作者承认的ONR的MURI奖（NN00014-06-1-1176）和美国陆军医学研究和装备司令部（批准号:W81XWH-10-1-0806）的资金支持。这种材料是根据工作支持的美国国家科学基金会根据格兰特第EEC 0552584号和0851519。

1。组合聚合物图书馆的合成高分子化学变化-请参阅图1为机器人设定<ol><li>各单体溶解在适当的溶剂中（浓度为25毫克/毫升），和加载每个成10cc的气密注射器。 </li><li>将附加抗溶剂的诱惑锁毛细管各注射器的端部。 </li><li>将注射泵的注射器（新时代可编程注射泵），并锁定到位。 </li><li>线性致动器（Zaber）的起始位置。 </li><li>使用环形支架夹的两个毛细管，定位结束进入首发瓶/单体沉积。 </li><li>启动LabVIEW程序导入各孔中根据所需的共聚物组合物的各单体的可变体积的泵。这是通过在程序中指示的Z轴致动器降低到小瓶/以及，然后每个泵的毛细管，以分配所需的量的。氖XT，程序指示的Z-致动器返回到其起始位置的X-和Y-致动器移动到的位置的下一个小瓶/孔。这是进行，直到每个井的所需体积的单体存入。 </li><li>单体的沉积之后，多井或多瓶单体库转移到一个预先加热的真空烘箱中，并培养在真空下的缩合聚合反应的持续时间。对于CPH:SA的合成反应进行了在180℃下，0.3乇，1.5小时，但这些反应条件下将不同的聚合物系统之间变化。 </li></ol> 2。组合空白和蛋白质加载聚合物纳米电影图书馆的制造-机器人设置，参见图1 <ol><li>填充在第一可编程注射器泵的注射器用溶剂（空白图书馆）或与蛋白质的溶剂中分散在它（蛋白质的加载库），而在第二注射器的注射器泵为空。在相邻的样品架中的纳米粒子的制造管填充有非溶剂（溶剂非溶剂比例是1到100）。对于膜的制造中，利用一个空的多井板在相邻的样本保持器的管代替。 </li><li>溶剂使用的LabVIEW程序，存入所有聚合物小瓶/井的库（浓度= 20毫克/毫升），并温育1-5分钟。可以引入一个可选的超声处理工序（30秒，在40赫兹），以确保完整的聚合物溶解。 </li><li>接着，通过启动一个独立的LabVIEW程序中，样品被撤回到空的注射器中，并沉积到其相应的管的非溶剂（纳米颗粒）或空井（电影），在相邻的样本保持器。 </li><li>此过程进行的每个组合物中的离散聚合物图书馆。 </li><li>的纳米粒子或薄膜库，然后放置在一个真空室中的溶剂和非溶剂的除去的纳米粒子（天秤座Ry的，也可以使用真空过滤回收）。 </li></ol> 3。高通量蛋白释放动力学<ol><li>为了研究聚合物库，高通量蛋白释放动力学的一个96油然而生（2ml /孔），聚丙烯板被修改，这样的前1/3的孔壁在相邻的列（如:A和B，除去C和D，E和F，G和H）的相邻孔中。的蛋白质 - 加载膜必须制造或纳米粒子转移到该板来执行高通量的释放动力学研究。蛋白质微纳米粒子或薄膜样品只能放置在一个相邻的列（如:A，C，E，和G）。有关详细信息，请参阅图4。 </li><li>继转移的纳米颗粒96深涌出释放板，颗粒被让其沉淀。 