Name: a method for selecting structure-switching aptamers applied to a colorimetric gold nanoparticle assay
Uploaded: 2015-02-28T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay at JoVE.com

This research was performed while the author (JAM) held a National Research Council Research Associateship Award at Air Force Research Laboratories. Co-author MW held a Wright Scholar Fellowship supported by the Air Force Office of Scientific Research. This work was supported by Air Force Office of Scientific Research, Air Force Research Laboratory, and Bio-X Strategic Technology Thrust.

1.库和引物设计与合成<ol><li>设计的初始库和引物（这个话题已经被广泛审查了以前的出版物）25,26。 <ol><li>合成使用标准的亚磷酰胺化学研究以下序列: 图书馆:GAATGGATCCACATCCATGG-N 40 -TTCACTGCAGACTTGACGAAGCTTGACGAA 正向引物:GGAATGGATCCACATCCATGG 反向引物:生物素AAGCTTCGTCAAGTCTGCAGTGAA </li><li>设计基因捕获探针是互补图书馆的5&#39;区域。选择通过在线熔化温度的软件分析的初始长度，以提供一个熔化温度略高于室温，用每个后续碱供给增加的熔化温度。 滨海捕获探针:CCATTCC生物素 滨海捕获探针:TCCATTCC生物素 滨海捕获探针:ATCCATTCC生物素</li></ol></li><li>净化用标准HPLC库。脱盐sufficient为引物和捕获探针。重建在无核酸酶的水的寡核苷酸在-20℃所需的浓度，并存储长达几个月。 </li></ol> 2.缓冲区，图书馆和样品制备<ol><li>制成500毫升之缓冲液中的微孔或无核酸级水以50毫摩尔Tris，137 mM氯化钠，5mM的MgCl 2的，pH为7.4的浓度。过滤通过无菌0.2μm膜的缓冲器;存储能够在环境条件下个月。 <ol><li>可替代地，可使用其它缓冲剂，例如PBS（磷酸盐缓冲盐水），HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸） 等。 </li></ol></li><li>设置了两个thermomixers;设置1至95℃，并保持其他在环境条件（约20℃，在​​此工作）。准备一个冰桶。 </li><li>热/管理单元通过将100微升文库冷却该DNA文库（〜2.5纳摩尔为10 15 -10 16个序列），在恒温设定吨95℃下5分钟。将管在冰上而磁性珠粒制备（部分3.1-3.6）。确定由紫外吸收DNA文库中的λ= 260nm处的实际浓度和施加适当的转换因子为库。 </li><li>在培养基中适当目标溶解度准备目标原液。 <ol><li>准备100毫米分析股票DMSO。这些股票是可行的约1个月储存时在环境条件下，但不同的目标将表现出不同的降解分布。 </li></ol></li></ol> 3.准备磁珠和选择首轮<ol><li>涡旋6.7×10 8珠/ ml的链亲和素包被的磁珠并取出400微升到一个新的离心管中。将管在磁铁2分钟，除去上清液。 </li><li>加入400微升的结合缓冲液，涡旋，置后吸取上清液洗珠在磁体的管以分离珠。重复此过程三次。 </li><li>通过再悬浮于400μl的结合缓冲液将小珠用50微米（最终）捕获探针和孵育10分钟，在恒温在环境条件下缓慢搅拌固定于磁珠上的捕获探针。 </li><li>通过重复在第3.2节以去除游离捕获探针的洗涤步骤洗珠三次400微升结合缓冲液。 </li><li>重悬珠子在400微升的结合缓冲液。除去将100μl珠子溶液用于下一步骤，并保留剩余的300微升，在4℃用于进一步的选择轮次，最多三个星期。 </li><li>用200微升作为第3.2节结合缓冲液洗两次珠。 </li><li>从2.3节中添加100微升卡扣冷却的DNA的洗涤珠子，并使用恒温在环境条件下温育30分钟并轻轻搅拌。 