We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.

注:分子生物学级水在所有的缓冲和解决方案的准备工作时使用。所有一次性管（离心及PCR）和枪头是DNase-和无RNA酶的。请咨询使用前的所有化学品的材料安全数据表（MSDS）。强烈推荐合适的个人防护装备（PPE）的使用。 1.适体母液的制备<ol><li>购买2股多脱氧核苷酸的市场。通过高效液相色谱法（HPLC）命令与纯化两条链。 串1（GD-适体）: 5&#39; - / 56-FAM / AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; 链2（QS）: 5&#39;-GTCCCGAGAGCCT / 3Dab / -3&#39; </li><li>溶解每条链在水中使钆适体和的QS100μM的个别储备溶液。 </li><li>存放于-20°C这些解决方案。该溶液是稳定的迄今，为3年。 </li><li>为了尽量减少FREEZ电子解冻周期，存储解决方案，股价在10微升等分。 </li></ol> 2. 2倍的Gd-传感器溶液的制备<ol><li>制备测定缓冲液（20mM HEPES，2mM的MgCl 2的，150毫摩尔NaCl，5mM的KCl）中。调整用NaOH和HCl pH值至7.4〜，并通过一次性无菌瓶顶过滤器具有0.2微米PES膜过滤器。存放在无菌瓶中。如果过滤，并妥善保存（在常温下），缓冲稳定到目前为止，2年。 </li><li>稀释钆适体原液（从步骤1.2）在测定缓冲液497微升和QS原液（从步骤1.2）的2微升1微升。拌匀，用旋涡。其在2×钆传感器溶液浓度是200和400nm，分别。 注意:在此步骤中制备的2倍的Gd-传感器溶液的体积为500微升，这是足以测试6 - 7不同浓度的Gd 3+的校准曲线和造影剂溶液（步骤3）。每个样品将在384孔板给复孔。 <ol><li>根据需要被测试的Gd 3+解的数目调整2倍的Gd-传感器溶液的体积。 </li></ol></li><li>转移2×钆传感器溶液到9的PCR管，用50微升到每个管中。放置管在热循环仪。 </li><li>在热循环仪设置程序以加热在管至95℃的溶液中，保持5分钟，然后经〜15分钟缓慢地冷却该溶液至25℃（以速率〜0.05 - 0.1℃/ S）。加热和冷却循环是确保钆适体和QS之间最佳杂交。部分杂交结果不完全淬火和传感器的一个更高的背景荧光。如果热循环仪是不可用，进行利用热水浴代替这个过程。 </li><li>一旦冷却到25℃，立即使用该溶液，或保持在热循环仪（最高达约2小时），直到准备好是用过的。当水浴用于加热，离开该管在浴中当水慢慢冷却到室温。 </li></ol> 3.构建荧光校准曲线和钆造影剂的溶液检测未螯合GD的存在3+ <ol><li>溶解GdCl 3在测定缓冲液固体（相同的缓冲液如在步骤2.1）。 </li><li>通过连续稀释，在最终所需浓度的校准曲线（2×溶液）的双制备各6个不同的Gd 3+在微量离心管溶液100微升。 <ol><li>例如，为了构建一个校准为0，50，100，200，400（仅无GdCl 3缓冲液），和钆3+为800nm，制备含有0，100，200，400，800，和1600纳米的解决方案的离子。确保始终包括"空白"0纳米的Gd 3+作为阴性对照。 </li></ol></li><li>溶解造影剂是测试编在测定缓冲液中。准备2个或3个不同的浓度通过连续稀释的造影剂的解决方案。 注:测试3种不同浓度造影剂溶液的建议。这是为了确保这些浓度的线性范围内。如果测试没有显示线性关系的样品中，减少造影剂的浓度使用。 </li><li>服用含有从步骤2.5 2倍的Gd-传感器解决方案出来的热循环仪的PCR管。 </li><li>添加来自步骤3.2的每个的Gd 3+溶液的50微升到含有2倍的Gd-传感器溶液9 PCR管6。吹打向上和向下混合。每个PCR管现在包含钆3+的所需浓度进行测试，100nM的钆适体，和200nM的QS。 </li><li>到含有2倍的Gd-传感器溶液剩余的PCR管，从步骤3.3添加造影剂溶液的50微升。通过上下吹打几次拌匀。 </li><li>孵育在Solutions在PCR管在室温下约5分钟。它们可以被放置长达30分钟。 </li><li>传送每个管的45微升到384孔板中。每个PCR管会给重复孔中。 </li><li>记录的每个孔中的荧光在平板阅读器。在GD-传感器设计中使用的荧光团（FAM），分别有495和520 nm的激发和发射最大值，对供应商的网站上所列出。选择取决于在板阅读器是否monochromator-或基于过滤器的合适的激发和发射波长或过滤器。 </li><li>绘制荧光任意荧光单位（AFU）对钆3+的浓度曲线图。 </li><li>画出图作为对抗的Gd 3+的荧光浓度倍数变化。由"空白"解决方案的AFU（0纳米的Gd 3+）将每个浓度的AFU计算荧光倍数变化。荧光倍数变化将使n个结果ormalization，应AFU显示一些周期性的（不同天等 ）的变化。 </li><li>比较含有造影剂的溶液和"空白"，它是含0纳米GdCl 3（仅缓冲）该溶液的荧光发射。 注:GBCA溶液的更高的荧光意味着未螯合的Gd 3+的存在，这可能需要造影剂的进一步纯化。未螯合的Gd 3+的存在量可以利用在步骤3.10或3.11构建的校准曲线来估算。 </li></ol>

