支持这项工作是由国家科学基金会（NSF的EF-0623664）FIBR和IDBR（NSF在DBI-0754570）。部分支持这项工作是由国家科学基金会FIBR格兰特光子学中心，NSF科学与技术中心管理0623664，由加州大学戴维斯分校，根据合作协议的PHY 0120999管理的资金。莉萨·拉皮德斯，博士研究支持部分由一个在科学界面从巴勒斯欢迎基金事业奖。

1。微流控混合芯片的制造 图3显示了基本的制作步骤。 <ol><li> 4英寸（100毫米）的石英晶片清洁与新鲜Piranha溶液（3:1 H 2 SO 4：H 2 O 2的 ）：I）热硫酸130°C，然后倒在过氧化氢 ​​。请注意，表面粗糙度和石英晶片的平整度，发挥重要作用，最终粘接步骤。 ii）浸泡至少30分钟的晶圆。三）冲洗水和干燥5分钟。申请多晶硅（多晶硅）涂层，〜1微米厚的每人每旨在微通道的最后10微米的深度，利用低压化学气相沉积（LPCVD）炉。 </li><li>清洁晶圆和掩膜步骤1.1食人鱼协议。清洁面膜以同样的方式，然后用清水冲洗，丙酮，甲醇和异丙醇，并立即吹干。脱水热板15晶圆0℃〜10分钟（使用铝箔帐篷，保持微粒），或在120°C的烤箱至少120分钟（最好隔夜）。要增加光阻附着力，立即暴露在温暖的晶圆HMDS的蒸汽5分钟。旋涂在晶圆与900毫微米AZ5214光阻软烤厚层在90°C直接上烤盘1分钟或30分钟（ 图3，步骤1）在110°C的烤箱。 </li><li>对齐口罩和作出努力和真空接触。暴露在紫外线照射下，几秒钟（总能量：〜103兆焦耳/厘米2）（ 图3，第2步）。 AZ400K开发与DI水稀释4:1〜50秒，（光致抗蚀剂，直到被完全开发）的发展，同时轻轻搅拌。 2分钟后用清水冲洗。检查与显微镜的功能（ 图3，第3步）。如果功能不佳解决，去除光阻，并再次从1.2开始。硬烤115℃直接在烤盘2分钟。 </li><li>灰粉或O 2等离子体descum晶圆在100 W的射频功率为2.5分钟。用深反应离子蚀刻（DRIE），蚀刻多晶硅层的方式，通过下面的石英。剩下的光阻去除与PRS2000抵抗在75°C，10分钟的脱衣舞。冲洗和干燥。测量蚀刻深度，以确保它是通过聚涂料（ 图3，第4步）。 </li><li>使用ULVAC公司民盟-6000蚀刻，蚀刻石英每微米厚度的聚涂料（ 图3，第5步）〜10微米的深度，如氧化能力的DRIE。将穿在这聚涂层腐蚀掉，所以它可能需要在蚀刻过程中定期检查涂层的完整性。如果故意晶圆表面特征的地区已成为在蚀刻，剥离的裸聚的保护涂层，表面最有可能成为进站，将不再适合粘结在生产的最后步骤。在这种情况下，晶圆是最有可能不再可行。 4分钟，射频功率100瓦与矩阵舍清洁。剥去与XEF 2蚀刻（ 图3，第6步）的多晶硅。它可能需要重复步骤1.4这一步了几次，以消除所有的光致抗蚀剂和聚涂料。 </li><li>旋转外套晶圆光阻一个新的〜1微米层保护的渠道，在后续处理。钻在每个芯片的入口和出口孔，采用计算机控制与喷砂（ 图3，第7步）钻石尖钻或手动。如果使用钻头，安装上牺牲的玻璃钢板，使用水族债券55晶片（功能朝下），注意保持焊机无气泡。冷却钻头与微观-90清洗液。这使小块玻璃，从坚持的渠道，然后堵塞在使用过程中的芯片。 </li><li>直接用注射器注入瓦特水冲洗晶圆亚特到每个孔。在肥皂水里浸泡一小时。删除与减贫战略2000光刻胶和水族债券几个小时，直到表面是干净的，在60°C。 DI水和温暖的肥皂水冲洗晶圆。再次冲洗每孔用注射器，避免蚀刻的功能。