ñOTE:在第一回合中的DNA浓度比随后的几轮，以尽量减少在较早轮次，其中唯一的序列是理论上存在序列损失更高。 </li><li>定量中使用的紫外线吸收上清液中的DNA为2.3节和3.7节中加入，以评估的DNA结合至珠的量的初始量减去此。 <ol><li>用200μl结合缓冲液，然后用200微升结合缓冲液轻轻搅动在恒温5分钟，在环境条件下两个附加洗涤洗珠。 </li></ol></li><li>重悬珠子在200μl的100μM的靶稀释在结合缓冲液，并旋转在恒温30分钟，在环境条件下。制备工作溶液之前选择新鲜在每一天的开始，因为目标和控制观察到聚集的随时间水溶液出来。保留上清溶液含有靶 - 洗脱序列S代表进一步研究。 </li><li>加入200微升1μM孕酮在3.8.1节准备的珠子执行完成1-2轮后负选择。轻轻摇晃的恒温在环境条件下30分钟。 </li><li>用磁力架捕获珠子和200微升的结合缓冲液之前，皮质醇除了在第3.9节弃上清，洗珠两次。 </li></ol> 4.富集监控，PCR和单链DNA生成<ol><li>上清液溶液添加至透析盒并监控选择富集。透析是必要从洗脱的DNA取出皮质醇因为皮质醇存在高得多的浓度比的DNA，并且还具有UV吸收峰重叠的标准DNA的UVλ 最大 = 260纳米。 <ol><li>执行此步骤每隔轮（轮2，4，6，和8）在前几轮选择。这减轻样品洛杉矶S当每个序列的潜在少数拷贝数存在（最高分集）。对于更快的结果，可替代的方法，例如大小排阻柱可以用作良好。 </li></ol></li><li>透析盒与新鲜250毫升的结合缓冲液进行2小时，在环境条件下的两倍的样品。更换缓冲和孵化在4°CO / N。 </li><li>分析由紫外光谱的透析池，以确定DNA的靶中洗脱的百分比（比较3.8节的结果）。 </li><li>制备3％的琼脂糖凝胶，用1％w / v的溴化乙锭。在这项工作中，从1.5克琼脂糖，50毫升TAE缓冲液（40毫摩尔Tris醋酸，1mM EDTA中，pH值= 8.0），和10μl溴化乙锭的制备凝胶。 </li><li>通过利用〜5-15个周期的小规模的PCR（5×50微升等份）周期优化透析池中。对于PCR成分，可以使用1×PCR反应缓冲液（所有终浓度），200μM的dNTPs，DNA模板的10％的反应体积，500nM的各引物，和2.5ü聚合酶（2.5 U /μl的股票）。 <ol><li>运行的PCR使用优化的条件，在95℃进行2分钟，接着是95℃（30秒），55℃（30秒）重复循环，72℃（1分钟），然后72℃（10分钟）最后延伸并保持在4℃。 </li><li>采用25个基点的标准梯，比较和可视化的凝胶成像仪。选择产生最高强度带在正确的大小标记物，而不不需要的副产物的循环数。 </li><li>通过组合10微升每个PCR循环与2微升6X蓝/桔样染料，并装载到3％的琼脂糖凝胶在步骤4.4（125 V 45分钟），制备5个独立的孔中进行分析的周期最优化的结果。 </li></ol></li><li>进行利用的条件和相应的循环次数在4.5节确定了大规模PCR。通常被要求6-8毫升的PCR反应，以获得1-2.5纳摩尔用于每个轮在此选择。使用8条管，以简化处理所需的许多管子，以等分的该卷PCR扩增。 </li><li>制备3％的琼脂糖凝胶在部分4.4中描述，并验证该PCR产物带出现在正确的位置下面的部分4.5.2-4.5.3的步骤。 </li><li>整个双链DNA的产品从4.7节转换成通过下面的步骤进行单链DNA: <ol><li>添加300微升链霉包被的珠子（没有磁性），以一小柱堵塞筛板。用5ml结合缓冲液通过将路厄锁注射器并施加轻柔的压力，柱塞洗珠。从柱中取出注射器和每增加一个材料之后移除注射器离线柱塞所以珠粒不会被柱塞抽吸干扰。 </li><li>添加的PCR产物，并通过柱，使用在柱塞温和的压力传递给它。使用，使得不超过1-1.2毫升的PCR产物被加入到每个列数列。放弃最后的FL流通过自该DNA将被保留在由上所述反义链的生物素部分的列。 </li><li>用5毫升结合缓冲液洗涤该柱。 </li><li>通过加入0.5ml的0.2M的NaOH Dehybridize该DNA。保留洗脱物，因为它包含的单链正义DNA。 </li></ol></li><li>通过加入0.5毫升至通过用不含核酸酶的水洗涤脱盐柱脱盐的ssDNA中的0.2M NaOH中。放弃最初的0.5毫升液，再加入1 ml核酸免费的水和收集这些脱盐单链DNA样本。将需要对每个0.5毫升部使用多个脱盐柱。 <ol><li>确定的DNA在λ= 260nm处洗脱通过紫外吸收的浓度。 </li><li>真空干燥该样品和重组中的结合缓冲液的适当体积。当然，如果没有为下一轮选择得到足够的DNA，重复部分4.6-4.9.2。 </li></ol></li></ol>选择和排序5.