 273-284 (2016).</li><li>Nemiroski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+mobile+electrochemical+detector+designed+for+use+in+resource-limited+applications.">Universal mobile electrochemical detector designed for use in resource-limited applications.</a> Proc Natl Acad Sci U S A. 111, 11984-11989 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Socio-economic impact of mHealth- An assessment report for the European Union. , (2013).</li></ol>

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この作品で生物学: ナノボディでキャリア蛋白質としてブッ β-ラクタマーゼを使用しての高機能化する手法を提案し、電位差センサー アッセイで、結果として得られるハイブリッド タンパク質を正常に実装できることを示します。他のバイオ センサーの試金と比較して我々 の仕事の主な技術革新の側面は、電気信号を生成する酵素活性の抗体部分の共有カップリングです。このいわゆるタ...

バイオ センサー、トランスデューサー1と呼ばれる物理または化学のシグナリング デバイスと生体分子の相互作用を結合した分析機器です。記録された信号の解釈および固定化と無料のパートナー間の相互作用を監視に変換できます。グルコのほとんどは、ホルモンなど異なる病原体マーカー2検体を検出する抗体の使用を含みます。別のセンサー フォーマットは使用でき、質量ベース、磁気、光学または電気化学バイオ センサーが含まれます。後者は最も一般に使用されるセンサーとバインディング イベントを電気信号に変換することによって機能します。公演とすべて抗体を用いたバイオ センサーの感度は、本質的に 2 つのパラメーターに強く依存する: i) 抗体と ii)2の信号を生成するために使用するシステムのプロパティの品質。
抗体は 2 つの重鎖および 2 つの軽鎖から成る高分子質量二量体蛋白質 (150-160 kDa) です。光と重鎖間の相互作用を主に保存されたジスルフィド結合と同様に、疎水性相互作用によって安定させます。各チェインには、本質的に補完決定領域 (CDR1-2-3) という 3 つの超可変領域を介して抗原と相互作用変数の領域が含まれています。フィールド、大規模な低コスト式システム (例えば、エシェリヒア属大腸菌) とフルレングス抗体発現の多数の進歩にもかかわらず、しばしば不安定で集約された蛋白質の生産に 。だからこそ、さまざまな抗体フラグメント、単一鎖変数3 (ScFvs ≈ 25 kDa) の断片のように設計されてです。彼らは、それぞれ 1 つの重いと共有結合合成アミノ酸シーケンスによってリンクされている 1 つの軽鎖の可変領域で構成されます。ただし、これらのフラグメントはしばしば安定性の悪いを表示され、集計、溶剤4その疎水性領域の大きい部分を公開するために傾向があります。この中で、nanobodies または VHHs と呼ばれる、単一のチェーン ラクダ科動物抗体フラグメントは ScFvs に優れた選択肢と思われます。これらのドメインは、ラクダ科一本鎖抗体の可変領域に対応します。従来の抗体と対照をなしてラクダ科動物抗体軽鎖を欠いている、のみ 2 つの重鎖5が含まれます。したがって、nanobodies は、従来の抗体6のような親和性を持つ抗原に結合することができる最小単量体抗体フラグメント (12 kDa)。さらに、安定性の向上と他のフルレングスの抗体や抗体フラグメントと比較して溶解性を呈する。最後に、その小さなサイズと、拡張 CDR3 ループに、不可解なエピトープを認識し、酵素活性部位7、8にバインドする許可にします。今日では、これらのドメインはかなりの注目を受けているし、バイオ センサー技術に結合されています。たとえば、黄ら。検出と人間の前立腺特異抗原 (PSA)9の定量化のための生物学: ナノボディでベースのバイオ センサーを開発しました。
言及した上としてバイオ センサーの試金で重要なパラメーターは電気信号を生成するために使用するシステムの効率です。このため、酵素法による電気化学バイオ センサーはますます注目を集めている、健康管理、食品の安全性や環境モニタリングなど様々 な用途に広く使用されています。これらのバイオ センサーは、電気信号を生成する酵素による基質の触媒加水分解に依存します。この文脈では、β-ラクタマーゼより具体的より敏感とアルカリホスファターゼまたは西洋わさびペルオキシダーゼ10など多くの他の酵素よりも実験的に実装する方が簡単に示されていた。Β-ラクタマーゼは β-ラクタム系抗生物質に耐性菌の責任、それらを加水分解する酵素です。彼らは、単量体、非常に安定した、効率的、かつ小さなサイズが。さらに、β-ラクタマーゼ挿入ドメイン/ペプチドはタンパク質-リガンド相互作用を研究する効率的なツールであることが示された bi 機能のキメラ蛋白質を生成します。確かに、最近の研究はその標的抗原に高い親和性と結合することができるままキメラ蛋白質 TEM1 β-ラクタマーゼ結果に変数の抗体のフラグメントをその挿入を示しています。興味深いことに、抗原結合 TEM1 触媒活性11,12のアロステリック効果を誘導するために示されました。さらに、我々 はバチルス リチェニホルミスブッ β-ラクタマーゼの寛容なループにタンパク質ドメイン挿入が蛋白質-リガンド相互作用13 を監視に適しています機能のキメラ蛋白質を生成するいくつかの研究で示した ,14。生物学: ナノボディで、ブッ15のこの寛容な挿入部位にという名前の cAb Lys3、最近挿入した.この生物学: ナノボディで示した鶏卵白リゾチーム (リゾチーム) にバインドする16その酵素活性を阻害します。ブッ-タクシー-Lys3 の名前生成されたハイブリッド蛋白質は高い特異性を保持/親和性リゾチーム β-ラクタマーゼ活性のまま変更しないことを示した.そして我々 は正常に β-ラクタマーゼ技術のハイブリッド電気化学バイオ センサーを組み合わせるし、生成された電気信号の量がブッ cAb Lys3 と電極に固定化したリゾチームの相互作用に依存していたことを示した。確かに、ブッによる β-ラクタム系抗生物質の加水分解は、定量的な電気信号に変換することができますプロトン放出を誘導します。このハイブリッド β-ラクタマーゼ技術電気化学バイオ センサーとの組み合わせは、高速、高感度、定量、生成された信号のリアルタイム測定が可能.この方法を次に記載します。

