这项研究是由基础科学研究计划通过韩国国家研究基金会（NRF）由教育，科学和技术（NRF-2013R1A1A2012159和NRF-2013R1A1A2007561），建国大学部提供资金，化学工程与MCubed部支持节目在密歇根大学。

1.全细胞生物催化剂制备方法注:重组E.大肠杆菌细胞窝藏pET28a- SpNox 31或pET28a- HjLAD 28以下简称为E.大肠杆菌SpNox和E.大肠杆菌HjLAD分别。 <ol><li>接种大肠杆菌的单个菌落大肠杆菌 HjLAD在3ml的Luria-BERTANI（LB）补充有卡那霉素（50微克/毫升）的培养基中并孵育在培养箱摇床O / N在37℃，250转。 </li><li>由1稀释培养:在200ml新鲜LB的含有50μg/ ml卡那霉素和孵育在37℃下100，250转，直到OD 600达到〜0.6。 </li><li>通过加入0.1mM异丙基β-D-硫代半乳糖苷（IPTG）到培养基中诱导HjLAD蛋白表达，并在16℃，180rpm下16小时孵育。 <ol><li>另外，在25℃，200 RP执行感应在同一天进行的m表示6小时，如果L-木酮糖的生物合成（下面的步骤2）。 </li></ol></li><li>收获诱导E.大肠杆菌 HjLAD细胞离心3,200 XG 20分钟，在4℃弃上清，进行第2步处理细胞沉淀。 </li><li>与此同时，执行步骤1.1 - 1.4 E.大肠杆菌 SpNox。 </li></ol>通过耦合E. 2.合成L-木酮糖大肠杆菌 HjLAD和E.大肠杆菌 SpNox的辅因子再生<ol><li>悬浮大肠杆菌的细胞沉淀大肠杆菌 HjLAD和E.大肠杆菌 SpNox分别在5.0克干细胞重（gDCW）/ L的细胞密度的50mM Tris-HCl缓冲液（pH8.0）中。 注意:gDCW和在600nm（OD 600）测量的光密度之间的相关性，可以建立以促进实验。中所使用的公式该协议是1 gDCW / L = 0.722 * 600 OD - 0.0965，可在不同的光谱仪各不相同。 </li><li>混合600微升5.0 gDCW / L E.大肠杆菌 HjLAD，600微升5.0 gDCW / L E.大肠杆菌 SpNox，100微升的20mM NAD +的，和150微升的2M -L-阿拉伯糖醇在14毫升的圆底管并通过加入550微升的50mM Tris-HCl中使反应体积为2毫升（pH8.0）中。 注意:两个全细胞生物催化剂量的比例可以进行优化，以提高产品的生物合成。对于所描述的系统， 大肠杆菌的比率大肠杆菌 SpNox:E.大肠杆菌 HjLAD = 1:1被发现是最佳的L-木酮糖的生物合成（ 图1B）。 </li><li>孵育在30℃，200rpm下进行8小时，将反应混合物。 </li><li>收集离心后取上清液在摄氏 4 度 3200 XG 10分钟次继续如在下面的步骤3中描述的量化的L-木酮糖的生产。 </li></ol> 3. MTT法检测L-木酮糖定量<ol><li>抽吸将100μl来自步骤2.4收集到1.5毫升管的反应上清液。 </li><li>加入50μl1.5％半胱氨酸，900微升70％硫酸和50微升0.1％的咔唑溶解在乙醇中，并通过反相管3倍轻轻混匀。 </li><li>在37℃，200rpm下20分钟，将反应混合物。 </li><li>测量在使用分光光度计560纳米（A 560）的反应混合物的光学吸光度。 <ol><li>稀释反应混合物，如果A 560读数高于1。 </li></ol></li></ol>在海藻酸钙珠重组全细胞催化剂4.固定<ol><li>溶解4克藻酸钠在100毫升蒸馏水中。通过添加S准备藻酸盐溶液海藻酸钠水，以避免团块的形成。如果需要，加热所述混合物。 </li><li>添加5.0 gDCW / L E. 600微升大肠杆菌 HjLAD和600微升5.0 gDCW / L E.大肠杆菌 SpNox 1.2毫升4-％海藻酸钠在步骤4.1编写，并轻柔吹打混合细胞和海藻酸钠，以避免泡沫形成。 </li><li>吸藻酸盐/细胞悬液到用针头的注射器，并逐滴添加该混合物成酰氯的0.3M钙在100毫升的烧杯中在连续搅拌下（ 氯化钙 ）的解决方案。 注意:用于藻酸盐珠形成的CaCl 2溶液的量应足以使藻酸盐的液滴被完全淹没。此外，注射器针头和氯化钙溶液的表面之间的距离必须在一个最佳范围内被维持，以确保形成均匀的球形珠。的最佳距离范围可被确定实验同盟并取决于针的内径。作为估计，约15±5厘米的距离被发现是最优的一个0.6厘米（内径）的注射器针头。 </li><li>留在2所述的CaCl 2溶液中的珠-在RT 3小时，不进行搅拌，以允许交联和凝胶珠的形成。 </li><li>倒出的CaCl 2溶液，而不通过仔细倾倒的CaCl 2溶液到50毫升锥形管扰乱珠，并在氯化钙溶液中的剩余的珠转移到另一个50ml锥形管中。 </li><li>用10毫升50毫米的Tris-HCl（pH值8.0）洗珠缓冲区三次，除去过量的氯化钙和未封装的细胞。 注意: 在任何给定的步骤，不要离心珠，因为这样做会破裂它们。从溶液中分离磁珠，使悬浮液静置3 - 5分钟。珠将沉降在底部和洗涤缓冲液可以滗到另一个容器。 <ol><li> 不要丢弃用过的洗涤液。池所使用的Tris-HCl洗涤缓冲液（30毫升）与来自步骤4.5中收集的用过的CaCl 2溶液。 </li></ol></li><li>颗粒未固定的E. 3,200×g离心20分钟从步骤4.6收集大肠杆菌细胞被汇集的CaCl 2和的Tris-HCl溶液的离心分离。为了确定固定化效率，如在步骤2.1中所述计算gDCW / L时沉淀未包封细胞的密度。 </li><li>从步骤4.6转移洗涤珠粒成管。按照步骤2.2 - 3.4评价使用所有的洗涤珠粒代替细胞沉淀的L木酮糖的生物合成。 </li></ol> 5.固定化生物催化剂的稳定性测定对L-木酮糖生产<ol><li>从步骤4.8收集珠和不离心，用10ml的50mM的Tris-HCl（pH为8.0）缓冲液洗两次（如步骤4.6中所述）。 </li><li>所有使用3.4 - 的洗涤珠粒如步骤2.2中所述进行反应。 </li><li>重复步骤5.1 - 5.2的生产周期所需数目并测量在每个周期中的反应上清液中产生的L-木酮糖的量。 </li></ol>