随后的几轮<ol><li>调整取决于富集监测（4.1节）的结果的选择的条件的严格性。一般地，使条件在连续几轮选择更严格，以便从池中选择的最高亲和力的粘合剂。 注意:这可能涉及增加的DNA浓度，增加的数量，时间，或在洗涤步骤体积，降低靶浓度的组合，提高了负选择化合物的浓度，或各种其他方法27。 <ol><li>应用适当的控制适当，以保证用于目标分子的序列的特异性。在这项工作中，施加负选择分子（孕酮）至系统（ 表1）开始第3轮和中浓度在整个选择增加。在开始11轮，预孵化池DNA为30分钟，10〜20微米的孕激素之前，固定在珠（​​第3.7节之前）。 </LI&gt; <li>增加捕获探针的长度后最大富集是观察。 </li></ol></li><li>准备最后的游泳池（15轮）和其他代表池测序最大限度地丰富（13轮）和微创丰富（第6轮）的条件下通过PCR扩增兼容引物和高保真聚合酶。 <ol><li>设置一个50μl的反应体积中使用终浓度1×（反应缓冲液）中，200μM（每种dNTP），400纳米（各引物），0.5微升的DNA池，和0.5微升聚合酶。周期优化每一轮使用使用相同的PCR条件，除了一个7分钟保持在72℃最终延伸在4.5节中概述的步骤。 </li><li>送50微升制备的池来测序设施在微量离心管中与盖与非粘合剂包裹彻底密封。将管在一个15毫升的锥形管挤满了泡沫包装和船舶O / N用冰袋来测序设施。 </li></ol&gt; </li><li>确定每个序列的拷贝数来评估排序池（S）的富集。 <ol><li>使用代码编写/整理FASTA格式的步骤（见补充代码文件）的新一代测序数据，以确定每个序列中由测序设备提供的所有池的数据重复的频率/拷贝数: </li></ol></li><li>分析的最高拷贝数序列的特异性和亲和力。相互作用（蛋白质或小分子），所述适体结合后的结构性能，以及预期的亲和力的类型将决定哪一种方法是适当的。本主题中进一步详细审查了一些参考1,28-30中。 </li></ol> 6.金纳米粒子合成与检测<ol><li>使用合成柠檬酸还原法21金纳米粒子。混合98毫升的微孔水用2ml 50mM的氯金酸水溶液，并加热至沸腾。 </li><li>加入10毫升38.8 mM柠檬酸钠作为一旦开始回流。几分钟后颜色会变成红色。继续搅拌该溶液20分钟以热关闭。 </li><li>允许金纳米粒子溶液通过0.2微米的聚酯膜冷却至室温，然后进行过滤。存储所有的金纳米粒子悬浮在一个黑暗的琥珀色瓶子。 </li><li>计算金纳米粒子浓度通过UV吸收，利用2.4×10 8升摩尔-1 -1 31中的消光系数。浓度为10nM的在这项工作中，为17±0.6纳米的尺寸通过动态光散射21来确定。 </li><li>孵育用10nM金纳米粒子DNA的1小时在由铝箔保护环境条件。而变化金纳米粒子的体积，以提供足够的溶液以使将要执行所有所需的测试（〜1-2毫升）中。 73，120，和200的D / NP（每金纳米粒子的DNA分子）装载密度进行了检查在这项工作中，而且体积和浓度会相应地变化。 </li><li>使用HEP 1:稀释DNA /金纳米粒子溶液1ES缓冲液（20mM HEPES，2mM的MgCl 2的，pH值= 7.4）。使混合物平衡在室温覆盖铝箔。 注:该步骤所需的时间将需要由研究人员进行优化。在这项工作中，时间范围从几分钟到O / N进行了调查。 </li><li>制备分析物（皮质醇，胆酸，2-甲氧基萘）储备溶液在100毫在DMSO中并覆盖有铝箔。做出新的初始解决方案之前，每个实验稀释股票为100微米的1/3稀释皮质醇结合缓冲液（2.1节）的。进一步稀释1/3的结合缓冲液的初始解，使得靶的恒定体积（10微升）加入到生成的范围内的浓度。 </li><li>优化通过加入80微升的DNA /金纳米粒子溶液与10微升的1/3结合缓冲液所需要的盐浓度（称为"空白"）。 <ol><li>孵育20分钟，在RT下再加入不同体积的NaCl SOLUT的离子。寻找盐诱导勉强视觉上明显朝着加入盐（13-40微升1 M氯化钠在这项工作中使用）后，一个蓝色的色调变化量。这提供了一个很好的起点，但可能需要进一步的调整后的结果，包括目标添加。 </li></ol></li><li>结合80微升的DNA /金纳米粒子溶液与10微升靶溶液孵育20分钟，在室温。利用96孔板和多道移液器同时分析多个目标浓度，总是包括空（第6.8节）。 <ol><li>添加的NaCl溶液适当体积（13微升1M NaCl的是在这项工作中使用），并立即用酶标仪分析的吸光度在650和530纳米。 </li></ol></li><li>绘制结果作为吸光度值与目标浓度标准化相对空白测量的比例（E 650 / E 530）。 </li></ol>

