我们感谢国家自然科学基金 (21675112)、北京教育委员会科技计划重点项目 (KZ201710028027) 和首都师范大学燕京青年学者项目的资助。

<ol>
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作者声明没有竞争的财政利益。

适配子的一个显著好处是, 它们通过体外方法 SELEX 识别, 而抗体是通过体内免疫生成的. 因此, 在设计良好的实验条件下, 适配子可以选择理想的靶向特异性, 而抗体仅限于生理条件。
为了便于从自由序列分离绑定序列, 最近报告了几种修改后的 SELEX, 其中毛细管电泳30, 微流体 31, 磁性/丙烯酸珠/琼脂糖珠 14, 等, 已更换硝化棉过滤器或亲和柱, 以实现更有效的分离。在这些技术中, 基于珠基的方法在小分子靶的核酸适体选择中得到了广泛的应用, 因为其简单的设置, 操作简单, 易于进行小分子分析。
有两组方法通常用于核酸适体选择小分子目标:目标13 和库14 基于固定化的方法。在前一组的方法中, 小分子靶被固定在固相, 如功能性磁性珠或琼脂糖珠通过共价键耦合反应。图书馆是用小分子包覆的珠子孵化的, 而那些在固体相中不束缚或弱于目标的序列, 只需执行洗涤和离心或磁性分离步骤就可以去除。然后通过 PCR 洗脱和扩增了束缚序列。小分子靶必须至少有一个功能组可用于偶联反应。对于没有合适官能团的人, 必须通过有机合成将功能组纳入原目标。精心设计的目标的综合可能涉及多个步骤, 有时也相当具有挑战性。对目标的结构修改也会对结合点和适配子的亲和性产生强烈影响。为避免这些问题, 本文开发了基于库的固定化方法, 将该库与磁珠上的互补 DNA 捕获探针进行杂交, 并结合靶点将其绑定序列脱落。在这组方法中, 目标被添加到绑定缓冲区中, 并且不需要任何功能组。PAEs 是邻苯二甲酸酯衍生物, 典型的 PAE 由邻苯二甲酸酯组和一或两个烷基链 (图 1A-d) 组成。PAEs 没有功能组可用于固相固定。因此, 基于库的固定化方法对 PAEs 的核酸适体选择显得更具吸引力。
一个良好的控制分散状态是至关重要的, 以确保所期望的丰富的图书馆通过 SELEX。然而, PAEs 是非常疏水性和容易形成聚集在水溶液中浓度高于几个µM, 即使在存在多个表面活性剂, 以促进分散。因此, PAEs 的分散状态是难以控制的, 很难选择适配子, 具体地绑定到单个 PAE 分子。为了解决溶解度问题, 选择了靶固定化方法, 将 PAEs 固定在亲水珠上, 通过共价键合。使用这种固定化的策略, 目标应该是在珠表面上的一个单一的分子状态, 而不是聚合。
锚定靶的结构设计对于成功地选择群特适配子也非常关键。结构设计需要考虑三因素。第一个因素是核酸适体选择的目的。考虑到选择组特定适配子的目的, 必须公开 PAEs 的通用组以核酸适体绑定, 并防止其余部分参与核酸适体绑定。因此, 应在侧链终端上引入功能组。开始时, 我们设计了 OH 功能化的 DEHP (4-OH−DEHP) (图 1E) 作为锚定目标, 并将其固定在羧酸磁珠16上。因此, 烷基链暴露在基体表面。我们试图选择磁性珠子与羧基基团作为固体基质。五轮选择后没有明显的亲和性改善, 裸基质的非特异性吸收保持强烈。
因此, DBP-NH2后来是基于以下思想设计的: (1) 邻苯二甲酸酯组比烷基链更有可能与核酸适体通过π-π栈和氢键相互作用;(2) NHS 介导的 carbodiimide 反应温和, 具有非常有效的耦合效率, 因此, 在 DBP 的烷基链末端引入 NH2 ;(3) 磁珠作为固相分割, 易于操作, 但对库的非特异性吸附较强。虽然在分离过程中, 琼脂糖珠的损失比磁性珠子高, 但对图书馆的非特异性吸附则低得多。因此, DBP-NH2被固定在环氧活化的琼脂糖珠上, 而邻苯二甲酸脂肪酸酯组暴露在基体表面。
