由工程和物理科学研究理事会（EPRSC），在纳米技术的跨学科研究中心（IRC），英国皇家学会（RS）和Biano顾问（BNC），约瑟夫·Ndieyira支持。我们感谢，亚历杭德拉·多诺索巴雷拉，周德舰，曼努埃尔Vögtli，巴彻勒马修，马修A.库珀，托尔斯滕·施特龙茨，特雷弗·雷门和Gabriel Aeppli的，有益的讨论。

1。悬臂的制备<ol><li>使用聚四氟乙烯镊子浸泡选定数目的悬臂式芯片（每个悬臂测量到新鲜制备的Piranha溶液（比例为1:1的H 2 SO 4和H 2 O 2）的长500微米，宽100微米，0.9微米厚）20分钟。 </li><li>约20分钟后取出悬臂芯片食人鱼的解决方案，并用去离子水彻底冲洗，并立即转移到新鲜烹制的食人鱼的解决方案。重复上述步骤1.1，如果芯片包含斑点的污垢，否则继续下一步。 </li><li>在去离子水中彻底清洗后，用纯乙醇冲洗和干燥的悬臂芯片放在热板上，在75℃下，以除去任何痕量的水。用光学显微镜检查，以确认其清洁。 </li><li>清洗悬臂芯片转移到蒸发室和泵下来，目标实现了真空10 -7毫巴的压力。 </li><li>一旦达到所需的真空压力，外套的一侧的每个悬臂阵列2 nm的第一钛作为粘合层之前的附加层为20nm厚的金行为。为了证实自己的厚度，使用石英晶体显示器直接放在上面的目标源。 </li><li>保留镀金的悬臂梁传感器芯片在1-2小时，在真空条件下冷却开幕前室。 </li><li>转移新鲜蒸发的芯片，充氩气的真空储存容器，以防止任何形式的污染。 </li></ol> 2。悬臂芯片官能的<ol><li>首先，安排微毛细管一个官能阶段2根据悬臂间距250微米大小的。 </li><li>如果是，注入2 mM的乙醇硫醇表面目标分子的解决方案， 即的的细菌mucopeptides（D-丙氨酸和D-Lac的 ），和“惯性t&#39;的已知的抗蚀剂的生物分子吸附在三乙二醇（PEG）链烷硫醇终端16的毛细管应包含随机确定，以避免使用偏置的各类表面的捕获分子。 </li><li>下孵育20分钟，在微毛细管悬臂充满含有表面捕获分子与巯基的解决方案。确保表面捕获分子被限制到每一个人的悬臂梁传感器，以避免或减少交叉污染的解决方案。如果这个过程被雇用正确的，它应该有系统地改变新鲜烹制的黄金涂层悬臂到允许黄金涂层表面受体细菌粘肽的目标，形成自组装形成的化学活性传感器。 </li></ol> 3。解决方案的准备工作<ol><li> 0.1M的单 - 和二 - 基本的磷酸钠盐溶解在超纯水（18.2MΩ·cm的电阻率）和组合吨Ø产生的pH值为7.4。 </li><li>添加0.002％PS80的缓冲溶液，以最小化的玻璃器皿的药物分子的非特异性相互作用引起的聚集。 </li><li>药物稀释到所需的浓度不同，将使计算K d的新鲜缓冲的解决方案。 </li><li>使用0.2微米的过滤器过滤新鲜配制的药物溶液，超声前5分钟，在室温下用氩气清洗。 </li><li>重复此步骤缓冲血清使用全血清药物。振荡15分钟，用5分钟，以确保完全溶解。 </li></ol> 4。表面应力检测<ol><li>加载官能悬臂梁传感器芯片成液体流动的细胞室。 </li><li>激光点对准到每个传感器的自由端上，并确认对应，通过加热的液体腔室1℃。所有八个镀金的悬臂梁传感器阵列应该接受COMPREssive向下弯曲，因为双金属的差异所造成的影响在硅和黄金膨胀率。 </li><li>加热约10分钟后，允许悬臂进一步冷却10分钟。 </li><li>计算在个人之间的悬臂式传感器和最大弯曲信号的弯曲信号的相对标准偏差为≤5％然后接受希望以其他方式重复该过程对应的弯曲变化。 </li><li>接着，测量所有8个悬臂的谐振频率，以计算它们的弹簧常数。如果于一个芯片上的每个悬臂梁传感器之间的弹簧常数的变化≤1％具有一致的力学性能，否则取代悬臂式芯片上的传感器芯片上，然后接受。 </li><li>接下来，使用流体系统（型号精灵+）通过六通阀，以实现内的流动池的液体交换，而数据采集应使用自动LabView的，所以可以实现ftware。 </li><li>要监视的悬臂弯曲数据，请使用以下的测量协议：1）无论是缓冲溶液或血清注入没有药物控制测量，持续5-30分钟，以建立一个基线;二）注入药物溶液30-60分钟; 3）注入的10mM盐酸洗涤10〜60分钟，离解绑定的药物复合物，iv）最后的另一个5-30分钟的缓冲液中使用重新生成的表面肽，并恢复基线信号进一步注入的洗涤步骤。始终确保所有信号都在恒定的液体流速为30至180微升/分钟，并在固定的温度为25℃的温度控制柜下取得的。 </li><li>所有八个悬臂绝对弯曲信号应使用串行时间复用一个单一的位置灵敏探测器的光束法监测。 </li><li>来分析每个浓度的药物的弯曲信号，所产生的差动弯曲转换成差分伸缩性s之间的上部和下部两侧的悬臂使用斯托尼方程。 </li></ol>

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没有利益冲突的声明

这些结果表明，悬臂阵列传感器的灵敏度进行检测和定量的药物靶结合的相互作用，特别是在删除一个单一的药物的结合口袋的氢键与万古霉素耐药性的变化。我们展现出的纳摩尔检测灵敏度凡在协议与以前的表面等离子体共振（SPR）的研究18,19，揭示悬臂方法可以直接检测和量化药物分子在血液中的浓度临床相关常规应用在临床实践中。我们的数据表明，表面应力差，可以说是由一个产品形式（1）式， 即 （一）化学反应的一个化学术语，描述具体的药物靶结合事件，及（ii）之间的机械连接的几何术语，描述面点，这意味着当地的化学作用，从全球的技工去耦人相互作用的悬臂。虽然经典的Langmuir吸附等温化学名词由下式给出，几何术语揭示了渗流机制的药物靶诱导的表面应力的变化。临界阈值个〜10％（ 图5）检测到的差分悬臂弯曲，表示该表面应力集体转导时，一个比较大的表面分数占用的抗生素分子。对于p≥化学转化表面之间的机械连接件，逐步建立，如立体相互作用的纳米机械的网络节点之间的短距离的排斥作用产生增加整个悬臂向下弯曲。据推测，我们的纳米机械的渗流模型可能发挥重要的作用，在真正的细菌糖肽类抗生素的作用方式。这些发现突出的悬臂式技术的高灵敏度为studying抗生素的作用方式和研究的药物，以改善了解抗生素在纳米尺度的操作通知，使强大的药物来控制超级细菌感染的问题，发现代表了一种新的研究工具。要准备悬臂系统，重复性好，灵敏的测量，我们已经解决了一组在协议的目标，尤其是对微细胞样品装载和标准作业程序，让奈米定量检测。 悬臂技术相对于现有方法中的意义综上所述，我们指出，而这项技术是超过25年前提出的，它没有发现它的方式进入诊所，因为缺乏医疗相关目标的小心和重复测量。在这里，我们演示的程序建立了相关的，使用奈米悬臂调查机械师人抗生素对细菌细胞壁的目标并检测抗菌性的影响。传统的药物筛选方法需要某种类型的荧光或放射性标记的报告分子，测量到它的目标分析物的结合，往往在具有竞争力的或酶的结合分析法和蛋白质检测20。标记生物分子不仅费时且昂贵的，但该标签也可以干扰与阻碍分子间的相互作用的结合位点，从而导致假阴性。此外，荧光化合物往往是疏水性的，这可能会导致背景结合和假阳性。由于这些限制，是新的标签技术，使几乎任何分子复合体进行甄别，以最小的试验开发的兴趣日渐浓厚。