我们还要感谢小玉苗和Ben威尔逊发展的最内所述的方法。这项工作是由国家科学基金会（DBI 0454324）和国家卫生研究所（R21 EB005183）和ECK NIGMS小灵通NRSA的T32 GM07270。

1。随机的Au纳米粒子阵列的制备 8,10,12,14 <ol><li>首先创建一个模板，是一个随机吸附乳胶球，平均直径454纳米的致密层形成的Au纳米粒子阵列。这是通过首先蒸发20铬纳米粘附层的厚度玻璃盖玻片黄金。 </li><li>聚苯乙烯球暴露镀金基板的混合物，然后自组装单层是1 - 乙基-3 - （3 - dimethylaminopropyl）碳二亚胺盐酸盐（EDC），乳胶球体悬浮液和去离子水。 </li><li>吸附过程是可以持续约一小时，非吸收的领域是用大量清水冲洗带走。 </li><li>形成单层允许空气干燥。 </li><li>最后，20纳米的黄金蒸发乳胶球单层形成随机的黄金纳米粒子阵列。 </li><li>如果SEM是可用的，可以看到金纳米粒子阵列，根据教育统筹局局长，看起来像图1的流程图如图8所示。 </li></ol> 2。生物样品制备9,11 <ol><li>现在显示样品准备，为光学诱捕鼠标细胞核。 </li><li>吖啶橙染料标记3T3老鼠细胞的细胞核，得到的弗雷德哈钦森癌症研究中心的Tewari组。 </li><li> 10％的牛血清白蛋白（BSA）的磷酸盐缓冲液（PBS）被添加到小鼠细胞细胞核中的浓度约1：10（BSA：鼠标细胞核）。 BSA有助于防止粘基材的原子核。 </li><li>使用超声混合的解决方案。 </li><li> 5 UL是我们的解决方案上沉积的铝光栅阵列盖玻片。这是更好地执行这一步显微镜舞台上的铝光栅，使你不必运输样品后的解决办法是存入。 </li><li>两个堆栈的两个1“1”盖玻片用于支持通过查看该样本第五盖玻片。 </li><li>样品放置在显微镜下观看。 </li></ol> 3。诱捕方法<ol><li>通过发送通过蔡司Axio上Imager.D1M的一个35兆瓦的氦氖激光与GFP的修改，以允许为633纳米的激光辐射到达样本的17个过滤器设置配备光学镊子的构造。 </li><li>蔡司LD EC Epiplan - NEOFLUAR 50X的目标是用形象是直径约5微米的细胞核。 </li><li>后样品是根据客观的，重点的黄金纳米粒子阵列或光栅显微镜。 </li><li>直到实现你的愿望陷阱原子核的重点是垂直翻译显微镜。 </li><li>激光对粒子和粒子陷阱当场位置应保持其在激光光斑的位置，甚至当舞台翻译。 </li></ol> 4。代表性的成果： 随机的黄金纳米粒子阵列的程序应存款，可在一个扫描电镜看类似于图1的金纳米粒子的单层。这些电浆镊子创建的捕获力可以由标准的光镊产生的力量的10-20倍。电浆镊子所需的最低强度，实现粒子约束为不同大小的颗粒在图4所示。9,10光栅实现20倍更高的捕集效率的路线和诱捕比黄金纳米点，并可以实现诱捕少17 UW / UM 2（图7）11。 <img alt="图1" src="/files/ftp_upload/3390/3390fig1.jpg" /> 图1 10（一）自组装金纳米粒子形貌的扫描电镜。个别的黄金纳米粒子的直径约450纳米。 （二）纳米粒子的分布稀疏的电浆基板的NSOM的形象，显示了近场辐射。激发激光的波长为633纳米。 （C）（b）中红色正方形的面积高倍镜视野。 （四）电浆基板的散射频谱效率，在624纳米的高峰期。 （E）的吸收效率的电浆基板频谱，显示在668纳米的高峰。 <img alt="图2" src="/files/ftp_upload/3390/3390fig2.jpg" /> 图2 13（一）金微球随机分布在2D域1 × 1 微米 2。