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The authors declare that they have no competing financial interests.

使用基于适体的Gd-传感器，荧光发射的增加正比于观察未螯合的Gd 3+的浓度。为了最大限度地减少样品的使用量，该测定可以在384孔微量板以每孔45微升总样品体积运行。在此设计中，荧光素（FAM）和DABCYL（DAB）的选择主要基于试剂的成本;修改发射波长的荧光团不同的配对和猝灭剂，可以使用11。 要注意的是，以获得与该传感器最好的结果是很重要的，过程的...

在临床诊断中，这是由该技术的固有敏感性限制磁共振成像（MRI）的重要性日益增加，已导致在研究的快速增长到新的基于钆的造影剂（GBCAs）1的开发。 GBCAs是被给药，以提高图像质量的分子，并且它们通常具有配位的多齿配体的三价钆离子（钆3+）的化学结构。这络合作为未螯合的Gd 3+是有毒至关重要;它已经牵涉于肾源性系统纤维化的一些患者的肾疾病或衰竭2的开发。因此，检测该水性自由离子是确保GBCAs的安全工具。未螯合的Gd 3+的GBCA溶液存在往往是在配体和离子的复合物的解离，或者displacemen之间不完全反应的结果其他生物金属阳离子3吨 。 在目前用于确定的Gd 3+，那些在通用性和适用性4方面依靠色谱法和/或光谱法秩最高的存在下的几种技术。在他们的优势是较高的灵敏度和准确度，分析各种样品基质（包括人血清5，尿液和头发6，废水7和造影剂配方8），和多个钆3+络合物的同时定量（一个列表的能力的研究之前，2013是在全面审查Telgmann 等人 ）4所述。唯一的缺点是，一些这些方法需要仪器仪表（如电感耦合等离子体质谱）4，一些实验室可能无法访问。在小说GBCA发现在研究和论证的概念层面，AR上下文elatively更方便，快速和成本有效的基于分光法（如紫外吸收或荧光）可以作为一种有价值的替代。考虑到这些应用中，水的Gd 3+的荧光基于适体的传感器被开发9。 适体（钆适体）是与通过指数富集（SELEX）9的配体的系统进化的过程中分离出的碱基序列特异性的44碱基长的单链DNA分子。适应适体为荧光传感器，荧光团被连接到线料，然后将其与经由13互补碱基（ 图1）淬火链（QS）杂交的5&#39;末端。了QS标记有在3&#39;末端的暗淬灭剂分子。在不存在的Gd 3+，所述传感器（钆传感器）的，由1:分别钆适体和QS的2摩尔比，将具有最小的荧光发射是由于吨从荧光基团淬灭Ø能量转移。在加入含水的Gd 3+的将从钆适体置换的QS，从而增加荧光发射。 <img alt="图1" src="/files/ftp_upload/55216/55216fig1.jpg" /> 图 1. 传感器（GD-传感器），由标有荧光素（荧光基团），并标有DABCYL 13个碱基长的淬链（QS）的44个碱基长的适体（GD-适体）（暗淬灭剂）的。在不存在未螯合的Gd 3+的，传感器的荧光是最小的。与另外的Gd 3+的，发生的QS位移和增加的荧光发射是观察。 <a href="http://ecsource.jove.com/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 有目前，人们常用的基于分光法检测ING水的Gd 3+。该测定使用了分子二甲酚橙，其经历在其最大吸收波长从433至573纳米时螯合离子10的换档。这两个最大吸收的比率可以用来量化未螯合的Gd 3+的量。适体传感器是一种替代（也可以是互补的），以在二甲酚橙法，因为这两种方法具有不同的反应条件（如pH值和所使用的缓冲溶液的组合物），目标的选择性，定量的线性范围，和检测方式9。

