欧洲研究理事会根据欧洲联盟第七框架方案提供的资金 (FP/2007-2013)/紧急救济委员会赠款协议 615458, 瑞典研究理事会和 Pronova 卓越蛋白质技术中心 (VINNOVA-瑞典语政府机构的创新系统) 是感激地承认。

根据收集国的法律和伦理要求, 获得了人体血清样本, i. e。经过伦理委员会 (或类似) 的批准, 并得到捐助者的书面同意。
1. 增强解决方案准备:
<ol><li>在去离子水中悬浮 mes 钠盐, 制备10毫米 mes pH 6 缓冲液。使用4米氢氧化钠溶液将 pH 值调整为6。</li>
	<li>在10毫米 MES pH 值6缓冲器 (解决方案 1) 中准备一个 5 mm HAuCl4解决方案。</li>
	<li>在10毫米 MES pH 值6缓冲区 (解决方案 2) 中准备一个1.027 米 H2O2解决方案。 注意: 两个解决方案1和解决方案2的卷取决于要增强的微阵列的数量和大小。例如, 10 x 10 毫米的微阵列需要 100 ul 总容量 (解决方案 1 + 解决方案 2), 以确保微阵列区域与增强解决方案保持联系。稀释后, 应在大约30-45 天内使用 H2O2 。</li>
</ol>2. 纳米微粒的制备:
<ol><li>按照 Bastús et al的描述, 准备 40 nm AuNPs。16简要地, 将1毫升水中25毫米 HAuCl4前驱体注入2.2 毫米柠檬酸钠 (149 毫升) 的沸腾溶液中。等到红葡萄酒的颜色被观察到。将纳米微粒的溶液作为种子生长步骤的种子。</li>
	<li>在90摄氏度, 注入1毫升的60毫米柠檬酸钠后, 1 毫升25毫米 HAuCl4进入含有溶液的种子。30分钟后, 反应完成。重复种子生长步骤五次, 直到得到40纳米纳米粒子, 如 Bastús et al所述。16
</li>
	<li>测量纳米粒子最终溶液的紫外可见光吸收量, 以确定纳米粒子的大小。17或者, 透射电子显微照片 (TEM) 可以获得并用于确定纳米粒子的大小。</li>
	<li>按照 Rivero et al的描述, 准备 90 nm AgNPs。18简要地, 将 dimethylaminoborane (DMAB) 添加到聚合 (丙烯酸、钠盐) (临机) 和 AgNO3的强力搅拌溶液中。解决方案的最终体积为50毫升, DMAB、临机局和 AgNO3的最终浓度分别为1.6 毫米、10毫米和3.33 毫米。 注: 100 纳米 IONPs 改性羧基组和 50 nm SiNPs 改性羧基组购买。</li>
	<li>Functionalize 使用 Puertas et描述的协议后的抗体 AuNPs。19简要地, 将1毫克的 COOH (5000 g·mol−1) 添加到 1.2x10-9 M AuNPs (10 光密度、OD) 的解决方案中。用4米氢氧化钠溶液将 pH 值调整为12。<ol>
<li>在温和的搅拌条件下, 让溶液在室温下 (22 摄氏度) 孵化一夜。</li>
		<li>通过将溶液离心 13800 x g 以15分钟的方式除去多余的 PEG。</li>
		<li>用 500 ul 的去离子水并用重悬颗粒。</li>
		<li>用5μmol n-(3-Dimethylaminopropyl)-n '-ethylcarbodiimide 盐酸盐 (AuNPs) 和7.5 μmol 的 n-hydroxysulfosuccinimide 钠盐 (磺-NHS) 在10毫米 MES 中孵化出 PEG 修饰的, 6 摄氏度为30分钟。</li>
		<li>通过离心 13800 x g 的溶液, 以5分钟的方式去除磺和亚胺酰胺的过量。</li>
		<li>并用重悬颗粒在500µL 10 毫米 MES pH 6 缓冲和添加 100g·mL -1 抗体。让溶液孵育2小时, 在37摄氏度。</li>
