这项工作部分由国家普通医学科学研究所（NIGMS）T32计划（T32 GM133369）和国家科学基金会（NSF 1454230）资助。此外，西弗吉尼亚大学共享研究设施和应用生物物理学的援助和支持也得到了认可。

1. ZIF-8合成
<ol><li>在本例中，使用1：10：100（金属：接头：溶剂）的质量比来合成ZIF-8。为此，测量出六水合硝酸锌，并记录测量结果。利用示例质量比计算接头、2-甲基咪唑和溶剂（即甲醇）所需的量。</li>
	<li>将六水合硝酸锌和接头放入两个不同的玻璃小瓶中。将计算量的甲醇量的一半加入六水合硝酸锌中，另一半加入接头中。让每种溶液溶解。</li>
	<li>将两种溶液（即分别含有金属和接头的溶液）结合起来。将装有反应组合溶液的容器放在搅拌板上，并在室温（RT）下以700rpm搅拌24小时。</li>
</ol>2. ZIF-8系列
<ol><li>上述反应结束后（即24小时后），将溶液放入50mL离心管中，并以3,075× g 离心5分钟（使用等量并平衡转子）。从离心管中取出任何上清液。</li>
	<li>向离心管中加入甲醇 （5-15 mL） 并重新分散颗粒。</li>
	<li>重复离心和洗涤步骤至少两到四次。最后一次洗涤后，排出上清液。 注意：洗涤步骤去除任何非沉淀物质。</li>
	<li>取下管子的盖子，将封口膜胶带放在顶部;随后，在封口膜上戳洞。将装有样品的试管置于室温的真空室中干燥24小时。</li>
</ol>3. ZIF-8表面形貌（扫描电子显微镜[SEM]）
<ol><li>将ZIF-8的干粉安装到碳带上，并用金钯溅射120秒，以防止在SEM分析过程中可能发生的任何电荷。</li>
	<li>将显微镜的加速电压设置为15 kV。聚焦粒子并调整放大倍率（使用500x，1,000x，1,500x，4,000x，10,000x，20,000x，30,000x和40,000x的放大倍率）。</li>
	<li>在放大镜下对颗粒进行成像，以便进行清晰的颗粒大小和形态评估（建议考虑 5 μm、10 μm 和 20 μm 颗粒的范围）。</li>
	<li>从至少三个不同的视野收集数据，对于每个视场，考虑不同的放大倍率。</li>
</ol>4. ZIF-8元素组成
<ol><li>按照 SEM 成像的步骤操作。</li>
	<li>使用SEM显微镜的能量色散X射线（EDX）光谱单元，分析样品元素组成。</li>
	<li>确保 SEM 加速电压和放大倍率与 EDX 监视器匹配。关闭 SEM 显微镜上的红外相机。</li>
	<li>在 EDX 监视器上，转到收集 频谱 并输入 200 秒的收集时间。建议这样做以避免可能导致错误评估的粒子充电和分解。</li>
	<li>从至少三个不同的视野收集数据。收集完成后，分析数据并确定感兴趣的元素。</li>
</ol>5. 细胞培养
<ol><li>在补充有5%胎牛血清（FBS）和0.2%青霉素/链霉素的培养基中培养永生化的BEAS-2B细胞。 注意：应使用5-10之间的传代，以确保先前使用的细胞批次中没有发生表型变化37 （所有试剂均列在 材料表中）。</li>
	<li>取一个 100 mm 细胞培养板，将 10 mL 培养基放在板上。向平板中加入 1 mL BEAS-2B 细胞溶液。</li>
	<li>将板放入培养箱（37°C，5%CO2）中，让细胞生长。</li>
	<li>24小时后检查板以确定细胞是否达到80%汇合度。本文中，80%的汇合度是指被粘附细胞覆盖的表面的百分比。如果没有，请更换培养基并让细胞生长，直到达到 80% 的汇合度水平。</li>
</ol>6. 细胞计数
