Name: development of an electrochemical dna biosensor to detect a foodborne pathogen
Uploaded: 2018-06-03T12:30:00.000+00:00
Duration: 17 min 16 s
Description: Watch this Scientific Journal Video about Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen at JoVE.com

作者希望感谢马来西亚马来西亚的支持。

注: 所有使用的化学药剂和生化试剂均应采用分析级, 无需进一步纯化。使用无菌去离子水制备所有溶液。灭菌前的所有玻璃器皿。
警告: 在进行实验室活动时, 请使用所有适当的安全做法, 包括使用工程控制 (油烟机、glovebox) 和个人防护设备 (安全眼镜、手套、实验室大衣、全长长裤、闭合脚趾鞋)。
1. 用 PLA-AuNPs 修饰电极的制备与表征
<ol><li>
PLA-AuNPs 的制备与表征
	<ol>
<li>快速添加10毫升柠檬酸钠溶液 (38.8 毫米) 到100毫升的沸腾水四氯金酸溶液 (1 毫米), 并冷却到周围温度。观察准备好的 AuNPs 溶液中的颜色变化, 开始为黄色, 变黑, 最后作为暗红宝石红色。</li>
		<li>将 PLA 颗粒放入不锈钢模具中进行熔化, 并在温度为190摄氏度时将其预热10分钟. 按照按下的程序: 将其置于 2.2 MPa 的压力下, 作为 PLA 板料制备的一部分。在冷却大约3小时后, PLA 板将准备就绪。</li>
		<li>在室温下搅拌, 在5毫升氯仿中溶解0.68 毫克的 PLA 片。将溶解的 PLA 与先前制备的 AuNPs 溶液的10毫升混合, 在室温下均匀搅拌。均匀的混合物可以表示为 PLA-AuNPs, 其特征是使用紫外可见光、XRD、TEM 和 FESEM EDX。</li>
	</ol>
</li>
	<li>
改性丝网印刷炭电极的制备与表征 注: 本研究使用的 SPCE 包括三电极系统: 计数器电极、碳工作电极和银/AgCl 参考电极。<ol>
<li>快速吸管25µL 的均匀溶液的 AuNPs 到 SPCE, 然后空气干燥24小时之前使用。</li>
		<li>电化学特征的修饰电极, SPCE/PLA-AuNPs, 在氰化钾 (K3[Fe (CN)6]3-/4-), 以测量有源表面积, 电化学阻抗谱, 重复性,重现性和稳定性30。</li>
	</ol>
</li>
</ol>2. 电化学 DNA 生物传感器的研制
<ol><li>
探头准备 注: ssDNA 探针和互补 DNA 序列是根据国家生物技术信息中心 (NCBI) 数据库的信息选择的。<ol>
<li>购买合成寡核苷酸 (20 ssDNA 探针, 20 的互补 dna, 20 不匹配的 dna, 21 的非互补 dna) 作为冻干粉从商业实验室, 根据以下序列: thiolated ssDNA 探针: 5′-/5ThioMC6-d-/CGGATTATGCAGAAGCACTG-3′ 补充 DNA: 5′-CAGTGCTTCTGCATAATCCG-3′ 单基地不匹配 DNA: 5′-CAGTGCTTCTGC ṪTAATCCG 3′ 三基不匹配的 DNA: 5′-CAGTGCTTCT ĊṪṪTAATCCG 3′ 非互补 DNA: 5′-CGCACAAGGCTCGACGGCTGA-3′</li>
		<li>用无菌 TE 溶液 (10 毫米三盐酸, 1 毫米 EDTA, pH 7.5) 制备所有寡核苷酸 (100 µM) 的库存溶液, 分为分析部分。保持这个在-4 摄氏度, 然后做适当的稀释在使用之前。</li>
	</ol>
</li>
	<li>
固定化与杂交的优化 注: 用 pla-AuNPs 修饰的 SPCE, 以 SPCE/PLA-AuNPs 为研究对象, 采用滴注法进行 DNA 固定化和杂交。<ol>