每个蓝点代表（A = 60 nm）的纳米微球的中心。观察平面平行随机纳米球阵列的散射场分布在（b） - （E）。由平面波的波长为540 nm的纳米球阵列均匀照明。周围介质的折射率为1.33。 “波拉rization方向沿X轴（横（一））平面波点。事件电场的幅度是假设在计算1。观察平面和纳米球阵列之间的分离是定义为H. B）H =一个 。 C）H = 2A。 D）H =λ。 E）H =2λ。 <img alt="图3" src="/files/ftp_upload/3390/3390fig3.jpg" /> 图3 9定制的荧光显微镜的配置，包括一个绕过激发滤光片和取代分色光束分离器的原理图。这是同时诱捕和荧光成像使用的配置。 <img alt="图4" src="/files/ftp_upload/3390/3390fig4.jpg" /> 图4 10最小的激光强度，保持利用电浆诱捕周围流体的流动速度的函数的陷阱。所有的光信号强度测量样品在显微镜下的目标平面。 （一） - （F）显示为单一的聚苯乙烯微球的直径为7.3，6.3（非球面），5.0，3.9，2.5和1.1微米的测量结果分别。插图显示了相应的颗粒显微图像。所有图像比例尺代表长度在5微米。 <img alt="图5" src="/files/ftp_upload/3390/3390fig5.jpg" /> 图5 10通过拟合线图来源的斜率。 4与粒径电浆诱捕。误差线显示线性拟合的标准偏差。拟合直线的斜率（光的强度阈值和流量之间的比例）图。 4颗粒大小约如本图所示的线性关系，表明优势，尤其是更小的粒子的电浆诱捕。 <img alt="图6" src="/files/ftp_upload/3390/3390fig6.jpg" /> 图6月11日 （一）增强光学诱捕利用一维周期性纳米结构的示意图。入射光是由周期性纳米结构在远场的衍射。 （二）与两个正交极化纳米结构在远场光强度分布。 （二）使用的FDTD模拟得到一个417纳米的铝光栅表面的两个正交偏振的光强度分布。分布在平坦的铝表面强度归。 （c）项和（d）为（c）在350 nm的聚苯乙烯珠和（d）在1微米的聚苯乙烯珠颗粒直接光栅表面对粒子的位置以上的套印潜力。白色圆圈说明粒子的大小。插图显示了相同的粒子的大小作为比较平坦的铝表面以上的诱捕潜力。每个粒子的大小值正常化。对于所有FDTD模拟数字视场为10 × 8微米2。 <img alt="图7" src="/files/ftp_upload/3390/3390fig7.jpg" /> 图7月11日 （一）陷阱效率和最小捕获强度与偏振垂直于光栅线各种大小的聚苯乙烯珠测量。陷阱插图不对称捕集效率转换为一个3.87微米的聚苯乙烯珠垂直与平行光栅规则。实线（大不对称）获得与入射光偏振垂直的光栅，虚线（小不对称）与入射光的偏振平行光栅获得。单位为（PN [MW /微米2] -1）。 （二） - （四）有荧光590 nm的聚苯乙烯珠补漏示范。红色圆圈表示的激光光斑的位置，激光灯太暗可见。起初粒子被困在现场，在更高的功率，电源降低克服阱力的粒子的布朗运动，让粒子逃脱。 （E） - （G）的一个荧光卵巢癌的细胞核补漏示范。启动诱捕所需的最低强度16μW/ 2微米获得使用20X物镜。 <img alt="图8" src="/files/ftp_upload/3390/3390fig8.jpg" /> 图8月14日的帽形的黄金纳米粒子的制备过程：蒸发的Cr和非盟的玻璃盖玻片上薄薄的一层）。 B）暴露1小时领域的聚苯乙烯球悬挂和吸附。 C）拆除非吸附聚苯乙烯球和表面干燥。 D）模板领域上的另一个金层的蒸发。 E）示意图帽状纳米金阵列，其中坳只涵盖模板球顶偏。