目前历史最悠久的无标签的表面处理技术是SPR和石英晶体微天平（QCM）。到SPR相反，测量第ë键介电常数，标签 - 悬臂技术检测到的配体 - 受体相互作用的，它能够直接测量表面的受体特异性结合的配位体所产生的纳米机械力所产生的表面应变。这些传感器的独特性，其灵敏度不依赖于由于SPR和QCM而是在一个微小的平面应力引起的变化的分析物结合的质量变化引起的介电性能，使该技术特别适合研究药物分子在临床相关的抗生素浓度（3-27微米）17纳米力学。悬臂也特别适合于小的分子（如DNA片段和药物）检测在生理条件下，包括复杂的环境中，这在很大程度上是制药业的基础，因此，作为在药物发现中的补充工具。悬臂式技术已经成功地应用在基因组学领域的3,5，气体传感21 22，蛋白质组学，药物1。此外，低成本的硅技术，由于其兼容性与微细加工流程都采用悬臂，悬臂可以小型化，以提高灵敏度和并行为大型阵列传感器，多种药物的化合物筛选和更高的吞吐量信息丰富的筛选试验。仪器和实验设计的改进将允许在实时帮助推动解决的问题多药耐药感染的新一代superdrugs寻找各种各样的相互作用进行分析。 在协议的关键步骤强大的测量协议的发展是这项技术的应用。为了达到令人满意的定量的药物靶测量，并确定最低浓度的抗生素吨帽子缓冲区或血液中可检测到血清，在协议的关键步骤解决。第一项任务涉及到调整和优化表面化学捕获提升悬臂检测的特异性和敏感性。不可否认，表面应力传导是一个集体现象，需要一个比较大的部分表面进行覆盖，以建立化学反应区域之间的连通性。我们表明，通过改变下垫面肽的密度，临界阈值，〜P≥10％时，确定规模的肽密度的函数，否则为零（ 图5）的表面应力。重要的是，实验旨在探讨沿悬臂应力的均匀性，敏感的测量，以确保最大的信号偏转。此外，高效的表面恢复协议需要的地方，从而使多个周期的测量，并降低成本，为每个测试通过万吨。在设计使用巯基化的疏水性端的受体表面的受体分子和它们之间的间距，方向是至关重要的允许范明镜范德华分子之间的相互作用，由于密实填充自组装，以减少非特异性相互作用。此外，应包含捕获分子的聚乙二醇（PEG）的连接器，以允许传感矩阵的某些部分是亲水性的，以防止插入待测溶液中的分子与未涂覆的金表面直接反应。 PEG连接分子，必须采取行动，作为间隔，以减少空间约束的，因此允许专门与互动解决方案的分析物的表面受体诱导可测量的表面应力变化。的悬臂阵列传感器必须与至少一个在原位参考测量来测定，在实时显示的信号是一个差分偏转，通过以下方式获得从S中减去绝对的参考悬臂偏转悬臂ensing。因此，参考悬臂考虑到的非特异性相互作用，如温度变化​​，折射率变化或相互作用的悬臂上的非官能化的底面是必不可少的。优化的流动速率（〜30-150微升/分钟）形成于该协议的一个关键步骤，因为它确保高效率地交换液体和溶液的材料有足够的稳定的大量运输。液体电池的设计必须允许最佳体积（5-80微升）采用快的流速，使完美的液体交换，克服大众运输的限制。的流率是特别重要的，同时进行动力学测量23。大容量液体室要求不可控的高流速，导致大样本量的要求，增加不必要的检测价格。此前，我们已经使用了重力流进入测量液体腔注入不同的样品。重力流的优势在于它的母鹿不要求任何机械部件，因此不会引入额外的噪声进入系统。然而，其显着的缺点是，它不仅可以可靠地工作在相对高的流率（约200微升/分钟）。显然，一个较低的流动速率（≤1微升/分钟）将需要的每单位时间的有限体积的样品，但在另一方面，它使反应更慢，因此需要较长的接触时间。此外，重力流有一个大的差别，它的流速，因为它依赖于，从而降低在实验过程中被消耗样品溶液的入口和出口之间的高度差。