与氮，在真空烘箱干燥晶圆设置为120°C。检查孔，以确保它们的砂砾和重复清洗的必要步骤。衡量一个光学或机械表面轮廓（ 图3，第8步）渠道的深度。 </li><li>清洁晶圆和同等数量为170微米厚的石英盖玻片1）Piranha溶液（1-2小时，按1.1的程序），2）RCA（5时01分01秒的DI：盐酸H 2 0 2的RCA 1解决方案（5时01分01秒的DI：22分钟，在75C H 2 O 2））：NH 4 OH）溶液在75℃10分钟，3）。 DI水冲洗5分钟，每个解决方案之间。 </li><li>顶部朝上放置一个晶片功能一摞3-4洁净室抹布放在平坦的表面硬上，如一个小窗格玻璃，。晶圆至少5分钟，用氮气彻底干燥。重复与玻璃盖。边拿起玻璃盖，倒置等打磨方朝下举行了晶圆对准平面边缘。仔细拖放到晶圆的玻璃盖，并允许它来解决。只有微调横向调整对齐。向下推，用一个手指晶圆中心。每个人都应该看到粘接前开始向外辐射。如果有必要，申请额外的压力，晶圆周围的其他职位，以推进粘接前（ 图3，第9步）。 </li><li>设置坡道从室温到800℃超过3小时的高温烤箱放置在密封的晶圆，然后从800 - 1100℃以上，另1.2小时。保持在1100℃2小时，然后急剧下降，超过1.5小时，室温。 </li><li>到我看到了一个晶圆切割的骰子ndividual芯片（ 图3，第10步）。 </li><li>协议描述制造相当不寻常的石英衬底紫外​​检测是必要的渠道。但是，这个协议也可以适用于硅衬底，这就要求只有一个蚀刻步骤2。可以结合使用阳极键合晶圆玻璃（但不熔融石英）玻璃盖。 </li></ol> 2。安装和载入芯片<ol><li>多方面的持有混合芯片和解决方案，可制成塑料的Lexan或有机玻璃，或温度控制的铝（ 见图4），如。如果使用铝，解决水库应涂以聚对二甲苯，在水库的盐溶液，以防止铝解散。热电装置安装两侧和电子控制器，可以控制整个多方面的，解决方案和芯片的温度。 </li><li>该芯片是交配的玛尼小O形圈和一个挡圈，允许一个清晰的光学芯片中心在地方举行倍。 O型环槽较浅的芯片没有施加太大压力，以确保良好的交配应该是比标准，即1 002 O形圈应该是深0.025“，一旦芯片的安装，添加解决方案中心和侧面水库，照顾释放被困在井底部有气泡。 </li><li>因为在此混频器所使用的量是如此之小，可以控制流量，与上面的解决方案，井应用于空气压力。 图4显示芯片多方面的顶部由O形圈的压力多方面的交配。压力歧管连接到计算机控制的压力传感器可以保持〜2-80 PSI的压力。该芯片已经设计等平等中心和侧通道的压力产生了〜100倍，比线性流速。在20 PSI的，在退出渠道的线性流速约1米/秒。 </li><li>放置在一个倒置显微镜多方面的，检查混合目镜或摄像机输出的地区。多方面的顶部放置一个白炽灯手电筒将提供足够的光线。在中心通道使用染料或高指数的折射解决方案（即6个M盐酸胍）查看射流。 </li><li>在平等中心和侧通道的压力，喷射将难以看到眼睛，所以开始在0 PSI侧通道的压力，慢慢增加至等于压力。如果一方的通道被堵塞，飞机将被推迟对出口通道的墙壁之一。如果出现一个可见的堵塞，它可能会脱落，通过施加压力或真空用注射器的出口通道。 </li><li>如果蛋白质或其他有机物质堵塞的芯片，它可以清洁浸泡过夜Pirhana解决方案，。 </li></ol> 3。数据采集 图5显示了基本的光学仪器布局。