作者承认比利时的瓦隆地区在 SENSOTEM 和 NANOTIC 研究项目的框架内, 以及国家科研基金 (储备-f.n.r.) 的财政支持。

1. 蛋白质样品制备
<ol><li>生产和纯化杂交蛋白 BlaP-cAb-Lys3 如我们以前的研究报告15。将蛋白质储存在50毫米磷酸盐缓冲液 pH 值7.4 以下组成: 8 克氯化钠, 0.2 克氯化钾, 1.44 克 Na2HPO4和 0.24 g 的下一2PO4溶解在800毫升蒸馏水中修复溶液的 pH 值为7。4将溶液的最终体积调整到 1 l. 过滤器对蛋白质溶液进行杀菌。</li>
	<li>准备一个母鸡卵白溶菌酶 (HEWL) 库存解决方案。溶解100毫克 (4万单位/毫克) 的商业购买 HEWL 在10毫升磷酸盐缓冲盐水 (PBS 见步骤 2.1.1)。用0.22 μ m 截止过滤器对蛋白质溶液进行消毒。</li>
</ol>2. 生物传感器检测
<ol><li>
解决方案和缓冲准备
	<ol>
<li>准备50毫米 PBS 通过溶解 8 g 氯化钠, 0.2 克氯化钾, 1.44 g Na2HPO4, 和 0.24 g 的, 在2毫升的蒸馏水4PO800 。调整到 pH 值7.4 与 1 n HCl 或 1 n 氢氧化钠, 然后调整解决方案的体积到 1 l. 过滤消毒和存储在4° c。</li>
		<li>准备一个饱和/堵塞解决方案, 溶解3克的酪蛋白水解物在100毫升的 PBS 制备如上文所述 (见步骤 2.1.1)。过滤消毒, 贮存在4° c。</li>
		<li>准备一个结合的解决方案, 溶解1克的酪蛋白水解物在100毫升的 PBS 制备如上文所述 (见步骤 2.1.1)。过滤消毒, 贮存在4° c。</li>
		<li>准备一个洗涤解决方案 (0.1% 补间-pbs), 添加100μ l 吐温 20 (100%) 在100毫升的 pbs 准备如上文所述 (见步骤 2.1.1)。贮存在4° c。</li>
		<li>准备一个电极准备解决方案 (1% 海卫 X-100-pbs), 增加1毫升的海卫 X-100 (100%) 在100毫升的 PBS 制备如上所述 (见步骤 2.1.1)。贮存在4° c。</li>
		<li>准备一个电极再生解决方案 (3.5 米氯化钾) 溶解26克的氯化钾在蒸馏水到最后一卷100毫升。过滤灭菌, 贮存在4° c。</li>
		<li>准备一个5毫米氯化钠溶液溶解0.29 克氯化钠在蒸馏水到最后的容量 1 l. 过滤器消毒并且存放在4° c。然后, 准备一个检测解决方案 (4 毫米青霉素) 溶解26.7 毫克的青霉素在20毫升5毫米氯化钠溶液。过滤消毒, 贮存在-20 ° c。</li>
	</ol>
</li>
	<li>
传感器的制备和再生 注: 聚苯胺涂层传感器芯片由 Dr. p Bogaerts、Dr. s ·尤努斯和 Prof. Glupczynski 的天主教大学 (鲁汶-拉-Neuve-楚-Godinne) 提供。对传感器的描述以及用于合成这些传感器的聚苯胺电聚合协议在以前的工作17中进行了详细介绍。简单地说, 该系统使用的是由传统印制电路板 (PCB) 技术制造的八单个芯片的可重新使用的传感器。单个芯片由三电极圆点组成。顶部是聚苯胺电合成的工作电极。中间一个是参考电极, 底部电极构成计数器电极。两个, 参考和计数器电极功能化使用固体银/氯化汞合金的顶部的碳层。<ol>
<li>执行3冲洗的电极, 浸渍的秘诀, 96-井板包含300µL/井的电极制备解决方案 (1% 海卫 X-100-PBS, 见步骤2.1.5。每洗2分钟, 在室温下进行温和搅拌。</li>
		<li>冲洗电极, 将小贴士浸入含有300µL 的96井板的井中, 并在室温下温和搅拌2分钟。</li>
		<li>在室温下, 在4° c 或 1 h 的条件下, 通过将尖端浸入96个井板 (3.5 M 氯化钾, 见步骤 2.1.6), 重新生成电极。</li>
		<li>执行3冲洗的电极, 把小费浸入96井板含300µL/井的磷酸盐缓冲盐水 (见步骤2.1.1。每洗2分钟, 在室温下进行温和搅拌。</li>
	</ol>
</li>
	<li>
在传感器上进行的结合化验
	<ol>
<li>通过将 PBS 制备的15µL 滴40µg/mL HEWL 沉积到电极表面, 将 HEWL 涂在电极上的聚苯胺表面。在室温下在4° c 或1小时孵育过夜。</li>
		<li>用磷酸盐缓冲盐水 (见步骤 2.1.1) 将传感器芯片的电极部分浸入含磷酸盐缓冲盐水300µL/井的96井板的井中, 执行三的电极洗涤。每洗2分钟, 在室温下进行温和搅拌。</li>
		<li>通过在电极表面加入50µL 滴的堵塞溶液 (见步骤 2.1.2) 使电极饱和。室温下孵育1小时。然后洗三次, 如前一步所述 (参见步骤 2.3.2)。</li>
		<li>将 BlaP-cAb-Lys3 溶液稀释到20µg/毫升的结合溶液中 (见步骤 2.1.3), 并将该稀释溶液的15µL 滴放到电极上。室温孵育10分钟。在抗原-nanobody 反应之后, 洗涤三次被描述在前步使用洗涤解答 (参见步 2.1.4)。然后用 PBS 冲洗电极 (见步骤 2.1.1)。</li>
		<li>用于检测, 将传感器芯片通过铜电路部件插入数字万用表。然后, 启动传感器的反应, 应用50µL 下降的检测解决方案 (见步骤 2.1.7) 到积极的电极和应用50µL 滴氯化钠5毫米溶液到负极 (见步骤 2.1.7)。室温孵育30分钟。用数字万用表监测电导率。 注: 万用表是由 Dr. p Bogaerts、Dr. 和 Prof. Glupczynski (鲁汶-拉-Neuve-楚 Godinne 天主教大学提供的。此电位是通过 USB 端口进行计算机控制的, 同时分析八种不同的传感器芯片。由尤努斯和同事们创建的软件17创建了一个实时图, 它表示参照和采样电极对时间的电导差的测量。</li>
	</ol>
</li>
</ol>