作者宣称没有竞争经济利益。本文针对报告的详细方法，生成固定在藻酸盐耦合全细胞生物催化系统。科学新奇已经报道在先前的研究16。

最近的技术进步已经使重组生物治疗的商业化的激增，导致其市场价值逐渐上升的生物技术产业。一个这样的进步是在重组微生物代谢工程，这表明在建立可扩展的工业系统38一诺大的来临。如同大多数工艺，通过遗传工程微生物生产的重组生物分子的成功的商业化是高度依赖于系统39的生产率。这样导致的"蛮力"遗传学39，其中，生物催化的酶的定向进化已经为了提高生?...

用微生物还原全细胞生物转化已成为商业上和治疗上重要的生物分子1化疗酶合成的普遍做法- 3。它呈现比使用分离的酶的几个优点，特别是成本密集型的下游纯化过程的消除和延长寿命4的示范- 7。用于需要对产物形成辅因子的生物催化途径，全细胞系统具有通过加入廉价供电子共基质5,8,9 原位辅因子再生提供的潜力。然而，这种能力被减少为需要稀有或昂贵的共同基片10的化学计量浓度的反应- 13。与整个细胞的可重用性差在一起，这阻碍了建立一个可扩展的和连续的机生产线ction系统。这些辅助因子依赖性的生物转化的全细胞系统的战略的修改都需要克服上述的局限性。具体地，工作协同全细胞生物催化剂的组合已显示出显著增强窝藏酶14的生产率和稳定性。这些因素，这往往是用于实现的商业上可行的产品的大规模生产的关键，可以通过共固定生物催化的微生物15进一步优化。我们最近开发出一种简单和可重复使用的全细胞生物催化系统，使两个辅因子再生和生物催化剂的固定化用于L木酮糖生产16。在这项研究中，该系统被用作一个例子来说明应用这两种策略，以提高生物转化产率和生物催化剂可重用性的实验程序。 的L-木酮糖属于共轭亚油酸生物有用分子的SS命名罕见的糖。稀有糖是在自然界中很少出现单糖独特或糖衍生物，但起关键作用作为生物活性分子17,18识别元件。它们有各种各样的应用，从增甜剂，功能食品，以潜在治疗19。的L-木酮糖可用作多种α葡糖苷酶的潜在抑制剂，并且还可以被用作肝炎或肝硬化17,20的一个指标。木糖醇在全细胞系统的L-木酮糖高效率转换已经在泛菌属先前报道ananatis中 21,22， 产碱菌 701B 23， 苍白球芽孢杆菌 Y25 24,25和大肠杆菌 26。在E.大肠杆菌，然而，它只是实现使用低（&lt;67毫摩尔）的木糖醇浓度26由于初始木糖醇浓度高于1潜在抑制作用00毫米木糖醇4脱氢酶活性21,26。木酮糖和木糖醇之间热力学平衡已表明强烈支持木糖醇25,27的形成。此外，木酮糖产量是由具有在原位辅因子再生系统的情况下的要被提供昂贵的辅助因子的量的限制。总之，这些因素限制了用于缩放到可持续系统对L-木酮糖的生物合成的潜力。 为了克服这些限制，提高的L-木酮糖的生物转化产率，辅因子再生的策略在建立一个耦合全细胞生物催化系统首先使用。具体地说，L-阿拉伯糖醇4-脱氢酶（EC 1.1.1.12）从红褐肉座菌 （HjLAD），在真菌的L-阿拉伯糖分解代谢途径的酶，被选择催化L-阿拉伯糖醇转化成L-木酮糖28,29 。像许多生物合成酶的一大limitatioHjLAD的n是它需要价格昂贵的烟酰胺腺嘌呤二核苷酸辅因子（NAD + NADH的氧化形式）的化学计量的量来进行这种转换。在酿脓链球菌 （SpNox）发现的NADH氧化酶已经显示出，以显示高辅因子再生活性30,31。以SpNox， 大肠杆菌的这个属性的优势表达HjLAD用于生产L-木酮糖的大肠杆菌细胞偶合与E.表达SpNox为NAD +的再生的大肠杆菌细胞，以提高由图1A中所示的耦合反应中所描绘的的L-木酮糖的生产。在最佳条件下，用150毫-L-阿拉伯糖醇的初始浓度，最大的L-木酮糖收率达到96％，从而使该系统比在文献中报道的有效得多​​。 全细胞固定化的策略采用下，进一步提高了耦合biocatalyt的可重用性IC系统。对于全细胞固定化的常用方法包括吸附/共价连接到固体基质，在聚合物网络32交联/包封和封装。在这些方法中，对细胞固定化的最合适的方法是封装在藻酸钙珠。其温和凝胶化性能，惰性水性基质和高孔隙度有助于保持包封的生物制品33的生理特性和功能。因此，耦合的生物催化剂系统含有大肠杆菌大肠杆菌细胞窝藏HjLAD或SpNox被固定在藻酸钙珠，使L -木酮糖生产的多个循环（ 图2）.The固定的生物催化剂体系表现出良好的操作稳定性，保持了第一次循环的转化率的65％的7次循环后连续再利用，而将无细胞系统几乎完全失去其催化活性。

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immobilization of multi-biocatalysts in alginate beads for cofactor regeneration and improved reusability

我们最近开发出一种简单，可重复使用以及耦合全细胞生物催化系统，辅因子再生和生物催化剂固定化，以提高产量和持续的综合能力。描述兹为这样一个系统由两个大肠杆菌发展的实验程序大肠杆菌菌株表达功能互补的酶。一起，这两种酶可起到共可操作以介导昂贵辅因子的再生用于提高生物反应的产物产率。此外，据报道通过在海藻酸钙珠全细胞的包封合成耦合生物催化系统的固定形式的方法。作为一个例子，我们通过偶合大肠杆菌呈现的L-木酮糖的改进的生物合成从L-阿拉伯糖醇大肠杆菌细胞中表达的酶的L-阿拉伯糖醇脱氢酶或NADH氧化酶。在最佳条件下和使用的150的初始浓度毫-L-阿拉伯糖醇，最大的L-木酮糖收率达到96％，这是比在文献中报道的更高。耦合全细胞生物催化剂的固定化形式表现出良好的操作稳定性，保持连续重复使用的7个周期后的第一个周期中获得的产率的65％，而将无细胞系统几乎完全失去催化活性。因此，这里所报告的方法提供了两种策略可以帮助提高工业生产的L-木酮糖，以及需要在一般的使用辅因子的其他增值化合物。

启用辅因子再生，L-木酮糖合成在含有大肠杆菌耦合全细胞生物催化系统中进行大肠杆菌 HjLAD和E.大肠杆菌 SpNox细胞。以下的各种参数的最佳化，该系统的可重用性是通过固定它在海藻酸钙珠（ 图2）的改善。 L-木酮糖的生产与辅因子再生偶合 <strong...