<ol><li>把使用显微镜外分色镜内或只是显微镜研究级的准直激光束。对齐，使激光焦点在目镜或混合区域附近有些相机可以看到。 </li><li>从搅拌机中蛋白质的荧光收集的目标和发送通过分色镜，由一个针孔镜头聚焦成像对光子计数器。芯片使用在x和y的压电扫描器扫描图像的混合地区（典型的步长是2微米）。选择一个位置上的喷气机和X和Z扫描，发现在垂直通道的中心焦点。在显微镜领域的深度远远超过航道水深和流速是在通道除了在1微米的墙壁统一。因此，确定的时空分辨率的观测荧光主要的聚焦点的大小。 </li><li>水平扫描范围内的出口通道（典型步骤sIZE是在整个喷射0.2微米，2微米100微米增量观察，沿射流〜1毫秒每个位置）沿射流（ 见图6）。沿着喷气显微镜阶段捕捉稍后的时间由80-90微米。 </li><li>另外，光可检测由光谱仪和CCD用镜头聚焦到狭缝后的分色镜。由于数据收集是比光子计数器，每个位置（1秒）慢，通常喷射第一次成像与光子计数器，然后沿射流光谱仪测量点（ 见图7）。 </li><li>从光子计数器的数据进行分析总结3-5像素周围喷射，在每个位置创建一个强度与距离的阴谋。时间的计算是从使用距离计算速度的基础上所施加的压力（ 见图8）。 </li><li>从光谱仪的数据进行分析的几种方法。 FRET，channe指定为“捐助”和“受体”的LS可以概括和接近率 E = /（我的D + 的 I D）计算（ 见图9）。另外，单值分解，可以执行独立的频谱分量与时间，如色氨酸的发射光谱作为一种蛋白质折叠的转变，来看看。 </li><li>退出通道的距离内，可以转换时间取决于所施加的压力侧通道使用恒定的流量。出口通道内的第2微米〜100倍的蛋白质射流的流速加快，并在该地区的时间必须与有限元分析计算的简化（生产时间在绘制图8）。这个加速度产生的偏移〜1-2微秒后的速度成为常数，通常可以忽略在测量动力学比混合慢得多。 </li></ol> 4。代表结果 图6显示了一个由光子计数器测量的混合区强度的等高线图。在射流外的退出渠道的背景应该是接近探测器的本底噪声，通常是不减去。较高的背景可能显示不协调或蛋白质坚持通道的墙壁。看起来扭曲或弯曲，特别是附近的混合区，喷气式飞机，表明穷人的喷嘴蚀刻。 图7显示了蛋白质的酰基辅酶A结合蛋白（ACBP）与488网站和Alexa 647标记的时间分辨光谱。这个数据已经背景减去删除黑暗的充电和激光信号。中心通道设置为0 PSI压力的背景信号。 搅拌时间可以测量通过测量的快速反应速度远远比混合，如色氨酸荧光猝灭bŸ碘化钾或崩溃的单链DNA标记与FRET的染料，加入NaCl。由于飞机是小于光学显微镜的分辨率，有喷气形式的混合区，在大强度下降。控制无解条件的变化。 图8显示的比例混合（碘化钾400毫米）和N-乙酰色氨酸酰胺荧光非混合实验测量，该仪器的响应可以被删除。该行显示从COMSOL模拟预测浓度的KI。搅拌时间，只要浓度减少80％的时间来衡量，是8微秒。 由于数据是在100微米增量沿500微米长的退出渠道收集，数据必须粘贴在一起，从多个视图。有这些意见，可能是由于在重点的小芯片是由移动显微镜阶段的变化之间的偶尔小信号偏移。由移动的舞台，80或90按次微米，这些偏移可以消除通过检查重叠的数据。通常情况下，数据收集至少有两个流速和数据相结合，以确认任何信号的变化是由于真正的光谱变化中的蛋白质，而不是光或流动的影响， 图9显示的共振稀释后的蛋白ACBP变化变性从6米至0.06研究这个数据不需要对照实验中，自共振已经是一个比和仪器的响应已被删除。 