作者希望感谢德国研究基金会 (DFG) 在赠款号 ur 70/10-01 和 ur 70/12-01 的部分资助这项工作。

1. 利用 DFR 技术制备微流控生物传感器 <ol> <li> PI 晶片的制备。
	<ol> <li> 将 PI 衬底切成6个圆片。将 PI 晶片放入烤箱中120和 #176; C 用于脱水烘烤约1小时. </li>
	</ol> </li> <li> 第一个光刻步骤用于剥离过程. 
	<ol> <li> 将自旋涂布机的30秒旋转时间定为 3000 rpm, 加速度为 2000 rpm/秒. 将 PI 晶片放在自旋涂布机上, 并将其固定, 应用真空。免除2毫升的抵抗, 使剥离过程, 并启动自旋涂布机程序。从自旋涂布机上取下晶片, 并在热板上 soft-bake PI 晶片2分钟, 在100和 #176; C. </li>
		<li> 调整所需的掩码以将 PI 晶片上的电极与肉眼进行阵列, 并将其暴露给400兆焦耳/cm 2 UV 光 (365 nm)。将晶片放在一个装有抗阻匹配显影液的碗里, 让它稍微摇动1分钟. </li>
		<li> 在去除多余的抗性后, 使用淋浴器在湿的长凳上用去离子水 (DI 水) 冲洗 PI 晶片, 然后用压缩空气将其烘干. </li>
	</ol> </li> <li> 沉积铂以形成电极。
	<ol> <li> 使用标准的物理气相沉积过程将200纳米铂沉积到晶片上 19 。
		<li> 将晶片放入碗中。添加匹配的卸妆到碗, 并删除剥离抵抗, 而轻微晃动的晶圆在一个轨道振动筛, 直到所有过剩的白金被删除。如步骤1.2.3 所述, 冲洗和烘干 PI 晶片. </li>
	</ol> </li> <li> 第二个光刻步骤: 形成保温层. 
	<ol> <li> 将 PI 晶片放入预热到120和 #176 的烤箱中; C 为5分钟, 使其脱水. </li>
		<li> 将自旋式涂布机的第一步按 500 rpm 的旋转时间的5秒进行编程。计划的第二步为三十年代在4000转每分钟, 以加速度700转/秒. </li>
		<li> 将 PI 晶片放在自旋涂布机上, 并将其固定, 应用真空。在启动自旋涂布机程序之前, 免除4毫升适当的氧光刻胶. </li>
		<li> 软烤涂覆的硅片在95和 #176; C 在热板上. </li>
		<li> 使用相应的校准标记和目镜调整晶片上的保温层的掩码。将硅片暴露为100兆焦耳/cm 2 UV 光 (365 nm). </li>
		<li> 对于后烘烤, 将晶片放在预热的热板上, 在95和 #176 处放置2分钟; C. </li>
		<li> 将晶片放入碗中, 并与合适的显影剂一起开发 ( 例如, 在 SU-8 上, 在轨道振动筛上的 1-甲氧基-2-丙基 acetat, 用于2分钟). </li>
		<li> 先用异丙醇冲洗 PI 晶片, 然后冲洗并干燥, 如步骤1.2.3 中所述. </li>
		<li> 硬-在烤箱中烘烤3小时的光刻胶, 在 150 #176; C. </li>
	</ol> </li> <li> 等离子辅助清洗电极。
	<ol> <li> 要除去光刻胶残留物, 请使用标准等离子单元的氧等离子体。启动清洁程序, 使用 200 W 低频电源与 100% O 2 流, 速率为 250 sccm, 总时间为 3 min. </li>
		<li> 将 PI 晶片放入等离子室, 用玻璃盖固定在接地板上的晶片, 并启动等离子过程. </li>
	</ol> </li> <li> 银和氯化银沉积: 创建芯片上的参考电极。
	<ol> <li> 钝化用 UV 敏感的胶带将晶片的铂接触垫。将铝箔切成5毫米宽和11厘米长的条纹, 并将其附着在晶片上, 以保护不被银沉积的部分. </li>
		<li> 对于银沉积, 将声波浴 (35 赫) 放入通风柜中, 并将银电解质溶液 (100 克/L Ag, pH 12.5) 放入浴缸中。将声波浴设置为室温和功率为 10%. 