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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>

作者没有什么可透露的。

在这项工作中, 我们提出了一种方法来化 nanobody 使用 BlaP β-酶作为载体蛋白, 我们表明, 我们可以成功地实现的结果混合蛋白的电位传感器检测。我们工作的主要创新方面与其他生物传感器的检测相比, 是抗体部分与产生电信号的酶活性的共价耦合。这种所谓的蛋白质插入技术带来的优势和局限性, 将是本节的主要重点。
混合β-酶技术的优势。
β-酶是高效...

生物传感器是一种与物理或化学信号装置相结合的分析设备, 称为传感器1。然后, 可以对记录的信号进行解释和转换, 以监测固定和自由伙伴之间的相互作用。大多数的生物传感器涉及使用抗体来检测分析如激素或不同的病原体标记物2。可以使用不同的传感器格式, 包括基于质量的、磁的、光学的或电化学的生物传感器。后者是最常用的传感器之一, 通过将绑定事件转换为电信号来发挥作用。所有基于抗体的生物传感器的性能和灵敏度主要取决于两个参数: i) 抗体的质量和 ii) 用于生成信号2的系统的属性。
抗体是由两个重链和两个光链组成的高分子质量二蛋白 (150–160 kDa)。光与重链之间的相互作用主要通过疏水相互作用以及一个守恒的二硫化键稳定。每个链包括一个变量域, 它通过三变区域 (CDR1-2-3) 来与抗原相互作用。尽管在该领域取得了许多进展, 但具有低成本表达系统 (例如、大肠杆菌) 的全长抗体的大规模表达往往导致不稳定和聚合蛋白的产生。这就是为什么各种各样的抗体片段被设计了例如单链可变片段3 (ScFvs ≈ 25 kDa)。它们由一个重和一个轻链的可变域组成, 由一个合成氨基酸序列共价键连接。然而, 这些碎片往往表现出很差的稳定性, 并有聚合的倾向, 因为它们将其疏水区域的很大一部分暴露给溶剂4。在这种情况下, 单链骆驼抗体片段, 称为 nanobodies 或 VHHs, 似乎是很好的替代 ScFvs。这些域对应于骆驼单链抗体的可变域。与常规抗体相比, 骆驼抗体缺乏光链, 只包含两个重链5。因此, nanobodies 是最小的单体抗体片段 (12 kDa) 能够绑定到一个抗原的亲和力类似于常规抗体6。此外, 与其他全长抗体或抗体片段相比, 它们具有更好的稳定性和溶解度。最后, 他们的小尺寸和他们的扩展 CDR3 循环允许他们辨认隐晦的表位并且束缚到酵素活跃站点7,8。目前, 这些领域得到了相当的重视, 并已与生物传感器技术相结合。例如, 黄等人。开发了一种基于 nanobody 的生物传感器, 用于检测和量化人类前列腺特异抗原 (PSA)9。
如上所述, 生物传感器检测的一个重要参数是用于产生电信号的系统的效率。因此, 基于酶的电化学生物传感器越来越受到人们的关注, 并被广泛应用于医疗保健、食品安全和环境监测等各种领域。这些生物传感器依赖于一种酶对基质的催化水解来产生电信号。在这种情况下, β-酶被证明是更具体, 更敏感和更容易实施实验比其他许多酶, 如碱性磷酸酶或辣根过氧化物酶10。β-酶是酶, 负责对β内酰胺抗生素的细菌耐药性, 通过水解它们。它们单体, 非常稳定, 高效, 体积小。此外, 域/肽插入β-酶产生双功能嵌合蛋白, 被证明是研究蛋白质-配体相互作用的有效工具。事实上, 最近的研究表明, 在 TEM1 β-酶中插入抗体可变片段的结果是嵌合蛋白, 仍然能够与其靶抗原的高度亲和性结合。有趣的是, 抗原结合被证明能诱发 TEM1 催化活性的构调节11,12。此外, 我们在几项研究中表明, 蛋白质域插入到一个允许循环的芽孢杆菌地衣BlaP β-酶产生功能嵌合蛋白, 非常适合监测蛋白配体的相互作用13 ,14。我们最近在 BlaP15的这个允许插入站点中插入了一个名为 cAb-Lys3 的 nanobody。这 nanobody 被证明绑定到母鸡-蛋清溶菌酶 (HEWL) 和抑制其酶活性16。我们表明, 生成的杂交蛋白, 命名为 BlaP-cAb-Lys3, 保留了高特异性/亲和力对 HEWL, 而β-酶活性保持不变。然后, 我们成功地将混合β-酶技术与电化学生物传感器相结合, 结果表明, 产生的电信号量与固定在电极上的 BlaP-cAb-Lys3 和 HEWL 的相互作用有关。事实上, β-内酰胺抗生素的水解 BlaP 诱导质子释放, 可以转化为定量的电信号。这种组合的混合β-酶技术与电化学生物传感器是快速, 灵敏, 定量, 并允许实时测量生成的信号。本文介绍了这种方法。

<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