<table border="0" cellpadding="0" cellspacing="0" width="2018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Gd-aptamer</td>
			<td style="width:187px;">IDTDNA</td>
			<td style="width:439px;">Input sequence and fluorophore modification in the order form</td>
			<td style="width:943px;">A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quenching strand</td>
			<td>IDTDNA</td>
			<td>Input sequence and quencher modification in the order form</td>
			<td>A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular biology grade water</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer, both DEPC or non-DEPC treated work equally well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gadolinium(III) chloride anhydrous</td>
			<td>Strem</td>
			<td>936416</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride anhydrous</td>
			<td>MP Biomedicals</td>
			<td>0520984480 - 100 g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Acros Organics</td>
			<td>327300025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P333-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide, pellets</td>
			<td>Fisher Scientific</td>
			<td>BP359</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>SA49</td>
			<td>Toxic and corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well low flange black flat bottom polystyrene NBS plates</td>
			<td>Corning</td>
			<td>3575</td>
			<td>Plates which are suitable for fluorescence reading are required.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene Rapid-Flow sterile disposable bottle top filter</td>
			<td>Thermo Scientific&#160;</td>
			<td>5680020</td>
			<td>The bottle top is fitted with&#160; 0.2 micron PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile bottles 250 mL</td>
			<td>Corning</td>
			<td>430281</td>
			<td>A larger or smaller bottle may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer, as long as they are DNAse and RNAse-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 mL PCR tubes</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer, as long as they are DNAse and RNAse-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipets</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipet tips (non filter) of appropriate sizes</td>
			<td></td>
			<td></td>
			<td>No specific manufacturer, as long as they are DNAse and RNAse-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biotek Synergy H1</td>
			<td></td>
			<td>Plate readers from other manufacturers would work equally well</td>
		</tr>
	</tbody>
</table>

an aptamer-based sensor for unchelated gadolinium(iii)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

在未螯合的Gd 3+的存在下的Gd-传感器溶液的典型荧光变化示于图2。发射可被绘制为荧光倍数变化（ 图2A）或在原始荧光读数（ 图2B）以任意单位（AFU）。两条曲线在&gt; 3μM收率低于1μM和饱和信号的线性范围为钆浓度3+非常相似的校准曲线。检测限为〜100纳米带3的信噪比。 ...

Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

对于未螯合钆基于核酸适配体传感器（III）

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

an-aptamer-based-sensor-for-unchelated-gadolinium-iii

Research

JoVE Journal

Chemistry

An Aptamer-based Sensor for Unchelated Gadolinium(III)

7.2K 次观看.  California State University, East Bay. The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor. 

