这项工作是支持 INBRE P20GM103418, 堪萨斯大豆委员会 (肯尼迪航天公司/电源 1663), ACS 脉冲 56629-UNI7 和电源聚合物化学启动基金, 所有的 SS。我们感谢大学摄影师, Mr. 雅各 Anselmi, 为他出色的视频工作。我们还感谢 Mr. 罗杰 Heckert 和 Mrs. 卡塔奥义 Heckert 对研究的慷慨支持。

1.Synthesis 多参量磁荧光纳米 (MFnS) 的功能化. 
<ol> <li> 合成顺氧化铁纳米粒子 (IONPs) <ol> <li> 准备 IONP 合成, 准备以下3解决方案: 解决方案 1: FeCl 3 (0.70 g) 和 FeCl 2 在 H 2 O (2 毫升), 解决方案 2: NH 4 OH (2.0 毫升, 13.4 米) 在 h 2 O (15 毫升) 和解决方案 3: 聚丙烯酸 (0.855 g) 在 h 2 O (5 毫升)。
		<li> 将90和 #181; 2 米盐酸 (HCl) 添加到溶液 1, 然后立即与溶液2混合, 而涡流在 875 rpm。然后添加解决方案3。继续涡流60分钟. </li>
		<li> 离心20分钟, 在 1620 x g. 离心机上清液20分钟, 在 2880 x g. </li>
		<li> 通过透析净化终末清液。将上清液添加到透析袋 (分子6和 #8722; 8 K), 并将其放在含有磷酸盐缓冲盐水 (PBS) (pH = 7.4) 和自旋棒的烧杯中。让它浸泡12小时, 用新的缓冲器每2-3 小时更换一次. </li>
	</ol> </li> <li> IONPs 的目标特定抗体与表面的共轭 <ol> <li> 准备以下四解决方案: 溶液 A: 5 毫克 1-乙基 3-(3-dimethylaminopropyl) 二盐酸盐 (氯甲酸) 250 和 #181; L MES [2-( N -吗) 磺酸] 缓冲液 (0.1 米, pH = 6.8), 溶液 B: 3 毫克 n-羟基琥珀酰亚胺 (NHS) 在250和 #181; MES 缓冲器 (0.1 米, ph 值 = 6.8), 溶液 C: 5 和 #181; IgG1, 大肠杆菌单克隆, 225 和 #181; 磷酸缓冲盐水 (PBS) (ph 7.4), 溶液 D: 4 毫升的 IONP (5.0 摩尔) 在1毫升 PBS (pH 7.4). </li>
		<li> 将 2-(N-吗) 磺酸 (MES) 缓冲区添加到解决方案 a 中, 然后在十五年代内立即将解决方案 a 添加到解决方案 D 中, 并在每次增加混合后反转解决方案. </li>
		<li> 立即开始在三分钟内将解决方案 B 添加到解决方案 D 的小增量中。在每次添加后通过反转解决方案进行混合. </li>
		<li> 将解决方案 C 添加到解决方案中, 增量为5和 #181; L, 在每增加混合后反转解决方案. </li>
		<li> 允许反应在室温下持续3小时, 然后在晚上4和 #176 继续孵化. </li>
		<li> 使用 PBS (pH = 7.4, 最终 [Fe] = 3.5 摩尔) 将产生的 Ab 共轭 IONPs 通过磁性柱净化, 以去除任何游离抗体。
		<ol> <li> 用 PBS (1 毫升) 清洗磁性柱。将柱连接到磁板上, 并添加 IONP 溶液. </li>
			<li> 再用 PBS (1 毫升) 清洗磁性柱。从磁性板中取出磁性柱。将 PBS 添加到列中并收集解决方案。存储在4和 #176; C </li> </ol> </li> </ol> </li> <li> 封装荧光11和 #39;-Dioctadecyl-3,3,3 和 #39;, 3 和 #39;-Tetramethylindocarbocyanine 高氯酸盐 (DiI) 染料的聚丙烯酸 (临机) 涂层的抗体共轭 IONPs 使用溶剂扩散法。
	<ol> <li> 到4毫升的 IONPs, 添加滴2.0 和 #181; DiI 染料 (2 摩尔) 在100和 #181 中的 l, 以连续混合在 1100 rpm 的亚砜 l. </li>
		<li> 透析结果解决方案 (分子 6-8 KDa, 12 h) 对抗 PBS, 创建最终的 MFnS 解决方案, 最终铁浓度为 [Fe] = 2 摩尔. </li>
	</ol> </li> <li> 特性 MFnS <ol> <li> 进行分光光度分析, 使用平板阅读器检测 595 nM 的封装 DiI 染料和荧光发射。
