This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

注意:放射性碘的氧化形式是相当挥发性的，且必须有足够的铅屏蔽和铅的小瓶处理。所有放射化学步骤应在通风良好的木炭过滤通风橱中进行，实验程序需要通过放射性检测装置进行监控。 1.化学品和反向相萃取柱的制备125 I标记的叠氮合成<ol><li> 在溶液试剂的制备 <ol><li>溶解1毫克150微升叠氮化物前体（2）无水乙醇（ 图1）。 注:叠氮化物前体的详细合成步骤（2）据报道，在以前的论文22。 </li><li>在20μl1×磷酸盐缓冲盐水（pH = 7.4）的溶解1毫克的氯胺T。 </li><li>溶解2毫克焦亚硫酸钠在20μlH 2 O </li></ol></li><li> Prepara墨盒化<ol><li>用10ml无水乙醇，然后用10毫升水2 O洗tC18柱不干燥与空气筒的矩阵。 </li></ol></li></ol> 2. 125 I标记的叠氮化辅基的放射合成<ol><li> 前体的放射性碘化反应 <ol><li>添加叠氮化物前体溶液（1毫克150微升绝对乙醇）和乙酸（10微升）到1.5ml微量离心管中。 </li><li>在0.1M的NaOH（50微升）添加的[125 I]碘化钠150活度到反应混合物中。 </li><li>添加氯胺T溶液（1mg在20μl1×磷酸盐缓冲盐水），并关闭含有微量离心管反应混合物。 </li><li>在室温下孵育该反应混合物15分钟，直到放射性碘化反应完成。 </li><li>一个焦亚硫酸钠溶液（2毫克在20μlH 2 O）加入到该反应混合物以猝灭放射性碘化反应。 </li><li>撤回0.2微升粗产物，然后用100微升的溶液稀释（H 2 O / CH 3 CN，1:1）进行HPLC分析。 注:对于所有的HPLC实验中，使用含有H 2 O（溶剂A）0.1％甲酸和0.1％甲酸含酸乙腈（溶剂B）作为洗脱剂。 </li><li>通过使用反相分析放射性HPLC（分析稀释粗产物C18反相柱;流速:1ml /分钟;洗脱剂梯度:0-2分钟，20％溶剂B，20-80％溶剂B为2-22分钟，为22-23分钟80-100％溶剂B，和100％溶剂B为23-28分钟;保留时间:16.4分钟）（ 图2）。 </li></ol></li><li> 用制备HPLC纯化粗产物 注意:提供围绕型HPLC份足够铅屏蔽诸如喷射器，柱，检测器，收集瓶，并在其中流出物被收集在容器。 <ol><li>第撤Ë整个反应混合物倒入HPLC小瓶。冲洗，用乙腈（0.5毫升）的反应管中，加入漂洗到相同的注射小瓶中。稀释用H 2 O（1毫升）中收集的溶液。 </li><li>注入粗产物到制备放射性HPLC（C18反相柱;流速:10ml /分钟;洗脱剂梯度:20％溶剂B为0-2分钟，20-80％溶剂B为2-22分钟，为22-23分钟80-100％溶剂B，以及用于23-28分钟100％溶剂B）。 </li><li>收集表示125 I标记的叠氮化物（1）的放射性峰（ 叔 R这些HPLC条件下是17.8-18.8分钟）中的玻璃试管（ 图2）。 </li><li>测量使用根据制造商的协议的放射性剂量校准馏分的放射化学产率。 </li><li>注入纯化产品上使用，用于确定产物的放射化学纯度的相同HPLC条件分析放射性HPLC。 </li></ol></li><li> 固相萃取的产物 <ol><li>稀释含用40ml纯H 2 O所需的产物（1）的级分</li><li>添加稀释的溶液成预处理tC18柱。 </li><li>用另外的15毫升水2 O洗盒</li><li>洗脱被困在盒用2毫升丙酮到由一铅屏蔽保护的10-ml玻璃管制瓶内的产品（1）。测量使用根据制造商的协议的放射性剂量校准洗脱产物的放射性。 注:二甲基亚砜（DMSO）或无水乙醇也可用于从盒中的产品的溶出。放射性的约5-10％，通常粘附在盒中，而剩余的放射性标记的产物不能通过使用过量的有机溶剂完全洗脱。 </li><li>蒸发用氮气或氩气的气流的丙酮。 </li><li>化解重用DMSO（100-200微升）为下放射性标记步骤sidue。 </li></ol></li></ol> 3. DBCO群结合的金纳米粒子的合成<ol><li> 13纳米大小的金表面改性与DBCO含基的聚乙二醇纳米粒 <ol><li>制备钠-柠檬酸盐稳定的金纳米粒子（3）（平均粒度= 13纳米）根据先前的报告24。 </li><li>添加吐温20（1毫米，1.5毫升）的水溶液的柠檬酸盐稳定的金纳米粒子（10纳米，15毫升）。摇上轨道摇床进行20分钟的溶液中。 </li><li>添加DBCO含有基团的聚乙二醇硫醇（平均分子量= 5,000，100μM，1.5毫升）的水溶液。摇上轨道摇床2小时的溶液中。 </li></ol></li><li> 所述DBCO团改性金纳米颗粒的纯化 <ol><li>净化DBCO团改性的金纳米粒子（4）&lt;/ STRONG&gt;是通过连续离心（11400 XG，15分钟×3）。 </li><li>倒出上清液并加入纯水金纳米颗粒粒料再悬浮。 </li></ol></li></ol> 4.通过无铜点击反应DBCO团改性金纳米粒子的放射性标记<ol><li> 使用125 I标记的叠氮化物125 I标记的金纳米颗粒的合成（1） <ol><li>通过使用离心分离（11400 xg离心，15分钟）制备DBCO团改性金纳米颗粒的浓溶液，并调整金纳米颗粒的浓度为2μM。 </li><li>添加在DMSO（5微升）的125 I标记的叠氮化物（1）的4.1活度的悬浮液金纳米粒子（4）（2μM，50微升）。 </li><li>孵育在40℃下将所得反应混合物60分钟。 </li><li>从粗产物撤回等分试样（0.2微升），并将其应用到二氧化硅 - 共ated薄层色谱（TLC）板。 </li><li>开发使用乙酸乙酯作为流动相的TLC板上。 </li><li>放置在无线电型TLC扫描器TLC板上并运行扫描器监视根据制造商的方案的放射性标记反应（ 图3）。 </li></ol></li><li> 粗产物的纯化 <ol><li>净化含有125 I标记的金纳米颗粒（4）通过离心（11400 xg离心，15分钟），将反应混合物。 </li><li>倒出上清液并加入纯水金纳米颗粒粒料再悬浮。 </li><li>从纯化的产物撤回等分试样（0.2微升），并将其应用到一个二氧化硅包覆TLC板上。 </li><li>开发使用乙酸乙酯作为流动相的TLC板上。 </li><li>放置在无线电型TLC扫描器TLC板上并运行扫描器，以确定125 I标记勒的放射化学产率和放射化学纯度ð纳米颗粒（4）（ 图3）根据制造商的协议。 </li></ol></li></ol>