<ol><li>一旦板上的细胞达到80%的汇合度，从平板上取出培养基。用 5 mL 磷酸盐缓冲盐水 （PBS） 清洗板。</li>
	<li>从盘子上取下PBS。向板中加入2mL胰蛋白酶，并将其置于培养箱（37°C，5%CO 2）中2分钟。</li>
	<li>向板中加入 2 mL 培养基，上下移液培养基以从平板中取出细胞。将溶液转移到 15 mL 管中，并以 123 x g 离心 5 分钟。</li>
	<li>从管中取出上清液并加入 2 mL 培养基。上下移液培养基以重新分散细胞。</li>
	<li>使用 1.5 mL 管，加入 20 μL 台盼蓝，然后加入 20 μL 细胞溶液（台盼蓝：细胞溶液的比例为 1：1）。</li>
	<li>将 10 μL 细胞混合物放在载玻片上，然后将其放在自动细胞计数器上。等到屏幕成为焦点，然后选择 捕获。</li>
	<li>屏幕显示活细胞数量和细胞活力。测量范围从 1 x 104-1 x 107 个电池。</li>
	<li>或者，使用血细胞计数器手动计数细胞。<ol><li>为此，向血细胞计数器中加入 15-20 μL 台盼蓝处理的细胞溶液。</li>
		<li>使用正置显微镜，对血细胞计数器的网格线进行10倍物镜聚焦。</li>
		<li>计算四个外部方块中的单元格数，然后将数字除以四。然后，乘以 10,000 以获得活细胞计数数。</li>
		<li>要计算细胞活力，请将活细胞计数和死细胞计数相加以获得总细胞计数，然后将活细胞计数除以总细胞计数。</li>
	</ol></li>
</ol>7. ZIF-8剂量制剂
<ol><li>制作 1 mg/mL 的 ZIF-8 储备溶液。为此，测量ZIF-8的量并向颗粒中加入去离子（DI）水以达到1mg / mL浓度。</li>
	<li>在水浴中以30秒的间隔对ZIF-8储备溶液进行超声处理（将超声仪设置为声波）2分钟。</li>
	<li>使用公式C1 V1 = C 2 V2计算达到所需剂量所需的储备溶液和 Dulbecco 的改良鹰培养基 （DMEM） 的量，为细胞暴露制作不同剂量的 ZIF-8。剂量范围为 0-950 μg/mL。 注意：在本实验中，选择0μg/mL，1μg/ mL，10μg/ mL，50μg/ mL，100μg/ mL，250μg/ mL，500μg/ mL，750μg/ mL和950μg/ mL的剂量。</li>
</ol>8. 半最大抑制浓度 （IC 50）
<ol><li>细胞计数后，将 BEAS-2B 细胞以 4 x 104 个细胞 /mL 的密度接种在 96 孔板中，体积为 100 μL/孔。<ol><li>要达到 4 x 104 个细胞/mL 的密度，请使用 C 1 V1= C 2 V2计算需要与培养基结合的细胞溶液量。</li>
		<li>确保每次剂量保持空白孔;将这些孔留空，直到ZIF-8暴露。</li>
	</ol></li>
	<li>将96孔板放入37°C和5%CO2 的培养箱中，并使细胞生长至汇合的单层24小时。</li>
	<li>从孔中取出培养基，并将BEAS-2B细胞暴露于0-950μg/ mL的ZIF-8剂量下。将 100 μL ZIF-8 加入各自的空白孔和细胞孔中。</li>
	<li>将96孔板放回培养箱中，暴露时间为48小时。</li>
	<li>暴露后，向每个孔（空白孔和细胞孔）中加入 10 μL 4-[3-（4-碘苯基）-2-（4-硝基苯基）-2H-5-四唑咯基]-1,3-苯二磺酸酯 （WST-1） 并孵育 2 小时。媒体仍然在井里;比例为1：10（试剂：培养基）。</li>
	<li>使用485nm吸光度在读板器上读取吸光度的变化。</li>