		<li> 动态光散射 <ol> <li> 将 MFnS 溶液的样品放到 zetasizer 中, 以确定 MFnS 的平均尺寸和表面电荷. 
			注意: 在这里, 功能 MFnS 的平均大小和表面电荷分别被发现为 77.09 nm 和-22.3 mV. </li>
		</ol> </li> </ol> </li> </ol> 2。细菌培养和砧木溶液制备 <ol> <li> 细菌培养 <ol> <li> 水合 freeze-dried 颗粒 ( 大肠杆菌 O157:H7) 在1毫升的营养液中, 然后再加入5毫升的肉汤。
		<li> 应用100和 #181; 此解决方案的 L 使用无菌接种循环, 然后在37和 #176 中孵化; C 为 24 h. </li>
		<li> 24 小时后, 从琼脂板中选择一个孤立的菌落, 并将其添加到15毫升的培养液中。在37和 #176 孵育, C 为4-6 小时, 同时监测光学密度值 (吸光度在 600 nm). </li>
		<li> 一旦获得0.1 的光学密度值, 停止培养, 并执行串行稀释. </li>
	</ol> </li> <li> 串行稀释 <ol> <li> 添加900和 #181; L 营养液为八无菌离心管。
		<li> 添加100和 #181; 细菌悬浮的 L 对第一个管子 (稀释 10 -1 ). </li>
		<li> 取100和 #181; L 从第一个管, 并添加它到第二个, 获得稀释 10 -2 。继续此模式的其余管, 最后稀释 10 -8 . </li>
		<li> 传输100和 #181; 从每个试管中分离琼脂板, 并在37和 #176 孵育24小时; C. </li>
		<li> 第二天, 计算每个板块上的菌落形成单位 (CFUs)。选择 100 CFUs 的板材。使用相应的稀释进行进一步的实验 (100 和 #181; L = ~ 100 CFUs). 
		注意: 这里是 10 -6 稀释, 导致 100 CFUs. </li>
	</ol> </li> </ol> 3。利用 MFnS 快速检测大肠杆菌 O157:H7 <ol> <li> 使用各种 PBS 解决方案 (1X、pH 值7.4、300和 #181; L), 10 -6 细菌的数量增加, 导致 CFU 范围从1-100。在这些解决方案中添加一致的 MFnS (100 和 #181; L). </li>
	<li> 创建一个基线解决方案, 其中仅包含 PBS (1X、pH 7.4、300和 #181; l) 和 MFnS (100 和 #181; l). </li>
	<li> 在37和 #176 孵育溶液30分钟, 然后让它们冷却到室温. </li>
	<li> 将单个解决方案转移到磁 relaxometer (0.47 泰斯拉), 并在每个解决方案中的 CFUs 中记录弛豫时间 (T2) 的变化。
	<ol> <li> 若要记录 T2 值的更改, 请首先测量包含 PBS 和 MFnS 的基线解决方案的 T2 值。
		<ol> <li> 将解决方案和 #160 放置在磁性 relaxometer 中。打开相应的软件, 选择和 #34; T2 放松和 #34; 设置和按下 #34; 测量. #34; </li> </ol> </li> <li> 在基线 T2 的集合之后, 测量附加解决方案的 T2 值, 这些解是用各种细菌的浓度. 
		注: T2 的变化相当于从尖刺 T2 中减去的基线 T2. </li>
	</ol> </li> <li> 从磁性 relaxometer 中取出样品, 并将试管离心 2880 x g, 10 分钟. </li>
	<li> 醒酒100和 #181 中的上清和重细菌颗粒; PBS (1X, pH 7.4). </li>
	<li> 添加80和 #181; 每个悬浮为96个井板, 记录荧光强度在 595 nM. 
	注: 测试可以重复使用不同的溶剂, 包括湖泊水, 牛奶和其他如在结果部分所述 </li> </ol>

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这一纳米技术的示范应用尚未得到 FDA 的批准。

本协议的设计目的是尽可能简单地生成完全功能的 MFnS。但是, 根据用户的最终目标, 更改协议可能有用的许多关键点。例如, 使用不同的抗体可以使许多其他病原体成为靶向。此外, 本议定书不限于使用抗体作为靶分子。任何对目标病原体有特定约束力的分子, 如宿主细胞受体, 也可用作靶分子。只要靶向分子有一个主要胺或酒精组, 或可以功能化有一个, 它可能被共轭到表?...