T = 0的附近迅速蹿代表在共振信号混合的时间内迅速变化。 <img alt="图1" src="/files/ftp_upload/3976/3976fig1.jpg" /> 图1。电子显微镜测量混合区的布局。 <img alt="图2" src="/files/ftp_upload/3976/3976fig2.jpg" /> 图2。水力混合研究蛋白质折叠的示意图。蛋白质，开展高变性溶解，流下来，对混合区的中心通道，满足缓冲流动快〜100倍。层流强制蛋白质从变性的分子能迅速扩散成一个窄窄的喷射流。由于喷气流下来的退出渠道，可以观察到蛋白质与高空间和时间的使用共聚焦显微镜的分辨率。 <img alt="图3" src="/files/ftp_upload/3976/3976fig3.jpg" /> 图3。熔融石英制造。 1）过程开始于一个高度抛光的500微米厚的石英衬底的多晶硅层的顶部和底部表面上沉积（聚）（一），（b）和旋涂一层光阻（三）。 2）光罩（五）被带来的诠释<html> o与晶圆和光致抗蚀剂的硬接触，暴露在紫外光（D）。 3）晶圆被放置在一个开发浴和模式显示，在光致抗蚀剂。 4）晶圆被放置在一个的DRIE蚀刻进入前聚涂层的功能。光致抗蚀剂，然后删除。 5）聚然后充当面膜当晶圆被放置在氧化物蚀刻和功能蚀刻10 - 40微米的石英衬底。 6）聚涂层，然后在XEF 2蚀刻去除。 7）晶圆保税牺牲玻璃板与热活化胶（G），功能的一面朝下，并从背面的通孔的金刚石钻头（F）abrasively演习。 8）表面轮廓（H），然后直接测量蚀刻的功能深度。 9）170微米厚的熔融石英盖玻片晶圆对晶圆的顶部表面直接结合。 10）晶圆被切割成个别的微流控芯片。 3976/3976fig4.jpg“/&gt; 图4。混频器多方面的。微流体芯片的安全解决方案与加工铝面板和四个O形圈〜200μL，水库下多方面的。通过一个大洞，加工中心，直接在上面贴的微芯片的混合地区多方面的，使过量的紫外线光散射，抑制任何从多方面本身的自动荧光逃脱，不用允许用肉眼直接照明芯片或相机对准。空气歧管密封各水库等，在每一个空气压力可以通过外部的压力控制箱控制。 <img alt="图5" src="/files/ftp_upload/3976/3976fig5.jpg" /> 图5。光学共聚焦显微镜的原理图。 <img alt="图6" src="/files/ftp_upload/3976/3976fig6.jpg" /> 图6。轮廓色氨酸的阴谋荧光微搅拌机。混合区位于y轴〜90微米。 <img alt="图7" src="/files/ftp_upload/3976/3976fig7.jpg" /> 图7。时间依赖性荧光光谱收集的搅拌机内。红色和绿色的矩形显示指定为供体和受体通道的波长，分别。 <img alt="图8" src="/files/ftp_upload/3976/3976fig8.jpg" /> 图8。测定色氨酸的荧光猝灭碘化钾（点和右轴）和有限元分析（线和左轴）计算的混合时间。 <img alt="图9" src="/files/ftp_upload/3976/3976fig9.jpg" /> 图9。FRET技术在折叠的蛋白质ACBP测量的变化。 T = 0附近的迅速崛起，代表了一阵搅拌的时间内和较慢的上升阶段代表一个渐进的累加TION作为蛋白质折叠结构。数据采取了两种不同的流速和覆盖，从而导致在不同的时间不同密度的测量。

