我们非常感谢国家科学基金会（瑞士- 0847145），和亨利德雷福斯教师学者奖慷慨支持这项研究计划。我们想特别承认，她与横截面成像的援助主任在里士满大学生物系的显微镜和成像恭戴维斯。特别感谢给博士。 R. Kanters，D.凯洛格，R.米勒，和W. T.利奥波德，以及拉斯科林斯，菲尔约瑟夫，卡罗琳商标，小敏马洛里和约翰Wimbush - 所有的人在大学本科生科研的可能里士满。一个非常个人的感谢你，是给所有的过去，现在，和未来的本科研究人员在利奥波德研究实验室。

1。 Hexanethiolate单​​层保护金团簇合成 Hexanethiolate官能单层保护金团簇（储值卡）是合成2:1 1 hexanethiol（C6）的摩尔比黄金生产的平均坳225（C6），75结构。4-9 Brust反应具体的修改，如配体类型，具体的巯基金的比例，温度，反应分娩率，或后期合成处理，9-11可以产生各种各样的不同核心的大小和功能的保护群体的多用途储值卡，分别为4。MPC近似（平均）与各硫醇团体功能的多用途储值卡的组成可以由质子（1H）核磁共振（NMR）的碘分解样品的分析。 <ol><li>在30毫升的甲苯溶解1.1克四辛基溴化铵（TOABr）与适当的通风柜通气。 </li><li>溶解0.38克硼氢化钠（硼氢化钠4）〜20毫升18MΩultrapurified的水（向上H 2 O），并允许在冰上冷却至少30分钟。 </li><li> 〜20毫升的H 2 O溶解0.31克氢tetrachloroaurate（HAuCl 4）和TOABr甲苯溶液定量转移溶液混合使用额外的H 2 O〜5毫升，以相转移水金非水溶液的解决方案。搅拌严格而掉以轻心，使焦橙色的水和明确非水​​相混合以及上限为30分钟。 </li><li>明确水和烧毁橙色非水相转移至分液漏斗。倒掉水（底）层和倒出的非水层（顶部）到一个干净的瓶中。 </li><li>添加HAuCl 4的比例为2:1的非水溶液C6 。搅拌30分钟，形成一个凹（一）聚合物，从红橙色的颜色变化，淡黄色，近无色的解决方案检测。 </li><li>反应混合物转移到一个绝缘的冰浴和冷却至0 ° C带搅拌至少30分钟。 </li><li>定量，迅速 ​​补充冷冻硼氢化钠反应混合物溶液 ，为了减少金属黄金AU（一）在硫醇的存在，瞬间形成了厚厚的黑色的多用途储值卡的解决方案后，除了。搅拌反应过夜在0 ° C。 </li><li>将反应混合物转移至分液漏斗，丢弃到一个废物烧杯中的水（底）层，旋转蒸发接近完成干燥烧瓶中留下一个沉重的黑色污泥的非水（顶部）甲苯层。 </li><li>沉淀加入乙腈和允许坐通宵的储值卡。 </li><li>收集真空过滤使用橡胶配件和侧武装烧瓶与抽吸的介质孔隙度的玻璃粉的储值卡，并具有丰富的乙腈冲洗。 </li><li>允许多用途储值卡的空气干燥，称重产品，特点是透射电子显微镜（TEM）和核磁共振分析，并储存供日后使用上限。获得溶解在甲苯下降到formvar /碳支持膜的铜网（400目）和经营80-100千伏TEM仪器铸造的多用途储值卡的TEM照片。可以预计的平均核心大小，使用图像分析软件，如图像J（免费）。 </li></ol> 2。电影大会:二硫联蛋白单分子膜的电化学的MPC电影大会第一黄金基板电化学清理和修改与C6 SAM沉浸在交替二硫连接分子和C6修改储值卡的解决方案，以弥补"浸周期"，这是反复几次，最终形成一个二硫联MPC电影大会之前。正如在以前的研究中所述，绿脓杆菌阿苏林（AZ）蛋白的质粒客客气气科里博士威尔逊莱斯大学和AZ里士满大学教授，博士乔纳森提供的纯化和冻干粉Dattelbaum，水化，后来用4.4毫米的磷酸钾缓冲液（KPB，pH值= 7.0，μ= 10毫米），创建一个5-10μM解决方案，紫外可见（UV - VIS）分析核实。 <ol><li>聚集在以下顺序从底部到顶部的电化学（echem）夹心细胞:第一个有机玻璃固定板，金基底作为工作电极，黄铜金工作电极的电接触，第一橡胶垫片，氟橡胶O形圈，它定义电极面积（0.32厘米2），玻璃细胞体，第二个橡胶垫圈，第二有机玻璃固定板。整个单元一起举行螺纹杆和仔细拧紧蝶形螺母。细胞是装有一个商业购买的参考电极，房屋1米饱和KCl，银/氯化银参考线，和铂辅助电极丝玻璃桶。 </li><li>电化学清洁日E的金基底潜在的Windows执行中的循环伏安法（CV）从0.2到0.9 V，0.2至1.2 V和0.2至1.35 V（相对于Ag / AgCl电极，氯化钾）100 mV / s的0.1氢2 SO 4和0.01 M氯化钾。 </li><li>通过执行"标准条件"，包括潜在的窗口，从0.1到0.4 V（相对于Ag / AgCl电极，氯化钾）扫描在100 mV / s的在KPB 1丢弃KPB CV测量的清洁的裸金基板的充电电流和冲洗先后与UP H 2 O，乙醇（EtOH）的H 2 O和乙醇。 </li><li>公开已清理的金基底〜300μL5毫米酒精C6解决方案，并允许坐过夜，以形成一个有序的C6的SAM。丢弃从细胞C6解决方案和冲洗先后与乙醇UP乙醇，H 2 O的H 2 O ， </li><li>在标准条件下测量充电电流的SAM。丢弃KPB，先后向上乙醇，H 2 O的H 2 O，和乙醇冲洗。充电电流应明显减少裸金测量（步骤2.3）1。 </li><li>公开的SAM修饰的金基板5毫米〜300μL乙醇1,9 - nonanedithiol（NDT）的解决方案，允许坐1小时interdisperse无损检测分子内C6 SAM联系起来。丢弃的无损检测解决方案，并先后与彻底冲洗UP乙醇，H 2 O的H 2 O，和二氯甲烷（CH 2 CL 2），与乙醇。 </li><li>公开金基板MPC解决方案的CH 2氯2（〜1毫克/毫升），搅拌1小时慢慢地与 N 2气体鼓泡。如有必要，更换蒸发与CH 2 CL 2 MPC解决方案。这是电影大会MPC层锚固。丢弃MPC解决方案，并先后与CH 2 CL 2，H 2 O的向上，并KPB再次冲洗。 </li><li>在标准条件下测量的MPC层的充电电流。丢弃KPB和冲洗的H 2 O和CH 2 Cl 2等先后。 </li><li> 20分钟慢慢地与 N 2气体鼓泡公开金基底〜300μLCH 2 Cl与搅拌 2 5毫米的无损检测解决方案。 </li><li>放弃无损检测并彻底冲洗CH 2 CL 2。重复步骤2.7和2.8存第二MPC的一层薄膜大会。 </li><li>存入额外MPC层，步骤2.9和2.10是重复的。随着每个额外的MPC层，在充电电流相应增加。 </li><li>网络MPC电影完成后，冲洗与KPB膜修饰的基板。排列的蛋白质被吸附在MPC电影大会echem夹心细胞注入〜150μL〜5-10μM的AZ的KPB解决方案，并允许坐封顶，冷藏至少1小时。 </li><li>从冰箱中取出的echem细胞，并允许它返回到接近室温。与N 2气体彻底冲洗10分钟KPB，笔芯echem KPB细胞和泡沫KPB。 </li><li>蛋白质单层的电化学研究简历，从-0.25 V至0.25 V的电位窗（相对于Ag / AgCl电极，氯化钾）100扫描在KPB毫伏/秒。 </li></ol> 3。电影大会:二硫联MPC光学跟踪电影大会增长光学评价MPC电影之前，载玻片部分预清洗食人鱼解决方案（CAUTION! 2:1浓H 2 SO 4和H 2 O 2）和（3 -巯基）丙基三甲氧基硅烷（3 - MPTMS处理然后组装这些修改后的玻璃上）。1-2 MPC电影幻灯片使用"浸循环"技术，如前所述。 <ol><li>冲洗与CH 2 CL 2 3 MPTMS的修改后的玻片上，MPC解决方案中的CH 2氯2（〜1毫克/毫升），1小时，在低速搅拌，而在振动筛。这就完成了电影大会第一锚多用途储值卡硅烷硫醇endgroups MPC层。彻底冲洗幻灯片与CH 2 CL 2，干与N 2气体。紫外光谱的幻灯片（从400至1000 nm），并再次冲洗CH 2 CL 2。 </li><li>放置1小时的幻灯片，在低速搅拌，而在振动筛在CH 2 CL 2 5毫米的无损检测解决方案。与CH 2 CL 2冲洗的幻灯片。 </li><li>在MPC解决方案的幻灯片，而在低速上摇床搅拌，放置1小时。这就完成了电影大会的货币政策委员会第二层。彻底冲洗幻灯片与CH 2 CL 2，干与N 2气体，并采取一个紫外光谱的幻灯片（从400至1000 nm）。在整个频谱的吸光度应增加额外MPC层吸附到电影大会。 </li><li>存款额外MPC层，STEPS 3.2-3.3是重复的。 </li></ol> 4。横截面透射电子显微镜表征单层保护金集群电影大会的TEM横截面是准备重新嵌入EN面对嵌入式电影2，12，这是由第一附加MPC电影3 - MPTMS修改后的载玻片上组装到一个干净的，标准的显微镜幻灯片使用嵌入812环氧树脂，以便改善在下面的程序处理。谨慎使用与应用的热量，为更高的温度下会分解电影内的储值卡。 <ol><li>混合嵌入812环氧树脂，并允许增厚至少12小时。 </li><li>填充环氧树脂与"00"BEEM胶囊和反转MPC薄膜样品的顶部（第3条编制）。胶囊，使气泡上升胶囊顶部，创造之间的环氧树脂和MPC薄膜样品密封处的压力。允许聚合至少18小时在60 ° C，然后冷却至室温安装幻灯片。 </li><li>热装在200 ° C，以便去除块附有MPC EN面对电影，持续20秒的幻灯片上铸铝热板。 </li><li>切割样品含有BEEM囊块使用一个珠宝商的看到电影。 </li><li>重新嵌入了面临的硅内部的MPC片方在硅平面模具拆除部分。环氧树脂填充硅在室温下使聚合至少18小时，在60 ° C。样品冷却至室温。 </li><li>使用钻石刀垂直切割部分在刀刃上获得一个莱卡UCT ultramicrotome 60-80 nm的薄样品的部分。 </li><li>切片formvar /碳支持膜的铜网（400目）的部分路段和MPC的薄膜组件的准备截面的TEM照片。 </li></ol> 5。代表性的成果: <img alt="图1" src="/files/ftp_upload/3441/3441fig1.jpg" /> 图1双层充电电流监测共5浸渍周期（交替暴露MPC和无损检测解决方案）MPC的薄膜生长。充电电流增加系统与每个浸渍周期，加入MPC的"层"的电影（图2）。循环伏安收集使用一个潜在的窗口，从0.1到0.4 V（相对Ag / AgCl电极，氯化钾）扫描100 mV / s的4.4毫米磷酸钾缓冲液（pH值= 7.0，μ= 10毫米）。 ML瓦戈，CP Gulka，JK Gerig，招商Manieri，JD Dattelbaum，CB标志，NT劳伦斯，ML Trawick，和MC利奥波德， 兰米尔26（1），560-569许可重印。 2010年美国化学学会版权所有。 <img alt="图2" src="/files/ftp_upload/3441/3441fig2.jpg" /> 图2（A）示意图排列蛋白吸附到二硫联MPC电影大会。 （二）排列的典型循环伏安吸附MPC电影大会收集的一个潜在的窗口从-0.25至+0.25 V（相对于Ag / AgCl电极，氯化钾）100 mV / s的扫描4.4 mM磷酸钾缓冲液（pH = 7.0， μ= 10毫米）。 <img alt="图3" src="/files/ftp_upload/3441/3441fig3.jpg" /> 图3代表一个二硫联MPC 3 MPTMS修改后的载玻片上的薄膜生长的紫外-可见光谱监测。浸周期由载玻片无损检测连接器解决方案，MPC解决方案曝光的曝光。每个随后浸在增长中膜厚度和并发吸收增加的结果。由于浸周期的数量增加，表面等离子体带正逐渐被定义在〜520 nm。 ML瓦戈，CP Gulka，JK Gerig，招商Manieri，JD Dattelbaum，CB标志，NT劳伦斯，ML Trawick，和MC利奥波德， 兰米尔26（1），560-569许可重印。 2010年美国化学学会版权所有。 <img alt="图4" src="/files/ftp_upload/3441/3441fig4.jpg" /> 图4透射电子显微镜（TEM）跨一个二硫联MPC电影大会的断层图像分析。插图:在电影大会hexanethiolate官能多用途储值卡的典型TEM图像。 TEM分析确定的储值卡〜2纳米，使用图像J分析平均黄金核心直径。 ML瓦戈，CP Gulka，JK Gerig，招商Manieri，JD Dattelbaum，CB标志，NT劳伦斯，ML Trawick，和MC利奥波德， 兰米尔26（1），560-569许可重印。 2010年美国化学学会版权所有。