PBS缓冲液中（0.1毫摩尔，pH7.4）中慢慢地添加到每个样品（列A，C，E和G），然后每个相邻阱（列B，D，F和H）的孔，直到充分和缓冲区是自由流动的在相邻的井。为纳米粒子，这个过程需要格外小心进行，以确保颗粒保持在底部的样品以及（列A，C，E和G），而不是转移到毗邻的版本以及（列B，D，F和H）。在某些情况下，需要离心本地化纳米粒子的样品，在样品的底部（列A，C，E和G）。 </li><li>接着，释放板用盖子密封，并放置在所需的温度（ 即 37℃），搅拌的条件下的实验的持续时间。 </li><li>在增加的时间点（ 即 0.04，1，2，3，5，7，9，12，15，19，24及30日）发布的荧光标记的蛋白质的量是量化使用9,400台风平板荧光扫描仪（ GE医疗）。释放板被放置在扫描仪上表面，使用适当的激励激光和发射滤清器扫描遥感，和释放的蛋白的释放井（列B，D，F和H）的量化（图像定量）的荧光强度。有人建议，已知浓度的标准蛋白被包括在用于计算蛋白质的量和荧光猝灭占孔板。 </li></ol> 4。代表性的成果制造的聚合物库后，表征已进行GPC，1 H NMR和FTIR来验证这个组合的方法1,7,8,11。分子量范围从10,000-20,000 g / mol的，多分散性指数范围从1.5-3.0，和化学组合物已被证明是准确和一致的与常规方法的聚酐合成12-15。同样，SEM图像的纳米颗粒的图书馆表明类似的表面形态，大小和粒度分布的常规制作的纳米粒子2。蛋白释放kinetics的聚酐纳米粒子或薄膜的改性孔板中进行，如先前描述1。结果显示一个近似的带或不带一个脉冲串取决于蛋白质加载和高分子化学（ 图2和图3）1,12,14,16的零级释放。 <img alt="图1" src="/files/ftp_upload/3882/3882fig1.jpg" /> 图1。组合的聚合物膜和纳米粒子的制造装置。 <img alt="图2" src="/files/ftp_upload/3882/3882fig2.jpg" /> 图2。得克萨斯红牛血清白蛋白（TRBSA）的高通量的释放从CPH:SA聚合物纳米粒子库。 SA丰富的聚合物释放封装的最迅速的TRBSA，，而CPH丰富的聚合物化学品释放最慢的。误差线表示标准偏差TION和n = 4。转载许可Petersen 等。人 1。版权所有2010年美国化学学会。 <img alt="图3" src="/files/ftp_upload/3882/3882fig3.jpg" /> 图3。高通量释放从CPTEG得克萨斯红牛血清白蛋白（TRBSA）:CPH聚合物膜库。 CPTEG丰富的聚合物释放封装的最迅速的TRBSA，，而CPH丰富的聚合物化学品释放最慢的。误差棒表示标准偏差和n = 3。 <img alt="图4" src="/files/ftp_upload/3882/3882fig4.jpg" /> 图4。图像显示两个相邻的井"之前"和"之后"修改中的96深涌出的聚丙烯板。在右侧的"之后"的修改图像还描绘了另外的聚合物膜（底部以及左）与封装的荧光分子成的缓冲液的情况下被释放的两口井之间。将释放的荧光分子，然后检测以及在右。