	<li>通过软件计算细胞活力以确定IC 50值（参见第10 节）。</li>
</ol>9. 电电池基板阻抗传感 （ECIS）
<ol><li>使用 96 孔板 （96W10idf） 进行 ECIS 分析。该板28 包含指间指指连接电极，覆盖面积约4毫米2。</li>
	<li>首先，测试传感器是否正在读取所有孔。为此，将 96 孔板放在井站上，然后单击 设置 按钮。</li>
	<li>如果井连接正确，将出现绿色;任何未连接的井将被标识为红色。如果出现这种情况，请取出并重新插入孔板，然后再次单击 “设置 ”按钮，直到所有孔都提供可行的绿色读数。</li>
	<li>用每孔 200 μL 培养基将电极稳定 40 分钟，以防止潜在的漂移。向使用的每个孔中加入 200 μL 培养基，然后将 96 孔板放在 ECIS 工作站上。单击 设置/检查 以确保板已正确连接。点击 稳定。</li>
	<li>稳定完成后，从工作站上取下板并从每个孔中取出培养基。</li>
	<li>将密度为 4 x 104 个细胞/mL（每孔体积为 100 μL）的 BEAS-2B 细胞接种到包含细胞的孔中，并将板放在培养箱中的 ECIS 站上。</li>
	<li>在监视器上，单击“ 检查 ”以确定工作站上的传感器是否读取了所有电极。</li>
	<li>通过选择 多个频率/时间 （MFT） 来设置时程度量。该程序将以七个不同的频率测量每个井。</li>
	<li>单击 开始 并让它运行 24 小时，以便细胞可以形成汇合的单层。</li>
	<li>24小时后，含有细胞的孔将在监视器上显示增加的电阻。这是细胞附着在电极上的指标。</li>
	<li>按显示器上的 暂停 以停止数据收集。</li>
	<li>按照上述第 7 节中概述的步骤，使 ZIF-8 剂量等于、高于和低于计算的 IC50 值。</li>
	<li>将ZIF-8纳米颗粒以每孔100μL的体积暴露于各自的空白孔和细胞孔中，并将96孔板放回工作站上。</li>
	<li>单击监视器上的“ 再次检查 ”，以确保传感器读取所有电极。单击 恢复 并允许系统运行 72 小时以监控蜂窝行为和附件。</li>
	<li>数据收集完成后，单击监视器上的 完成 。若要保存文件，请单击 “文件”>“另存为”。将数据另存为电子表格文件。</li>
	<li>要获取 alpha 参数， 请单击模型。在屏幕弹出窗口中，单击“仅媒体井”，然后选择 仅具有媒体的井。</li>
	<li>单击设置 参考。 在显示器上，单击 查找。</li>
	<li>如果系统中显示的孔与所选孔不同，请重复步骤 9.16。</li>
	<li>单击 设置。单击拟 合 T 范围以确定数据收集所需的时间范围。</li>
	<li>在处理过程中，单击 阿尔法。系统完成分析后，单击 保存 RbA 并将其另存为电子表格文件。 注意：请参阅 ECIS 手册，该手册可在制造商的网站38 上找到。</li>
</ol>10. 数据分析
<ol><li>搜索引擎优化<ol><li>打开成像软件以分析SEM图像。单击“ 文件>打开”，然后选择要分析的图像。</li>
		<li>单击 图像>键入 8 位> 将图像转换为灰度以执行阈值操作。</li>
		<li>将像素的距离测量值校准为微米。单击 直线 工具并跟踪要测量的每个粒子的长度。</li>
		<li>单击 分析 ，然后单击 设置比例。已知距离将设置为 SEM 图像上比例尺大小的长度。已知的长度单位将设置为微米。检查 全局 并按 确定。</li>
		<li>单击直线工具并追踪粒子的直径，然后选择分析和测量。记录长度结果。</li>