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+protein+folding+kinetics+studied+by+ultrarapid+mixing.">Submillisecond protein folding kinetics studied by ultrarapid mixing.</a> PNAS. 94, 1779-1784 (1997).</li><li>Shastry, M. C. R., Luck, S. D., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous-flow+capillary+mixing+method+to+monitor+reactions+on+the+microsecond+time+scale.">A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.</a> Biophysical Journal. 74, 2714-2721 (1998).</li><li>Pollack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compactness+of+the+denatured+state+of+a+fast-folding+protein+measured+by+submillisecond+small-angle+x-ray+scattering.">Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 10115-10117 (1999).</li><li>Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+cytochrome+c+initiated+by+submillisecond+mixing.">Folding of cytochrome c initiated by submillisecond mixing.</a> Nature Structural Molecular Biology. 4, 44-50 (1997).</li><li>Knight, J. B., Vishwanath, A., Brody, J. P., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+focusing+on+a+silicon+chip:+Mixing+nanoliters+in+microseconds.">Hydrodynamic focusing on a silicon chip: Mixing nanoliters in microseconds.</a> Physical Review Letters. 80, 3863-3866 (1998).</li><li>DeCamp, S. J., Naganathan, A. N., Waldauer, S. A., Bakajin, O., Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Downhill+Folding+of+lambda-Repressor+in+a+Microfluidic+Mixer.">Direct Observation of Downhill Folding of lambda-Repressor in a Microfluidic Mixer.</a> Biophysical Journal. 97, 1772-1777 (2009).</li><li>Waldauer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+in+the+folding+landscape+of+protein+L.">Ruggedness in the folding landscape of protein L.</a> Hfsp Journal. 2, 388-395 (2008).</li><li>Shastry, M. C. R., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+barrier-limited+protein+folding+kinetics+on+the+microsecond+time+scale.">Evidence for barrier-limited protein folding kinetics on the microsecond time scale.</a> Nature Structural Biology. 5, 385-392 (1998).</li><li>Uzawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+and+search+dynamics+of+apomyoglobin+folding+revealed+by+submillisecond+observations+of+alpha-helical+content+and+compactness.">Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 1171-1176 (2004).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Multiple+Folding+Pathways+for+the+Villin+Headpiece+Subdomain.">Evidence of Multiple Folding Pathways for the Villin Headpiece Subdomain.</a> The Journal of Physical Chemistry B. 115, 12632-12637 (2011).</li></ol>