		注意: 银电解质溶液是剧毒的, 应格外小心处理。烟雾不应该被吸入. </li>
		<li> 将参考电极的大容量触点连接到恒定的电流源。使用标准连接电缆将计数器电极、银电解液中浸入的银线连接到当前源. </li>
		<li> 将当前源设置为 DC, 并将电流密度设为-4.5 毫安/厘米 2 , 导致银沉积速率约0.3 和 #181; m/分钟启动声波浴, 让电流源运行10分钟. 用 DI 冲洗硅片
		<li> 对于参考电极的氯化, 在声波槽中插入0.1 米氯化钾 (氯化钾) 溶液的容器。将晶片的本体接触和在氯化钾溶液中浸泡的铂电极与恒流源连接. </li>
		<li> 将当前源设置为 DC, 并将电流密度设为 +0.6 毫安/cm 2 , 从而使沉积速率大约为0.075 和 #181; m/分钟启动声波浴, 让当前源运行 7.5 min. </li>
		<li> 冲洗和烘干 PI 晶片, 如步骤1.2.3 中所述. </li>
		<li> 在 uv (365 nm) 曝光300兆焦耳/cm 2 并冲洗并烘干 PI 晶片后, 再按步骤1.2.3 中的描述删除 uv 敏感磁带. </li>
	</ol> </li> <li> 第三个光刻步骤: 使用 DFRs 创建微流体通道. 
	<ol> <li> 将所需的 DFR 层剪切到与硅片 (20 x 20 cm 2 ) 类似的大小。将所需的掩码固定在曝光单元上, 并使用肉眼将 DFR 层对准蒙版。用250兆焦耳/cm 2 UV 光 (365 nm) 照亮抵抗. </li>
		<li> 取下光刻胶的保护箔, 并在1% 碳酸钠 (Na 2 CO 3 ) 中使用标准声波槽 (35 kHz) 预热至42和 #176, 以大约2分钟 (根据结构) 开发抗蚀剂; C 和声波功率100%。为了立即停止反应, 在一个轨道振动筛的 1% HCl 浴中摇动抵抗1分钟。冲洗和干燥每个 DFR 层, 如步骤1.2.3 所述. 
		注: 总共需要处理四 DFR 层, 如步骤1.7 所述: 一个通道层、一个盖层和两个背面层 (防止生物传感器在末端弯曲). </li>
	</ol> </li> <li> 在 PI 衬底上的 DFR 层的分层。
	<ol> <li> 将 PI 晶片放在透明的架空箔上, 并用标准胶带固定。使用相应的对齐结构在显微镜下调整 PI 晶片上的通道 DFR 层. </li>
		<li> 对于层压, 使用标准热辊式贴合机, 并将顶辊预热至100和 #176; c 和底部辊至60和 #176; c. 将压力设置为 3 bar, 向前速度为0.3 米/分. 将晶片与固定的 DFR 层放在层压机的中间启动它;DFR 是通过层压机推的。在将硅片旋转180和 #176 后重复该步骤;. </li>
		<li> 为了防止生物传感器的弯曲, 将两个开发后的背面层层压到硅片上, 步骤 1.8. 1-1. 8.2. </li>
	</ol> </li> <li> 使用疏水阻挡屏障并密封芯片。
	<ol> <li> 从 DFR 通道的前侧卸下保护层将标准胶带放在 DFR 的一端, 向上拉。使用带有0.004 个管的手分配器, 将被溶解的聚四氟乙烯的小水滴分配到绝缘层的井中。要密封微流控芯片, 将盖 DFR 层层压到通道层上, 如步骤1.8 所述. </li>
	</ol> </li> <li> 对微流体传感器进行硬烘烤. 
	<ol> <li> 卸下盖上的所有保护箔和背面 DFR 层。使用普通剪刀把生物传感器切成条。在160和 #176 的烤箱中治疗芯片; C 为 3 h. </li>
	</ol> </li> </ol> 2。片上试验固定化程序 <ol> <li> 在通道的固定区中吸附素. 
	<ol> <li> 准备100和 #181; 在10毫米磷酸盐缓冲盐水 (PBS) 中素溶液, pH 值为 7.4. </li>
		<li> 免除2和 #181; 将素溶液的 L 放到生物传感器的入口. 