		<li>对所有收集的图像重复此操作。平均至少60个颗粒的所有直径，以进行适当的统计评估。</li>
	</ol></li>
	<li>EDX<ol><li>从所有收集的图像中平均每个元素的所有权重百分比（请参阅上面的EDX部分）。</li>
		<li>使用电子表格，在 x 轴上绘制每个元素的条形图，在 y 轴上绘制其平均元素权重百分比。</li>
	</ol></li>
	<li>集成电路50<ol><li>平均空白孔并平均每个重复的剂量孔。</li>
		<li>通过从剂量空白平均值中减去对照空白平均值来计算调整后的空白。通过从平均剂量值中减去调整后的空白来计算每个剂量的真实细胞值。</li>
		<li>从平均对照值中减去每个剂量的调整空白值。使用以下公式计算每个剂量的细胞活力：细胞活力=（真实细胞值/（对照值 - 调整空白值））x 100。</li>
		<li>对所有其他仿行重复此操作。</li>
		<li>平均每个重复的细胞活力，以获得每个剂量的最终细胞活力。</li>
		<li>在软件中，选择屏幕上的 XY 选项卡。</li>
		<li>在X列中输入浓度（剂量）值，在Y列中输入每个剂量的细胞活力（%）。</li>
		<li>通过单击“ 分析>转换”>“确定”和“转换 X = Log（X）”来转换 X 值。</li>
		<li>通过选择转换后的数据表，单击分析，然后选择归一化来规范化 Y 值。</li>
		<li>选择 变换归一化 数据表，单击 分析，然后选择 非线性回归（曲线拟合）。选择 剂量 -反应-抑制，然后单击 对数（抑制剂）与反应-可变斜率（四个参数）。单击 “确定 ”查看结果。</li>
	</ol></li>
	<li>欧易斯<ol><li>从电子表格中，获取每个重复（空白孔和剂量孔）的电阻（欧姆）列，并从4,000 Hz频率将它们平均在一起。该频率允许细胞间接触评估38。</li>
		<li>通过从每次重复的平均剂量中减去平均空白来计算校正的阻力。平均每个剂量的重复校正阻力。</li>
		<li>对 alpha 值重复相同的步骤以计算平均 alpha 参数。将 x 轴绘制为时间 （h），将 y 轴绘制为每个剂量值的平均阻力/α 值。</li>
	</ol></li>
</ol>11. 统计分析
<ol><li>搜索引擎优化<ol><li>在计算ZIF-8颗粒平均直径的电子表格文件中执行标准偏差。</li>
		<li>利用电子表格中的 STDEV 函数，突出显示 60 个粒子的数据，然后单击 Enter。绘制计算的每个平均直径的标准偏差图表。</li>
	</ol></li>
	<li>EDX<ol><li>与SEM（步骤11.1.1-11.1.2）类似，使用电子表格中的 STDEV 函数计算每个平均元素组成的标准偏差。绘制每个平均元素组成的标准偏差。</li>
	</ol></li>
	<li>集成电路50<ol><li>打开用于计算IC50 的软件，然后单击 分组数据表。</li>
		<li>在标有 A 组、B 组等的行中输入 ZIF-8 剂量。输入每个剂量的平均细胞活力，并在与ZIF-8剂量对应的列中重复。</li>
		<li>单击“分析”，然后在“列分析”下，选择单因子方差分析（以及非参数或混合）。</li>
		<li>在“实验性设计”选项卡中，选择“无匹配或配对”。在残差的高斯分布下，选择是使用方差分析。最后，在假设 SD 相等下选择是使用普通方差分析检验。</li>
		<li>在“ 多重比较 ”选项卡下，选择“ 将每列的平均值与控制列的平均值进行比较”。单击 “确定 ”查看结果。</li>
	</ol></li>
</ol>