我们什么都没有透露。

快速搅拌一直是多年的蛋白质折叠领域的发展目标，因为它早已认识到生物大分子的分子过程，范围从皮秒到几秒钟的时间尺度发生。传统的停流混频器有死的动荡主要是由有限的时间为1-5毫秒。由一个小的群体数量在过去15年来已连续流混合器湍流混合时间30-300微秒，但一般需要高流速实现这些混合时间，因此使用了很多样品6-8。 这个协议描述了微流控混合芯片的使用，在层流制度，实现快速稀释。有多重优势，在这一制度工作，但主要是高精度模拟整个搅拌过程中，它允许一个修改的混合反应的能力。简单GEOM其他群体开发的T型搅拌机etries已经证明混合时间100-200μs的主要渠道9-10大小的限制。通过渠道的规模扩大到10微米的骑士〜10μs的11减少搅拌时间。姚明和Bakajin表明，在几何上的小变化可能导致显着改善，在搅拌时间4。然而，这项工作表明，加速混合，也可以导致速度较慢的阶段，以及。 12日至13日在这项工作中使用的设计是参考文献4中最好的两个设计之间的妥协，以尽量减少在变性剂浓度指数衰减较慢，也使功能更重现的DRIE蚀刻。 已经有一些在外地超速超过混合搅拌时间的定义争议。重要的是要认识到“混合时间”和“死时间”之间的区别。其间measuremen的时间，死区时间是指在动荡的混频器T不能进行，实际上可能大大低于实际的搅拌时间较长。它通常由多次测量伪一阶双分子反应在不同浓度（如色氨酸荧光猝灭由N bromosuccinate）。观察指数衰减拟合收敛在一个单一的值假定为T = 0秒，这一点，并第一个测量点之间的时间是死的时间。层流混合器，测量可在搅拌过程中，所以没有死的时候，只有一个混合的时间。搅拌时间仅仅是时间，结合了两种解决方案，直至达到足够的均匀性。我们先前已定义的搅拌时间是90/10的时间，时间为变性剂的浓度从90％降低到10％的纯种值。然而，混合曲线的形状是不完全对称的尾部有一个小的指数分量的衰减。此外，蛋白质折叠的有高度非变性剂浓度的线性关系，所以可以经常展开，折叠的变性，即使只减少两倍。因此，我们定义为最近的时间减少了80％的变性剂浓度的混合时间。当然，其他定义，可用于根据实验的要求。 此混频器的使用，研究蛋白质折叠一贯显示出令人吃惊的结果。细胞色素c和​​apomyoglobin折叠，长期以来一直研究，有多个连续流混合器折叠步骤和早期结果表明发生崩溃〜100微秒的时间尺度10,14-15。与此混频器的测量表明，至少有2个步骤，这个时间表，在搅拌机的搅拌时间较慢的阶段（如一个非常快的，可能非特异性，崩塌（如色氨酸荧光光谱位移测量）通过了淬火总色氨酸排放的测量），很可能在F的IRST形成的原生结构5。 protein L的B1域，我们也观察到了50微秒的过程中，推翻了传统的解释，这种蛋白质是2状态的文件夹13。 这种快速混合器的优点是，它可以探测纳秒的T-跳跃文书通常只可测量最快的折叠蛋白质折叠。 T型跳通常需要观察折叠/展开放宽蛋白质的熔融温度附近，因此从来没有如下的整个人口的折叠。与此混频器，我们看着折叠（λ 至86），γ-阻遏两个色氨酸排放总量和光谱位移，并已发现的证据表明，蛋白质折叠强折叠条件下有没有屏障，没有以往的T访问跳测量12。最后，我们测量的绒毛头饰HP-35的折叠，一个他最快的文件夹尚未测量，发现混合后测量，折叠速度比的T-16相同的条件下下跳慢〜5倍。这表明，折叠动力学取决于初始条件，推翻了在该领域的主要假设。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> AZ P4110光阻</td><td> AZ电子材料</td><td></td><td></td></tr><tr><td>排列400ķ开发</td><td> AZ电子材料</td><td></td><td></td></tr><tr><td>贝克减贫战略2000光阻剥</td><td> avantor性能材料</td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBond技术</td><td> AquaBond 55 </td><td></td></tr><tr><td> 500微米盖晶圆</td><td>传感器准备服务</td><td></td><td>直径：100毫米±0.5毫米 厚度：0.5 mm±0.025毫米 标准公差（除非另有说明） 表面处理：SI4-8A;精工：8-12A 单位：一小学半性病;抄写 清洁和抛光，双面</td></tr><tr><td> 170微米盖晶圆</td><td>传感器准备服务</td><td> 7980 2G晶片</td><td>直径：100毫米±0.5毫米 厚度：0.17毫米±0.025毫米 标准公差（除非另有说明） 表面处理：司：4-8A;精工：8-12A 单位：一小学半性病;抄写 清洁和抛光，双面</td></tr><tr><td>深反应离子蚀刻氧化物</td><td> ULVAC公司</td><td> ULVAC公司NL-6000 </td><td></td></tr><tr><td>氩离子激光器</td><td>剑桥激光实验室</td><td> LEXEL 95的倍频</td><td> λ= 258 nm处</td></tr><tr><td>氩离子激光器</td><td> Melles Griot的</td><td></td><td> λ= 488 nm的</td></tr><tr><td>显微镜</td><td>奥林巴斯</td><td> IX-51</td><td></td></tr><tr><td>物镜</td><td>托尔实验室</td><td> OFR的慕尼黑-40X-UVB的0.5不适用</td><td> λ= 258 nm处</td></tr><tr><td>物镜</td><td>奥林巴斯</td><td> UPLSAPO 60XW 1.2不适用</td><td> λ= 488 nm的</td></tr><tr><td> nanopositioner </td><td>疯狂的城市实验室</td><td>纳米-LP100 </td><td></td></tr><tr><td>电动平移台</td><td> semprex公司</td><td>吉隆坡的系列12-6436 </td><td></td></tr><tr><td>砷化镓光子计数器</td><td>滨松</td><td> H7421-40 </td><td></td></tr><tr><td>单色</td><td> HORIBA仪器公司</td><td> MicroHR </td><td></td></tr><tr><td> CCD相机</td><td>安道​​尔技术</td><td> IDUS 420A-卜</td><td></td></tr><tr><td>盐酸胍（GuHCl） </td><td> Sigma-Aldrich公司</td><tD&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

