作者们承认，海军研究办公室，美国大豆委员会，国家科学基金会资助。

1。季铵盐改性的Nafion <ol><li>摇动一瓶5％W / V的Nafion暂停约大力。被暂停30秒，确保Nafion膜均匀的解决方案。 </li><li>吸取了2毫升的Nafion膜，现在又重新悬浮到一个玻璃小瓶（瓶量可能包含从2.5毫升到10毫升）。 </li><li>测量了3倍摩尔过量盐（ 见表1）适当群众的烷基三甲基溴化铵（相对的Nafion聚合物磺酸基），并加入到小瓶含2毫升的Nafion。 </li><li>涡小瓶10-15分钟1500转。 </li><li>粘性溶液倒入塑料称重托盘约措施。 3×3英寸，并使用吸管传送任何残留的解决方案，从小瓶称重托盘。 </li><li>允许溶剂蒸发出的权衡船，离开称重托盘的底部（ 连接黄色/棕色，透明薄膜古雷2）。溶剂的挥发速度，应该是这样的，它需要所有溶剂蒸发超过6小时。如果溶剂蒸发过快，将形成一个白色的，硬皮材料，而不是一个透明的薄膜，表明聚合物胶束的结构已被破坏，你必须重新启动程序。如果溶剂蒸发速度太慢，一个去加湿器可能是必要的，因为过于缓慢蒸发通常会导致俗气，橙色的凝胶，您必须重新启动程序。典型的溶剂蒸发温度范围是20 - 37°C干燥的实际情况是在房间的相对湿度和温度的功能，但重要的是，干燥缓慢保持胶束结构，但不能太慢，使凝胶形成。 </li><li>填补了权衡与18MΩcm去离子水的水（10-20毫升），盖船，并允许浸泡12-24小时，以消除过剩烷基三甲基溴化铵盐和HBr。 </li><l我倒（或吸管）出的水，并有足够的DI水冲洗3次，每次填补托盘。要小心，不要失去任何的聚合物薄膜在这一步。 </li><li>聚合物允许的称重托盘坐下发现，直到聚合物在这一点上是完全干燥，应该是明确的，有点脆的塑料薄膜。再次，如果空气很潮湿，一个去加湿器可能有必要及时完成蒸发。 </li><li>使用铲，小心地取出称重托盘干片，它转移到一个干净的玻璃瓶。 </li><li>加入2毫升乙醇和3混合陶瓷珠，4小时旋涡或直到完全重新悬浮聚合物薄膜。 </li></ol> 2。到TBAB的改性Nafion膜活性测定酶的固定化<ol><li>干酶，掂量出1-10毫克到1.​​5 mL离心管中的酶，并添加1毫升100毫米的pH 7磷酸盐缓冲创建一个1-10毫克/毫升酶液。火炭ŕ1酶的溶液中，用二喹啉酸（BCA）检测13，以确定蛋白质的数量，并添加适量带来100毫米的磷酸盐缓冲蛋白浓度1-10毫克/毫升。 1-10毫克/毫升，通常会对应到1-50纳摩尔/毫升。 </li><li> 120μL的1毫克/毫升酶液，添加10秒60μL的烷基铵改性的Nafion溶液，和旋涡。 （这种混合物可以扩展为大量的重复。酶聚合物溶液比例保持在2:1。） </li><li>吸取60μL的酶/聚合物溶液成3个独立的1厘米2试管底部，允许干通宵。 </li></ol> 3。固定的NAD依赖型脱氢酶酶的含量测定<ol><li>到试管中，加入NAD（新鲜配制）的15毫米50毫米焦磷酸钠（pH值8.8）1.3毫升，1.5毫升，0.1毫升的水。 </li><li>将试管中的紫外/可见分光光度计（即ThermoScientific Evolut离子260生物和热Spectronic Genesys的20）设置为340纳米的波长。 </li><li>零光谱仪，然后加入0.1 mL乙醇。混合试剂，轻轻吹打溶液和5倍。为一片空白，使用更多的水，而不是0.1毫升0.1毫升的乙醇。 </li><li>试剂后，添加到试管和20分钟后，记录在5分钟，在340 nm的吸光度。画出两个数据点，得到一个可以用来计算活动的斜坡。 </li></ol> 4。固定吡咯喹啉醌依赖性脱氢酶的含量测定<ol><li>到试管中，加入1.5毫升的磷酸钠（pH值7.3）和600微米的PMS 200μL。 </li><li>试管放置在设定波长600纳米的紫外/可见分光光度计，然后零谱仪。 </li><li>加入100μL，700μLDCIP和200μL利益基板（乙醇，乙醛，甘油，葡萄糖，或甘油），由轻轻吹打solut的试剂混合离子起来下降了5倍。