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分配表:获准公开发行;发行分布是无限的（88 ABW-2014-4103）。 作者宣称，他们有没有竞争的财务权益。

一个事实，即小分子是生物兴趣的，而只是构成报告的所有适体的19％，使设计用于选择适体的适用于具有重大意义的小分子的方法。小分子的SELEX是因为更少的官能团，结构基序，和表面积可供与相比蛋白1序列相互作用特别具有挑战性的，并且已经估计，选择用于尝试所有目标的不少于30％已导致适体38。因此，人们必须以成功地选择适体的小分子靶分外知道实验设计和执行。 在这项工作中所描述的适体的选择方法是有利的，因为它适用于小分子，而不需要任何化学修饰可以改变它们的结合性质。它也是基于洗脱，这意味着自吨他的DNA，而不是目标，最初绑定然后从磁珠由目标洗脱的DNA与珠粒基质相互作用不会被扩增为下一轮选择的，因为粘合剂到感兴趣的分子被释放到上清液中的缓冲和非特异性基质粘合剂保持绑定。相反地，是必需的固定目标到固体支持物每轮因为DNA中与基质相互作用将除了目标粘合剂1被放大的方法对基质的负选择方法。当前方法的目的是作为一个Tm值有限的选择策略。这意味着，该cDNA /池杂交的Tm值保持接近室温。莫尔斯使用类似的策略，但施加的6聚体cDNA探针用的Tm &lt;10℃下用选择条件在4℃下17。我们的应用是在室温进行的生物传感检测，所以该cDNA的长度是根据调整LY。这样可以使选择足够低的严格性，使得因为目标结合相互作用中发生不诱导能够破坏的cDNA结合的构象变化的远程位置的潜在的粘合剂不会丢失。然而，该方法的分割效率低，因为有些序列会dehybridize完全基于热力学。与此相反，Nutiu 等人施加的15聚体的cDNA，并且是唯一的能够鉴定适体为2的四个目标，可能是因为在15聚体的Tm值太高，以允许释放由小分子靶18。因此，在当前选择的策略将需要多轮去除背景序列，通过控制选择的严格和减少潜在的粘合剂成功的可能性将增加的损失。 许多研究人员对新适配子的选择是不知道相关的PCR的重要方面潜在的粘合剂。周期优化（第4.5节）是必需的，因为过度的扩增DNA文库的产生不需要的副产物（通常在尺寸大）在高循环数，以及所期望的产品可能完全用过量只有5个循环39的消失。在过度放大的情况下，将PCR产物不再代表那些由目标洗脱序列，和选择成功的机会都显著减少。 图4示出的周期最优化的例子，从第5轮在此工作。最低循环产生一个最小的产物条带，该带的增加量，通过8个周期;在10个周期的频带开始到较高尺寸过的扩增产物的过渡，并且几乎完全由过度扩增产物的内12个周期。另一个细节，许多研究人员在SELEX涉世可能会忽视的是，整个池必须在大规模的PCR扩增放大的FIR（第4.6节）第一轮，以减轻粘合剂的损失。从寡核苷酸可能独特序列的数目为4 N，其中4表示的四个核酸碱基，而N是在库的随机区域的碱基数。对于这项工作中，N = 40，造成了1.2×10 24个可能的独特序列的序列空间。 2.5纳摩尔在第一轮选择中使用的库对应于〜10 15分子，这意味着DNA的每个副本是有可能在初始池作为一个独特的序列表示。因此，DNA的整个体积由目标从珠粒中洗脱，必须被放大到保留每个潜在的粘合剂的副本为下一轮选择。一旦这个初始放大已经发生，多个副本可供选择在假定均匀溶液取样以下回合。 对象的浓度也应该仔细考虑在每一轮。 Nutiu 等人使用整个选拔过程1 mM的目标达浓度，选择了ATP适体具有K D = 600μM18。无论是当前的工作和莫尔斯17的研究始于100μM的目标，在整个选拔过程中减少，导致适体低亲和力微摩尔。正是这一轮紧缩应增加（低目标浓度）取决于富时观察到的。另一个关键步骤是应用适当的阴性选择步骤，以便适体展示特异性靶分子。其控制用于为所选择的适体的预期应用而定。例如，适体15-1被设计用作在对皮质醇生物传感检测一个识别元件，所以孕酮用作阴性选择分子，因为它是一种代谢的前体，以皮质醇是在生理液体40中。 ATP的适体通过Nutiu 等选择。&lt;/ em&gt;的未包括阴性选择步骤，用结构上相似的分子，包括ADP，AMP，腺苷，和的dATP 18相互作用。 最后需要说明的考虑是，金纳米粒子检测往往需要大量的优化每一个适配子/目标对。寻找盐诱导勉强视觉上明显朝着加入盐后，一个蓝色的色调变化量是一个很好的起点，但可能需要观察响应调整的起点。我们还发现，在测定产生取决于缓冲液的浓度（在选择缓冲液可能需要的稀释，因为缓冲器的高盐浓度经常引起凝集）和组合物，DNA的覆盖度（ 图3），样品制备（一些有机大大不同的反应用于溶解靶溶剂可能引起高的背景下，掩模定位响应），温度，盐的类型和浓度，以及incuba化时间（包括DNA与金纳米粒子和DNA /金纳米粒子与目标）。下完全优化的条件下，结果通常观察到与目标的温育时间&lt;5分钟，显示出金纳米粒子的生物传感平台的快速目标响应的优点。例如， 图5中的适体的温育时间/金纳米粒子步骤是从O / N（ 图3）减少到30分钟，并立即盐溶液中加入（&lt;10秒）目标添加后，而不是20分钟后（ 图3 ）以73的D / NP的装载密度。这增加了使用以前的条件（ 图3）的皮质醇响应于〜比空白（ 图5A）高82％，在10微米的目标浓度与〜40％。这种反应可以区分用肉眼（ 图5B）。需要注意的是皮质醇检测的线性范围比使用以前的条件，这表明在这些条件可以为所希望的优化不同检测范围。胆酸和2-甲氧基萘（2MNP）对照没有产生显著响应（ 图5A-B）。研究人员必须认识到，减少这些孵育时间可能有利于分析与金纳米粒子表面，这将有助于整体增强的信号（目标）或背景（非目标分子）的反应增加。因此，仔细分析金纳米粒子的设计和性能特性是必要的每一个适配子/目标对。 这个过程描述的协议，用于选择功能的生物传感平台的小分子结构切换适体，如所描述的金纳米粒子测定中，其中所需要的DNA的构象变化，以检测目标的存在。然而，该方法可以适用于其他生物传感器系统，如电化学或荧光发挥功能上相同的前提下对几乎任何尺寸的目标。该协议的功率可进一步由实验开发几种方法。首先，在选择自身可以潜在地通过调查优化的方法，例如确定的cDNA /文库杂交，提供了一个相互作用是弱到足以用小分子靶相互作用，但足够坚固，以减少量的理想的Tm提高背景序列中，从热力学dehybridized单纯。这会凝结所需选择循环的次数，从而节省时间和劳动减少试剂消耗。进一步调查的修改的选择是要确定最有利的浓度的珠子和DNA，使序列的多样性群体被暴露于目标，尤其是在第一轮。证明 - - 原则这些实验的成功提供了基础投入更多的资源对优化和调整金纳米粒子缓冲区分析生理液体。有合法需要一个快速，强大的生物传感平台，可以赋nction作为个体的生理状态的诊断工具在人血清中的金纳米粒子测定的，并进一步发展，汗液或唾液将满足该电流间隙。