		注: 通道由毛细管力填充, 直到流体到达疏水阻挡屏障. </li>
		<li> 确保射流在整个孵化时间内在屏障上不停地停留, 免除2和 #181; 在芯片出口处的 DI 水。将芯片在25和 #176 上孵育; 在封闭容器中, C 为1小时. </li>
		<li> 通过应用真空, 将多余的试剂通过芯片的入口移除。使用50和 #181 清洗通道; 洗涤缓冲器 (PBS 与0.05% 吐温 20), 在真空应用时, 在插座上分配。干燥的渠道, 三十年代, 而真空正在应用. </li>
	</ol> </li> <li> 阻塞通道表面以抑制不绑定. 
	<ol> <li> 在10毫米 PBS 中制备1% 牛血清白蛋白 (BSA) 溶液, pH 值为 7.4. </li>
		<li> 吸管2和 #181; 1% BSA 溶液在入口和2和 #181 上的 l; 在通道出口处的 DI 水。将芯片在25和 #176 上孵育; C 为1小时, 在密闭容器中。去除多余的试剂和干燥的通道, 如步骤2.1.4 所述. </li>
	</ol> </li> <li> 寡标记有生物素和6个家族的 DNA 的孵化。
	<ol> <li> 在10毫米 PBS 中准备不同浓度 (0.001-5 和 #181; M) 的 DNA 溶液, pH 值为 7.4. </li>
		<li> 免除2和 #181; 在入口和2和 #181 上的一个浓度的 DNA 溶液的 l; 出水口的 l。在25和 #176 中孵育芯片; C 为封闭容器中的15分钟. </li>
		<li> 使用额外的生物传感器芯片对其他 DNA 浓度重复步骤 2.3.2. </li>
		<li> 删除多余的试剂和干燥的通道, 如步骤2.1.4 所述. </li>
	</ol> </li> <li> 固定化氧化标记的6抗体。
	<ol> <li> 制定一个5和 #181; 在 10 mM PBS 中, 6 个抗体的浓度, 在 pH 值为 7.4. </li>
		<li> 引入2和 #181; 对入口和2和 #181 的抗体溶液 l; 向通道的出水口。在封闭容器中孵育25和 #176 的标记抗体; C 为15分钟。删除多余的试剂, 如步骤2.1.4 中所述. </li>
	</ol> </li> </ol> 3。使用停止流技术的安培信号检测 <ol> <li> 用于电化学检测的基底溶液的制备. 
	<ol> <li> 在 0.1 M PBS 中制定40毫米葡萄糖溶液, pH 值为 7.4, 超纯水. </li>
		<li> 对于初步处理, 请在超纯水中以7.4 的 pH 值制备0.1 米 PBS 溶液. </li>
	</ol> </li> <li> 对工作电极的初步处理。
	<ol> <li> 使用 custom-made 芯片托架可以方便地放置芯片和一个射流和电连接. </li>
		<li> 将生物传感器电连接到电位。确保微流体通道的射流接触到注射器泵和使用 custom-made 适配器和管的试剂库。启动连接到电位的笔记本电脑, 启动注射器泵控制器, 控制流经芯片的射流流. </li>
		<li> 手动启动注射器泵, 流量为20和 #181 的0.1 米 PBS;在工作电极与芯片上的参考电极, 适用于 5 s 的交流电压为0.8 和-0.05 V 为30周期。在这之后, 使用 0.8 V 的电压在六十年代氧化工作电极. </li>
	使用停止流技术的 </ol> </li> <li> 检测读数。
	<ol> <li> 启动检测信号读数, 等待测量的电流信号稳定。停止注射器泵, 将试剂从0.1 米 PBS 切换到 40 mM 葡萄糖溶液, 并在注射器泵控制器上启动软件;它将自动停止1、2或5分钟的流, 然后重新启动流。观察所产生的当前峰值. 
		注: 对于数据的分析, 可以考虑电流峰值或峰值电荷, 如电化学信号读出模型所描述的 ( 图 2 ). </li>
	</ol>
	</li>
</ol>