选择缓冲器的选择是很重要的, 特别是对于具有不同溶解度的小分子靶点。需要仔细准备绑定缓冲区, 以避免聚合, 并确保持久性有机污染物在整个核酸适体选择和描述过程中的分散状态良好。在我们的研究中, 我们发现 DEHP 不能溶解在常规缓冲器没有表面活性剂, 显示两个不同的层次。在优化数量中添加多个表面活性剂后, 该层消失。明确的解决方案需要存储在温度高于20摄氏度, 以保持其状态。详细信息载于《议定书》步骤3.1。
双链 PCR 产物单链 DNA 的生成是 SELEX 过程中的一个关键步骤。目前在文献32、33中描述了几种不同的方法, 包括不对称 PCR、λ exonuclease 消化、链亲和素涂层珠的磁性分离和变性的粒度分离。尿素聚丙烯酰胺凝胶。
不同的方法有自己的长处和短处。目前, 最常用的 ssDNA 生成方法是用链亲和素包覆的珠子进行磁分离。该方法具有省时、操作简单等优点。缺点是它的高成本与其他方法相比。与此相反, 采用带 GC stem–loop 结构的反向引物进行尺寸分离的方法是最便宜的方法之一, 而单链 DNA 的收率在这些方法中是最低的32。在本协议中, 我们描述了使用λ exonuclease 消化产生单链 DNA, 这是最便宜的方法之一。结果表明, 单链 DNA 的屈服率与磁分离法和链亲和素涂层珠的两种方法相比较。此外, 我们发现, exonuclease 反应是由高浓度的盐抑制。在文献33中报告的 pcr 产物的不完全消化可能是由于 pcr 产物中存在太多的盐。此外, λ exonuclease 非常活跃且价格低廉 (图 4)。
适配子的特性和验证非常费力, 耗时, 并且在核酸适体发现管线33中体现了一个主要的瓶颈。大多数技术都是对较大的核酸适体结合伙伴 (> 1万) 有很好的质量敏感的方法, 但对于测量低分子量目标 (< 1000)15 之间的相互作用不够敏感。像 PAEs 这样的疏水性小分子适配子的特性和验证更加困难。它们的水溶性差导致滴定曲线的不饱和度, 从而防止了K d 在溶液中或力场适配子表面上的测定.因此, 我们通过在亲水性磁珠上固定的 DBP-NH 2 , 通过高吞吐量排序确定了已识别适配子的 K d, 以避免溶解度问题。适配子对其他 PAEs 的相对亲和性和选择性由竞争性化验确定, 核酸适体候选者被预先限定在 DBP- NH 2 附着的磁性珠子上, 并在孵化后释放到上清。与测试的 PAEs 或其他潜在的干扰物质。竞争试验的应用, 因为它提供了一个简便的亲和力比较 PAEs 之间没有功能组的表面固定化。此外, 磁性珠基荧光检测方法适用于小分子核酸适体相互作用的亲和性研究34。然而, 我们发现, 磁珠有时会导致荧光淬火的原因不明。因此, qPCR 测定用于亲和性测量。
本研究描述的电化学生物传感器的一个关键技术提示是电极35的表面钝化。由于 DEHP 的高疏水性, 它有很强的 nonspecifically 吸收到金电极上的倾向, 导致检测失败。最常用的表面钝化剂, 6-巯基-1-hexanol (MCH)36,37, 不足以防止 DEHP 的非特异性吸收, 而我们发现 HS-(CH2)2-[2 CH 2]6-DEHP3的有效性足以启用对38的敏感检测.
本程序描述了一种用于选择高度疏水性小分子的组特定 DNA 适配子的协议, 以及在电化学生物传感器中选择核酸适体的应用。该协议有助于选择其他疏水小分子, 并提供了关于高疏水性小分子的传感器发展的见解。核酸适体选择过程属于目标固定化方法的范畴。此类方法的局限性也存在于此协议中, 例如需要复杂的锚定目标综合, 以及固相对核酸适体结合的影响。本协议所描述的电化学生物传感器具有很大的优越性, 包括其简单的设计和高灵敏度。主要的缺点是它们的精度有限, 因为它们的动态范围非常广泛。因此, 这里描述的生物传感器更适合于筛选试验, 而不是定量测量目标。