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Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

呈现为共固定化全细胞生物催化剂用于辅因子再生和改善的可重用性，使用生产L-木酮糖的作为例子的协议。辅因子再生是通过偶合表达功能上互补酶2 大肠杆菌菌株实现;全细胞生物催化剂固定化是通过细胞封装在藻酸钙珠来实现。

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

immobilization-multi-biocatalysts-alginate-beads-for-cofactor

Research

JoVE Journal

Chemistry

17.2K 次观看.  Konkuk University. 呈现为共固定化全细胞生物催化剂用于辅因子再生和改善的可重用性，使用生产L-木酮糖的作为例子的协议。辅因子再生是通过偶合表达功能上互补酶2 大肠杆菌菌株实现;全细胞生物催化剂固定化是通过细胞封装在藻酸钙珠来实现。 

视频: Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

的凹孔隙PDMS珠微泡制作

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O substrates outside vacuum at low temperature. The performance of photovoltaic devices featuring atmospherically fabricated ZnO/Cu2O heterojunction was dependent on the conditions of AP-SALD film deposition, namely, the substrate temperature and deposition time, as well as on the Cu2O substrate exposure to oxidizing agents prior to and during the ZnO deposition. Superficial Cu2O to CuO oxidation was identified as a limiting factor to heterojunction quality due to recombination at the ZnO/Cu2O interface. Optimization of AP-SALD conditions as well as keeping Cu2O away from air and moisture in order to minimize Cu2O surface oxidation led to improved device performance. A three-fold increase in the open-circuit voltage (up to 0.65 V) and a two-fold increase in the short-circuit current density produced solar cells with a record 2.2% power conversion efficiency (PCE). This PCE is the highest reported for a Zn1-xMgxO/Cu2O heterojunction formed outside vacuum, which highlights atmospheric pressure spatial ALD as a promising technique for inexpensive and scalable fabrication of Cu2O-based photovoltaics.

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Here we present a protocol for synthesizing Zn1-xMgxO/Cu2O heterojunctions in open-air at low temperature via atmospheric pressure spatial atomic layer deposition (AP-SALD) of Zn1-xMgxO on cuprous oxide. Such high quality conformal metal oxides can be grown on a variety of substrates including plastics by this cheap and scalable method.

在铜改善异质结质量<sub&gt; 2</sub&gt; O型的太阳能电池通过大气压力的空间原子层沉积的优化<br /&gt;锌<sub&gt; 1-X</sub&gt;镁<sub系列&gt; X</sub&gt; 0

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Surface Functionalization of Metal-Organic Frameworks for Improved Moisture Resistance

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. However, the main issue limiting their applicability is their poor stability in humid conditions. The common methods to overcome this problem involve the formation of strong metal-linker bonds by using highly charged metals, which is limited to a number of structures, the introduction of alkylic groups to the framework by post-synthetic modification (PSM) or chemical vapour deposition (CVD) to enhance overall hydrophobicity of the framework. These last two usually provoke a drastic reduction of the porosity of the material. These strategies do not permit to exploit the properties of the MOF already available and it is imperative to find new methods to enhance the stability of MOFs in water while keeping their properties intact. Herein, we report a novel method to enhance the water stability of MOF crystals featuring Cu2(O2C)4&#160;paddle-wheel units, such as HKUST (where HKUST stands for Hong Kong University of Science &#38; Technology), with the catechols functionalized with alkyl and fluoro-alkyl chains. By taking advantage of the unsaturated metal sites and the catalytic catecholase-like activity of CuII ions, we are able to create robust hydrophobic coatings through the oxidation and subsequent polymerization of the catechol units on the surface of the crystals under anaerobic and water-free conditions without disrupting the underlying structure of the framework. This approach not only affords the material with improved water stability but also provides control over the function of the protective coating, which enables the development of functional coatings for the adsorption and separations of volatile organic compounds. We are confident that this approach could also be extended to other unstable MOFs featuring open metal sites.

Robust functional catechol coatings were produced in one step by direct reaction of the material known as HKUST with synthetic catechols under anaerobic conditions. The formation of homogeneous coatings surrounding the entire crystal is ascribed to the biomimetic catalytic activity of Cu(II) dimers on the external surface of the crystals.

金属-有机框架的表面功能化改善防潮性能

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

纳米金刚石的生物激发多巴胺表面改性及其对银纳米粒子的还原

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

聚五氟丙烯酸酯 (五氟丙烯酸酯) 功能化 sio2 珠的蛋白质纯化

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...