小分子长期以来被认为是在不同的生物过程，如生理毒性或营养，细胞信号传导，并作为药物治疗疾病1发挥至关重要的作用。各种小分子也被建议作为表示生理条件包括压力2,3，疲劳4和5疾病的生物标志物。例如，升高的皮质醇水平与应力，这可能导致降低的生理性能和其他健康条件6-8。同样地，在变体的某些肽的唾液中的比率是预测疲劳，其中生理表现包括注意力不集中，受损的反应时间，并减少认知功能4。因此，目标特定的生物传感器的发展，监测小分子的水平可用于评估一个逐张的健康和性能提供了宝贵的指标超大超大超大超大。 在历史上，小分子检测已经由劳动密集的分离技术或通过基于抗体的识别6,7执行。最近，核酸适体9,10已经成为了具有超过在特定应用中的抗体显着的优点识别元件。与它们结合大小从金属离子11对组织12上的目标的能力，适体已被广泛地在生物传感平台13,14识别元件施加。相比于抗体，适体是化学合成的，并且它因此简单地介绍重现的化学修饰对于表面固定或报告。适体可与通过修改所使用的选择条件所需的条件下的高靶特异性进行选择，而抗体被限制为在生理条件15,16。此外，适体能够高密度配体由于THEIR尺寸更小，从而导致更高的目标信令效率上的生物传感器平台，和适体的增强的稳定性允许重复使用和长期贮存传感器16。 使用被称为SELEX方法，其中序列的初始库是从10 13 -10 15个不同的分子进化到包含若干（10 1 -10 2）序列与潜在的目标分子结合的富集最终池的过程中产生的适体。然后最终的富集池测序，所述解离常数（K d）的自结合的最高拷贝数序列的测定与目标分子获得。池至最终富集群体的进化是通过监测池结合在每一轮靶的百分比，直到获得最大的富集跟踪。这发生在若干进化轮，其中结合序列是从nonbinders和AMPL分配后指定使用聚合酶链式反应（PCR）。以有效地进行分区和保留的粘合剂的能力是选择和直接影响所选择的适体15的特性的效率的主要决定因素之一。在SELEX划分阶段为小分子更具挑战性，因为它们不具有大小或多种官能团的辅助蛋白质靶1,15分区过程。例如，许多蛋白质分区平台依靠尺寸或基于电荷的分离，但是对结合的DNA小分子靶复合物的特性，一般不高于非结合序列的显著不同，从而导致无效分区1。替代SELEX分区方法可以涉及固定或目标的标记，这可能改变一个小分子的结合特性。因此，选择程序设计来产生适体的小分子靶向řequires特殊考虑。 各种修改已经被应用到原始的SELEX方法，以提高该过程的分隔效率，提高了序列的亲和力或特异性在最终池，或使之更适合于不同的应用程序15,16。一变形例的SELEX能够选择为小分子的设计，以产生结构切换适体，适体或该经过在与靶分子17,18相互作用的构象变化。在这种方法的一个例子中，文库杂交以一小段的非结合互补DNA（cDNA）固定于磁珠17。在靶结合，结合序列的构象变化使它们从cDNA到dehybridize，释放到上清液溶液，而其余nonbinders杂交到的cDNA /珠粒磁性分区和丢弃。这种方法是ADVANtageous为小分子，因为它不需要固定化的靶分子或标记来实现分离。 适体，其能够构造切换的具有对任何潜在的生物传感器平台，需要在适体结构的变化来检测目标分子的存在下一个有价值的特性。具体地，适体可以用金粒子（AuNPs）被组合以创建该功能结构的基础上切换原理，以产生盐挑战19,20后在靶分子存在下的比色反应检测。在一个金纳米粒子测定中，在单链DNA（ssDNA）的适体最初吸附至金纳米粒子表面，并从盐挑战保护它。靶的存在诱导暴露该金纳米粒子表面上，从而产生红至蓝的颜色变化之后加入盐的适体的构象变化。金纳米粒子分析也被证明对放大信号，其中微摩尔亲和力的适体S能探测目标的幅度比解离常数（K D）21低级别的订单。此功能对于小分子目标，其中亲和力通常范围从低到微摩尔毫摩尔浓度1,15特别有吸引力。一般来说，实验也快速和相对简单的执行，鼓励适体，金纳米粒子作为检测生物传感器检测平台的进一步研究。 这项工作的目的是提供一种用于选择结构切换适体的小分子用于使用应力标记皮质醇作为代表性分子生物传感应用的通用协议。一个金纳米粒子的生物传感器检测方案感兴趣，因为它的操作和比色读出的简单，但结构切换适体都适用于替代传感平台的产出，例如电化学22或23的荧光也通过在适体conformatio变化提供检测ñ对目标具有约束力。相比于以前的方法中，多个负选择步骤均纳入设计17,18，和一个简单的基于UV的富集检测方案被实施（ 图1）。该协议是在与传统的方法17中使用的放射性配体富集检测方案的对比。所提出的方法还掺入的增加在选择用于增加分区效率并有效地调整选择的严格的最大富集观察24后的cDNA捕获探针的长度。调谐严紧导致DNA的目标在最终池洗脱较低的总比例，但产生了一个序列的是结合有增强的亲和性相比从较早轮的最高拷贝数序列更高的拷贝数。从最终池的最高拷贝数序列被施加到金纳米粒子测定来说明，该序列是适合于一个平台这需要一个结构性的变化来表示目标结合。这种基于缓冲器测定表明，该金纳米粒子测定的响应可以通过改变的DNA的密度施加于表面进行改性，并作为验证的原则工作投入未来的研究努力，朝向显影小分子基于适配子的生物传感器金纳米粒子在生理性液体。