<li>首先, 固定25µL 的 thiolated ssDNA 探针 SPCE/pla AuNPs, 然后空气干燥24小时室温, 然后它将最后被称为 SPCE/解放军-AuNPs/ssDNA。用三因素确定固定条件的优化: ssDNA 的浓度 (从0.2 到1.4 µM), 时间 (从30到210分钟不等), 温度 (从25到75摄氏度不等)。</li>
		<li>吸管25µL 的补充 DNA 的表面上 SPCE/解放军-AuNPs/ssDNA 进行杂交事件后, 它可以最后被称为 SPCE/解放军-AuNPs/dsDNA。通过两个时间因素 (从5到30分钟不等) 和温度 (范围从25到75°c) 确定杂交条件的优化。</li>
		<li>将固定化和杂交电极浸泡在20µM MB 30 分钟内, 通过洗涤0.5 米 ABS/20 毫米氯化钠 (pH 4.5) 去除非特异吸附的 DNA 和过量 MB, 然后用去离子水冲洗。使用 DPV 技术测量 MB 还原的峰值电流。使用不含指示器的0.1 米 PBS (pH 7) 执行 DPV 测量。</li>
	</ol>
</li>
	<li>
制备 DNA 生物传感器的特性研究
	<ol>
<li>浸泡 SPCE/解放军-AuNPs 在20µM MB 30 分钟, 洗0.5 米 ABS/20 毫米氯化钠 (pH 4.5), 然后用去离子水冲洗之前测量。对所有的相互作用, 包括探针 dna, 互补 dna, 不匹配的 dna 和非互补的 dna 样本, 遵循类似的程序。</li>
		<li>测量电化学还原。 注意: 我们用一个商业制造的 Autolab 与接口软件测量 DPV。 MB 电化学还原的 DPV 测量在可能范围从-0.5 v 到 0.25 v 进行, 具有 0.005 v 的步长电位, 调制振幅为 0.05 V, 以及 7.73 M PBS (pH-1) 中0.1 个 mv7的扫描速率, 不含指示器。进一步研究了制备的 DNA 生物传感器的选择性、灵敏度、重现性和热稳定性。报告的结果作为平均价值测量被提出了三复制。</li>
	</ol>
</li>
</ol>3. 用真实样品验证制备的 DNA 生物传感器
<ol><li>细菌菌种的制备
	<ol>
<li>根据海鲜的严重污染, 选择细菌样本31,32 。 注:副溶血性为参考菌株和其他8株细菌 (空肠弯曲菌、李斯特氏细胞增生症、沙门氏菌伤寒、沙门氏菌肠炎、克雷伯杆菌肺炎、本文研究了常见食源性致病菌的大肠杆菌 O157:H7、蜡样芽孢杆菌、弧菌携带溶藻) 的电化学 DNA 生物传感器验证。</li>
		<li>每两周在各自琼脂上进行细菌菌株的例行亚文化以保持其生存能力, 请参阅表 1中的区域性条件。</li>
		<li>保持长期保存的细菌菌株在其各自生长的汤含有 20% (v/v) 甘油在-80 摄氏度, 因为甘油保护细菌, 防止冰晶体的形成, 可以破坏他们在冻结过程中。其次, 将甘油类中保存的细菌细胞培养循环接种到各自的汤中。在需要恢复细菌时, 根据各自的生长时间和温度孵化汤。</li>
		<li>使用扩展板技术 (33) 确定v. 副溶血性细胞的数量, 这是一种用于列举一系列稀释的微生物的标准和众所周知的方法。<ol>
<li>文化v. 副溶血在胰酪胨大豆汤 (3% 氯化钠) 在37°c 在 140 rpm 震动情况。然后, 将细菌细胞培养的1毫升转化为9毫升的3% 氯化钠, 以获得 10-1稀释因子。</li>
		</ol>
</li>
		<li>重复此步骤九次以获得多达10个-10稀释因子的完整系列。接下来, 分别将每个稀释因子的0.1 毫升 (从 10-1到 10-10) 传播到一个弧菌的选择性琼脂板上进行菌落计数。孵化的板块孵化在37°c 24 小时。</li>
		<li>使用蚁群计数器计算单个菌落数, 然后返回计算 (计数 CFU/pipetted 量 x 稀释因子) 以获得每毫升密度的菌落形成单位 (CFU mL-1)。从 CFU mL-1的定标图中获得菌落形成单元 (CFUs) 在细菌细胞悬浮液中的浓度。</li>
	</ol></li>
	<li>PCR 法的制备
	<ol>
<li>在经过改良的煮沸裂解过程中提取基因组 DNA34。首先, 对琼脂培养基上的单个细菌株进行条纹, 并将其接种在肉汤培养基中, 然后在其相对生长条件下孵化, 140 rpm 震动状态。</li>