		<li>去除抗体的过量通过离心溶液在 13800 x g 10 分钟两次和并用重悬颗粒在 500 ul 10 毫米磷酸磷酸钠 pH 值 7.5, 0.3 M NaCl 缓冲。让溶液孵育30分钟, 在37摄氏度。</li>
		<li>去除不特异的绑定抗体通过离心溶液在 13800 x g 10 分钟两次, 并并用重悬在 500 ul 1% 牛血清白蛋白 (BSA) 在10毫米 MES pH 值6缓冲区的颗粒。在4摄氏度一夜之间孵化样品。</li>
		<li>通过离心溶液在 13800 x g 10 分钟两次清洗 BSA 的过量, 并在 500 ul 10 毫米 MES pH 值6缓冲器中 resuspending 颗粒。</li>
		<li>遵循步骤2.5.1 通过2.5.9 的修改5米 AgNPs, 0.5 毫克 IONPS 和0.5 毫克 SiNPs 与抗体, 调整的数量和磺-NHS 相应。</li>
	</ol>
</li>
</ol>3. 垂直流纸的化验方法:
<ol><li>用机器人打印机将蛋白质 G 的梯度浓度沉积在硝化棉纸膜上, 制备微阵列。打印后, 让微阵列在室温下过夜 (摄氏22摄氏度)。</li>
	<li>用过滤器内所附膜进行垂直流动试验。使用超注射器泵, 以 1 mL·min−1的速率, 以8米的 AuNPs 溶液进行 IgG 修饰。重复此步骤, 解决方案为 17 M IgG 修饰 AgNPs, 0.1 mg·mL−1 IgG 修饰 SiNPs 和 0.1 mg·mL−1 igg 修饰 IONPs。</li>
	<li>让微阵列在室温下干燥 (约22°c) 30 分钟, 然后在平板扫描仪中扫描 4800 dpi 分辨率。将图像另存为24位彩色 TIFF 文件。</li>
</ol>4. 参照商用玻璃的化验方法:
<ol><li>在室温下孵化两张120分钟的玻璃滑梯, 用于检测4种不同人体血清样品的过敏原成分。</li>
	<li>用去离子水冲洗微阵列。</li>
	<li>用荧光共轭抗人 ige 抗体在室温下孵化一张幻灯片 (~ 22°C) 30 分钟. 用抗人 IgE 抗体修饰 AuNPs 在室温下孵化第二张幻灯片。</li>
	<li>用去离子水冲洗微阵列。</li>
	<li>用激光扫描仪测定用荧光抗人 IgE 抗体孵化的微阵列的荧光强度。数字化的基因芯片, 与抗人 IgE 抗体修改 AuNPs 与一个桌面扫描仪的比色信号采集。</li>
	<li>数字化后, 在室温下 (~ 22°C) 用增强溶液孵育微阵列5分钟。</li>
	<li>用去离子水冲洗传感器, 使其在室温下干燥10分钟 (摄氏22摄氏度)。</li>
	<li>数字化在 4800 dpi 的分辨率下, 用桌面扫描仪将微阵列用于色度信号采集。将图像另存为16位灰度 TIFF 文件。</li>
</ol>5. 采用增强解决方案的微阵列孵化:
<ol><li>预混合相同数量的解决方案1和解决方案2或直接混合在传感器的表面上, 并确保芯片上感兴趣的领域涵盖了解决方案的混合物。</li>
	<li>让传感器在室温下孵育5分钟以上的增强溶液, 可提高灵敏度和较高的噪声/信号比, 尤其是纸基微阵列。</li>
	<li>在5秒内将微阵列浸入去离子水中, 将其洗净. 在室温下保持干燥。 注意: 干燥步骤的时间取决于芯片的材料。对于基于纸张的微阵列, 干燥步骤需要大约30分钟。对于基于玻璃的微阵列, 干燥步骤需要大约5-10 分钟。</li>
</ol>6. 成像分析:
<ol><li>用软件工具分析荧光强度。根据传感器制造商的使用说明 (DfU), 考虑所有相等或大于 0.3 Isac 标准化单元的结果。</li>
	<li>使用软件工具分析色度强度。反转图像文件, 测量每个光斑的强度, 计算平均像素强度。使用三个点的值计算最终信号值作为三平均光斑像素强度的平均值。</li>
</ol>