<table><tbody><tr><td>Dynabeads M280 Streptavidin</td><td>Invitrogen</td><td>112.06D</td><td>Compatible with DynaMag-2 Magnet</td></tr><tr><td>DynaMag-2 Magnet</td><td>Invitrogen</td><td>12321D</td><td>Compatible with Dynabeads M280 Streptavidin</td></tr><tr><td>Streptavidin Sepharose High Performance</td><td>GE Healthcare</td><td>17-5113-01</td><td>This is a high density streptavidin product for ssDNA preparation</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H4001</td><td>&amp;ge;98%</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P0130</td><td>&amp;ge;99%</td></tr><tr><td>Cholic Acid</td><td>Sigma</td><td>C1129</td><td>&amp;ge;98%</td></tr><tr><td>NanoDrop ND-1000</td><td>NanoDrop</td><td>ND-1000</td><td>Replaced by ND-2000 model</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>&amp;ge;99.9%</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S9888</td><td>&amp;ge;99.5%</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Fluka</td><td>63068</td><td>&amp;ge;98%</td></tr><tr><td>500 ml Filter System</td><td>Corning</td><td>430769</td><td>0.22 &amp;micro;m cellulose acetate membrane</td></tr><tr><td>DNA Library</td><td>Operon</td><td>Custom</td><td>HPLC purified</td></tr><tr><td>All other DNA</td><td>IDT</td><td>Custom</td><td>Capture probes and primers &amp;#8212; desalted; Aptamers &amp;#8212; HPLC</td></tr><tr><td>Thermomixer&amp;nbsp;</td><td>Eppendorf</td><td>R</td><td>Any model with&amp;nbsp; temperature and mixing speed control will work</td></tr><tr><td>PfuUltra II Fusion HotStart DNA Polymerase, 400 rxn</td><td>Agilent</td><td>600674</td><td>Taq polymerase can be used as well</td></tr><tr><td>Deoxynucleotide Mix, PCR-Grade&amp;nbsp;</td><td>Agilent</td><td>200415</td><td>Other brands will work here</td></tr><tr><td>10x Cloned Pfu DNA Polymerase Buffer</td><td>Agilent</td><td>200532</td><td>This buffer was optimized for PfuUltra II polymerase</td></tr><tr><td>GeneAmp PCR System</td><td>Applied Biosystems</td><td>9700</td><td>Any model that allows researcher to open lid for cycle optimization will work</td></tr><tr><td>Agarose-LE</td><td>Ambion</td><td>AM9040</td><td>&amp;ge;99.9%</td></tr><tr><td>Ethidium Bromide</td><td>Sigma</td><td>E-1510</td><td>Acute toxicity, inhalation; suspected carcinogen</td></tr><tr><td>Empty Synthesis Columns, 1 &amp;micro;m Expedite Style</td><td>Glen Research</td><td>20-0021-01</td><td>This model is ideal because of the Luer-Lok fitting to couple with syringe</td></tr><tr><td>Replacement Filters Expedite</td><td>Glen Research</td><td>20-0021-0F</td><td>1 &amp;micro;m size</td></tr><tr><td>Illustra NAP-25 Columns</td><td>GE Healthcare</td><td>17-0852-01</td><td>Any brand with DNA grade resin gel filtration column will work</td></tr><tr><td>Slide-A-Lyzer Dialysis Cassette</td><td>ThermoFisher Scientific</td><td>66205</td><td>2k MWCO used</td></tr><tr><td>Gold(III) chloride hydrate</td><td>Sigma</td><td>254169</td><td>99.999% purity is important</td></tr><tr><td>Sodium Citrate Dihydrate</td><td>SAFC</td><td>W302600</td><td>We have found that the compound produced from different manufacturers greatly affects AuNP assays</td></tr><tr><td>SpectraMax&amp;nbsp;</td><td>Molecular Devices</td><td>M5</td><td>Results were similar to Bio-TEK system</td></tr><tr><td>Synergy</td><td>Bio-TEK</td><td>HT</td><td>Results were similar to Molecular Devices system</td></tr><tr><td>HEPES Buffer</td><td>Amresco</td><td>J848</td><td>Any brand that makes a sterilized product will work</td></tr><tr><td>250 ml Filter System</td><td>Corning</td><td>430767</td><td>0.22 &amp;micro;m cellulose acetate membrane</td></tr><tr><td>UV Spectrophophotometer</td><td>Varian</td><td>Cary 300&amp;nbsp;</td><td>Other brands will work here</td></tr><tr><td>5 ml Syringe</td><td>Becton-Dickinson</td><td>309646</td><td>This model is ideal because of the Luer-Lok fitting to couple with Expedite column</td></tr><tr><td>ThermoEC Minicell Primo</td><td>ThermoFisher Scientific</td><td>EC320</td><td>Compatible with Power Supply</td></tr><tr><td>Power Supply</td><td>Fisher Scientific</td><td>FB300</td><td>Compatible with ThermoEC Minicell Primo</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D8418</td><td>Flammable liquid</td></tr><tr><td>2-Methoxynaphthalene</td><td>Sigma</td><td>148245</td><td>99%</td></tr><tr><td>Assay Plate</td><td>Corning</td><td>3370</td><td>96-well Flat Bottom, Sterile</td></tr></tbody></table>

a method for selecting structure-switching aptamers applied to a colorimetric gold nanoparticle assay

小分子由于其生理意义，作为人类健康和性能的各个方面的生物标志物为生物传感应用丰富的目标。核酸适体已作为生物传感器平台的识别元素被越来越多地应用，但选择的适体对小分子的目标，需要特殊的设计考虑。这项工作描述的修改和设计选择结构切换的适体小分子目标的方法关键步骤。从杂交到互补DNA捕获探针上的磁性珠粒的DNA文库结合序列是从nonbinders通过在构象的靶引起的变化分开。这种方法是有利的，因为序列结合的载体基质（珠）将不会被进一步放大，并且它不需要在目标分子的固定。然而，捕获探针的熔解温度和库保持在或略高于室温，以使得序列吨基于热力学帽子dehybridize也将存在于上层清液。这有效地限制了分区效率（能够单独靶结合从nonbinders序列），并且因此许多选择轮将要求去除背景序列。报道的方法不同于前面的结构切换适配子的选择，由于执行的阴性选择步骤，简化富集监测，并扩展捕获探针以下选择富集，以提供增强的严格的长度。所选的结构切换适体在金纳米粒子测定平台，报告一个靶分子通过适体的构象变化的存在是有利的。金纳米颗粒测定应用，因为它提供了一种简单，快速比色读出是有利的临床或部署环境。设计和优化方面的考虑提出了实验的证明，principl的评估工作在缓冲区提供对生理体液的小分子生物传感器的工作进一步延伸奠定了基础。

