This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

动物实验是由动物研究伦理委员会麦克马斯特大学按照加拿大议会关于动物饲养（廉政公署）的指导方针批准。 1. 99m锝TZ-齿配体的放射性标记注意:以下步骤需要使用放射性化合物。工作只应在有执照的实验室进行与遵守安全和处置法规。微波反应应该专门为化学合成设计的微波炉进行。 <ol><li> 合成[99m锝（CO）3（H 2 O）3] + 19,20 <ol><li>在微波小瓶中，合并8毫克K 2 [BH 3 CO 2]，15毫克的 Na 2 CO 3，20毫克的 Na 2 B 4 O 7·10H 2 O和25毫克KOCO [CH（OH）]2 COONa·4H 2 O吹扫小瓶10分钟用氩气。 </li><li>在0.9％的盐水（〜1100活度，〜30毫居里）到小瓶-添加4mL的标99m TCO 4。 </li><li>热在微波反应在110℃下10秒后搅拌3.5分钟，以确保试剂的彻底混合。 </li><li>使用〜400微升的1M HCl调节溶液的pH至3.5-4。验证使用pH试纸。 </li></ol></li><li> 四嗪的放射性标记配体1-5 <ol><li>在250μL甲醇溶解21毫克2每配体（化合物1-5）。 </li><li>添加250微升[99m锝（CO）3（H 2 O）3] +（〜74活度，〜2毫居里）向每个溶液中。 </li><li>加热在60℃下用20分钟微波反应混合物中。 注:此步骤是所有5个四嗪相同。 </li><li>对化合物2-5，使溶剂蒸发并重新dissol已经将所得产物在1毫升1:1的体积/体积的DCM:TFA。 </li><li>在60℃下加热溶解反应产物（2-5）在6分钟（2-4）或10分钟（5）一台微波炉。 </li><li>在冷却至室温后，蒸发用蒸发器（36℃，8毫巴，3分钟，6000转）的溶剂，并溶解在1干燥化合物:1的ACN:H 2 O或1:1的MeOH:H 2 O，之前HPLC纯化。 </li><li>净化99m锝标记的化合物（1-5），包括使用HPLC（C18反相）分离未标记的四嗪配体的标记产物。通常情况下，使用30:70的ACN梯度洗脱:H 2 O（均含有0.1％TFA），以40:60的ACN:H 2 O在20分钟内（18分钟）和C 18的分析4.6×100毫米柱。同时使用UV（254纳米）和伽马探测。 <ol><li>每次取标记产品的一个小样本及其HPLC保留时间比较，一个共同的注射ected，无放射性，重新标记的标准（0.125毫克20％甲醇- H 2 O）。 [重新标记的标准是在UV HPLC迹识别，并且将在同一时间，在γ-HPLC跟踪该99m锝标记的化合物洗脱。此共注射示出了在可比较的保留时间的峰，确认了99m锝标记的化合物的身份。 </li></ol></li><li>蒸发从用蒸发器（36℃，8毫巴，3分钟，6000转）的HPLC级分的溶剂。 </li><li>配制纯化的化合物在7.4 KBQ /微升在PBS中的浓度，含有0.5％BSA和0.01％吐温-80。 </li><li>为了确保标记的化合物是稳定的，执行的体外稳定性研究。孵育在37℃配制的化合物为1,4和6小时，在每个时间点注射该混合物在HPLC上少量（3.7活度）评估稳定性。 </li></ol></li></ol> 2.预靶向生物分布研究<ol><l我&gt; 动物的制备 <ol><li>使用7-9周龄，雌性BALB / c小鼠（N = 3），管理TCO-BP生理盐水（20毫克/公斤）（5微克/μL）制定的，经尾静脉注射。 </li><li>放置鼠标在物理约束装置，和识别位于尾部的横向表面上的静脉和用酒精棉签擦拭。在从尾部的端部大约2厘米，以小角度，平行于静脉插入一个30号针头。慢慢地压低柱塞注入，去掉针头和注射部位有轻微的压力施加干净的纱布海绵，直到出血停止。 </li><li>在1小时后注射的TCO-BP，辖〜99m锝四嗪0.74活度（20微居里）的100微升0.5％的BSA的配制，0.01％吐温80在PBS中，通过尾静脉注射。 </li></ol></li><li> 生物分布研究 <ol><li>在期望的时间点（T = 6小时），麻醉用3％异氟烷和2％的氧的气体混合物的小鼠。表现出以在麻醉鼠标ê捏撤离，以确保它们麻醉手术平面之下。 </li><li>通过使用注射器肝素预处理心脏穿刺收集血液（1mL）中。它与鼻子的鼻锥为继续回麻醉将鼠标和定位对动物剑突。 <ol><li>插入地下25针，略向剑突下动物的中线的左边，在一个20°的角度。完全插入针，慢慢向后拉柱塞看到血针毂如果心脏被刺破。略微调整针的同时保持柱塞如果必要，刺穿心脏。慢慢地抽取血液到注射器中。 </li></ol></li><li>通过颈脱位安乐死的动物，而在麻醉下。 </li><li>将每个动物在一个塑料袋，并使用剂量校准（99m锝设置）来测量全身活性水平。 </li><li>收集在下面的组织和体液的预称重ED计数管:血液，骨（膝盖和肩部），胆，肾，肝，胃（有内容），小肠（有内容），大肠和盲肠（有内容），甲状腺和气管，有尿膀胱和尾部。 </li><li>在冲洗PBS适当组织（不包括血液，胆囊和膀胱）将组织以适当的计数管之前去除血液和吸干。 </li><li>放置动物胴体在一个塑料袋中并使用剂量校准测量剩余全身活性。 </li><li>称量每个管含有组织样品。该管的减去初始重量以获得组织的质量。 </li><li>使用剂量校准（99m锝设置）在注射为每只小鼠的时间来测量酶活性的测试样品（100μL）中的量。 注意:此测试样本等于注射体积，从而在注射时给予活动计数。 </li><li>在组织的测量，一个时liquot5μL的以前使用的测试样品。使用多检测器γ计数器（99m锝设置）并计数，以获得每分钟（CPM）的计数为5微升试样。 </li><li>使用所获得的两个值中2.2.9和2.2.10计算使用等式1获得的换算因子（CPM微居里-1）的活性和CPM关系。 （1） <img alt="式（1）" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li>使用γ计数器测定在每个组织或流体样品的放射性的量。 </li><li>使用方程1在相对于总注射剂量的测量的时间来计算每个组织或流体的活动量。此值然后通过器官重量归一化并且报告为组织的每克百分比注射剂量（ 即，％ID /克）。 </li><li>按照步骤2.1.2 2.2.13在艾比森用99m锝标记的四嗪配体进行阴性对照实验TCO-BP的行政长官。牺牲小鼠（n = 3）为0.5，1,4和6小时后注射，将获得的组织或流体，如上所述。 </li></ol></li></ol>