在商业生产的食物和水源中, 细菌污染的持续发生, 已使人们对越来越快速和具体的诊断平台产生了需求。1,2一些更常见的细菌污染物是由沙门氏菌、葡萄球菌、李斯特菌、弧菌、志贺氏杆菌和大肠杆菌引起的。3,4这些病原体的细菌污染通常会导致发烧、霍乱、肠胃炎和腹泻等症状。4水资源的污染往往对社区造成严重和不利的影响, 无法获得足够的过滤水, 食品污染导致大量疾病和产品召回努力。5,6
为了减少因细菌污染引起的疾病的发生, 已经作出了许多努力, 以制定方法, 在销售或消费之前, 可以有效地对水和食物进行扫描。3技术, 如 PCR、1、7、8、9、10 ELISA、11、12循环介导的等温放大 (灯),13,14 ,15,16,17,18,19,20,21, 22、23、24最近被用于检测各种病原体。与传统的细菌培养方法相比, 这些技术在特异性和时间方面更有效率。然而, 这些技术仍在与虚假的正面和负面、复杂的程序和成本斗争。1,3,25正是由于这个原因, 多磁荧光纳米 (MFnS) 被提出作为细菌检测的替代方法。
这些纳米独特的配对磁松弛和荧光模式, 允许双检测平台, 既快速又准确。使用大肠杆菌O157:H7 作为样品的污染物, MFnS 在几分钟内检测到 1 CFU 的能力被证实。病原体特异抗体用于增加特异性, 并且磁性和荧光方式的组合允许检测和定量细菌污染物在两个低 high-contamination 范围。16在细菌污染的情况下, 由于特定病原体抗体的靶向能力, 纳米将围绕细菌群。磁性纳米和细菌之间的结合限制了磁性铁芯与周围的水质子之间的相互作用。这会导致 T2 松弛时间的增加, 如磁 relaxometer 所记录的那样。随着溶液中细菌浓度的升高, 纳米随细菌数量的增加而分散, 导致 T2 值降低。反之, 由于纳米直接与病原体的数量增加, 荧光辐射与细菌浓度的比例会增加。离心的样品, 和分离的细菌颗粒, 将只保存纳米颗粒直接附着在细菌, 消除任何自由漂浮的纳米, 并直接关联的荧光辐射与数量溶液中存在细菌。此机制的示意图表示形式在图 1中表示。
这个 MFnS 平台已经设计了点的筛选, 导致低成本和便携的特点。MFnS 在室温下是稳定的, 只有在非常低的浓度下才能准确检测细菌污染物。此外, 在合成后, 使用 MFnS 是简单的, 不需要在实地使用训练有素的专业人员。最后, 这个诊断平台允许高度可定制的目标, 提供一种手段, 这一平台可以用来检测各种病原体, 在许多不同的设置。

<table><tbody><tr><td>Ferrous Chloride Tetrahydrate</td><td>Fisher Scientific</td><td>I90-500</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Fisher Scientific</td><td>I88-500</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Polyacryllic Acid</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>EDC</td><td>Thermofisher Scientific</td><td>22980</td><td></td></tr><tr><td>NHS</td><td>Fisher Scientific</td><td>AC157270250</td><td></td></tr><tr><td>Anti-E. coli O111 antibody&amp;nbsp;</td><td>sera care</td><td>5310-0352</td><td></td></tr><tr><td>Anti-E. coli O157:H7 antibody [P3C6]&amp;nbsp;</td><td>Abcam</td><td>ab75244</td><td></td></tr><tr><td>DiI Stain</td><td>Fisher Scientific</td><td>D282</td><td></td></tr><tr><td>Nutrient Broth</td><td>Difco</td><td>233000</td><td></td></tr><tr><td>Freeze-dried E. coli O157:H7 pellet</td><td>ATCC</td><td>700728</td><td></td></tr><tr><td>Magnetic Relaxomteter&amp;nbsp;</td><td>Bruker</td><td>mq20</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern</td><td>NANO-ZS90</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Magnetic Column&amp;nbsp;</td><td>QuadroMACS</td><td>130-090-976</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 Series</td><td></td></tr><tr><td>Centrifuge (accuSpin Micro 17)</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>Floor Model Shaking Incubator</td><td>SHEL LAB</td><td>SSI5</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Open-Air Rocking Shaker</td><td>Fisher Scientific</td><td>02-217-765</td><td></td></tr></tbody></table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