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没有利益冲突的声明。

诱捕这些方法的意义是，他们10μW/ 2微米的顺序 10 3 /μW的2微米到某处为了减少光的强度从某处持续诱捕所必需的。 10,11对这些技术的局限性黄金纳米粒子阵列经验暖气，必须克服的问题。为了克服这个问题，一个是电介质材料组成的二维光子晶体结构可以使用。这种结构在理论上可以产生在低光的强度和控制诱捕微型和纳米粒子在精确地控制输入偏振的旋转和位?...

<table border="1"><tr><td>材料名称</td><td>类型</td><td>公司</td><td>产品编号</td><td>评论</td></tr><tr><td> Axio上成像仪显微镜</td><td> D1M </td><td>蔡司</td><td> D1M </td><td>蔡司Axio上Imager.D1M </td></tr><tr><td>显微镜物镜</td><td> 50x/0.55 </td><td>蔡司</td><td></td><td> LD欧共体Epiplan - NEOFLUAR 50x/0.55高清DIC的</td></tr><tr><td>蔡司显微镜摄像机</td><td> AxioCam MRC </td><td>蔡司</td><td></td><td></td></tr><tr><td>氦氖激光器</td><td> 35兆瓦</td><td>研究光电</td><td></td><td></td></tr><tr><td>可变衰减器</td><td>连续可变ND </td><td> ThorLabs </td><td> NDC - 100C - 4M </td><td>对于调整显微镜强度</td></tr><tr><td>蔡司过滤器集</td><td>过滤器设置＃17 </td><td>蔡司</td><td> 488017-9901-000 </td><td>过滤器设置＃17 </td></tr><tr><td>显微镜载玻片</td><td>厚度为0.5毫米</td><td> VWR </td><td></td><td></td></tr><tr><td> 3T3小鼠细胞的细胞核</td><td></td><td colspan="2">弗雷德哈钦森癌症研究中心</td><td>存储为尽可能冷</td></tr><tr><td>吖啶橙染料</td><td></td><td colspan="2">弗雷德哈钦森癌症研究中心</td><td></td></tr><tr><td>牛血清白蛋白</td><td> 1到10的PBS的口粮</td><td colspan="2">弗雷德哈钦森癌症研究中心</td><td></td></tr><tr><td> 454 nm的聚苯乙烯乳胶球</td><td></td><td> Polysciences，公司</td><td></td><td></td></tr><tr><td>碳二亚胺盐酸盐（EDC） </td><td> 1 - 乙基-3 - （3 - dimethylaminopropyl） </td><td> G - Biosciences公司</td><td> BC25 - 1 </td><td></td></tr><tr><td>金（沉积） </td><td></td><td></td><td></td><td></td></tr><tr><td>反光裁定光栅</td><td></td><td>埃德蒙光学</td><td></td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td>贝科的磷酸盐缓冲液（D - PBS）（1X） </td><td> Invitrogen公司</td><td> 14190-144 </td><td></td></tr></table>

utilization of plasmonic and photonic crystal nanostructures for enhanced micro- and nanoparticle manipulation

非破坏性的方法来操纵亚微米粒子的位置和方向将基础生物学研究的一个非常有用的工具。也许使用最广泛的物质力量来实现小颗粒的无创性操作已介（DEP）[1]然而，对自己的DEP缺乏通用性和精度，操作时所需的细胞，因为它是传统与固定电极。光镊，利用一个三维电磁场梯度小颗粒施加的力量，实现这一目标所需的通用性和精度。然而，这种方法的主要缺点是需要达到必要的武力来捕获一个粒子的辐射强度高可以破坏生物样品可诱捕和较低的光强度排序的解决方案光电镊子（OET），但OET的有小颗粒的精细操纵限制;。。被DEP的，基于技术，也使该解决方案的财产约束4 5 此视频文章将介绍两种方法，减少光学操纵活细胞所需的辐射强度，还描述了定向控制方法。第一种方法是使用一个随机的黄金纳米粒子（金纳米粒子）阵列，如图1所示样品基板电浆镊子。金纳米粒子阵列转换成本地化的表面等离子体（LSP），包括谐振偶极矩的事件光子辐射和产生的细胞液中的一个大梯度与图案的辐射场。表面等离子体增强诱捕Righini等和我们自己的造型的初步工作表明电浆基板所产生的领域，减少所需的初始强度，提高梯度场的粒子陷阱。6,7,8电浆方法允许罚款由于更高效的光能量转换成机械能和依赖偶极子辐射场的低光信号强度的椭球颗粒与细胞的定向控制。这些领域都显示在图2和图4和5中详细诱捕强度低。电浆镊子的主要问题是，LSP的产生大量的热量和诱捕只有二维。这种热产生对流流动和热泳可以强大到足以驱逐从陷阱的亚微米颗粒。9,10，我们将介绍第二种方法是利用周期性的电介质纳米结构非常有效地散射入射光成衍射模式，如图6所示11，理想的情况下，人会做出这种结构，电介质材料与电浆镊子，以避免相同的加热遇到的问题，但我们的做法镀铝光栅是作为一维周期介质纳米结构。虽然它不是一个半导体，它没有显著暖气经验和有效被困诱捕强度低的小颗粒，如图7所示。对准光栅基板的粒子概念验证的命题，一个2 - D光子晶体可以允许非球面微米大小的粒子的精确旋转10这些光学陷阱的效率增加，由于在纳米结构制作的增强的领域。本文。

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Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

电浆镊子和光子晶体纳米结构的光学诱捕微型和纳米粒子的生产效率和方向的控制非常有用的增强。

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

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JoVE Journal

Bioengineering

12.2K 次观看.  University of Washington. 电浆镊子和光子晶体纳米结构的光学诱捕微型和纳米粒子的生产效率和方向的控制非常有用的增强。

视频: Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

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

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

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 ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

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 ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

单域液晶的弹性体和液晶橡胶纳米复合材料的制备

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...