<ol>
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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> Handbook of Physical Vapor Deposition (PVD) Processing. , (2010).</li></ol>

作者没有什么可透露的。

在这里提出的用于制造微流体电化学生物传感器的协议, 使开发一个低成本, 紧凑, 易于使用的平台, 用于检测分子。根据随后在生物传感器上使用的化验, 可以检测到几种不同的生物标志物。这使得该平台非常通用, 可以广泛地访问各种应用程序领域, 从标准诊断测试 (例如,确定医生办公室的特定疾病的存在) 到点应用程序 (例如,对患者进行治疗性药物监测以进行个体化药物治疗)。特?...

在过去20年中, 诊断应用已成为 in-depth 研究全球公共卫生发展的基础。传统上, 实验室诊断工具用于检测疾病。尽管它们在疾病诊断方面仍然发挥着关键作用, 但在病人或病人附近进行的点试验 (POCT) 近年来已变得越来越普遍。特别是在需要立即治疗的情况下, 如急性心肌梗塞或糖尿病监测, 临床发现的快速确认是必不可少的。因此, 越来越需要 POCT 设备, 可以由非操作, 并且同时能够在短时间内执行精确的体外诊断测试,1,2,3,4.
在 POCT 领域已经取得了显著的进步。但是, 仍有许多挑战需要克服5、6、7、8。为了使 POCT 平台能够成功地投放市场并具有实验室诊断的竞争力, 该设备必须严格满足以下要求: (i) 提供与实验室相符的精确和定量的测试结果结果;(ii) 有较短的 sample-to-result 时间, 使病人可立即接受治疗;(iii) 功能简单, 易于操作, 即使由未经训练的个人操作, 并需要尽量减少用户干预;(iv) 由一个低成本的传感器单元组成, 用于一次性应用。此外, 无设备诊断是有利的, 主要在资源贫乏的环境中3,4,6。
由于这些苛刻的要求, 只有两个基于电化学检测的 POCT 系统 (例如,血糖试纸) 和侧流免疫 (例如,家庭妊娠测试) 已经成功投放市场, 以便远.然而, 这两个系统都有缺点, 如性能不佳 (即,血糖监测有不准确的测试结果和侧流分析只提供定性 (阳性或阴性) 测量结果)4, 6。传统的 POCT 系统的这些缺点导致了对开发新技术的需求不断增加, 这些技术提供了快速、低成本和定量检测, 在4,5。
为了应对 POCT 设备面临的这些挑战, DFR 技术最近被用于制造一次性和低成本的生物传感器9,10,11,12,13,14. 与软、液体平版印刷材料相比, 如 SU-8、DFRs 等, 具有许多优点: 它们 (i) 有多种组合物和厚度 (从几微米到几毫米);(ii) 有一个非常粗糙的表面积, 便于与各种材料粘合;(iii) 具有优异的厚度均匀性;(iv) 为大规模生产提供廉价、轻便和高通量的制造;(五) 容易用各种低成本的工具切割, 像一把简单的剪刀;和 (vi) 允许创建三维结构, 如微流控通道, 通过堆叠在彼此顶部的多个 DFR 层。
另一方面, DFRs 一般有一个相对较差的分辨率相比, 液体光刻, 这主要是由薄膜厚度和增加距离之间的面具和 DFR 由于保护箔, 这还使光散射.然而, 对于集成微流体传感器的制造, DFRs 是非常适合低成本的大规模生产。
因此, 我们在本工作中介绍了一种 DFR-based 电化学微流控生物传感器的制备和应用。详细的协议描述了生物传感器平台的每一个生产步骤, dna 模型检测的片上固定, 以及使用停止流技术的电化学读出。这个通用平台能够检测多种生物分子, 使用不同的化验技术 (例如,基因组学, cellomics, 蛋白质组学) 或化验格式 (例如,竞争性, 三明治, 或直接)。基于这样一个 DFR 平台, 我们的小组以前成功地证明了各种各样的分析的快速和敏感的定量, 包括抗生素13,15,16 (四环素,pristinamycin, 和？内酰胺抗生素), 肌钙蛋白 i17, 和物质 P18。