<table border="0" cellpadding="0" cellspacing="0" width="1471">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Reagents</td>
			<td style="width:215px;"></td>
			<td style="width:128px;"></td>
			<td style="width:615px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td>Sigma-Aldricht</td>
			<td>V000225&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td>Sigma-Aldricht</td>
			<td>1551128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldricht</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8211;HCl</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldricht</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>NIST2186II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Sigma-Aldricht</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alanine</td>
			<td>Sigma-Aldricht</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HClO4</td>
			<td>Fluka</td>
			<td>34288</td>
			<td>1M HClO4 solution, distributor : Sigma-Aldricht</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">casein hydrolysate</td>
			<td>Sigma-Aldricht</td>
			<td>22090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzylpenicillin sodium</td>
			<td>Sigma-Aldricht</td>
			<td>B0900000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">hen egg white lysozyme</td>
			<td>Roche</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">heptane</td>
			<td>Sigma-Aldricht</td>
			<td>246654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">methanol</td>
			<td>Sigma-Aldricht</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium hydroxide solution</td>
			<td>Sigma-Aldricht</td>
			<td>380539</td>
			<td>28% NH3&#160;in H2O, purified by double-distillation (concentrated?)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laboratory consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657165</td>
			<td>CELLSTAR 6-Well Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>WTW</td>
			<td>1AA110</td>
			<td>Lab pH meter inoLab pH 7110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vacuum and filtration system</td>
			<td>Nalgene</td>
			<td>NALG300-4100</td>
			<td>Filter holders with receiver, distributor : VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">potentiometric sensor chips</td>
			<td></td>
			<td></td>
			<td>manufactured by Yunus and colleagues (ref 16)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGSTAT30 Autolab</td>
			<td>Metrohm Autolab</td>
			<td></td>
			<td>discontinued, succesor Autolab PGSTAT302N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital multimeter, METRAHit 22M</td>
			<td>Gossen Metrawatt</td>
			<td></td>
			<td>discontinued, successor Metrahit Base</td>
		</tr>
	</tbody>
</table>