条件的严格最初保持在较低水平，以维持低拷贝数存在粘合剂在第一几轮。第一轮，特别是实施了最低严格保留通常呈现为独特的序列，由高皮质醇和DNA的浓度，并且缺乏阴性选择步骤展示了粘合剂。该适体的选择的成功证明了池从低百分比由目标（第2轮），以较高的分数通过保留在轮12-13（ 表1）相对恒定目标洗脱洗脱的进化。的DNA的高原由目标洗脱是传统上在选择（"浓缩"）的端点。然而，该方法进一步通过增加的cDNA捕获探针的长度（ 图1）中提出的选择的严格富集。延长该探针增加了CDN之间的杂交的强度A和池，增加了复合物的熔化温度（T M），以及规定的目标和序列之间的较强的相互作用，以释放结合序列到溶液中。第一轮选拔的实施，由于对图书馆的5&#39;末端少了一个G碱基较弱基因的相互作用。这是符合一般较低严格性条件在第一轮，并且预期不会显著影响选择比其它通过允许更多的潜在的粘合剂，以及背景序列（dehybridize基于热力学）传递到下一个选择轮。这些背景序列最小化的选择通过实现更高的严格条件，在随后的几轮选择继续走向致富。当该cDNA是从7聚体，延长至8聚体在轮14中，DNA的目标溶出的百分比从15.3％降低到11.1％的Tm值从19.2℃升高至26.7℃。该基因瓦特为进一步扩展至9个碱基的15轮与31.3℃，Tm值，洗脱至3.3％的总下降。 上富集的进一步信息可以通过测序几个池，既富集和非富集的例子中，为了跟踪在每一轮的池的进程来获得。新一代测序（NGS）已成为一个强有力的方法进行测序的选择，主要是因为较高的顺序读取（序列总数报道），得到比较Sanger测序。这些更高的序列读数呈现出更大的覆盖范围，在池中的序列的总数，提供的序列的多样性的更全面的了解。 NGS还删除克隆偏压32，以及允许多个池在单个批次用加入了条形码24,33-测序。 NGS用于分析富集的进展，从在该样本选择三个独立的池（ 图2）。泳池从第6轮（轻微富集相比轮2％洗脱），圆13（最高的富集）和圆15（最高严格性）被选择作为选择的代表点。每个独特序列的拷贝数被绘制为序列的总数量的百分比读取每个池。排名靠前的（最高拷贝数）序列只占在6轮的所有序列（33435共序列，22463独特的序列）的0.1％。由于池将变成最大富集轮13，排名靠前的顺序增加到包括整个池的15.4％（17681共有序列，2987独特的序列），比6轮高150倍，尽管只有5倍〜在富集增加UV观察。第15轮（28919全序列读取，2,980独特的序列）跃升到由单排名靠前的顺序代表了所有序列的44.9％，尽管UV富集监测显示，由目标溶出量较圆13〜5倍降低（ 表1 </str翁&gt;）。富集也证明了独特序列的较低百分比（67％在轮6〜10％的圆15）为池产生于许多到多个序列与潜在靶标结合。此外，在第二轮13（15-3）排名靠前的顺序在15轮第三高的拷贝数序列中的基因长度延长后，在13轮排名第二的顺序成为最高拷贝数序列中的15轮（15 -1）: 15-1 GGAATGGATCCACATCCATGGATGGGCAATGCGGGGTGGAGAATGGTTGCCGCACTTCGGCTT CACTGCAGACTTGACGAAGCTT 15-3 GGAATGGATCCACATCCATGGGAGGGTTGGAAGGGAGGGGCCCGGGGTGGGCCATCGTTCGTT CACTGCAGACTTGACGAAGCTT 以往的研究探讨这两个序列的结合通过微型热泳和平衡透析24。结果表明显著皮质醇的顺序15-1（K D结合= 6.9±2.8μM通过平衡透析; 16.177; 0.6μM由微型热泳），而最小的约束力是皮质醇和顺序15-3的观察。这表明，该捕获探针的轮15中的增加的长度受助于提供更高的严格性条件，以解析出较高的亲和力的粘合剂。此外，既没有表现出序列观察到的结合的阴性选择分子，孕酮，显示出另外的阴性选择步骤是对程序是有益的。 即一个较高的亲和性粘合剂出现以下的最大的浓缩后升高的严格条件是在进一步轮选择施加（通过分析由目标洗脱DNA的百分比的传统方法来确定）的事实证实延长的cDNA捕获的长度的方法探测后浓缩。这一结果表明，拷贝数从排序的池和亲和力的靶可以只在某些条件下24,34相关联，在CON对比度与以前的报告中暗示有直接的关系35。它还突出监测富集的利益由NGS，而不是仅仅由％洗脱策略中最常见的选择，它可从一个圆到下自然变化为高严紧条件被应用。然而，当在相似条件跨几轮施加％洗脱是用于监视富集有用。例如，轮数6-10所有涉及的类似条件下，用在富集无显著变化（ 表1）。当这是观察到在几轮，条件可能过于严格，以促进富集，和研究者应在下一轮降低严格性。例如，皮质醇浓度从35μM增加到100微米轮11-13，并且％洗脱，从2.5％在轮10上升至14.5％，在第二轮12。因此，％洗脱可以作为用于调节导向simil当一个选择的过程中严格性AR条件从圆到圆比较，并且可以提供互补的信息，以NGS用于确定选择的端点。 序15-1被施加到金纳米粒子检测，以确定是否在一个方式中的序列，以产生​​结构切换适体将在依赖于下列靶结合以产生一个响应的结构变化的测定工作。以前最初的研究表明，与15-1的金纳米粒子测定响应了应力生物标记皮质醇，但应力，肾上腺素和去甲肾上腺素，或胆酸其它不两个标记，肝病结构相似的标记皮质醇24。该反应中观察到报告的人血清（〜150-600纳米）6速，确认后皮质醇结合选定的结构变化的适体产生的金纳米粒子测定的响应游离皮质醇的正常范围内。在当前的工作中，该系统的特征是更进了一步，通过调节15-1上的金纳米粒子表面的覆盖率（密度）。使用相同的一组条件，将DNA的覆盖物，从73的D / NP（DNA分子/金纳米粒子）的增加，到120的D / NP和200的D / NP（ 图3）。胆酸产生一个最小的响应，在200的D / NP除外10微米。测定减少的覆盖面的反应增加一样的检测限（LOD）。该LOD为29.5纳米的73 D / NP，145.2纳米120 D / NP和27.3μM200 D / NP。该结果与Smith等谁说明，利用可卡因适体的金纳米粒子测定的响应降低当适体覆盖从60的D / NP增加到300的D / NP 36的工作。这意味着可以基于优化的DNA的表面覆盖的金纳米粒子测定调节检测范围为感兴趣的靶。比较，例如，当这可能是有益的，游离皮质醇在人血清（〜150-600纳米）与人的唾液（〜5-25纳米）的范围&lt;SUP&gt; 6,37。而生理流体远远比这个缓冲器测定更复杂的，结果提供证明型的原理工作的延伸表明金纳米粒子的条件可以调节取决于应用，并朝扩大介质向生物流体将是进一步的工作感兴趣的生物传感器的社区。 <img alt="图1" src="/files/ftp_upload/52545/52545fig1highres.jpg" /> 图的结构切换选择方法1.概述，一个小的生物素化的cDNA捕获探针被固定在链亲和素包被的磁珠。该cDNA互补于库的5&#39;-区，杂交的库珠。当目标被添加，构象变化被诱导以释放结合序列从胎圈，允许分区施加的磁铁容易。上清液被保留并监控富集（％脱氧核糖核酸洗脱通过第Ë目标）紫外从DNA透析的目标之后。该步骤仅必要的，如果在目标分子吸收在相同的UV范围内的DNA。序列然后PCR扩增并为下一轮选择的准备。作为选择的进行，负选择被应用，其中通过阴性选择分子洗脱序列被丢弃，并与潜在目标的粘合剂的珠粒，然后用靶孵育。的cDNA探针的长度增加以下最大富集（％洗脱），以增加选择的严格性。这个数字已重印许可24。