出大肠杆菌O157:H7 已与水和食源性疾病联系在一起, 尽管目前使用的食品和 water-screening 方法仍然存在威胁。虽然传统的细菌检测方法, 如聚合酶链反应 (PCR) 和酶联免疫吸附试验 (ELISA) 可以明确检测致病污染物, 他们需要广泛的样本准备和漫长的等待期。此外, 这些做法需要先进的实验室仪器和设置, 必须由训练有素的专业人员执行。在此, 提出了一种简单的诊断技术的协议, 其特点是磁性和荧光参数在纳米平台的独特组合。所提出的多磁荧光纳米 (MFnS) 可以检测到在小于1小时内, 在溶液中存在的1菌落形成单元的大肠杆菌O157:H7 污染。此外, 还证实了 MFnS 在诸如牛奶和湖水等复杂介质中保持高度功能的能力。另外的特异性化验也被用来证明 MFnS 只检测特定目标细菌的能力, 即使在存在类似的细菌种类的情况下。磁性和荧光模式的配对允许检测和定量的病原体污染在广泛的浓度, 表现出其在早期和晚期污染检测的高性能。MFnS 的有效性、可承受性和可移植性使他们成为点筛选各种环境中的细菌污染物的理想候选者, 从水生水库到商业包装食品。

MFnS 操作的机制在图 1中表示。细菌污染物表面周围 MFnS 的聚类干扰了 MFnS 和周围氢核的磁性核心之间的相互作用。由于这种聚类, 磁松弛值增加。随着细菌污染物浓度的增加, 聚类减少, T2 值的变化减小。因此, 增加一个荧光形态是至关重要的。随着细菌浓度的增加, MFnS 所产生的荧光信号的强度增加, 从而使得对低浓度和高浓缩范围内细菌污染物的敏感检测成为可以。
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Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

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

本协议的总体目标是通过磁性松弛和荧光发射方式的结合, 合成功能性纳米, 用于便携式、cost-effective 和快速检测特定靶向致病菌。

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 ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

7.6K 次观看.  Pittsburg State University. 本协议的总体目标是通过磁性松弛和荧光发射方式的结合, 合成功能性纳米, 用于便携式、cost-effective 和快速检测特定靶向致病菌。

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

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色纸为基础的检测<em&gt;大肠杆菌</em&gt;<em&gt;沙门氏菌</em&gt;属，和<em&gt;单核细胞增生李斯特氏菌</em&gt;从农业用水大容量

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

科研

环境科学

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

从个人污秽苍蝇检测食源性细菌病原体

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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 ...

生物工程

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

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

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 ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

连续流PCR微流控芯片中 大肠杆 菌的扩增及毛细管电泳系统的检测

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

生物学

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

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

摘要

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此视频中的章节

比色纸为基础的检测

从个人污秽苍蝇检测食源性细菌病原体

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

光学检测

电化学 DNA 生物传感器检测食源性致病菌的研究进展

沙门氏菌筛选的高通量平台.

连续流PCR微流控芯片中大肠杆菌的扩增及毛细管电泳系统的检测

检测现场的

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

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

摘要

探索更多视频

此视频中的章节

比色纸为基础的检测

从个人污秽苍蝇检测食源性细菌病原体

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

光学检测

电化学 DNA 生物传感器检测食源性致病菌的研究进展

沙门氏菌筛选的高通量平台.

连续流PCR微流控芯片中 大肠杆 菌的扩增及毛细管电泳系统的检测

检测现场的

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

连续流PCR微流控芯片中大肠杆菌的扩增及毛细管电泳系统的检测