通过电电池-衬底传感评估 MOF 诱导的电池行为变化

作者报告这项工作没有利益冲突。

先前的分析表明，ECIS可用于评估暴露于分析物（即碳纳米管35，药物43或纳米粘土16）的细胞的行为。此外，Stuekle等人使用ECIS评估暴露于纳米粘土及其副产物的BEAS-2B细胞的毒性，发现细胞行为和附着取决于此类材料的物理化学特性42。在此，我们提出确定BEAS-2B细胞响应暴露于MOFs的可能变化，从而有助于旨在区分ZIF-8对 体?...

金属-有机骨架（MOF）是通过金属离子和有机接头1,2在有机溶剂中结合而形成的杂化物。由于这种组合的多样性，MOFs具有结构多样性3，可调孔隙率，高热稳定性和高表面积4,5。这些特性使它们在各种应用中具有吸引力的候选者，从气体储存6,7到催化8,9，以及从造影剂10,11到药物输送单元12,13。然而，在此类应用中实施MOF引起了人们对其对用户和环境安全性的担忧。例如，初步研究表明，当细胞暴露于用于MOF合成的金属离子或接头时，细胞功能和生长会发生变化1,14,15。例如，Tamames-Tabar等人证明，ZIF-8 MOF，一种基于Zn的MOF，相对于基于Zr和Fe的MOF，导致人宫颈癌细胞系（HeLa）和小鼠巨噬细胞系（J774）的更多细胞变化。这种效应可能是由于ZIF-8的金属成分（即Zn），它可能在框架崩解和Zn离子释放1时诱导细胞凋亡。同样，Gandara-Loe等人证明，当浓度为10μg/mL或更高时，基于铜的MOF的HKUST-1导致小鼠视网膜母细胞瘤细胞活力的降低最高。这可能是由于在该框架的合成过程中掺入的Cu金属离子，一旦释放，可能会在暴露的细胞中诱导氧化应激15。
此外，分析表明，暴露于具有不同理化特征的MOFs会导致暴露细胞的不同反应。例如，Wagner等人证明，用于永生化人支气管上皮细胞暴露的ZIF-8和MIL-160（一种基于Al的框架）导致细胞反应取决于框架的物理化学性质，即疏水性，大小和结构特征16。作为补充，Chen等人证明，暴露于人正常肝细胞（HL-7702）的浓度为160μg/mL的MIL-100（Fe）导致细胞活力的最大损失，可能是由于该特定框架的金属成分（即Fe17）。
虽然这些研究根据其物理化学特性和暴露浓度对MOFs对细胞系统的有害影响进行分类，从而引发了对框架实施的潜在关注，特别是在生物医学领域，但大多数这些评估都是基于单时间点比色测定。例如，结果表明，当使用（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）四唑（MTT）和水溶性四唑盐（WST-1）测定时，这些生化试剂在与细胞也暴露于的颗粒相互作用时可能导致假阳性18。四唑盐和中性红试剂在颗粒表面具有很高的吸附或结合亲和力，导致试剂信号干扰19。此外，对于其他类型的测定，例如流式细胞术，以前被证明用于评估暴露于MOFs 20,21的细胞的变化，研究表明，如果要考虑对颗粒的有害影响进行可行的分析，则必须避免主要问题。特别是，必须解决颗粒大小的检测范围，特别是在混合群体中，例如MOF提供的检测范围或细胞变化前用于校准的颗粒参考22。还表明，在细胞标记期间用于此类细胞术测定的染料也可能干扰细胞暴露于23的纳米颗粒。
本研究的目的是使用实时、高通量评估测定来评估暴露于选定 MOF 时细胞行为的变化。实时评估有助于深入了解与曝光窗口相关的时间相关效应16.此外，它们提供了有关细胞 - 底物相互作用，细胞形态和细胞 - 细胞相互作用的变化的信息，以及这些变化如何取决于感兴趣的材料的物理化学性质和暴露时间分别为24,25。
为了证明所提方法的有效性和适用性，使用了人支气管上皮（BEAS-2B）细胞、ZIF-8（沸石咪唑酸盐16的疏水框架）和电池-基底阻抗传感（ECIS）。BEAS-2B细胞代表肺暴露的模型26，以前已用于评估细胞暴露于纳米粘土及其热降解副产物26,27,28时的变化，以及评估纳米材料的毒性，例如单壁碳纳米管（SWCNTs）18。此外，这些细胞已被用作肺上皮功能的模型30多年29。之所以选择ZIF-8，是因为其在催化30中广泛应用，并作为造影剂31用于生物成像和药物递送32，因此在此类应用中具有扩展的肺部暴露潜力。最后，ECIS是一种非侵入性实时技术，以前用于实时评估细胞粘附性、增殖、运动性和形态的变化16,26，这是分析物（材料和药物）与暴露细胞之间各种相互作用的结果16,18,28.ECIS使用交流电（AC）来测量固定在金电极上的电池的阻抗，阻抗变化可以深入了解电池 - 金基板界面处电阻和电容的变化，由细胞 - 细胞相互作用引起的屏障功能以及此类金电极的过电池层覆盖率33,34。使用ECIS允许以非侵入性，实时的方式以纳米级分辨率进行定量测量26,34。
本研究评估并比较了实时评估MOF诱导的细胞行为变化的简单性和易用性与单点测定评估。这样的研究可以进一步外推，以评估细胞谱，以响应暴露于其他感兴趣的颗粒，从而允许安全的设计颗粒测试并随后帮助实施。此外，这项研究可以补充单点评估的遗传和细胞测定。这可能导致对颗粒对细胞群的有害影响的更明智的分析，并可用于以高通量方式筛选此类颗粒的毒性16,35,36。