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyralux</td>
			<td>DuPont</td>
			<td>AP8525R</td>
			<td>Used as polyimide substrate</td>
		</tr>
		<tr>
			<td>MA-N 1420</td>
			<td>Micro Resist Technology</td>
			<td>MA-N1420</td>
			<td>Lift-off resit to define the platinum depostion</td>
		</tr>
		<tr>
			<td>Ma-D 533s</td>
			<td>Micro Resist Technology</td>
			<td>MaD533S</td>
			<td>Developer for MA-N1420</td>
		</tr>
		<tr>
			<td>Platinum</td>
			<td>-</td>
			<td>-</td>
			<td>Electrode and contact pad material</td>
		</tr>
		<tr>
			<td>Ma-R 404s</td>
			<td>Micro Resist Technology</td>
			<td>MaR404S</td>
			<td>Remover for MA-N1420</td>
		</tr>
		<tr>
			<td>SU-8 3005</td>
			<td>MicroChem Corp.</td>
			<td>SU-8-3005</td>
			<td>Photoresist to define the electrode area and as insulation</td>
		</tr>
		<tr>
			<td>1-methoxy-2-propanol acetate</td>
			<td>Sigma-Aldrich</td>
			<td>108-65-6</td>
			<td>Developer for SU-8 3005</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>VWR</td>
			<td>8.18766.2500</td>
			<td>Removing of the SU-8 developer</td>
		</tr>
		<tr>
			<td>1020R</td>
			<td>Ultron Systems Inc.</td>
			<td>1020R</td>
			<td>UV sensitive adhesive tape for protection of contact pads</td>
		</tr>
		<tr>
			<td>Arguna S</td>
			<td>Degussa</td>
			<td>1935</td>
			<td>For Silver depostion on reference electrode</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Methrom</td>
			<td>62308.020</td>
			<td>For chloridation of the silver reference electrode</td>
		</tr>
		<tr>
			<td>Pyralux</td>
			<td>DuPont</td>
			<td>PC1025</td>
			<td>Dry film photoresist</td>
		</tr>
		<tr>
			<td>Sodium carbonat</td>
			<td>Fluka</td>
			<td>71352</td>
			<td>Developer for Pyralux PC1025</td>
		</tr>
		<tr>
			<td>Hydrogen chloride</td>
			<td>Sigma-Aldrich</td>
			<td>30720</td>
			<td>To top the development of the DFR</td>
		</tr>
		<tr>
			<td>Teflon AF 1600</td>
			<td>DuPont</td>
			<td>AF1600</td>
			<td>For employing the stopping barrier</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PA104</td>
			<td>Mega Electronics</td>
			<td>-</td>
			<td>Bubble etch tank</td>
		</tr>
		<tr>
			<td>FED 53</td>
			<td>Binder</td>
			<td>9010-0018</td>
			<td>Oven</td>
		</tr>
		<tr>
			<td>SPIN150</td>
			<td>APT</td>
			<td>-</td>
			<td>Spin coater</td>
		</tr>
		<tr>
			<td>Pr&#228;zitherm</td>
			<td>Harry Gestigkeit GmbH</td>
			<td>PZ 28-2</td>
			<td>Hot plate</td>
		</tr>
		<tr>
			<td>Hellas</td>
			<td>Bungard Elektronik</td>
			<td>40000</td>
			<td>Exposure unit</td>
		</tr>
		<tr>
			<td>Tetra30-LF-PC</td>
			<td>Diener</td>
			<td>-</td>
			<td>Plasma unit</td>
		</tr>
		<tr>
			<td>Univex 500</td>
			<td>Leybold</td>
			<td>-</td>
			<td>Physical vapor deposition unit</td>
		</tr>
		<tr>
			<td>Shaker S4</td>
			<td>ELMI</td>
			<td>-</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Sonorex Super 10 P</td>
			<td>Bandelin</td>
			<td>783</td>
			<td>Sonic bath</td>
		</tr>
		<tr>
			<td>6221 DC and AC</td>
			<td>Keithley</td>
			<td>-</td>
			<td>Current source</td>
		</tr>
		<tr>
			<td>HRL 350</td>
			<td>Ozatec</td>
			<td>-</td>
			<td>Laminator unit</td>
		</tr>
		<tr>
			<td>Vaccum pen</td>
			<td>EFD</td>
			<td>-</td>
			<td>Vacuum pen</td>
		</tr>
	</tbody>
</table>

dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation

近年来, 生物标志物诊断成为诊断人类疾病的不可或缺的工具, 特别是对点诊断。一个易于使用和低成本的传感器平台是非常理想的测量各种类型的分析 (例如,生物标记物, 激素, 和药物) 定量和具体。由于这个原因, 干膜光刻胶技术-使廉价, 轻便, 和高通量制造-被用于制造微流体生物传感器在这里提出。根据随后使用的生物测定, 多功能平台能够检测各种类型的生物分子。在制造的设备, 铂电极是结构在一个灵活的聚酰亚胺 (PI) 箔在唯一的洁净室工艺步骤。PI 箔担当基体为电极, 用氧抗蚀剂绝缘。微流控通道随后由干膜光刻胶 (DFR) 箔在 PI 晶片上的发展和层压产生。通过在通道中使用疏水阻挡屏障, 将该通道分为两个特定区域: 酶链法的固定化切片和安培信号读出的电化学测量单元。
通过生物分子对通道表面的吸附, 进行了片上生物测定的固定化。葡萄糖氧化酶是一种用于电化学信号产生的传感器。在基体的存在下, 葡萄糖, 过氧化氢被生产, 在白金工作电极被发现。在快速检测的同时, 采用了停止流技术来获得信号放大。通过引入微流控系统, 可以定量地测量不同的生物分子, 从而指示不同类型的疾病, 或在治疗药物监测方面, 促进个性化治疗。

微流控生物传感器平台的设计与制作:
采用多 DFR 层的标准光刻技术, 在晶片级上实现了微流体生物传感器芯片的制备。这种制作策略依赖于铂图案的 PI 基底上的 DFRs 层的分层, 形成微流控通道。在图 1中给出了描述不同制造步骤的简短摘要。一个单一的6个晶片包括130微流控生物传感器, 每一个尺寸为 8 x 10 mm...

Watch this Scientific Journal Video about Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation at JoVE.com

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

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

利用低成本干膜光刻胶技术对各种分析进行快速、灵敏的定量化设计, 制备了微流控生物传感器平台。这种一次性系统允许通过停止流动技术在片上固定的酶联用的电化学读出。

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

dry-film-photoresist-based-electrochemical-microfluidic-biosensor

Research

JoVE Journal

Bioengineering

11.6K 次观看.  University of Freiburg. 利用低成本干膜光刻胶技术对各种分析进行快速、灵敏的定量化设计, 制备了微流控生物传感器平台。这种一次性系统允许通过停止流动技术在片上固定的酶联用的电化学读出。

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

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

丝绸电影文化系统在体外分析和生物材料设计

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

科研

生物工程

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...