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作者没有什么可透露的。

目前, 以 nanoprobe 为基础的检测的信号增强技术是基于酶的12, 需要第二组纳米粒子来瞄准检测 nanoprobes21 , 或者, 在染色技术的情况下, 仅限于使用 AuNPs 或 AgNPs 作为检测探头。22这里描述了一种简单、快速、无酶的方法, 用于 nanoprobe 检测的信号增强。利用这种方法, 可以提高4种 nanoprobes 的色度信号: AuNPs、AgNPs、IONPs 和 SiNPs。
所观察到的每组 nanoprobes 的放大系数为100倍, 但 SiNPs 由于缺乏预增强信号而无法量化增强因子。这种增强因素表明, 协议的效率在所有类型的 nanoprobes 中都是相似的。此外, 当增强协议是在一个纸支持上进行的, 其中一个稀释系列的股票 AuNPs 悬浮被打印, 它被观察到10000倍放大系数。在增强之前, AuNPs 数量最少的可见斑点被印在大约10000粒纳米粒子上, 在增强了少于10纳米粒子的斑点后, 可见。20
在这里提出的增强协议的条件允许利用将 au3 +减少到 au0以改善信号, 同时将背景噪音保持在最低限度。这种增强可以在进行化验后进行, 以及在化验完成后储存了数月的微阵列进行。增强解决方案由1:1 混合解决方案1和解决方案2组成。两种解决方案在 pipetted 到微阵列时都可以混合或直接混合。预混合或直接混合的区别只影响增强溶液的保质期。当预混合的货架寿命是5-7 天增加到30-45 天, 如果不预先混合。
进一步分析结果表明, 这种增强方法不会干扰生物传感器的定量分析。维持观察到的强度与分析物浓度之间的线性相关性 (表 1)。采用基于玻璃的过敏原组分微阵列免疫分析法, 用标准荧光检测和比色法检测4例临床样品, 显示了两种检测方法之间的良好一致性。比较平均荧光强度 (多边基金会) 和平均比色强度 (MCI), 当数据在两个轴上记录10时, 就得到了平均值 R2 = 0.79 +/-0.08。实验表明, 这种增强方法可以应用于商用套件, 如果它使用 nanoprobes 进行检测, 或者可以适应 nanoprobe 的检测。
增强方法的有效性依赖于两个关键方面。必须保证溶液1和溶液2的混合物是均匀的。如果没有均匀混合, 传感器将与增强解决方案的分数相联系, 其中解决方案1或2相对于另一种优势。这将降低 au3 +到 au0的降低效率, 从而损害增强的效率。同样重要的是, 在潜伏期内, 在与增强溶液接触时, 芯片的整个感兴趣区域都有。
为了达到超灵敏增强的信号, 需要更长的潜伏期 (约5分钟) 与增强解决方案将是必要的。为了避免开发可能干扰信号采集的背景噪声, 应事先阻止微阵列表面 (例如BSA、PEG), 以防止 Au0的快速不特异沉积, 并由此形成一个金层.
对增强方法的限制是检测的内在功效。该化验要求有消极和积极的控制, 以确保所观察到的信号增强可以归因于真正的积极结果。如果 nanoprobes 与目标的非特定交互作用, 则在增强后获取的信号将无效。
其他增强技术是基于纳米粒子的聚合或纳米微粒的染色, 这种材料允许改进信号的采集。通过促进在检测现场收集纳米微粒的数量, 信号的采集将可能是视觉上的或紫外的测量。5这些信号增强技术依赖于两步检测系统, 初始检测感兴趣的分析物, 第二步是要求记者绑定到初始检测构造。由于对每种检测方法的特定增强协议优化要求, 这种策略缺乏通用性。
染色增强技术, 如银染色, 很像在这里提出的协议, 允许直接增强信号检测结构。然而, 与此处显示的协议不同, 银染色仅被证明适用于 AuNPs 或 AgNPs。6,7,8,9
这种方法的适用性可能跨越任何使用 AuNPs、AgNPs、IONPs 或 SiNPs 作为检测剂的化验。目前正在开展进一步的工作, 研究该方法在其他材料组成的传感器中的应用。