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没有利益冲突的声明。

知识库的必要的合成条件和所合成的聚合物的玻璃化转变温度（T 克 ）是必不可少的图书馆制造。如果的 T g是低于室温下，纳米粒子的制造工序中，可能需要进行在受控温度以下的环境中的 T g的聚合物。此外，应采取谨慎，以确保所有设备在接触高温和溶剂必须适当处理这些情况。此协议的一些参数可以调整，以适应不同的聚合物体系（ 即 ，温度，真空度，孵育时间，溶剂，非溶剂，聚合物浓度，溶剂，非溶剂的比率，等）合成或颗粒/膜制造。在某些情况下，纳米粒子是稳定的溶剂中进行长时间（足以通过真空干燥除去溶剂），以便两个备用溶剂雷姆椭圆形的方法都可以使用。 1）该颗粒可以被缓慢地离心分离，滗析上清液溶剂，和剩余的颗粒干燥，或2）的颗粒，可以通过真空过滤分离，然后干燥。继制造空白或蛋白质加载纳米颗粒/膜，高通量表征或测试的可以进行筛选的生物材料，蛋白质，细胞，或宿主的相互作用。这种高通量的方法可以快速生物材料的性能优化所需的应用程序。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录号 </td></tr><tr><td>电动XYZ阶段:了3倍T-LSM050A，每轴50 mm行程</td><td> Zaber技术</td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000单注射泵</td><td>新的时代水泵系统</td><td> NE-1000 </td></tr><tr><td>高硼硅* Vista *的无框可重复使用的玻璃文化管</td><td>康宁</td><td> 07-250-125 </td></tr><tr><td>玻璃比色皿</td><td>科学战略</td><td> G102 </td></tr><tr><td> LabVIEW的</td><td>美国国家仪器公司</td><td> 776671-35 </td></tr><tr><td>的SGE气密性注射器，露儿禄</td><td> Sigma Aldrich公司</td><td> 509507 </td></tr><tr><td> U96深孔板1.3 ML＆2.0毫升的</td><td>赛默飞世尔科技:Nunc公司</td><td> 278743 </td></tr><tr><td>井帽垫</td><td>赛默飞世尔科技:Nunc公司</td><td> 276000 </td></tr><tr><td>台风9400 </td><td> GE医疗集团</td><td> 63-0055-79 </td></tr><tr><td> Whatman公司级50圈90毫米</td><td> Whatman公司</td><td> 1450-090 </td></tr></tbody></table>

combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries

聚酸酐的生物材料是一类具有优良的生物相容性和药物输送能力。虽然他们已经被广泛研究，与传统的单样本在每次一个合成技术，最近的高通量方法已经开发使聚酐1的合成和试验的大型图书馆。这将有利于更有效地优化和设计过程中这些生物材料，药物和疫苗交付应用。在这项工作中描述的方法的组合合成的可生物降解的聚酐膜和纳米颗粒的图书馆和从这些库的蛋白释放的高通量的检测。在这种机器人的操作方法（ 图1），线性致动器和注射器泵控制的LabVIEW，它使免提自动化协议，消除了用户错误。此外，这种方法可以快速制造微观尺度聚合物图书馆，红色ucing创造多元聚合物系统而导致的批量大小。这个组合的聚合物的合成方法便于用相当量的时间，将采取以往合成聚合物中的15种不同的聚合物的合成。此外，聚合物组合库，可以制作成空白或蛋白质加载的几何形状，包括在溶剂中和沉淀到非溶剂（纳米粒子），或通过真空干燥（薄膜）的薄膜或纳米颗粒溶解时的聚合物库。在加载到聚合物中的图书馆荧光染料共轭蛋白，蛋白质的释放动力学可以在高吞吐量的评估使用基于荧光的检测方法（ 图2和图3），如前面描述1。组合平台已验证常规方法2和聚酐膜和纳米库的特点<suP&gt; 1 H NMR和FTIR。蛋白释放动力学，稳定性和抗原性的图书馆已筛选， 体外细胞毒性，细胞因子的产生，表面标志物的表达，黏附，增殖和分化，以及体内生物分布和粘膜粘附1-11。开发本发明的组合的方法，使高吞吐量的聚合物合成和加载蛋白质纳米粒子的制造和膜的图书馆，可以反过来， 在体外和体内筛选的生物材料的性能的优化。

Watch this Scientific Journal Video about Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries at JoVE.com

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

该方法描述了组合化学合成的可生物降解的聚酸酐薄膜和纳米粒子库，，蛋白释放这些库和高通量检测。

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 ...

combinatorial-synthesis-high-throughput-protein-release-from-polymer

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.3K 次观看.  Iowa State University. 该方法描述了组合化学合成的可生物降解的聚酸酐薄膜和纳米粒子库，，蛋白释放这些库和高通量检测。

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

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 ...

科研

生物工程

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

基于几丁聚糖，激光激发薄膜手术胶粘剂，"SurgiLux的制备及示范

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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 ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

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 ...

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 ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

薄膜挤出<em&gt;海甘蓝abyssinica</em&gt; /谷朊粉共混物

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

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 ...

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

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