生物传感器在病原检测、分子诊断、环境监测、食品安全控制等各个领域的应用越来越重要。在这方面, 我们使用β-酶作为有效的报告酶在几个蛋白质-蛋白质相互作用研究。此外, 他们接受多肽或结构蛋白/域插入的能力强烈鼓励使用这些酶产生嵌合蛋白。在最近的一项研究中, 我们在芽孢杆菌地衣BlaP β-酶中插入了一个单域抗体片段。这些小的域, 也称为 nanobodies, 被定义为骆驼单链抗体的抗原结合域。就像常见的双链抗体一样, 它们具有很高的亲和力和特异的靶向。所产生的嵌合蛋白在保持β-酶活性的同时, 对其靶向性具有较高的亲和力。这表明, nanobody 和β-酶基保持功能。在目前的工作中, 我们报告了一个详细的协议, 结合我们的混合β-酶系统的生物传感器技术。由于酶的催化活性释放出的质子的电导测量, 可以检测到 nanobody 对其目标的特定约束力。

嵌合蛋白 BlaP-cAb-Lys3 的设计与工程
图1表示将 cAb-Lys3 插入到 BalP 类的一个允许循环中, 从地衣芽孢杆菌中的β-酶。在残余 Asp198 和 Lys199 之间进行插入。在 cAb-Lys3 的两侧引入了凝血酶劈裂部位。用本构表达质粒对 BlaP-cAb-Lys3 嵌合蛋白进行转化的细胞能够在氨苄青霉素的小浓度下生长。这一结果表明, 杂交β-酶是可溶性的, 正?...

Watch this Scientific Journal Video about The Use of a Î²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

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

在这项工作中, 我们报告了一个新的方法来研究蛋白质-蛋白质相互作用的电导生物传感器的基础上的混合β-酶技术。这种方法依赖于质子在β-内酰胺水解时的释放。

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.9K 次观看.  University of Liège. 在这项工作中, 我们报告了一个新的方法来研究蛋白质-蛋白质相互作用的电导生物传感器的基础上的混合β-酶技术。这种方法依赖于质子在β-内酰胺水解时的释放。

视频: 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.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

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

科研

生物工程

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.

生物分子检测的干涉反射成像传感器（IRIS）

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.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

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.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

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.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

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.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

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.

大鼠模型在腹腹疝修补术中的应用研究

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:蛋白质 - 磷酸肌醇相互作用的无标记研究

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.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

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

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

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