使用纳米材料, 如金纳米粒子 (AuNPs) 或氧化铁纳米粒子 (IONPs), 使其在生物传感中的应用具有改进的灵敏度和通用性。1对于各种配体的纳米粒子表面的装饰, 为了充分利用它们的高表面体积比, 设计了大量的方法, 使传感器能够提高灵敏度。2然而, 生物传感工具依赖于实现可检测信号所需的 nanoprobes 数。例如, 在 40 nm AuNPs 的情况下, 通过紫外可见光谱获取信号, 需要大约 90 x 106 nanoprobes, 以便有效地检测出感兴趣的目标。3通过使用信号放大, 可以规避此 nanoprobe 密度限制。此类策略4可以基于粒子间聚合或聚集, 其中初始 nanoprobes 的密度通过在第一个集合中累积第二组 nanoprobes 来增加。5在传感器表面的给定位置增加纳米粒子的数量, 允许视觉或紫外-可见光信号采集。然而, 这种检测的敏感性将与第二套纳米粒子对初始 nanoprobes 的靶向能力有内在的联系。其他的策略依赖于黄金和银 nanoprobes 的染色。6,7染色是通过将银离子还原到纳米粒子表面, 从而实现视觉或紫外可见光检测。8这些方法通过 potentiating 表面共振等离子体信号来增强现有金或银 nanoprobes 的信号, 而不依赖于粒子间聚类方法中的次级靶向事件。然而, 银染色方法只被报道使用金或银 nanoprobes。8,9
在 2005年, Zayats et al。10报告将 Au 离子还原到金 nanoprobes 表面, 以增加表面等离子体共振信号。在这种依赖于酶的工作中, 过氧化氢是由葡萄糖氧化酶催化产生的, 与铵氯化物一起允许四氯金酸的还原。
最近, 王et。报告了一种增强方法, 在现有 nanoprobes 表面上产生金层。11这些扩大的 nanoprobes 显示过氧化物酶样的催化活性对基板 3′, 5,5′-tetramethylbenzidine (TMB), 使明亮的蓝色的可视化肉眼。
史蒂文斯et 。报告了等离子 ELISA 检测的发展情况。12在通过传统的 ELISA 策略检测到前列腺特异抗原 (PSA) 后, 用传感器将传感器浸入含有过氧化氢的溶液中, 并将其标记为过氧化氢酶的二级抗体, 并四氯金酸。二次过氧化氢酶修饰抗体 (阳性结果) 的存在将促进过氧化氢的消耗, 减缓四氯金酸的降低, 并产生具有红色颜色的准球形分散 AuNPs。过氧化氢酶修饰抗体 (阴性结果) 的缺乏, 使过氧化氢浓度保持不变, 从而促进快速四氯金减少, 并产生 AuNPs 与病态的形态负责蓝/紫颜色.过氧化氢的浓度, 由过氧化氢酶的活性决定, 被证明是与感兴趣的分析物的浓度相关。对过氧化氢酶修饰的二级抗体的需要和催化活性条件的控制是阻碍这种方法普遍性的两个因素。此外, 金簇的形成, 独立于现有的 AuNPs, 可能会引入背景噪声问题的放大策略。
上述技术以及其他几个13、14、15, 使得基于 nanoprobe 的生物传感器能够达到与传统技术同等的检测限制。
在这里, 我们展示了一种新的金增强方法, 即通过 potentiating 或引入表面等离子体共振信号来放大现有 nanoprobe 的信号。在使用金、银、二氧化硅或氧化铁纳米粒子进行检测后, 该传感器被允许在 2-(n-吗啉代) 磺酸 (MES) pH 6 缓冲液中以过氧化氢和四氯金酸混合物的溶液孵化。增强溶液中各组分的浓度经过优化, 有利于在现有 nanoprobes 表面沉积 Au0 。对于所有研究过的 nanoprobe 类型, i. e。AuNPs, 银纳米粒子 (AgNPs), 二氧化硅纳米粒子 (SiNPs) 和 IONPs, 形成一个病态定义的层产生或增强散射的光, 导致可检测或增加可见信号。在纸和玻璃微阵列中, nanoprobes 实现了100倍放大因子的信号增加, 该过程需要少于5分钟来获取信号。信号采集可以通过肉眼、紫外可见光谱或成像低端工具 (如数码相机或台式扫描仪) 来完成。此外, 据证明, 这种增强协议可以很容易地应用于商业上可用的过敏诊断化验, 而不需要具体的优化。