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The authors declare they have no competing financial interests.

不同极性的四嗪联三齿螯合物的集合被制备，并且与在体内的TCO衍生物IEDDA反应其99m锝复合物的效用进行评价。一种有效的和可再现的99m锝标记方法是五四嗪螯合物，其中该配位体的浓度为10 -3 M的标记步骤之后是叔丁基基（化合物2-5）的脱保护的发展。配体的高浓度使用以提高放射化学产率，并减少反应时间，这减少了四嗪21的...

99m锝保留在核医学诊断中使用的主导放射性同位素，每年全球1，2，3进行超过5000万的成像程序。大多数临床使用99m锝剂是灌流型的放射性药物。有在其中99m锝涉及通过结扎特定生物标志物结合至靶向构建积极靶向化合物的数量有限。靶向99m锝放射性药物的创建通常是由99m锝-配体复合的对靶向分子的能力的影响阻碍了对感兴趣的生物标记物结合，或同位素半衰期不够长为具有更高分子量的生物分子用如抗体。后者通常需要数天的图像，以获得前的生物分子从非目标清除做卷烟<html>的UE。预靶向提供了一种替代的方法来克服这些挑战。 预靶向结合生物正交化学已被证明是开发用于两个荧光和放射成像4，5，6，7，8个新的分子成像探针的有效方法。 -1,2,4,5-四（TZ）和反式 -cyclooctene（TCO）的衍生物之间的逆电子需求狄尔斯-阿尔德（IEDDA）反应， 如图1，已被证明是特别有效的6。这些组件的IEDDA反应可在PBS呈现快速动力学（K 2≈6000 M -1秒-1）和高选择性，因此非常适合在体内预先指定的应用9,10。 e_content"&gt;使用的最常见的方法包括施用的TCO源性靶向载体和以下足够长的延迟时间，放射性标记的四嗪是基于11℃，（18）F，64的Cu，89 Zr和111在已经施用。放射性标记的四嗪报告11，12，13，14，15，相反，仅存在一个一个99m锝标记Tz的，其使用一个HYNIC型配位体需要使用共配体的，以防止在体内蛋白结合和降解制备的报告16。作为替代方案，我们在这里报告99m锝（Ⅰ）的合成中使用的是家族，其形成具有稳定的三齿络合物的配体的标记的四嗪[99m锝（CO）3] +核心。 图1:四嗪和反 -cyclooctene之间的生物正交反应IEDDA。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 制备配体的家族包含三齿螯合物，在极性和连接基团的金属结合区和Tz的（ 图2）之间的性质不同而不同。该目标是确定一个99m锝-四嗪构造，可以有效地定位和在体内的TCO标记位点和当未结合迅速明确反应，以产生高靶-非靶比率。为了测试配位体，二膦酸盐的TCO-衍生物（TCO-BP），使用17。之前我们已经表明，TCO-BP定位于活跃的骨代谢领域，可以与反应放射性四嗪在体内 18。它是一种方便的试剂测试新四嗪，因为它可以在一个单一的步骤进行制备和实验可以在本地化主要发生在关节（膝和肩）正常小鼠进行。