随着经济的快速发展, 工业化的加速, 城市建设的不断加快, 环境污染问题也日益严重。典型的环境污染物包括重金属离子、毒素、抗生素、杀虫剂、内分泌干扰物和持久性有机污染物 (pop)。除了金属离子和毒素, 其他污染物是小分子, 通常由各种同源物组成。例如, 最有毒的持久性有机污染物包括多氯联苯、多环芳烃 (多环芳烃)、多溴联联苯醚 (二苯醚)、聚氯氯苯并 (PCDDs)、多氯氧芴 (呋喃) 和苯二甲酸酸酯 (PAEs)1,2, 它们都由许多同源项组成。小分子检测主要是以色谱/质谱技术为基础进行的, 因为其应用的多样性3,4,5,6。对于现场检测, 最近开发了基于抗体的方法7,8,9。但是, 由于这些方法对某些同类具有高度的特异特性, 因此必须执行多个测试。更严重的是, 新的同源生长得如此之快, 以至于他们的抗体不能及时生成。因此, 在一项试验中, 开发组特定的生物传感器来监测所有同源物的总水平, 可为评估环境污染状况提供一个宝贵的指标。
近年来, 核酸适配子在各种生物传感平台中被广泛应用为识别元素, 因为它们能够识别各种各样的目标, 从离子和小分子到蛋白质和细胞10, 11 ,12。适配子是通过一个体外方法来识别的, 所谓的配位系统进化的指数富集 (SELEX) 13, 14.SELEX 从随机合成单链寡核苷酸库开始, 其中包含大约 1014-1015序列。随机库的大小确保了 RNA 或 DNA 候选结构的多样性。典型的 SELEX 过程包括多轮的浓缩, 直到图书馆丰富的序列, 高度亲和性和特异性的目标。最后的浓缩池然后排序, 离解常数 (Kd) 和选择性反对潜在的干扰物质是由不同的技术, 如过滤器结合, 亲和层析, 表面等离子共振 (SPR),等。15
由于水的溶解度极差, 且缺乏用于表面固定的功能基团, 因此在理论上很难核酸适体选择持久性有机污染物。SELEX 的重大进展加速了适配子的发现。但是, 尚未报告持久性有机污染物的组特定适配子的选择。到目前为止, 只有对某些同类的高特异性的 PCB 结合 DNA 适配子被识别为 16.PAEs 主要应用于聚氯乙烯材料中, 将聚氯乙烯从硬塑料改成弹性塑料, 从而充当增塑剂。一些 PAEs 已被认定为内分泌干扰物质, 可对肝肾功能造成严重损害, 减少男性精子的运动, 并可能导致畸形精子形态和睾丸癌的发生17。未报告复合类或组特定的 PAE 绑定适配子。
这项工作的目标是提供一个代表性的协议, 以选择组特定的 DNA 适配子高疏水性小分子, 如 PAEs, 一个代表性的持久性有机污染物组。我们还演示了选定的核酸适体在环境污染检测中的应用。该协议为核酸适体发现其他疏水小分子提供了指导和见解。