<table><tbody><tr><td>Chloroauric acid</td><td>Sigma-Aldrich</td><td>254169 ALDRICH</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>S4641 SIGMA-ALDRICH</td><td></td></tr><tr><td>Dimethylaminoborane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>476668 ALDRICH</td><td></td></tr><tr><td>Poly(acrylic acid, sodium salt)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>416037 ALDRICH</td><td></td></tr><tr><td>Silver nitrate</td><td>Sigma-Aldrich</td><td>204390 ALDRICH</td><td></td></tr><tr><td>100 nm magnetic nanoparticles</td><td>Ademtech</td><td>021111</td><td></td></tr><tr><td>50 nm silica nanoparticles</td><td>Corposcular</td><td>140510-10</td><td></td></tr><tr><td>COOH-PEG-SH&amp;nbsp;</td><td>RAPP Polymere</td><td>135000-4-32</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>1.06462&amp;nbsp;EMD MILLIPORE</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;rsquo;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>03449&amp;nbsp;SIGMA</td><td></td></tr><tr><td>N-Hydroxysulfosuccinimide sodium salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>56485&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>95313 SIGMA-ALDRICH</td><td></td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid hydrate</td><td>Sigma-Aldrich</td><td>M8250 SIGMA</td><td></td></tr><tr><td>IgG antibody</td><td>Abcam</td><td></td><td></td></tr><tr><td>Sodium phosphate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>342483&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Bovine serum albumin</td><td>VWR&amp;nbsp;</td><td>422351S</td><td></td></tr><tr><td>Protein G</td><td>Thermo Scientific</td><td>21193</td><td></td></tr><tr><td>Nitrocellulose paper membrane</td><td>Whatman</td><td>10402096</td><td></td></tr><tr><td>Nanoplotter 2.1 robotic printer</td><td>GeSIM</td><td></td><td></td></tr><tr><td>Filter holder</td><td>Merck</td><td>XX3001200</td><td></td></tr><tr><td>PhD2000 Ultrasyring pump</td><td>Harvard</td><td></td><td></td></tr><tr><td>Flatbed scanner CannonScan 9000F Mark II</td><td>Cannon</td><td></td><td></td></tr><tr><td>Anti-human IgE antibody-modified AuNPs&amp;nbsp;</td><td>Thermofisher</td><td></td><td></td></tr><tr><td>Laser scanning LuxScan 10/K</td><td>Capital Bio</td><td></td><td></td></tr><tr><td>Table-top scanner Image Scanner Epson Expression 1600 pro</td><td>Epson</td><td></td><td></td></tr><tr><td>Glass slide ImmunoCAP ISAC sIgE 112</td><td>Thermofisher</td><td>81-1011-01</td><td></td></tr><tr><td>Fluorescent-conjugated anti-human IgE antibody&amp;nbsp;</td><td>Thermofisher</td><td>81-1011-01</td><td></td></tr><tr><td>Phadia Microarray Image Analyzer MIA 1.2.4&amp;nbsp;</td><td>Thermofisher</td><td></td><td></td></tr></tbody></table>

rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis

使用 nanoprobes, 如金, 银, 二氧化硅或氧化铁纳米粒子作为检测试剂在生物化验可以使高灵敏度和方便的色度读数。然而, 高密度的纳米粒子通常需要检测。现有的基于合成的增强协议要么限于金和银纳米粒子, 要么依赖于精确的酶控制和优化。在这里, 我们提出了一个协议, 以提高比色读数的黄金, 银, 二氧化硅, 氧化铁 nanoprobes。据观察, 比色信号可以提高高达10000倍的因素。此类信号增强策略的基础是将 au3 +的化学还原为 au0。有几种化学反应可以使 au3 +减少到 au0。在协议中, 使用了好的缓冲区和 H2O2 , 并且可以支持将 Au0沉积到现有 nanoprobes 的表面上, 这有损于新的金纳米粒子的形成。该协议包括了微阵列的孵化, 其解决方案由四氯金酸和H2O 2 在 2-(n--吗啉代) 磺酸 pH 6 缓冲器中组成, nanoprobe 的检测方法之后。该增强方案可应用于纸张和玻璃基传感器。此外, 它可以用于商业上可用的免疫, 证明了该方法应用到一个商业化的过敏原微阵列。信号的发展需要少于5分钟的孵化与增强解决方案和读数可以评估的肉眼或低端图像采集设备, 如台式扫描仪或数码相机。