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没有利益冲突的声明。

蛋白质单层的电化学是一种有效的的技术，用于研究氧化还原蛋白质和合成吸附平台之间的相互作用。然而，这一战略的有效性依赖于工程师的能力与高度的分子水平上控制的吸附接口。本协议产生的平台MPC为基础的代表专门设计的平台，能够提供更均匀的蛋白质吸附环境和促进对较大的距离相比传统PME系统alkanethiolate地空导弹ET。实力作为一种电化学接口MPC电影大会是它的多功能性和适?...

<table border="1"><tr><th>试剂和设备的名称</th><th>公司</th><th>目录编号</th></tr><tr><td>四辛基溴化铵</td><td> Sigma - Aldrich公司</td><td> 294136 </td></tr><tr><td>硼氢化钠</td><td> Sigma - Aldrich公司</td><td> 213462 </td></tr><tr><td>氢tetrachloroaurate </td><td> Sigma - Aldrich公司</td><td> 254169 - 5G </td></tr><tr><td> 1，Hexanethiol </td><td> Sigma - Aldrich公司</td><td> 234192 </td></tr><tr><td>透射电子显微镜</td><td> JEOL </td><td> 1010 </td></tr><tr><td>核磁共振波谱仪</td><td>布鲁克AVANCE </td><td> 300兆赫</td></tr><tr><td> Formvar /碳支持膜的铜网（400目） </td><td>电子显微镜科学</td><td> FCF400 -铜</td></tr><tr><td>金基底</td><td>蒸发金属Films公司</td><td>自定义</td></tr><tr><td>银/氯化银参比电极</td><td>微电极，公司</td><td> MI - 401F </td></tr><tr><td>恒电位</td><td> CH仪器公司</td><td> CHI650A，CHI610B </td></tr><tr><td> 1,9 - Nonanedithiol </td><td> Sigma - Aldrich公司</td><td> N29805 </td></tr><tr><td> （3 - 巯基）-三甲氧基硅烷</td><td> Sigma - Aldrich公司</td><td> 175617 </td></tr><tr><td>紫外可见分光光度计</td><td>安捷伦</td><td> 8453 </td></tr><tr><td>嵌入812环氧树脂</td><td>电子显微镜科学</td><td> 14120 </td></tr><tr><td> "00"BEEM胶囊</td><td>电子显微镜科学</td><td> 70000 - B </td></tr><tr><td>硅平面模具</td><td>电子显微镜科学</td><td> 70900 </td></tr><tr><td>钻石刀</td><td> Diatome </td><td> 21模块，S12801 </td></tr></table>

synthesis, assembly, and characterization of monolayer protected gold nanoparticle films for protein monolayer electrochemistry

纳米胶体金alkanethiolate配体称为单层保护金团簇（储值卡），合成和蛋白质单层电化学（PME）的吸附平台服务的电影集会，随后被纳入保护。 PME是利用模型系统研究氧化还原蛋白质的电化学性能，限制了他们吸附在修饰电极，这也为电子转移（ET）反应的氧化还原合作伙伴的平台。有研究表明，这种性质的金纳米粒子薄膜组件提供更均匀的蛋白质吸附环境，促进无距离的依赖相比，更为传统的醇自组装单分子膜（SAM）修改系统的ET。1-3在本文中，储值卡官能与hexanethiolate配体合成使用修改Brust反应4与紫外可见（UV - VIS）光谱，透射电子显微镜（TEM），和质子（1H）核磁共振（NMR）的表征。 MPC电影组装SAM修饰的金电极接口使用的是"浸周期"交替MPC层和二硫连接分子的方法。通过测量充电电流的系统更改双层薄膜生长在金电极是电化学跟踪。硅烷改性玻片上组装的类似电影让光学薄膜生长和横截面TEM分析监测，提供了一个估计的薄膜厚度。电影大会期间，MPC配体保护的操纵以及间的联动机制，让网络电影，容易适应，界面氧化还原蛋白具有不同的吸附机理。例如， 绿脓杆菌阿苏林（AZ）可吸附疏水hexanethiolate储值卡和细胞色素 C（ 细胞色素c）静电可以固定在修改MPC界面层羧酸二硫联片。在这份报告中，我们完全专注于电影的AZ系统协议。调查涉及蛋白质合成MPC修改的平台上的吸附作用，可进一步了解生物分子和人造材料之间的相互作用，因此援助的生物传感器的计划，ET建模系统，合成生物相容性材料发展。5-8

Watch this Scientific Journal Video about Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry at JoVE.com

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Alkanethiolate稳定的黄金被称为单层保护集群（储值卡）胶体合成，特点，并组装成薄膜作为一个简单的氧化还原蛋白的蛋白质单层的电化学像吸附接口<em&gt;绿脓杆菌</em&gt;阿苏林（AZ）和细胞色素<em&gt; C</em&gt;（CYT<em&gt; C</em&gt;）。

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

synthesis-assembly-characterization-monolayer-protected-gold

Research

JoVE Journal

Bioengineering

14.4K 次观看.  University of Richmond. Alkanethiolate稳定的黄金被称为单层保护集群（储值卡）胶体合成，特点，并组装成薄膜作为一个简单的氧化还原蛋白的蛋白质单层的电化学像吸附接口绿脓杆菌阿苏林（AZ）和细胞色素 C（CYT C）。

视频: Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...