一种蛋白质折叠成其天然构象的过程，是高度相关的生物和人类健康仍然知之甚少。原因之一是，折叠的地方接管的时间表广泛，从纳秒到秒或更长，取决于蛋白1。传统的停流混频器都允许折叠动力学的测量，在约1毫秒。我们最近开发的微流体混合器，稀释变性〜〜8微秒2 100倍。停止流混频器不同，这种混频器在层流制度，在这种动荡不会发生。动荡的情况下，允许在混频器具有优良的协议，实验3-4的所有流量的精确数值模拟。 重 ≤100雷诺数层流实现。对于水溶液，这需要微米尺度的几何形状。我们用硬的基材，如硅或熔融硅CA，使5-10微米渠道，宽，深10微米（ 见图1）。最小的尺寸，在混合区的入口处，是在1微米大小的顺序。该芯片是密封的薄玻璃或石英光纤接入盖玻片。典型的共线性流速1米/秒，产生再 〜10，但蛋白质的消费量只有0.5 NL / s或1.8μL/小时。蛋白浓度的检测方法取决于：色氨酸荧光典型的浓度是100微米（1 /色氨酸蛋白）和FRET技术是典型的浓度约100 nm。 折叠过程的开始，从6米至0.06 mol盐酸胍变性剂快速稀释。在高变性的蛋白流下来的中央通道，并会见了混合区两边的缓冲区而不变性移动快〜100倍（ 见图2）。这个几何导致迅速收缩成一个窄的蛋白质流喷气〜100纳米宽。光变性分子的扩散非常迅速，而沉重的蛋白质分子的扩散是非常慢，在1毫秒的扩散小于1微米。在不断扩散，迅速稀释从蛋白质的流变性的变性剂和蛋白结果的差异，各地的蛋白质的变性剂的有效浓度降低。蛋白喷气流率固定下来的观测通道和荧光蛋白质在折叠可以使用扫描共聚焦显微镜观察。

Watch this Scientific Journal Video about Microfluidic Mixers for Studying Protein Folding at JoVE.com

Microfluidic Mixers for Studying Protein Folding

在这项工作中，我们解释的制造和使用微流体混合器混合〜8微秒的两个解决方案的能力。我们也证明了这些混频器的使用与光谱紫外荧光检测和荧光共振能量转移（FRET）的使用。

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

microfluidic-mixers-for-studying-protein-folding

Research

JoVE Journal

Bioengineering

15.0K 次观看.  Michigan State University. 在这项工作中，我们解释的制造和使用微流体混合器混合〜8微秒的两个解决方案的能力。我们也证明了这些混频器的使用与光谱紫外荧光检测和荧光共振能量转移（FRET）的使用。

视频: Microfluidic Mixers for Studying Protein Folding

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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 ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

研究蛋白质功能和改变的蛋白的表达由源抗体干扰和三维重建中的作用

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...