the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions

バイオ センサーは、病原体検出、分子診断、環境監視、および食品安全性の管理などさまざまな分野でますます重要かつ実装になっています。この文脈では、いくつかのタンパク質間相互作用研究の効率的なレポーター酵素と β-ラクタマーゼを使用しました。さらに、強くペプチッドまたは構造化蛋白質/ドメインの挿入を受け入れる能力はキメラ蛋白質を生成するこれらの酵素の使用を奨励しています。最近の研究ではバチルス リチェニホルミスブッ β-ラクタマーゼに単一ドメイン抗体フラグメントを挿入されます。Nanobodies とも呼ばれるこれらの小さなドメインは、ラクダから一本鎖抗体の抗原結合ドメインとして定義されます。共通の二重鎖抗体のよう彼らは高い親和性と特異性の目標を表示します。結果のキメラ蛋白質 β-ラクタマーゼの活性を維持しながらその目標に対して高い親和性を展示しました。機能生物学: ナノボディで、β-ラクタマーゼの鎖を維持することが示唆されました。現在の仕事では、バイオ センサー技術に当社のハイブリッド β-ラクタマーゼのシステムを組み合わせた詳細なプロトコルを報告します。生物学: ナノボディでターゲットの特定の結合は、酵素の触媒活性が発表したプロトンの伝導度測定のおかげで検出できます。

デザインとエンジニア リングのキメラ蛋白質ブッ cAb Lys3
図 1 は、バチルス リチェニホルミス由来 BalP クラス A の β-ラクタマーゼの寛容なループに cAb Lys3 の挿入を表します。残基 Asp198 と Lys199 間挿入を行った。トロンビン切断部位は、cAb Lys3 の各側に導入されました。ブッ cAb Lys3 キメラ蛋白質発現プラスミドで形?...

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The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

この作品は、ハイブリッドの β-ラクタマーゼ技術に基づく伝導度センサーを用いたタンパク質間相互作用を研究する手法を報告する.このメソッドは、β-ラクタムの加水分解時に陽子のリリースに依存しています。

生体分子相互作用を検出する β-ラクタマーゼによる伝導度バイオ センサー法の使用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

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8.9K ビュー.  University of Liège. この作品は、ハイブリッドの β-ラクタマーゼ技術に基づく伝導度センサーを用いたタンパク質間相互作用を研究する手法を報告する.このメソッドは、β-ラクタムの加水分解時に陽子のリリースに依存しています。

ビデオ: The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

癌細胞とヒアルロン酸の相互作用を研究するマイクロパターン表面

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

生物工学

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

干渉反射イメージングセンサー（IRIS）を用いた生体分子の検出

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

核酸、タンパク質と低分子のReagentless検出用電気化学DNAバイオセンサーの作製

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Gタンパク質共役受容体によるカリウムチャネルの調節を検出するために、ラベルフリー光学バイオセンサの使用

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

ハイスループットの使用<em&gt;インビトロ</emオーラル多種バイオフィルムを開発するために&gt;マイクロ流体システム

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

腹側腹部ヘルニア修復術を勉強するラットのモデルの使用

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip：タンパク質 - ホスホイノシチド相互作用のラベルフリー研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

ドライ フィルムのレジストを用いた電気化学マイクロ流体バイオ プラットフォーム: デバイス作製、チップの試金の準備、およびシステム操作

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

生体分子相互作用を検出する β-ラクタマーゼによる伝導度バイオ センサー法の使用

生体分子相互作用を検出する β-ラクタマーゼによる伝導度バイオセンサー法の使用