		<li>将1毫升的肉汤培养成微离心管。离心机这在 4830 x g 和4°c 为1分钟获得药丸。使用约500µL 的无菌 TE 缓冲器, 以重新悬浮与颗粒。在加热块中按住所产生的悬浮10分钟, 在98到2摄氏度, 并立即冷却它在-18 摄氏度为10分钟溶解细胞和释放 DNA。</li>
		<li>离心机的基因组 DNA 在 4830 x g 和4°c 3 分钟, 以获得明确的悬浮和保持在-20 °c 的上清, 进一步使用。使用 biophotometer 测量与紫外线吸收在波长260毫微米 (作为核酸吸收波长的光) 并且在波长280毫微米 (作为蛋白质吸收波长的光) 确定提取的浓度和纯度基因组 DNA, 然后用标准生化化验方法确定所有菌株35并通过 16S rRNA 基因序列36进行验证。最后, 变性基因组 DNA 在92°c 2 分钟, 并在冰水中迅速冷却, 然后再应用到生物传感器。</li>
		<li>通过 PCR 对 toxR 基因的靶向进行进一步确认. 使用底漆对 (5 '-GTCTTCTGACGCAATCGTTG-3 ' 和 5 '-ATACGAGTGGTTGCTGTCATG-3 ') 在 thermocycler 中执行此过程。优化 PCR 放大的总反应量25µL 由不育水 (15.5 µL), 缓冲器 (5 µL), 底漆 (1 µL, 10 µM), dNTP 混合 (1 µL), dna 模板 (2 µL), Taq dna 聚合酶 (0.2 µL, 10x)。</li>
		<li>混合的成分, 并安排 PCR 放大的目标序列在 thermocycler 30 周期的放大程序。在我们的研究中, 每个周期包括三步反应i. e., 初始变性 (94 °c, 3 分钟), 其次为30循环变性 (94 °c, 1 分钟), 退火 (63 °c, 1.5 分钟) 和延伸 (72 °c, 1.5 分钟), 后跟最后的延伸 (72 °c, 7 分钟)。</li>
		<li>通过1.5% 琼脂糖凝胶电泳法测定扩增产品及其尺寸。用商业制作的凝胶文件系统捕捉凝胶图像。</li>
	</ol></li>
	<li>蛤皮样品的制备
	<ol>
<li>获取新鲜样品。 注: 我们的新鲜的蛤 (Anadara 蚶) 是从雪兰莪沙登训练班的湿市场获得的, 并迅速带到实验室的冰冷却箱进行分析。将样本分成2组, 即被治疗和未处理的组, 假设在收割开始时, 它们会均匀地收获, 直到放入冷库。</li>
		<li>在治疗组中, 先将其贮存在-20 摄氏度, 24 小时, 然后在 DNA 提取前, 在20摄氏度的紫外线照射下, 进行4小时的治疗。 注: 用于对照组的冷冻温度和紫外线照射会杀死或至少限制在蛤中自然积聚的v. 副溶血。在本研究中, 不要采用更高的巴氏杀菌70摄氏度, 目的是模拟新鲜的蛤的实际情况。同时, 直接从未经治疗的组中分析样品, 一旦到达实验室就不进行任何预处理。</li>
		<li>亚文化系一种v. 副溶血性 ATCC 17802 的菌株 (3% NaCl)。通过在 CA 上适当稀释 (3% NaCl), 确定接种中可行细胞的水平, 以获得接种 104 CFU mL-1 v. 副溶血性。在冷凝水中清洗每一个蛤, 然后在层流柜中使用无菌钳将其从外壳中取出, 然后将其擦洗干净。</li>
		<li>六十年代, 在90毫升无菌的融汇 (3% NaCl) 中, 对10克的蛤皮组织样品进行了测定. 将已知量的v. 副溶血性添加到9毫升的匀质样品汤中。将 unspiked 示例用作负控制。估计v. 副溶血性的细胞计数通过电镀0.1 毫升样品在 CA, 并且随后, 吹打1毫升样品入离心管为脱氧核糖核酸样品提取, 将被使用在生物传感器和 PCR 试验。</li>
		<li>在经过改良的煮沸裂解过程36后从尖刺和 unspiked 样品中提取出v. 副溶血性的基因组 DNA。最后, 确定 DNA 浓度和纯度的生物光度测量与紫外线吸收在波长 260 nm (作为核酸吸收波长的光), 也在波长 280 nm (作为蛋白质吸收波长的光)。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