为一片空白，用200μL的水，而不是利益的基板。 </li><li>试剂后，添加到试管和20分钟后，记录在5分钟，在600 nm的吸光度。 </li></ol> 5。固定化葡萄糖氧化酶的含量<ol><li>到试管中，加入2.0毫升含有0.2 M P-羟基酸，0.02％（W / V）的叠氮化钠，128×过氧化物酶，4 -氨基安替比林0.3毫米，1 M磷酸钾，50 mM葡萄糖溶液。混合的解决方案，通过吸取和5倍。 </li><li>试管放置在波长510 nm的紫外/可见分光光度计集。 </li><li>记录后试剂的试管，并在20分钟后再次被添加到5分钟，在510 nm的吸光度。 </li></ol> 6。代表结果改性的Nafion聚合物胶束的结构可以破坏干燥原盐/聚合物共同铸造电影太FAST。 图2显示正确在一个透明，浅棕色电影已经干涸的盐/聚合物混合物。 A片，干燥过快可能会导致不透明，白色聚合物薄片由于干燥过程中，可以摧毁胶束结构的事实。 一旦已修饰的Nafion聚合物和酶混合到试管底部共同投，酶的活性检测可用于评估内酶聚合物膜的稳定性。 表2-4显示两个脱氢酶的检测结果葡萄糖氧化酶固定成各种改性的Nafion薄膜，分别。注意缓冲溶液中的酶与固定化酶活性较高，表明改性的Nafion聚合物实际上可以增强某些酶的活性（称为超活性）。其他酶有运输的限制，降低他们的具体活动时，固定在聚合物（即纤维素酶和淀粉酶，其基板是相当大的大分子）。 <table border="1"><tbody><tr><td> 使用了季铵盐 </td><td> 3倍过量 </td></tr><tr><td>四丙基溴化铵（T3A） </td><td> 32.37毫克/毫升</td></tr><tr><td>四丁基溴化铵（TBAB的） </td><td> 39.19毫克/毫升</td></tr><tr><td> TPAB（tetrapentylammonium甲基溴） </td><td> 46.​​01毫克/毫升</td></tr><tr><td> TEHA（triethylhexylammonium甲基溴） </td><td> 32.37毫克/毫升</td></tr><tr><td> TMHA（trimethylhexylammonium甲基溴） </td><td> 27.25毫克/毫升</td></tr><tr><td> TMOA（trimethyloctylammonium甲基溴） </td><td> 30.66毫克/毫升</td></tr><tr><td> TMDA（trimethyldecylammonium溴） </td><td> 34.07毫克/毫升</td></tr><tr><td> TMDDA（trimethyldodecylammonium甲基溴） </td><td> 37.48毫克/毫升</td></tr><tr><td> TMTDA（trimethyltetradecylammonium甲基溴） </td><tD&gt; 40.89毫克/毫升</td></tr><tr><td> TMHDA（trimethylhexadecylammonium甲基溴） </td><td> 44.31毫克/毫升</td></tr><tr><td> TMODA（trimethyloctadecylammonium甲基溴） </td><td> 47.71毫克/毫升</td></tr></tbody></table> 表1。四烷基季铵盐款项使用的Nafion聚合物改性。 <table border="1"><tbody><tr><td> Nafion的类型 </td><td> 酶的活性（U / G） </td></tr><tr><td>缓冲区（无聚合物） </td><td> 16.63±8.11 </td></tr><tr><td> Nafion膜（un-mod.） </td><td> 9.25±2.21 </td></tr><tr><td> TMTDA </td><td> 3.23±2.92 </td></tr><tr><td> TBAB的</td><td> 3.93±3.33 </td></tr><tr><td> TMDDA </td><td> 4.19±1.04 </td></tr><tr><td> TMOA </td><td> 3.51±1.11 </td></tr><tr><td> TMDA等</td><td> 8.00±4.53 </td></tr><tr><td> TMHA </td><td> 1.68±1.39 </td></tr><tr><td> TMHDA </td><td> 4.83±0.99 </td></tr><tr><td> TMODA </td><td> 10.45±3.20 </td></tr></tbody></table> 表2的NAD依赖的葡萄糖脱氢酶活性，固定在选定改性的Nafion聚合物（注:固定的活动是一种特定酶的活性初始功能）。 <table border="1"><tbody><tr><td> Nafion的类型 </td><td> 酶的活性（亩/ G） </td></tr><tr><td>缓冲区（无聚合物） </td><td> 7.18±0.51 </td></tr><tr><td> Nafion膜（un-mod.） </td><td> 70.1±0.5 </td></tr><tr><td> TMTDA </td><td> 133±6 </td></tr><tr><td> TBAB的</td><td> 244±4 </td></tr><tr><td> TMDDA </td><td> 221±6 </td></tr><tr><td> TMOA </td><td> 1.78±0.63 </td></tr><tr><td> TMDA等</td><td> 206±5 </td></tr><tr><td> TEHA </td><td> 40.1±50.6 </td></tr><tr><td> TMHDA </td><td> 0 </td></tr><tr><td> TMODA </td><td> 1.45±0.06 </td></tr></tbody></table> 表3固定在选定改性的Nafion聚合物（注:固定的活动是一种特定酶的活性初始功能）吡咯喹啉醌依赖性葡萄糖脱氢酶活性。 <table border="1"><tbody><tr><td> Nafion的类型 </td><td> 酶的活性（U / G） </td></tr><tr><td>缓冲区（无聚合物） </td><td> 103.61±3.15 </td></tr><tr><td> Nafion膜（un-mod.） </td><td> 19.93±10.10 </td></tr><tr><td> TMTDA </td><td> 247.25±12.49 </td></tr><tr><td> TBAB的</td><td> 152.27±5.29 </td></tr><tr><td> TMDDA </td><td> 262.05±6.26 </td></tr><tr><td> TMOA </td><td> 129.18±2.31 </td></TR&gt; <tr><td> TMDA等</td><td> 141.23±1.97 </td></tr><tr><td> TMHA </td><td> 131.75±2.89 </td></tr><tr><td> TMHDA </td><td> 132.50±1.18 </td></tr><tr><td> TMODA </td><td> 136.50±0.96 </td></tr></tbody></table> 表4代表葡萄糖氧化酶的具体活动固定在选定改性的Nafion聚合物（注:固定的活动是一种特定酶的活性初始功能）。 <img alt="图1" src="/files/ftp_upload/3949/3949fig1.jpg" /> 图1。TBAB的成立将酶固定化的Nafion聚合物和后续使用示意图。 <img alt="图2" src="/files/ftp_upload/3949/3949fig2.jpg" /> 图2。初步合作演员的Nafion薄膜和TBAB的光学照片。延缓干燥产生一个透明，浅棕色的薄膜覆盖的博称重托盘ttom的。

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没有利益冲突的声明。

在描述过程中，四烷基季铵盐用于修改商业的Nafion创建胶束聚合物，可用于固定和稳定的酶。实验中所述的程序表演，该聚合物可用于多种固定的酶具有高活性保留。如果感兴趣的酶具有非常低的活动，或者是不纯的，高浓度可能会被要求，不应影响固定化过程中，除非固定酶浓度大于10毫克/毫升。简单的程序区别于其他酶固定化技术没有合成步骤是必需的（如合成聚合物或交联）。此外，这些?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Nafion膜</td><td> Sigma-Aldrich公司</td><td> 70160 </td></tr><tr><td>利烷基溴化盐</td><td> Sigma-Aldrich公司</td><td> N / A </td></tr><tr><td>乙醇脱氢酶</td><td> Sigma-Aldrich公司</td><td> A3263 </td></tr><tr><td>烟酰胺腺嘌呤二核苷酸（NAD） </td><td> simga-Aldrich公司</td><td> N7004 </td></tr><tr><td>焦磷酸钠</td><td> Sigma-Aldrich公司</td><td> P8010 </td></tr><tr><td>嗪methosulfate（PMS）， </td><td> Sigma-Aldrich公司</td><td> P9625 </td></tr><tr><td> 2,6 - 二氯（DCIP） </td><td> SIGMA-Aldrich公司</td><td> D1878 </td></tr><tr><td>葡萄糖氧化酶</td><td> Sigma-Aldrich公司</td><td> G7141 </td></tr><tr><td> 4 - 羟基苯甲酸</td><td> Sigma-Aldrich公司</td><td> 240141 </td></tr><tr><td>叠氮化钠</td><td> Sigma-Aldrich公司</td><td> S8032 </td></tr><tr><td>过氧化物酶</td><td> Sigma-Aldrich公司</td><td> P8375 </td></tr><tr><td> 4 - 氨基安替比林</td><td> Sigma-Aldrich公司</td><td> 06800 </td></tr><tr><td>紫外/可见分光光度计</td><td>热</td><td> 260生物进化或Spectronic Genesys的20 </td></tr><tr><td>涡精灵</td><td></td><td></td></tr><tr><td>天平</td><td></td><td></td></tr></tbody></taBLE&gt;

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

在过去的十年中，已经有丰富的应用，固定和稳定的酶，包括生物催化，生物传感器和生物燃料电池的应用在大多数的生物电化学1-3，被固定到电极表面的酶或细胞器，与使用某种类型的聚合物基体中。这种聚合物支架应保持稳定的酶，并允许浅显的扩散和矩阵的分子和离子。多胺或多元醇的大部分用于这种类型的固定的聚合物 - 聚合物模拟酶的自然环境，封装和稳定通过氢键或离子键的酶。稳定酶的另一种方法涉及使用胶束，其中包含可以封装和稳定酶的疏水区域，尤其是4,5，敏提尔组制定了胶束聚合物市售的Nafion 6,7的Nafion本身就是一个胶束聚合物允许质子和其他小阳离子通道辅助扩散，但胶束和渠道是极其微小的聚合物是非常酸性的磺酸侧链，这是不利于酶的固定化。然而，当Nafion膜超过疏水烷基季铵盐如四丁基溴化铵（TBAB）混合，季铵盐阳离子取代质子磺酸基聚合物侧链（ 图1），并成为反离子。这将导致更大的胶束和聚合物内的通道，让烟酰胺腺嘌呤二核苷酸（NAD）的酶的功能，如必要的大型基板和离子的扩散。这个修改的Nafion聚合物已被用于许多不同的类型以及线粒体酶固定在生物传感器和生物燃料电池的使用。8-12本文介绍了一种新颖的程序，使这个麦克风ellar聚合物固定化酶膜，可以稳定酶。合成胶束酶的固定化膜，固定在膜的酶的过程，研究酶固定化酶的具体活动的检测详述如下。

Watch this Scientific Journal Video about Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization at JoVE.com

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

本文将介绍合成一种疏水改性Nafion膜固定化酶膜和固定在膜蛋白和/或酶和测试他们的具体活动的过程。

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

hydrophobic-salt-modified-nafion-for-enzyme-immobilization

Research

JoVE Journal

Bioengineering

16.1K 次观看.  University of Utah. 本文将介绍合成一种疏水改性Nafion膜固定化酶膜和固定在膜蛋白和/或酶和测试他们的具体活动的过程。

视频: Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

科研

生物工程

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT:生物质酶的发现和鸡尾酒优化的自动化平台

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

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. ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.