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 相互作用: (i) 通过静电相互作用的 mb 阳离子电荷与磷酸盐主干阴离子电荷的 ssDNA 和 dsDNA;(ii) 通过在 dsDNA 中的交替 G C 基对之间成功插入 mb, 通过 mb 插层;(iii) 由共价键到目标 ssDNA 的末端, 导致低标号密度 (每脱氧核糖核酸链的一个或二 MB 分子);和 (iv) MB 与未绑定的鸟嘌呤基在 ssDNA 的相互作用。我们应用了第四原则, 它准确地确定了v 副溶血性从细菌和新鲜的蛤的交叉反应。在特别感兴趣的氧化之后, MB 和 ssDNA 之间有更强的亲和力。ssDNA 与其互补序列的杂交取代了粘结的 MB 分子, 从而减少了信号。
本文的一系列实验结果重点研究了利用 DNA 杂交原理快速检测具有高选择性和灵敏度的v. 副溶血性的方法。第一步是合成和表征聚乳酸稳定的金纳米粒子 (PLA-AuNPs), 用于修饰电极 (图 1-6和表 2)。利用紫外可见光谱 (UV-可见光)、X 射线衍射 (XRD)、场发射扫描电子显微镜 (FESEM)、透射电镜 (TEM)、循环伏安法 (CV) 等方法对金纳米粒子的质量进行了评价, 并阻抗分析。开发的第二部分涉及修饰电极 (图 7-12和表 3-4) 的电化学特性, 它决定了修饰电极相对于裸电极的成功增强。检测杂交事件的术语。
DNA 生物传感器发展的第三步是基于杂交事件实验条件的优化, 以及已开发的生物传感器在实际病原体检测中的应用 (图 13-20)。利用差分脉冲伏安法 (DPV) 对确定病原体检测成功的杂交事件进行了评估。通过减少电流, 表明靶向病原体的阳性存在。通过构造标定图和 LOD 的计算 (图 15) 评估了所开发的生物传感器的灵敏度。制备的生物传感器能够根据不同的电流还原水平 (图17和 18), 从其他病原体中明确区分v. 副溶血性。在图19和 20中介绍了已开发的生物传感器在实际食品样品中检测v. 副溶血性的可行性, 并指出了靶向病原体的阳性存在.dna 生物传感器的成功发展, 高度依赖于 dna 探针的选择、改进电极的增强以及实验条件的优化。这里的 DNA 探针已经从浩67的以前的工作中进行了调整。采用聚乳酸稳定的金纳米粒子对改性电极进行了增强, 使纳米标准化。金纳米粒子的作用是电催化材料的68 , 并采取行动, 以提高电子的传输速率, 并提供一个表面上锚定 DNA 探针。实验参数根据温度和潜伏期进行优化, 对 DNA 形成双工具有重要意义。杂交温度影响非特异吸附之间的分离, 这增加了传感器的选择性。杂交的孵化时间增加了双相形成的几率。为了确保 DNA 探针在合适的方向上形成双相地层, 必须对固定化过程进行优化。
表 5比较最近开发的电化学 DNA 生物传感器69,70,71,72,73,74,75,.基于 DNA 的生物传感是一种很有希望的替代分子技术的方法, 虽然有一些限制, 必须解决之前, 该方法可以移植足够的现场使用。主要的限制是样本处理 dna 提取作为纯度和 dna 的数量在现场提取不太可能是那么好, 从标准的实验室方法提取。然而, 如果 PCR 和凝胶电泳可以用微流控样品预处理系统代替, 则是一种很有前景的选择。
基于 DNA 的生物传感器对于许多应用 (包括医疗诊断、环境监测和食品监测) 可能有用。这项技术将使在线监测的质量控制过程和护理点监测的病人。整个传感器系统的可移植性将使诊断过程分散, 消费者可以在舒适的家里使用检测系统。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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&#38;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&#38;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&#160;</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。

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

Nanyang Technological University

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

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 ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

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 ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

的发展<em&gt;体外</em&gt;眼平台，试验用隐形眼镜

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.