视频: An Aptamer-based Sensor for Unchelated GadoliniumIII

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物:用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

科研

化学

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

一个两亲性共聚物的阴离子聚合通过π-π堆积作用稳定嵌段共聚物胶束的制备

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

利用 iccd 摄像机对无氧环境下的三重收集有机化合物进行光物理表征

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

An Experimental Protocol for Femtosecond NIR/UV - XUV Pump-Probe Experiments with Free-Electron Lasers

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a free-electron laser. This includes methods to establish the spatial and temporal overlap between the optical and free-electron laser pulses during the experiment, as well as important aspects of the data analysis, such as corrections for arrival time jitter, which are necessary to obtain high-quality pump-probe data sets with the best possible temporal resolution. These methods are demonstrated for an exemplary experiment performed at the FLASH (Free-electron LASer Hamburg) free-electron laser in order to study ultrafast photochemistry in gas-phase molecules by means of velocity map ion imaging. However, most of the strategies are also applicable to similar pump-probe experiments using other targets or other experimental techniques.

This protocol describes the key steps for performing and analyzing pump-probe experiments combining a femtosecond optical laser with a free-electron laser in order to study ultrafast photochemical reactions in gas-phase molecules.

用自由电子激光器进行飞秒近红外/紫外 XUV 泵-探针实验的实验协议

This protocol describes key steps in performing and analyzing femtosecond pump-probe experiments that combine a femtosecond optical laser with a ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

通过活性表面生长机制合成基片束缚金纳米线

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Key Factors Affecting the Performance of Sb2S3-sensitized Solar Cells During an Sb2S3 Deposition via SbCl3-thiourea Complex Solution-processing

Sb2S3 is considered as one of the emerging light absorbers that can be applied to next-generation solar cells because of its unique optical and electrical properties. Recently, we demonstrated its potential as next-generation solar cells by achieving a high photovoltaic efficiency of &#62; 6% in Sb2S3-sensitized solar cells using a simple thiourea (TU)-based complex solution method. Here, we describe the key experimental procedures for the deposition of Sb2S3 on a mesoporous TiO2 (mp-TiO2) layer using a SbCl3-TU complex solution in the fabrication of solar cells. First, the SbCl3-TU solution is synthesized by dissolving SbCl3 and TU in N,N-dimethylformamide at different molar ratios of SbCl3:TU. Then, the solution is deposited on as-prepared substrates consisting of mp-TiO2/TiO2-blocking layer/F-doped SnO2 glass by spin coating. Finally, to form crystalline Sb2S3, the samples are annealed in an N2-filled glove box at 300 &#176;C. The effects of the experimental parameters on the photovoltaic device performance are also discussed.

Key Factors Affecting the Performance of Sb2S3-sensitized Solar Cells During an Sb2S3 Deposition via SbCl3-thiourea Complex Solution-processing

This work provides a detailed experimental procedure for the deposition of Sb2S3 on a mesoporous TiO2 layer using a SbCl3-thiourea complex solution for applications in Sb2S3-sensitized solar cells. This article also determines key factors governing the deposition process.

利用SbCl3-硫脲复合溶液处理锑23沉积物对锑2S3敏化太阳能电池性能影响的关键因素

Sb2S3 is considered as one of the emerging light absorbers that can be applied to next-generation solar cells because of its unique optical and electrical ...

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The cholesteric LC is composed of a nematic LC 4&#39;-pentyloxy-4-cyanobiphenyl (5OCB), redox-responsive chiral dopant (FcD), and a supporting electrolyte 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMIm-OTf). The most important component is FcD. This molecule changes its helical twisting power (HTP) value in response to redox reactions. Therefore, in situ electrochemical redox reactions in the LC mixture allow for the device to change its reflection color in response to electrical stimuli. The LC mixture was introduced, by a capillary action, into a sandwich-type ITO glass cell comprising two glass slides with patterned indium tin oxide (ITO) electrodes, one of which was coated with poly(3,4-ethylenedioxythiophene)-co-poly(ethylene glycol) doped with perchlorate (PEDOT+). Upon application of +1.5 V, the reflection color of the device changed from blue (467 nm) to green (485 nm) in 0.4 s. Subsequent application of 0 V made the device recover the original blue color in 2.7 s. This device is characterized by its fastest electrical response and lowest operating voltage among any previously reported cholesteric LC device. This device could pave the way for the development of next generation reflective displays with low energy consumption rates.

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

一种用于快速和低压彩色调制的电化学胆固醇液晶器件

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The ...