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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</ol>

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

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

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 ...

科研

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

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

化学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

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, ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

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. ...

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. ...

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

检测使用荧光脱氧核酶的细菌

光学检测

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

利用磁调制生物传感系统的生物检测的快速均质检测

电化学 DNA 生物传感器检测食源性致病菌的研究进展

纳米传感器检测体内蛋白酶活性的无创诊断

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

细菌利用试金石比色法检测

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

检测使用荧光脱氧核酶的细菌

光学检测

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

利用磁调制生物传感系统的生物检测的快速均质检测

电化学 DNA 生物传感器检测食源性致病菌的研究进展

纳米传感器检测体内蛋白酶活性的无创诊断

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

细菌利用试金石比色法检测

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