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			<td>Hitachi S-4700 Field emission scanning electron microscope equipped with energy dispersive X-ray&#160;</td>
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			<td>S4700 and EDAX TEAM analysis software</td>
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			<td>ImageJ software</td>
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			<td>Immortalized human bronchial epithelial cells</td>
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			<td>CRL-9609</td>
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electric cell-substrate sensing for real-time evaluation of metal-organic framework toxicological profiles

金属-有机骨架（MOFs）是通过金属离子和有机接头在有机溶剂中的配位形成的杂化物。MOFs在生物医学和工业应用中的实施引起了对其安全性的担忧。在此，在暴露于人肺上皮细胞时评估了选定的MOF（沸石咪唑框架）的轮廓。评估平台是一种实时技术（即电池-基板阻抗传感[ECIS]）。本研究确定并讨论了所选MOF对暴露细胞的一些有害影响。此外，这项研究证明了使用实时方法与其他生化测定相比进行全面细胞评估的好处。该研究得出的结论是，观察到的细胞行为变化可能暗示暴露于不同物理化学特征的MOFs以及正在使用的这些框架的剂量时可能引起的毒性。通过了解细胞行为的变化，人们预见了通过专门定制其物理化学特性来改进MOFs用于生物医学应用的安全设计策略的能力。

本研究使用常见的 体外 模型细胞系39 （BEAS-2B），旨在证明ECIS评估暴露于实验室合成的MOF后细胞行为变化的可行性和适用性。这些变化评估通过常规比色测定的分析得到补充。
首先评估框架的物理化学特性，以确保所采用方法的可重复性，获得的结果的有效性以及此类结果的相关讨论。例如，ZIF-8的表面形貌分析是通过SEM进行的;图像显示，这些框架?...

Watch this Scientific Journal Video about Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks at JoVE.com

作者聚焦：评估人肺上皮细胞对金属有机框架反应的进展 

以下研究利用电池-基板阻抗传感（ECIS）评估所选金属有机框架的毒理学特征，这是一种实时，高通量筛选技术。

用于实时评估金属有机框架毒理学特征的电池-基底传感

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

我的研究旨在评估人肺上皮细胞在暴露于金属有机框架时的行为变化，金属有机骨架是通过有机溶剂中的有机接头中的金属离子结合形成的杂交体。这项研究表明，金属有机骨架的物理和化学组成及其细胞暴露率以时间和剂量依赖性的方式影响人肺上皮细胞的行为。这可以实时记录高吞吐量。我们的研究有助于评估，了解金属有机框架暴露于细胞系统时的毒性机制。目前的方法允许以实时和高通量的方式连续监测电池。此外，该方法不依赖于任何外部试剂，因此它减少了它们对用于暴露的框架可能产生的任何干扰。我们的实验室将继续专注于理解和评估与纳米材料暴露相关的毒性机制，以及利用这些信息来合成新的安全系统，这些系统可以在生物医学应用中以对用户或环境的风险最小地实施。

electric-cell-substrate-sensing-for-real-time-evaluation-metal

Research

JoVE Journal

Bioengineering

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

752 次观看.  West Virginia University. 我的研究旨在评估人肺上皮细胞在暴露于金属有机框架时的行为变化，金属有机骨架是通过有机溶剂中的有机接头中的金属离子结合形成的杂交体。这项研究表明，金属有机骨架的物理和化学组成及其细胞暴露率以时间和剂量依赖性的方式影响人肺上皮细胞的行为。这可以实时记录高吞吐量。我们的研究有助于评估，了解金属有机框架暴露于细胞系统时的毒性机制。目?...

视频: 作者聚焦：评估人肺上皮细胞对金属有机框架反应的进展 

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

科研

生物工程

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

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

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

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

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

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

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...