这类电化学生物传感器成功发展的框架中的关键步骤是为传感器 (核酸或 DNA) 选择合适的生物识别元件;构造传感器传感层的化学方法转导材料;DNA 固定化与杂交的优化研究并通过实际样品对所研制的生物传感器进行验证。
核心到成功开发的敏感和选择性电化学 DNA 生物传感器, 是优化的固定化和杂交条件。在这项工作中, 我们用 MB 作为氧化还原络合物。有多种原则的 mb DNA 相互...

近年来公众和科学争论的一个主要话题, 食物中毒主要与3剂有关: 微生物1、化学试剂2和寄生虫3。受污染的食物会对人类造成严重的健康后果, 特别是在免疫系统薄弱、老年人、孕妇、婴儿和年幼儿童的高危人群中,4。由于非洲、亚洲和拉丁美洲5岁以下儿童每年发生100万多例急性腹泻, 食物中毒是全球主要疾病5,6 , 世界卫生组织已建立微生物作为最重要的贡献者7。副溶血弧菌在最广泛公认的毒株中脱颖而出。通常在沿海、河口和海洋环境中发现8, 它是一种革兰阴性菌, 在高盐环境中活跃, 在未充分烹调、处理不当或未加工的海洋中进食时引起严重的人胃肠炎。产品9。此外, 一些人现有的医疗条件使他们容易受到伤口感染、败血症或耳部感染, 这是由v 副溶血症10引起的。溶血素的毒力因子可分为两种类型, 分别为致病致病机制: TDH 基因编码的耐热直接溶血素 (TDH) 和 trh 基因11编码的 TDH 相关溶血素. 在临床标本中, 而不是在环境标本中, tdh 的毒力标志物 (trh 基因) 主要表现为副溶血性。
v. 副溶血性具有在广泛的条件下生存的能力, 迅速响应环境变化12。它的增殖机制随着细胞质量的增加, 其毒性也随之增大13, 从而提升其危险电位。更糟糕的是, 气候变化给这些细菌提供了充足的条件来加速他们的细胞数量增长14。由于其频率高, 需要在食品供应链上对v. 副溶血性进行监测, 特别是在海鲜的贸易和生产方面, 因为这些产品在很大数量上被发现,15,16在世界各地。目前, 细菌的识别和隔离使用了一系列的方法, 包括生物化学试验, 浓缩和选择性培养基17, 酶联磁吸附剂检测 (ELISA)18, 脉冲场凝胶电泳 (PFGE)19、乳胶凝集试验和聚合酶链反应 (PCR) 测试20。这些方法通常需要合格的人员, 先进的仪器, 和费力的技术, 没有立即提供污染的信息。这严重限制了及时检测有害污染和现场应用的可能性。快速检测工具仍然是一个突出的挑战。
生物传感正在成为检测食源性病原体的一个有希望的选择, 因为它提供了节省时间、经济高效、实用和实时分析方法 21, 22, 23, 24.然而, 尽管在尖刺样品和使用生物传感器的标准溶液中分析物检测有许多积极的结果, 但在水混合物或有机萃取物中, 仍缺乏对实际样品的研究25。最近, 利用直接和/或间接脱氧核糖核酸 (DNA) 检测的电化学生物传感器在科学家中得到了越来越多的关注, 因为它们通过杂交事件对互补靶的具体检测26,27,28,29. 与基于酶的生物传感器相比, 这些独特的方法更加稳定, 从而为小型化和商业化提供了一个有前景的技术。这项研究的目标是构建一个快速的工具, 能够检测出具有高选择性、灵敏性和实用性的、基于 DNA 序列特异性的杂交法. 识别策略包括聚乳酸稳定的金纳米粒子 (PLA-AuNPs)30和丝网印刷碳电极 (SPCEs) 在存在杂交指示剂, 亚甲基蓝 (MB)。利用细菌 DNA 裂解液和新鲜的蛤样, 进一步探讨了所研制的检测结构的潜力。