进行检测后, 使用 nanoprobes 作为检测代理, 传感器检测区域可能使用 nanoprobes (图 1a) 填充。如果 nanoprobes 的数量低于视觉检测的限制, 则对分析物的检测最初将被认为是阴性的。使用此处提供的协议 (图 1b), 通过引入不确定的 Au 层 (图 1c), nanoprobes 的信号增强。
为了展示增强协议 (图 2) 的有效性, 设计了一种用于检测蛋白质 G 的基于纸张的免疫分析方法。AuNPs、AgNPs、SiNPs 和 IONPs 用 IgG 抗体和使用和检测剂进行了修改。编写了纸微阵列, 以获得每点蛋白质 G 分子的梯度。
化验完成后, 据观察, igg 修饰 AuNPs (igg-AuNPs) 能够检测到 2 x 105分子每当场 (图 2a), igg 修饰 AgNPs (igg-AgNPs) 能够检测到低至 2 x 104每个点的分子 (图 2c), igg 修饰 IONPs (igg-IONPs) 能够检测到每点 2 x 107分子 (图 2e) 和 igg 修饰 SiNPs (igg-SiNPs) 无法提供视觉信号 (图 2g)。
通过用增强溶液孵化微阵列, 可以在使用的所有类型的纳米粒子上增加100倍的信号。在 SiNPs 的情况下, 增强解决方案使人们有可能观察到没有增强解决方案 (图 2b、2 d、2 f 和 2h) 时没有看到任何可视信号的信号。
评价了该方法在现有商业免疫分析中的应用可能性。对一种基于玻璃的过敏原组分微阵列免疫检测进行了改进。用微阵列 (样本被指定为 a、b、c 和 d) 对一组4例过敏患者的血清标本进行了评价。将该方法的标准荧光检测与抗 IgE 标记 AuNPs 的染色法进行比较, 然后再增强信号 (图 3)。在增强之前, 在用反 IgE 标记 AuNPs 进行检测的微阵列上没有发现斑点。增强后, 肉眼可见多个斑点, 其强度由图像数字化 (图 3) 进行注册。通过荧光检测, 检测到了各种斑点 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57297/57297fig1.jpg"> 图 1.microspot 从分析物检测到增强信号所经历的步骤的说明。() 两个抗体修饰 nanoprobes 在检测到固定在 microspot 上的分析分析后 (b), 利用增强协议和随后的抗体修饰 nanoprobes 的种子生长来孵化微阵列, (c) microspot 在通过增强解决方案孵化5分钟后, nanoprobes 的大小增加。此数字已从 Dias et aldapted。20<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57297/57297fig1large.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/57297/57297fig2.jpg"> 图 2.蛋白质 G. 检测用微阵列的数字化图像在每个微阵列的左侧是观察到的 microspots 在增强的检测之前进行 (a) igg-AuNPs, (c) igg-AgNPs, (e) igg-IONPs 和 (g) igg-SiNPs。在每个微阵列的右侧是增强后的 microspots (b) igg-AuNPs, (d) igg-AgNPS, (f) igg-IONPs 和 (h) igg-SiNPs。蛋白质 G 的每一个浓度都在微阵列上沉积。根据每个 microspot 中所印蛋白质的浓度, 对蛋白质 G 的数量进行了计算。此数字已从 Dias et中进行了调整。20<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57297/57297fig2large.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/57297/57297fig3.jpg"> 图 3.用商用过敏诊断法测定荧光强度与比色强度的相关性.测定样品 a、b、c 和 d 中的过敏原的平均荧光强度 (多边基金会) 和平均比色强度 (MCI). 镶嵌是记录10在两种方法之间的线性相关性的区间内转换的数据。根据荧光修饰抗体 (荧光) 检测的 microspots 强度为荧光发射。在对微阵列进行数字化后, 用成像软件对图像进行反转, 得到了基于 Ab AuNPs 检测的 microspots 的亮度。微阵列的设计是有垂直 triplicates 与积极的控制, 荧光检测在极右的底部。其他三个角用于软件评估。用16位灰度分析图像。此数字已从 Dias et中进行了调整。20<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57297/57297fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>R2以前的增强</td>
			<td>R2增强后</td>
		</tr>
<tr>
<td>IgG-AuNPs</td>
			<td>8.70E-01</td>
			<td>7.80E-01</td>
		</tr>
<tr>
<td>IgG-AgNPs</td>
			<td>9.10E-01</td>
			<td>9.60E-01</td>
		</tr>
<tr>
<td>IgG-IONPs</td>
			<td>9.17E-01</td>
			<td>9.30E-01</td>
		</tr>
<tr>
<td>IgG-SiNPs</td>
			<td>-</td>
			<td>8.50E-01</td>
		</tr>
</tbody></table>表1。R2 值, 用于检测蛋白质 G、之前和之后的增强, 使用不同的纳米粒子.R2值是在将斑点的强度与所测量的分析物的浓度关联后获得的。

Watch this Scientific Journal Video about Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis at JoVE.com

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

在这项工作中, 提出了一种基于 nanoprobe 的生物传感信号增强协议。该协议的基础是减少四氯金酸的表面上的现有 nanoprobes, 包括金, 银, 二氧化硅或氧化铁纳米粒子。

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 ...

rapid-nanoprobe-signal-enhancement-situ-gold-nanoparticle

Universidad San Sebastian

Research

JoVE Journal

Chemistry

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

7.5K 次观看.  KTH Royal Institute of Technology. 在这项工作中, 提出了一种基于 nanoprobe 的生物传感信号增强协议。该协议的基础是减少四氯金酸的表面上的现有 nanoprobes, 包括金, 银, 二氧化硅或氧化铁纳米粒子。

视频: Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

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

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

生物工程

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 ...

化学

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

金纳米棒基于对苯二酚合成

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

<meta charset="utf8"></head><body>DNA 罕见点突变的肉眼检测

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

DNA 罕见点突变的肉眼检测

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

金纳米粒子合成

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

通过等离子体 DNA 折纸纳米天线实现单分子 SERS 测量

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

作者聚焦:通过等离子体 DNA 折纸纳米天线实现单分子表面增强拉曼散射测量  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

等离子体DNA折纸纳米天线实现的单分子表面增强拉曼散射测量

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

原位金纳米粒子合成快速 Nanoprobe 信号增强

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