<table>
	<tbody>
		<tr>
			<td>UV-2550</td>
			<td>Shimadzu,Japan</td>
			<td></td>
			<td>protocol, section 3.8.2</td>
		</tr>
		<tr>
			<td>DNA Engnine Thermal cycler,PTC0200</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>section 3.5.1.2 and 3.5.2</td>
		</tr>
		<tr>
			<td>C1000 Touch</td>
			<td>BIO-RAD</td>
			<td></td>
			<td>section 5.3.6 and 6.3</td>
		</tr>
		<tr>
			<td>VMP3 multichannel potentiostat</td>
			<td>Bio-Logic Science, Claix, France</td>
			<td></td>
			<td>section 7.4,7.8 and 7.11</td>
		</tr>
		<tr>
			<td>Epoxy-activated Sepharose 6B</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>10220020</td>
			<td>argarose beads, section 2.3 and 3.3</td>
		</tr>
		<tr>
			<td>Dynabeads M-270 carboxylic acid magnetic beads</td>
			<td>Invitrogen, USA</td>
			<td>420420</td>
			<td>magnetic beads,section 5.2. and 5.3</td>
		</tr>
		<tr>
			<td>Premix Taq Hot Start Version</td>
			<td>Takara,Dalian,China</td>
			<td>R028A</td>
			<td>polymerase, section 3.5.1.1</td>
		</tr>
		<tr>
			<td>PARAFILM Sealing Membrane</td>
			<td>Bemis, USA</td>
			<td>PM-996</td>
			<td>section 3.6.5</td>
		</tr>
		<tr>
			<td>Lambda Exonuclease</td>
			<td>Invitrogen, USA</td>
			<td>EN0561</td>
			<td>section3.7.1.2.The 10 &#215; reaction buffer is provided along with &#955; exonuclease by the provider.</td>
		</tr>
		<tr>
			<td>Dr. GenTLE 
			Precipitation Carrier</td>
			<td>Takara,Dalian,China</td>
			<td>9094</td>
			<td>section 3.6.2 and 3.8.1</td>
		</tr>
		<tr>
			<td>UNIQ-10 PAGE DNA recovery kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B511135</td>
			<td>section 4.2</td>
		</tr>
		<tr>
			<td>SYBR Gold nucleic acid gel stain</td>
			<td>Invitrogen, USA</td>
			<td>1811838</td>
			<td>nucelic acid stain dye, section 3.5.1.5</td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq II</td>
			<td>Takara,Dalian,China</td>
			<td>RR820A</td>
			<td>polymerase mix contaning polymerase and dNTPs, section 5.3.5</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 1132-61-2</td>
			<td>section 5.2.1</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Invitrogen, USA</td>
			<td>CAS: 25952-53-8</td>
			<td>section 5.2.2</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>6066-82-6</td>
			<td>section 5.2.3</td>
		</tr>
		<tr>
			<td>mercaptohexanol (MCH)</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 1633-78-9</td>
			<td>section 7.7</td>
		</tr>
		<tr>
			<td>Gold electrode</td>
			<td>Shanghai Chenhua</td>
			<td>CHI101</td>
			<td>section 7.4. - 7.11</td>
		</tr>
		<tr>
			<td>tris(2-carboxyethyl) phosphine hydrochloride (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 51805-45-9</td>
			<td>section 7.5</td>
		</tr>
		<tr>
			<td>O-(2-Mercaptoethyl)-O&#39;-methyl-hexa-(ethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 651042-82-9</td>
			<td>section 7.7</td>
		</tr>
		<tr>
			<td>diethylhexyl phthalate (DEHP)</td>
			<td>National Institute of Metrology, China</td>
			<td>CAS: 117-81-7</td>
			<td>section 7.11</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 9005-64-5</td>
			<td>polyoxyethy-lene(20) sorbaitan monolaurate</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>CAS: 9002-93-1</td>
			<td>non-ionic surface active agent</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5368</td>
			<td>10 mM phosphate buffer containing 1 M NaCl, pH 7.4</td>
		</tr>
	</tbody>
</table>

phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor

邻苯二甲酸酯 (PAEs) areone 主要群体的持久性有机污染物。由于同源性的快速增长, PAEs 的群特异检测非常理想。DNA 适配子已越来越多地应用于生物传感器平台上的识别元素, 但很少报道适配子对高度疏水性的小分子靶点 (如 PAEs) 的选择。这项工作描述了一种基于珠子的方法, 用于选择组特定的 DNA 适配子 PAEs。将氨基甲酸二丁酯 (DBP-NH2) 合成并固定在环氧活化的琼脂糖珠上, 使其在固定基质表面显示邻苯乙酯基团, 并因此选择组特定的粘合剂。通过定量聚合链反应和磁分离, 确定了核酸适体候选者的离解常数。适配子对其他 PAEs 的相对亲和性和选择性由竞争性化验确定, 核酸适体候选者预先限定在 DBP- NH 2 附着的磁性珠子上, 并在孵化时释放到上清被测试的 PAEs 或其他潜在的干扰物质。竞争试验的应用, 因为它提供了一个简便的亲和力比较 PAEs 之间没有功能组的表面固定化。最后, 我们演示了电化学 aptasensor 的制备方法, 并将其应用于邻苯二甲酸二 (2-己酯) 的超灵敏和选择性检测。该协议为核酸适体发现其他疏水小分子提供了见解。

我们设计并合成了氨基甲酸二丁酯 (DBP-NH2) 作为定位靶 (图 1F)。然后, 我们进行 DNA 核酸适体选择 PAEs 使用 DBP-NH 2 作为锚定目标和遵循经典的目标固定化的方法(图 2)。在每一个回合中, 使用变性页来优化 pcr 的周期数 (图 3) 进行了先导 pcr。强烈建议变性页面, 而不是本机页, 因为我们和其他同事发现, 在较高的循环数上显示在本机凝胶上的疑似高分子量副产品实际上是交联复合物形成的正确大小的序列。因此, 变性页的频带强度可以真正反映出正确产品的数量。单链 DNA 生成是另一个关键步骤, 并且已报告了此步骤的许多方法28。λ exonuclease 消化法是最便宜的方法。单链 DNA 生成的成功可以方便地检查本机页, 其中 PCR 产品显示为单 (前几轮 SELEX) 或多波段, 而只有一个波段显示后, 消化 (图 4)。
SELEX 在选择的第五轮以后被停止了, 因为大量脱氧核糖核酸积累在页的顶部, 即使在变性治疗以后 (其中脱氧核糖核酸在95°c 被加热10分钟在 2 x TBE–containing 8.3 M 尿素), 表明 dna 之间存在严重的交联, 并表明这些序列是丰富的。因此, 为高通量排序发送了池4 。高通量结果表明, 前100最频繁发生的序列是高度保守的, 是从一个家庭。顶部序列 DBP-1 显示 nanomolar 亲和性 (图 5), 并通过方便的 qPCR 检测 (如 "协议" 部分所述), 对 PAE 同源 (DBP、BBP、DEHP) (图 6) 具有良好的组特异性。
DBP-1 已被用来构造一个基于线位移的电化学 aptasenor 相应的我们以前报告的工作29。DEHP 传感器可以敏感地、有选择地响应 DEHP (图 7)。常见的环境污染物, 如重金属离子、抗生素和具有类似功能基团的小分子, 对 DBP-1 的反应非常微弱。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56814/56814fig1.jpg"> 图 1: (A) PAEs 的化学结构, (B) BBP, (C) DBP, (D) DEHP, (E) 4-OH DEHP, (F) DBP−NH2.此数字已在汉 Y 的许可下转载. et22<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56814/56814fig2.jpg"> 图 2: 使用 DBP-NH2作为锚定目标的 PAEs 的组特定 DNA 核酸适体选择过程。此数字已在汉 Y 的许可下转载. et22<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56814/56814fig3.jpg"> 图 3: 使用变性页进行 PCR 循环优化的典型结果.从左到右, 车道代表:20 bp DNA 梯子标准, 15 个周期 (没有 dna 模板), 20 个周期 (没有 dna 模板), 25 个周期 (没有 dna 模板), 30 个周期 (没有 dna 模板), 15 个周期, 20 个周期, 25 个周期, 和30个周期。在25个循环中观察到了最佳条件, 单产品带在高强度的情况下没有不需要的副产品, 而且在相应的空白控制中没有观测到带。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56814/56814fig4.jpg"> 图 4: 使用本机页消化磷酸化链的λ exonuclease 反应的优化.从左到右, 车道代表:20 bp DNA 梯子标准, 阴性 (无λ exonuclease), 2U, 5U, 8U 和10U。在 2U, 双 PCR 产物成为单链 dna 时, 观察到酶的最小用量。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56814/56814fig5.jpg"> 图 5: 基于 qPCR 的检测方法对 DBP-1 的亲和性测量.Kd的错误 (标准偏差, SD) 是从同一样本的三测量中计算出来的。此数字已在汉 Y 的许可下转载. et22
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56814/56814fig6.jpg"> 图 6: 自由 DBP-NH2 (10 µM) DBP-1 的相对亲和性测量, DBP (10 µM), DEHP (10 µM), BBP (10 µM), 乙酸乙酯 (10 µM), 苯甲酸 (10 µM), 邻苯二甲酸 (10 µM), 其他通过竞争化验.其他: 潜在干扰 (葡萄糖, 卡那霉素, 氨苄西林, 乙醇) 的混合物在10µM。该比值 (相对亲和性) 是通过在 PAE 绑定缓冲区中从珠子中释放的适配子数除以样品存在时从珠子上释放的适配子的数量来计算的。酒吧代表的是卑鄙的。SDs 是根据三个单独的测量结果计算出来的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56814/56814fig7.jpg"> 图 7: DEHP 电化学生物传感器用于超灵敏和 ultraselective 检测 DEHP: 机制 (A)、SWV 曲线 (B)、校准曲线 (C) 和选择性试验 (D) 。这些误差 (SDs) 是根据三个单独的测量结果计算出来的。此数字已在汉 Y 的许可下转载. et22<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56814/56814fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