<table><tbody><tr><td>Acetic acid</td><td>Merck</td><td>100056</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>102445</td><td></td></tr><tr><td>Diaminoethane tetraacetic acid</td><td>Promega</td><td>E5134</td><td></td></tr><tr><td>Dibasic sodium phosphate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9763</td><td></td></tr><tr><td>Disodium hydrogen phosphate</td><td>Sigma-Aldrich</td><td>255793</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>16368</td><td></td></tr><tr><td>Gold (III) chloride trihydrate</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100317</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M44907</td><td></td></tr><tr><td>Monobasic sodium phosphate, monohydrate</td><td>Sigma-Aldrich</td><td>S3522</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma-Aldrich</td><td>P5119</td><td></td></tr><tr><td>Poly(lactic acid) resin, commercial grade 4042D</td><td>NatureWorks</td><td>4042D</td><td></td></tr><tr><td>Potassium chloride</td><td>R&amp;amp;M Chemicals</td><td>59435</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Potassium hexacyanoferrate III</td><td>R&amp;amp;M Chemicals</td><td>208019</td><td></td></tr><tr><td>Sodium acetate anhydrous salt</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Trisodium citrate</td><td>Sigma-Aldrich</td><td>S1804</td><td></td></tr><tr><td>Tris(hydroxymethyl) aminomethane</td><td>Fisher Scientific</td><td>T395-100</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>2X PCR Master Mix with Dual-Dye</td><td>Norgen Biotek</td><td>28240</td><td></td></tr><tr><td>Agarose gel</td><td>Merck</td><td>101236</td><td></td></tr><tr><td>Bolton Agar</td><td>Merck</td><td>100079</td><td></td></tr><tr><td>Bolton Broth</td><td>Merck</td><td>100079</td><td></td></tr><tr><td>CHROMagar Vibrio</td><td>CHROMagar</td><td>VB910</td><td></td></tr><tr><td>dNTPs</td><td>Promega</td><td>U1511</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Scientific</td><td>R0581</td><td></td></tr><tr><td>Eosin methylene blue agar&amp;nbsp;</td><td>Merck</td><td>101347</td><td></td></tr><tr><td>GelRed</td><td>Biotium</td><td>41001</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>104092</td><td></td></tr><tr><td>Go Taq Buffer</td><td>Promega</td><td>M7911</td><td></td></tr><tr><td>Loading dye 100 bp DNA ladder</td><td>Promega</td><td>G2101</td><td></td></tr><tr><td>Loading dye 1kb DNA ladder</td><td>Promega</td><td>G5711</td><td></td></tr><tr><td>Magnesium chloride</td><td>Promega</td><td>91176</td><td></td></tr><tr><td>Mannitol egg yolk polymyxin agar</td><td>Merck</td><td>105267</td><td></td></tr><tr><td>McConkey Agar</td><td>Merck</td><td>105465</td><td></td></tr><tr><td>Nutrient Broth</td><td>Merck</td><td>105443</td><td></td></tr><tr><td>Taq polymerase</td><td>Merck</td><td>71003</td><td></td></tr><tr><td>Trypticase Soy Broth</td><td>Merck</td><td>105459</td><td></td></tr><tr><td>Trypticase Soy Agar</td><td>Merck</td><td>105458</td><td></td></tr></tbody></table>