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The authors have nothing to disclose.

在一般情况下，纯化的125 I标记的叠氮化物（1）所观察到的放射化学产率为75±10％（N = 8）。放射性标记用放射性50-150活度完成，放射化学的结果是相当一致的。如果[125 I]碘化钠（T 1/2 = 59.4 D）将经历放射性衰变了一个多月的放射性碘化反应时，观察到1放化收益率将略有下降（53-65％）。因此，我们建议，因为它产生的[125 I]碘化钠?...

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloramine T trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">402869</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">[125I]NaI in aq. NaOH</td>
			<td style="width:101px;">Perkin-Elmer</td>
			<td style="width:160px;">NEZ033A010MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium metabisulfite&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">S9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formic acid</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">251364</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sep-Pak tC18 plus cartridge</td>
			<td style="width:101px;">Waters</td>
			<td>WAT036800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Dimethyl sulfoxide&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acetone</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gold(III) chloride trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">520918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DBCO PEG SH (MW 5000)</td>
			<td style="width:101px;">NANOCS</td>
			<td style="width:160px;">PG2-DBTH-5k</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">TLC silica gel 60 F254</td>
			<td style="width:101px;">Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Analytical HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1290 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Preparative HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1260 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Analytical C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">Zorbax Eclipse XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Preparative C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">PrepHT XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Radio TLC scanner</td>
			<td style="width:101px;">Bioscan</td>
			<td style="width:160px;">AR-2000</td>
			<td>Model number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Radioisotope dose calibrator</td>
			<td style="width:101px;">Capintec, Inc</td>
			<td style="width:160px;">CRC -25R dose calibrator</td>
			<td>Model number</td>
		</tr>
	</tbody>
</table>

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.

所述stannylated前体（2）的放射性碘化反应进行了使用[125 I]碘化钠，乙酸，和氯胺T 150活度在室温下15分钟，以提供放射性标记的产物（1）。粗混合物的制备HPLC纯化后，用放射化学产率的75±10％（N = 8）中得到所需产物。分析性HPLC显示，125 I标记产物的放射化学纯度是99％以上（ 图2），和产品1所观察到的?...

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

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

对于一个125 I标记的叠氮化物的合成和dibenzocyclooctyne（DBCO） -基共轭，13纳米大小的金使用无铜点击反应的纳米粒子的放射性标记的详细过程进行说明。

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 ...

an-optimized-protocol-for-efficient-radiolabeling-gold-nanoparticles

Research

JoVE Journal

Chemistry

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

8.1K 次观看.  Korea Atomic Energy Research Institute. 对于一个125 I标记的叠氮化物的合成和dibenzocyclooctyne（DBCO） -基共轭，13纳米大小的金使用无铜点击反应的纳米粒子的放射性标记的详细过程进行说明。 

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

稳定肝细胞表型采用优化合成表面

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

科研

化学

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

根据环境条件的金纳米粒子的稳定低聚簇的大小控制合成的一种简便方法

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

一种醇溶蛋白氰基丙烯酸酯纳米粒子为亲水涂料生产协议

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

功能性降冰片烯的催化聚合插入一种简单高效的协议

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

使用有机锂试剂的一个协议安全的锂化反应

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

土壤样品多涨价可能分析的 MPLEx 协议

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

结合金 (III.) 对牛血清白蛋白不同构象的 Luminophore 形成

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

两栖金纳米粒子的合成与表征

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...