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52545/52545fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 圆 </td><td> [皮质醇]（μM） </td><td> [孕酮]（μM） </td><td>结合的DNA（皮摩尔） </td><td> ％洗脱目标 </td><td> 基因长度 </td></tr><tr><td> 1 </td><td> 100 </td><td> 0 </td><td> 357.57 </td><td> N / A </td><td> 7个碱基</td></tr><tr><td> 2 </td><td> 50 </td><td> 0 </td><td> 145.49 </td><td> 1.4 </td><td> 7个碱基</td></tr><tr><td> 3 </td><td> 50 </td><td> 1 </td><td> 188.74 </td><td> N / A </td><td> 7个碱基</td></tr><tr><td> 4 </td><td> 50 </td><td>五</td><td> 336.4 </td><td> 2.3 </td><td> 7个碱基</td></tr><tr><td>五</td><td> 35 </td><td>五</td><td> 88.05 </td><td> N / A </td><td> 7个碱基</td></tr><tr><td> 6 </td><td> 35 </td><td> 10 </td><td> 303.89 </td><td> 3.1 </td><td> 7个碱基</td></tr><tr><td> 7 </td><td> 35 </td><td> 10 </td><td> 255.01 </td><td> N / A </td><td> 7个碱基</td></tr><tr><td> 8 </td><td> 35 </td><td> 10 </td><td> 236.81 </td> &lt;TD&gt; 2.1 </td><td> 7个碱基</td></tr><tr><td> 9 </td><td> 35 </td><td> 10 </td><td> 318.95 </td><td> 2.6 </td><td> 7个碱基</td></tr><tr><td> 10 </td><td> 35 </td><td> 10 </td><td> 297.18 </td><td> 2.5 </td><td> 7个碱基</td></tr><tr><td> 11 </td><td> 100 </td><td> 10 </td><td> 147.61 </td><td> N / A </td><td> 7个碱基</td></tr><tr><td> 12 </td><td> 100 </td><td> 10 </td><td> 154.36 </td><td> 14.5 </td><td> 7个碱基</td></tr><tr><td> 13 </td><td> 100 </td><td> 10 </td><td> 150.39 </td><td> 15.3 </td><td> 7个碱基</td></tr><tr><td> 14 </td><td> 75 </td><td> 20 </td><td> 247.71 </td><td> 11.1 </td><td> 8聚体</td></tr><tr><td> 15 </td><td> 50 </td><td> 40 </td><td> 409.63 </td><td> 3.3 </td><td>九聚体</td></tr></tbody></table> 表1.选择发展富集％洗脱24 &lt;<html> STRONG&gt;。N / A（不适用）报道了那里并没有在早期选择两轮进行透析/ UV富集的监测，以减轻低拷贝数序列的损失轮。该表已重印从24的许可。 <img alt="图2" src="/files/ftp_upload/52545/52545fig2highres.jpg" /> 通过新一代测序确定图2.选择富集（A）第6轮。 （B）轮13; （C）15轮序列是根据套数排名。从构成整个测序池的比例较低在6轮（0.1％），以高比例在第二轮15（44.9％），成为池富集皮质醇结合序列的最高拷贝数序列增加。这个数字已经再版24的许可。等="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52545/52545fig3highres.jpg" /> 从不同的适体15-1的覆盖图3.金纳米粒子测定的响应。孵育所述金纳米粒子的适体的密度为从73的D / NP（红色三角形）的增加，到120的D / NP（绿色正方形）和200的D / NP（蓝圈）。皮质醇反应是大胆的色彩，胆酸的反应是浅色。在测定反应在较低的密度提高为皮质醇，因此降低了检测限和目标可以在被检测的范围内。具有最高皮质醇响应（73 D / NP）的条件进行了进一步（特征的线性范围以所期望的正常范围游离皮质醇报道在人血清（〜150-500纳米）和唾液中提供更多的点5-25纳米）6,37。胆酸的应用相同的73的D / NP条件的最小响应是具有恩详见以前的工作24。所有地块代表平均值±SEM重复或三次测量。 <img alt="图4" src="/files/ftp_upload/52545/52545fig4highres.jpg" /> 从PCR循环优化图4代表性的结果从左至右的孔表示:4个周期，第6个周期，8个周期，10个周期，12个周期，阴性（无DNA模板），25 bp的DNA梯标准。最佳条件是在8个周期，其中该单一的产品带是在高强度而不过度扩增产物存在于更高的周期变化。 <img alt="图5" src="/files/ftp_upload/52545/52545fig5highres.jpg" /> 图5.金纳米粒子测定温育时间进行优化。在73 D中的金纳米粒子/ DNA的步骤的孵育时间/ NP被选自O减少/ N（ 图3），以30分钟，并在目标INCU插管紧接其后加入盐，而不是20分钟（ 图3）之后。 （A）的反应被观察到皮质醇（蓝色菱形），但尚未对胆酸（绿色圆圈）或2MNP（2-甲氧基萘;红色正方形）。所有地块代表平均值±SEM重复或三次测量。 （B）从左至右:空白，10μM皮质醇，10μM胆酸，10μM2MNP。视觉上的变化可以用肉眼对皮质醇可以观察到。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52545/52545fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay at JoVE.com

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

a-method-for-selecting-structure-switching-aptamers-applied-to

Universidad San Sebastian

Research

JoVE Journal

Biology

15.1K 次观看.  Wright-Patterson Air Force Base. A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

视频: A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

制造的UV-Vis和拉曼光谱免疫平台

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

科研

生物化学

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

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 rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

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 ...

化学

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 ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

生物工程

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

癌症研究

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

开发用于快速定量检测小分子化合物的横向流动免疫色谱带

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

生物学

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

直接合成金纳米颗粒用于蛋白质定位分析

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.