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			<td height="21" style="height:21px;width:367px;">Argon gas&#160;</td>
			<td style="width:255px;">Alphagaz</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:255px;">EMD Millipore</td>
			<td style="width:199px;">106395</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2B4O7.10H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">S9640</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KNaC4H4O6.4H2O</td>
			<td style="width:255px;">Anachemia</td>
			<td style="width:199px;">217255</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Technelite 99mTc generator</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>Source of 99mTcO4-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">0.9% Saline</td>
			<td style="width:255px;">Lantheus medical imaging</td>
			<td style="width:199px;">---</td>
			<td>To elute generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M HCl</td>
			<td style="width:255px;">Lab Chem</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MeOH</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACN</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Millipore H2O</td>
			<td style="width:255px;">Thermo Fisher Scientific&#160;&#160;</td>
			<td style="width:199px;">Barnstead Nanopure</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DCM</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TFA</td>
			<td style="width:255px;">Caledon</td>
			<td style="width:199px;">---</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PBS</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">10010023</td>
			<td>pH 7.4 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">BSA</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">A7906</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tween80</td>
			<td style="width:255px;">Sigma Aldrich</td>
			<td style="width:199px;">P8047</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane&#160;</td>
			<td style="width:255px;">CDMV</td>
			<td style="width:199px;">108737</td>
			<td>Supplier: Fresenius Kabi Animal Health&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:367px;">HPLC&#160;</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 2-4</td>
			<td style="width:255px;">Phenomenex</td>
			<td style="width:199px;">00G-4435-E0</td>
			<td>Gemini&#174; 5 &#181;m C18 110 &#197;, LC Column 250 x 4.6 mm,</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">HPLC column for analysis and purification of compounds 1 and 5</td>
			<td style="width:255px;">Waters&#160;</td>
			<td style="width:199px;">186003115</td>
			<td>XBridge BEH C18 Column, 130 &#197;, 5 &#181;m, 4.6 mm X 100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave Reactor&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Initiator 8</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Biotage V10 Evaporator</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">Serial # V1041</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dose calibrator</td>
			<td style="width:255px;">Capintec, Inc.&#160;</td>
			<td style="width:199px;">CRC-25R</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">Gamma counter</td>
			<td style="width:255px;">Perkin Elmer</td>
			<td style="width:199px;">Wizard 1470 Automatic Gamma Counter</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Animal room scale&#160;</td>
			<td style="width:255px;">Mettler Toledo</td>
			<td style="width:199px;">XP105 Delta Range</td>
			<td style="width:559px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microwave vials&#160;</td>
			<td style="width:255px;">Biotage&#160;</td>
			<td style="width:199px;">355629</td>
			<td>0.5-2 mL&#160;</td>
		</tr>
	</tbody>
</table>

preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

该配体通过一个简单的还原胺化的策略（ 图2），随后将产物的耦合到市售四嗪22,23使用不同的连接体和螯合剂合成。放射性标记物使用的所有化合物中相同的方法进行，并且是高度可重复的。 83％（1），45％（2），31％:该过程通过改变pH值，配体，反应时间和温度的量，随后在99m?...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Research

JoVE Journal

Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

8.1K 次观看.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

视频: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

科研

化学

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

生物聚合物气凝胶制备使用绿色溶剂

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

热可逆交联橡胶通过的Diels-Alder反应的化学制备与性能

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Simple Methods for the Preparation of Non-noble Metal Bulk-electrodes for Electrocatalytic Applications

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and composition of the material was characterized via powder X-ray diffraction (PXRD), M&#246;ssbauer spectroscopy (MB), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and energy dispersive X-ray spectroscopy (EDX). Two preparation methods of pentlandite bulk electrodes are presented. In the first approach a piece of synthetic pentlandite rock is directly contacted via a wire ferrule. The second approach utilizes pentlandite pellets, pressed from finely ground powder, which is immobilized in a Teflon casing. Both electrodes, whilst being prepared by an additive-free method, reveal high durability during electrocatalytic conversions in comparison to common drop-coating methods. We herein showcase the striking performance of such electrodes to accomplish the hydrogen evolution reaction (HER) and present a standardized method to evaluate the electrocatalytic performance by electrochemical and gas chromatographic methods. Furthermore, we report stability tests via potentiostatic methods at an overpotential of 0.6 V to explore the material limitations of the electrodes during electrolysis under industrial relevant conditions.

A facile preparation method of electrodes using the bulk material Fe4.5Ni4.5S8 is presented. This method provides an alternative technique to conventional electrode fabrication and describes prerequisites for unconventional electrode materials including a straightforward electrocatalytic testing method.

用于制备用于电催化应用的非贵金属体电极的简单方法

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

C 型60/石墨烯混合纳米结构的制备与表征

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

用 Bioinspired 法制备功能性二氧化硅

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

用快速 Bioorthogonal 反应生成抗体-药物共轭物的非规范氨基酸的遗传编码

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...