development of an electrochemical dna biosensor to detect a foodborne pathogen

副溶血性弧菌 (副溶血性)是一种常见的食源性致病菌, 在全球范围内造成很大比例的公共卫生问题, 严重影响人类死亡率和发病率。常规的检测v. 副溶血性的方法, 如基于文化的方法、免疫学化验和基于分子的方法, 需要复杂的样品处理, 耗时、繁琐、费用高昂。近年来, 生物传感器已被证明是一种有前景的综合检测方法, 具有快速检测、性价比高、实用性强等优点。本研究的重点是利用 DNA 杂交原理开发一种快速检测具有高选择性和灵敏度的v. 副溶血性方法。在工作中, 采用 x 射线衍射 (XRD)、紫外可见光谱 (紫外-可见光)、透射电镜 (TEM)、场发射等方法, 对合成聚乳酸稳定金纳米粒子 (PLA-AuNPs) 进行了表征。扫描电镜 (FESEM) 和循环伏安法 (CV)。并对 PLA-AuNPs 的稳定性、灵敏度和重现性进行了进一步的测试。研究发现, PLA-AuNPs 在水溶液中形成了稳定纳米粒子的良好结构。我们还观察到, 由于较小的电荷转移电阻 (Rct) 值和活动表面积 (0.41 厘米2) 的增加, 灵敏度得到了改善。我们的 DNA 生物传感器的发展是基于对 AuNPs 和以亚甲基蓝 (MB) 为氧化还原指标的丝网印刷碳电极 (SPCE) 的改性。用差分脉冲伏安法 (DPV) 对固定化和杂交事件进行了评估。我们发现, 互补, 非互补, 和不匹配的寡核苷酸是专门区分的捏造的生物传感器。它还显示了对各种食物传播病原体的交叉反应性研究和在新鲜的蛤中鉴定v. 副溶血的可靠敏感检测。

通过柠檬酸钠的水溶液的颜色变化, 揭示了 AuNPs 的形成。这使颜色从淡黄色变为深宝石红色。从紫外可见光谱 (图 1) 中证实了 PLA-AuNPs 的产生, 其中表面等离子体共振 (SPR) 峰值的增长约为 540 nm。AuNPs 的形成和存在是在 500-600 nm 波长范围内, 这取决于粒子大小37。AuNPs 和 PLA-AuNPs 的 XRD 模式显示在图 2中。在31.7、38...

Watch this Scientific Journal Video about Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen at JoVE.com

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

电化学 DNA 生物传感器检测食源性致病菌的研究进展

本文提出了一种电化学 DNA 生物传感器的研制方案, 包括聚乳酸稳定、金纳米粒子修饰、丝网印刷碳电极检测副溶血性弧菌。

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

development-an-electrochemical-dna-biosensor-to-detect-foodborne

Research

JoVE Journal

Bioengineering

13.0K 次观看.  Universiti Putra Malaysia. 本文提出了一种电化学 DNA 生物传感器的研制方案, 包括聚乳酸稳定、金纳米粒子修饰、丝网印刷碳电极检测副溶血性弧菌。

视频: Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫与感染

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

生物学

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

细菌利用试金石比色法检测

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...