本文提出了一种体外的协议, 用于对组特定邻苯二甲酸酯结合 DNA 适配子的筛选和表征。在电化学 aptasensor 中也包括了所选核酸适体的应用。

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

phthalic-acid-ester-binding-dna-aptamer-selection-characterization

Nanyang Technological University

Research

JoVE Journal

Chemistry

9.7K 次观看.  Capital Normal University. 本文提出了一种体外的协议, 用于对组特定邻苯二甲酸酯结合 DNA 适配子的筛选和表征。在电化学 aptasensor 中也包括了所选核酸适体的应用。

视频: Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

科研

化学

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

一个反分析方法化工运输的油漆表征

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

氯离子的动态电化学测试

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

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.

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 ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

电化学阻抗谱作为电化学速率常数估计的一种工具

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

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 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 ...

Electrochemical Roughening of Thin-Film Platinum Macro and Microelectrodes

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries of the metal. Using this method, a crack free, thin-film macroelectrode surface with up to 40 times increase in active surface area was obtained. The roughening is easy to do in a standard electrochemical characterization laboratory and incudes the application of voltage pulses followed by extended application of a reductive voltage in a perchloric acid solution. The protocol includes the chemical and electrochemical preparation of both a macroscale (1.2 mm diameter) and microscale (20 &#181;m diameter) platinum disc electrode surface, roughening the electrode surface and characterizing the effects of surface roughening on electrode active surface area. This electrochemical characterization includes cyclic voltammetry and impedance spectroscopy and is demonstrated for both the macroelectrodes and the microelectrodes. Roughening increases electrode active surface area, decreases electrode impedance, increases platinum charge injection limits to those of titanium nitride electrodes of same geometry and improves substrates for adhesion of electrochemically deposited films.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries. The electrochemical techniques of cyclic voltammetry and impedance spectroscopy are demonstrated to characterize these electrode surfaces.

薄膜铂金宏观和微电极的电化学粗加工

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries ...