The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for financial support within the Sonderforschungsbereich SFB 985 "Functional Microgels and Microgel Systems".

1.微凝胶合成注:N -isopropylacrylamide（NIPAM）从正己烷重结晶。原样使用其它试剂。 <ol><li>聚（NIPAM）矩阵微凝胶的常规合成批<ol><li>在过滤245毫升溶解1.8克NIPAM和24毫克N，N&#39;-bisacrylamide（BIS）（0.2微米的再生纤维素（RC）的膜过滤器）在装有回流冷凝器的500毫升三颈圆底烧瓶中双蒸水，搅拌器和橡胶隔片。 </li><li>插入温度计和120mm的针头穿过隔膜的氮气输入。 </li><li>加热该溶液至60℃，同时搅拌。通过用氮气吹扫40分钟脱氧溶液。 </li><li>同时准备155毫克过硫酸钾（KPS）的在过滤双蒸水和气泡用氮气将溶液以除去氧气加入5ml引发剂溶液。 </li><li>转让完成5毫升KPS小号olution在装有120mm的针氮气洗涤注射器。 </li><li>解除在三颈烧瓶中的溶液水平以上的氮针和迅速通过橡胶隔膜加入KPS溶液到反应器中。 </li><li>让聚合继续进行，在氮气流和缓慢搅拌1小时，在60℃。 </li><li>使用布氏漏斗和滤纸向热的反应溶液，以丢弃大的聚集体进行过滤。让分散体冷却下来。 </li><li>离心机和再分散的分散体40分钟三次在257,000 xg离心，最后再分散在双蒸水的最小可行量的沉淀物。通常，这是2-4毫升。 </li><li>冻干该分散存储。 </li></ol></li><li>荧光标记的聚（NIPAM）微凝胶的非搅拌合成<ol><li>在玻璃容器称量257.7毫克NIPAM，3.5毫克BIS和1.5毫克丙烯酰氧乙基硫代罗丹明B（染料），并加入10毫升的过滤双重蒸馏的编辑水。 </li><li> Ultrasonicate 15分钟的染料单体溶液以溶解染料在水中。 </li><li>制备不含染料的相同溶液到一个单独的玻璃容器中。 </li><li>制备与使用不含染料的单体溶液以获得具有各种染料浓度的浓度系列中的染料单体溶液的各种稀释液。在这项工作中，使用的染料在0.02-0.1毫摩尔/ L的浓度范围。 </li><li>溶解在10毫升过滤的双蒸水8.4毫克KPS以得到引发剂溶液。 </li><li>传送将0.5ml浓度系列和0.5ml的KPS溶液与直径为10毫米到试管以获得最终的反应溶液并用橡胶隔片密封。 </li><li>在连接到一个加热循环，以63℃的双层壁玻璃容器预热油浴中。 </li><li>通过120毫米针用氮气吹扫20分钟去氧反应液。 </li><li>将管放入自动对焦loating平台和浸入平台放入预热油浴。设定温度至60℃。在浴最初更高的温度是必要的，因为室温溶液冷却浴。用于高精密粒径调谐初始反应过程中的温度控制已成为严格，一般为±0.1℃。 </li><li>让反应继续进行适当的时间。通常1小时就足够了。 </li><li>迅速转移反应管至烘箱中在60℃，并把热分散一滴至10ml过滤预热过PNIPAM体积相转变温度（VPTT，32 -34℃）1双蒸水，为DLS表征在折叠状态。 </li><li>让分散体的其余部分冷却到室温，并将其转移到离心管中。 </li><li>对溶液离心40分钟三次在257,000 XG，最后稀释微凝胶在2毫升过滤的双蒸水FOr用作为示踪粒子。 </li></ol></li></ol> 2.光散射特性<ol><li>流体动力学半径测定折叠状态，通过动态光散射<ol><li>洗试管并用丙酮蒸气玻璃器皿。 </li><li>热的10ml过滤（ 例如，200纳米或更小的RC滤波器）双蒸水通过PNIPAM VPTT。 </li><li>使用预先加热的针（0.9×40毫米），注射器（1毫升）转移热分散的液滴向过滤水。 </li><li>脾气DLS测角仪指数匹配浴至50℃，不让它冷却下来的样品转移到仪器上。 </li><li>找到最大的散射角，其中的散射强度足以获得通过进行试验测量一相关图。 <ol><li>插入样品试管（10毫米直径的玻璃管用1ml颗粒分散体的）。移动至检测器臂到小散射角（这里30°）。 </li><li>检查FO光束轮廓r多个散射:主光束，没有多重散射等周围没有辉光检查计数范围是适于在最低散射角度测量（约30和600千赫之间;软件窗口的右上角。） </li><li>移动测角仪手臂最高的散射角（在这里选择120°）。检查计数率依然为测量足够高（30和600千赫之间）。如果强度过低，移动手臂以降低散射角。 </li></ol></li><li>检查光束通过直观的甲苯浴玻璃在最低的散射角，如果入射光束灼热周围观察到多重散射发生。在这种情况下，降低了激光强度或使用更高的稀释。 </li><li>获得的最小和最大散射角20之间相关图（ 例如 ，30° - 140°）用60秒的最小采集时间。增加了采集时间为弱强度大散射角如果必要的。 </li></ol></li><li>数据分析37 <ol><li>计算散射向量量值为按散射角<img alt="公式2" src="/files/ftp_upload/54419/54419eq2.jpg" /> ，其中n是分散体的折射率， <img alt="（3）式" src="/files/ftp_upload/54419/54419eq3.jpg" />在真空中激光的波长和<img alt="4式" src="/files/ftp_upload/54419/54419eq4.jpg" />散射角。 </li><li>在这种情况下测量软件提供了强关联函数<img alt="式5" src="/files/ftp_upload/54419/54419eq5.jpg" /> ，将其转换为电场相关函数<img alt="6式" src="/files/ftp_upload/54419/54419eq6.jpg" />根据<img alt="7式" src="/files/ftp_upload/54419/54419eq7.jpg" /> 。参数<img alt="8式" src="/files/ftp_upload/54419/54419eq8.jpg" />是关系到散射光奥雅纳的空间相干程度的无趣工具参数r是探测器面积。 </li><li>上相关图进行累积量分析， 即 ，适合二阶多项式的每个电场相关函数的对数<img alt="9式" src="/files/ftp_upload/54419/54419eq9.jpg" />通过线性最小二乘。 <img alt="8式" src="/files/ftp_upload/54419/54419eq8.jpg" />显示为拟合的截距和其精确值是不重要的在相对于数据分析。限制拟合 ​​到一个有意义的延迟时间τ 的值， 例如 ，这样的相关性幅度10 -最大振幅的20％。第一阶项的系数是相关函数的平均衰减速度， <img alt="10式" src="/files/ftp_upload/54419/54419eq10.jpg" /> 。 </li><li>找到的均值扩散系数的最可能的值<img alt="11式" src="/files/ftp_upload/54419/54419eq11.jpg" />通过线性最小二乘颗粒适合在<img alt="12式" src="/files/ftp_upload/54419/54419eq12.jpg" /> 。如果<img alt="10式" src="/files/ftp_upload/54419/54419eq10.jpg" />反对<img alt="13式" src="/files/ftp_upload/54419/54419eq13.jpg" />不出现的线性和经过误差范围内的原点，粒度分布宽和流体力学半径将被定义不佳。 </li><li>从斯托克斯 - 爱因斯坦关系计算平均流体动力学半径<img alt="14式" src="/files/ftp_upload/54419/54419eq14.jpg" /> ，其中<img alt="15式" src="/files/ftp_upload/54419/54419eq15.jpg" />是波尔兹曼系数， <img alt="16式" src="/files/ftp_upload/54419/54419eq16.jpg" />绝对温度和<img alt="17式" src="/files/ftp_upload/54419/54419eq17.jpg" />该分散体在粘度<img alt="16式" src="/files/ftp_upload/54419/54419eq16.jpg" /> 。传播的标准偏差<img alt="11式" src="/files/ftp_upload/54419/54419eq11.jpg" />至<img alt="18式" src="/files/ftp_upload/54419/54419eq18.jpg" /> 。 </l我&gt; </ol></li><li>颗粒结构测定静态光散射<ol><li>洗试管并用丙酮蒸气玻璃器皿。使用直径为20毫米或更大的比色杯，以尽量减少柱面透镜效果。 </li><li>过滤器（200纳米RC滤波器或更小）大约20毫升双蒸水至玻璃小瓶中，并纯化分散一滴转移到小瓶中。使用它进行样品制备，除去从制造工艺残留的杂质之前洗涤用10毫升水的过滤器。 </li><li>检查样品反对任何环境光源。如果观察到蓝色色调，样品很可能是过于浓缩。相应地稀释。 </li><li>用过滤后的水多次冲洗试管准备背景水样品，然后填充到合适的样品体积，这取决于反应杯，并在仪器中的激光的位置。激光必须穿过样品而不从弯液面被折射。 </li><li>校准仪器和设备耳鼻喉科用甲苯样本。 </li><li>测量整个可用的角度范围散水（背景）。 </li><li>测量从整个可用的角度范围的样品的散射强度优选在几个波长。归到正向散射强度的散射图案被称为形状因子。 </li><li>如果颗粒结构是已知的，使用适当的模型公式计算在不同波长测量数据集全球契合。 </li><li>为正则直接（如FitIt！33）未知颗粒结构使用或更一般的间接傅立叶逆变换21,22例程在结合一对距离分布函数（仅对于球形粒子）的解卷积23,24为粒子的近似分类类型。 </li><li>万一嵌合或反转例程提供粒子半径分布函数的估计，计算多分散性指数（通过其平均值除以分布的标准偏差）。 </li></ol></li></ol> 3.粒子跟踪由宽视场荧光显微镜注:在20℃的465±7 nm和405±7nm的水力半径示踪和基质颗粒，分别用于颗粒跟踪。 <ol><li>样品制备<ol><li>通过再分散冻干未标记微粒凝胶的已知量的双蒸水已知量制备浓缩基质微粒凝胶分散体。加标记的示踪粒子的小体积。 </li><li>在确认显微镜适当的示踪剂微凝胶浓度。最佳浓度是磁道最大数量的同时采集之间的折衷，同时具有示踪剂浓度足够低，使得该示踪粒子轨迹在采集过程中交叉的概率是可忽略的。 </li><li>准备通过蒸发浓缩分散液水在烘箱。通过蒸发之前分散体的重量比较样品的原始重量确定的重量浓度。 </li></ol></li><li>数据采集​​与分析<ol><li>使用所需的放大倍率的适当物镜和光圈为示踪剂的激发和荧光同时从收集样品。在这项工作中，使用100X / 1.3 NA油浸物镜。 </li><li>湿度室放置到一个XYZ-压电表，适合商用的显微镜​​。 </li><li>为了防止干燥样品，将等离子清洗盖玻片成所需浓度的保湿室和吸管10微升聚（NIPAM）的分散到滑。 </li><li>根据荧光染料的激发和发射光谱，使用合适的激光激发和适当地调整激光功率。的强度应足够低，以避免染料的快速光漂白，但在同时足够强的精确的单粒子定位（见下文）。在这项工作中，使用561纳米的二极管泵浦固体激光器，并保持激光功率恒定在16毫瓦（ 约 0.5千瓦厘米-2的样品）进行所有的测量。 </li><li>为了获得均匀的样品照明，使用这里描述的临界照明设置。为此，耦合激光器到多模光纤（NA 0.22±0.02为0.6mm纤芯直径），摇使用涡旋纤维以时间平均出来的激光斑点，并投射在纤维端插入样品平面。 </li><li>校准从盖玻片的背面反射的Z距离和通过移动物镜略微向上集中几微米到样品和使用的z补偿器固定的z位置。这避免了因盖玻片的界面效果。 </li><li>调整检测器的参数，如曝光时间，对荧光信号的强度。在这种情况下，使用相机EMCCD用0.1秒的曝光时间，电子倍增模式和50的增益。 </li><li>获得几部电影有帧的适当数量，以获得足够的滞后时间来计算在样品的不同区域中的微凝胶的均方位移。在这项工作中，使用500或1000帧采集帧数。 </li><li>通过使用高斯拟合25每帧中的粒子的定位数据进行分析，并使用恰当的粒子追踪算法26以获得均方位移。27计算通过平均在所有的电影所有曲目平均值和标准偏差。通过线性回归计算滞后时间长的扩散系数<img alt="19式" src="/files/ftp_upload/54419/54419eq19.jpg" /> ，其中<img alt="20式" src="/files/ftp_upload/54419/54419eq20.jpg" />是均方位移，D的平均扩散系数和τ的延迟时间。 </li><li>美东时间从反常扩散方程imate异常参数γ <img alt="21式" src="/files/ftp_upload/54419/54419eq21.jpg" />通过转换数据以对数标度，得到<img alt="22式" src="/files/ftp_upload/54419/54419eq22.jpg" /> 。这种异常的参数<img alt="23式" src="/files/ftp_upload/54419/54419eq23.jpg" />通过剧情的衍生给出。该衍生物可以通过数据点的有限差来估计，或者通过多项式函数拟合数据点和分析鉴别。通过绘制拟合残差和剩余范增加多项式阶决定了足够程度的多项式拟合函数。 </li><li>重复同样的步骤对不同浓度的微粒凝胶基质。 </li></ol></li></ol>

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The authors have nothing to disclose.

少量官能共聚单体的加成可对PNIPAM衍生微凝胶的粒度和结构的显著效果。同时小规模试管聚合是考虑到这种变化的好方法，有助于迅速找到目标粒度合适的反应剂组合物根据需要扩大该反应。该颗粒的质量是约指数依赖当热分解引发剂的聚合温度，例如KPS，使用20，因此，人们需要建立反应器中良好的再现内稳定和精确的温度...

刺激敏感聚（N -isopropylacrylamide）（PNIPAM）微凝胶1,2吸引过去二十年的不断兴趣，因为他们在各种智能应用的潜力。证明用例包括可切换的乳液稳定剂3-8，微透镜9中，为了便于细胞收集10,11细胞培养基材，以及低分子量化合物的智能的载体和其它生物医学用途12。从一个基础研究点这些颗粒已被证明是调查受试者有用诸如胶体相互作用13-15和聚合物-溶剂相互作用16-18。 成功使用PNIPAM微凝胶以及它们的衍生物中的任何给定的应用的典型地需要在粒度分布的平均粒径和宽的知识。对实验结果的涉及PNIPAM微正确解释凝胶，颗粒结构，这可以通过官能共聚单体的影响，必须已知。动态和静态光散射（DLS和SLS，分别）是唯一适用于因为这些方法是快速和相对容易使用的获取这些信息;它们探查颗粒性能非侵入在其天然环境中（分散液）。 DLS和SLS还收集广大避免由于样本量小，是典型的显微镜方法而产生的偏见颗粒的数据。因此，这项工作的第一目的是介绍关于对从业人员新的胶体特性的光散射很好的做法。 典型地，沉淀聚合在实验室规模进行，并找到合适的反应条件对于特定的颗粒属性可以是费力的和所需要的合成的许多重复。在对比大批量合成，无搅拌沉淀聚合19,20是芳APID程序，其中不同的反应物组合物的批次可以聚合粒径分布窄的同时屈服颗粒。同时聚合减少实验误差，产量大意味着正确的反应条件可快扩大该反应被发现。因此，我们的第二个目的是要证明在原型和在不需要大量产品的应用程序的非搅拌沉淀聚合的有用性。 合成与表征的不同方面走到了一起，在胶体相互作用的研究荧光标记的PNIPAM微凝胶的应用实例。这里我们使用高度精确的单粒子跟踪调查标记的示踪剂微凝胶在未标记的基质微凝胶分散在很宽的基质浓度范围扩散和解决集中胶体分散的笼效应。宽视场荧光显微镜是非常适合FOR此​​目的，因为它可以表征中的大量潜在的不同的矩阵种类的几示踪分子的特定行为。这是相对于技术如DLS，SLS和流变学，其测量的系统总体平均性能，因此无法解析在一个大系统的小数目的探针粒子的行为。此外，在该具体例常规的光散射方法不能也利用由于高粒子浓度，这会导致强烈的多次散射无效任何标准分析。自动数据处理和统计方法使用使整个系统的行为也对单粒子跟踪，当通过大样本平均分析。

<table><tbody><tr><td>Acetone</td><td>VWR Chemicals</td><td>KRAF13455</td><td></td></tr><tr><td>Bisacrylamid</td><td>AppliChem</td><td>A3636</td><td></td></tr><tr><td>n-Hexane</td><td>Merck</td><td>104374</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Isopropylacrylamide</td><td>Fisher Scientific</td><td>AC412785000</td><td>recrystallized from n-hexane</td></tr><tr><td>Methacryloxyethyl thiocarbamoyl rhodamine B</td><td>Polysciences</td><td>23591</td><td></td></tr><tr><td>Potassium peroxodisulfate</td><td>Merck</td><td>105091</td><td></td></tr><tr><td>Silicone oil 47 V 350</td><td>VWR Chemicals</td><td>83851</td><td></td></tr><tr><td>Toluene</td><td>Sigma Aldrich</td><td>244511</td><td></td></tr><tr><td>F12 Refrigerated/heating circulator</td><td>Julabo</td><td>9116612</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>XY(Z) Piezo System</td><td>Physik Instrumente</td><td>P-545.3R7</td><td></td></tr><tr><td>100X Oil immersion objective</td><td>Olympus</td><td>UPLSAPO</td><td></td></tr><tr><td>QuadLine Beamsplitter</td><td>AHF Analysentechnik</td><td>F68-556T</td><td></td></tr><tr><td>Cobolt Jive 150 laser</td><td>Cobolt</td><td>0561-04-01-0150-300</td><td></td></tr><tr><td>Multimode Fiber</td><td>Thorlabs</td><td>UM22-600</td><td></td></tr><tr><td>iXON Ultra 897 EMCCD camera</td><td>Andor</td><td>DU-897U-CS0-BV</td><td></td></tr><tr><td>Laser goniometer</td><td>SLS Systemtechnik</td><td>Mark III</td><td></td></tr><tr><td>CF40 Cryo-compact circulator</td><td>Julabo</td><td>9400340</td><td></td></tr><tr><td>Laser goniometer system&amp;nbsp;</td><td>ALV GmbH</td><td>ALV / CGS-8F</td><td></td></tr><tr><td>Multi-tau corretator</td><td>ALV GmbH</td><td>ALV-7004</td><td></td></tr><tr><td>Light scattering electronics</td><td>ALV GmbH</td><td>ALV / LSE 5004</td><td></td></tr><tr><td>Photon counting module</td><td>PerkinElmer</td><td>SPCM-CD2969</td><td>2 units in pseudo cross-correlation mode</td></tr><tr><td>633 nm HeNe Laser</td><td>JDS Uniphase</td><td>1145P</td><td></td></tr><tr><td>F32 Refrigerated/heating circulator</td><td>Julabo</td><td>9312632</td><td></td></tr></tbody></table>

controlled synthesis and fluorescence tracking of highly uniform poly(n-isopropylacrylamide) microgels

刺激敏感聚（N -isopropylacrylamide）（PNIPAM）微凝胶具有前瞻性的各种实际应用和基础研究使用。在这项工作中，我们使用单一粒子由一个快速无搅拌沉淀聚合过程跟踪的荧光标记PNIPAM微凝胶作为调谐微粒凝胶大小展示。这种方法非常适合原型新的反应的组合物和条件或用于不需要大量产品的应用程序。微凝胶合成，颗粒大小和结构测定动态和静态光散射在协议中详细介绍。它表明在加入官能共聚单体可具有在颗粒成核和结构有很大的影响。通过宽视场荧光显微镜单粒子跟踪允许标记的示踪剂微粒凝胶的扩散的非标记的微凝胶的浓缩矩阵调查，系统不易受调查其他方法，如动态光散射。

PNIPAM微粒凝胶颗粒的批次中从而最终颗粒体积的数量，而且，在成核阶段20疏水共聚单体染料甲基丙烯酰硫代若丹明B通过减少在间歇的粒子数密度影响成核过程中的反应初确定。为两个不同的初始NIPAM浓度颗粒浓度的降低可以被看作是在折叠状态与染料浓度的增加，平均最终颗粒的体积增加，在图1所示的?...

Watch this Scientific Journal Video about Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels at JoVE.com

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

非搅拌沉淀聚合提供了一种快速，可重复的成型方法来刺激敏感聚（N- -isopropylacrylamide）粒径分布窄的微粒凝胶的合成。在这个协议中合成，光散射表征和在宽视场显微镜设置这些微粒凝胶的单个粒子荧光跟踪被证明。

可控合成和高度统一聚荧光追踪（<em&gt;ñ</em&gt; -isopropylacrylamide）微凝胶

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

controlled-synthesis-fluorescence-tracking-highly-uniform-poly-n

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

10.2K 次观看.  RWTH Aachen University. 非搅拌沉淀聚合提供了一种快速，可重复的成型方法来刺激敏感聚（N- -isopropylacrylamide）粒径分布窄的微粒凝胶的合成。在这个协议中合成，光散射表征和在宽视场显微镜设置这些微粒凝胶的单个粒子荧光跟踪被证明。 

视频: Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

合成聚（<em&gt;ñ</em&gt; -isopropylacrylamide）剑锋Microhydrogels各向异性热响应能力和亲有机/亲水承载能力

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

科研

化学

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

生物工程

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

磁性和热敏性聚（<em&gt;ñ</em基于异丙基丙烯酰胺）的微凝胶用于磁触发控制释放

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

微流体合成微孔退火粒子支架用微凝胶构建块

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

可控合成和高度统一聚荧光追踪（

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