要避免重力流问题，注射器泵应该被使用。使用注射泵的优点在于，它允许以恒定的流速在很长一段时间，允许以一种更可控的环境中实现的实验。 的协议的限制获得重现的主要挑战ð特定的生物检测采用悬臂式传感器在于确保受体层的属性生物化学“有效”，对每个测定均匀。实验成功的秘密是一种谨慎的预处理的传感器芯片与连接器定位在其活性构象的受体分子的化学性质，以标准化的清洗和固定化的协议。在我们目前的体制，我们官能悬臂阵列使用小玻璃毛细管，这可能是受到一些缺点，在某些情况下，可能会出现问题的。在这些玻璃毛细管在两端开口，因此样品的溶剂可以很容易蒸发。例如，如果不能准确地控制官能化阶段的温度时，蒸发速度可以显着不同在不同的时间，尤其是在使用挥发性溶剂，如乙醇。有轻微的变化也有可能在孵育时间从一个悬臂到另一个克尔文感测液体必须连续加载到毛细血管。在毛细管官能化的其他限制因素是缺乏的能力，以确保悬臂总是插入到毛细管中完全相同的方式。另外，有时稍微被拉出，以防止交叉污染的液体样品可以流动到芯片上的身体从毛细血管悬臂。我们可以解决这些问题，作为一种替代的表面涂层过程大衣悬臂采用喷墨监视人。虽然这将允许样品涂层的精确控制的可能性扩大的大型阵列中，最常见的缺点是，沉积到悬臂的小滴可以在几秒钟内蒸发的和需要控制的湿度的环境中。因此，温育时间可以容易地调整，这对于某些应用可能是可取的。微妙的相互作用的因素，如样品VOlume明，孵化时间和蒸发速率官能样品曝光的悬臂上，有直接的影响，必须小心，以确保优化的表面化学悬臂试验，因为任何微小的变化将有很大的影响，在悬臂上的受体分子密度的响应。 实验设计修改（ 即替代技术或材料） 为了克服获得重现涂层过程的悬臂技术的未来发展面临的主要挑战，需要不同的策略，将使用集成在微流体通道的悬臂。这个想法是，特殊的悬臂芯片的设计应使每个悬臂被放置到自己的渠道，让在线渠道单独解决原位功能化程序。这些实验的设计修改将使涂装工艺执行在一个控制和封闭的环境中，暴露到溶剂的精确自动化悬臂芯片上固定化的捕获分子的培养时间和流量监测。然后，相同的信道可以被用于所有悬臂可能会暴露在相同的分析物溶液的实际的结合实验。除了的悬臂官能程序，悬臂读出机制还需要改进。的光束偏转方法是高度敏感的，它已被成功地用于原子力显微镜（AFM）技术多年。然而，为的自由站立悬臂式传感器的应用程序的光学读出有一些缺点，例如它并没有允许不透明的液体，如血液中的测量，激光器阵列的对应的时间，费时和乏味的，将当前配置不能区分倾斜和垂直偏转之间。因此，未来的发展cantilev呃将不得不考虑技术方面的一般传感器的设计（ 如悬臂几何）和悬臂读出强大的测量协议的领域和实验室环境。 Manalis和他的同事22发达的小说类型，其中有一个嵌入式的微流体通道内的梁悬臂空心悬臂传感器，允许在真空中进行操作的设备提供高品质的因素。在这种模式下，悬臂作为微量天平22，因此，它不再需要有一个相对于官能化的双方之间的不对称，因此，捕获分子可以被物理吸附或共价键直接连接在硅，通常使用的自组装膜或硅烷24。的悬臂也可以修改，用薄的聚合物层，然后共轭生化检测抗体25。 故障排除一个常见的​​亲事态进一步恶化的悬臂测量与气泡引入到微流体的流动池。必须小心，以确保没有气泡被引入到液晶单元而安装的芯片。信号的漂移也是样品的温度的差异所造成的另一个常见问题。悬臂必须是平衡稳定后再进行测量。所有的缓冲区，分析物溶液和再生溶液必须被存储在同一房间内的悬臂仪器，以允许所有的解决方案，具有相同的温度。虽然注射泵能提供非常精确的和非常低的流速，它一般是与机械噪声的悬臂测量。因此重要的是设计出一种简单而有效的降噪器，以避免不必要的偏转信号中的噪声。降噪器由注射泵和液体的细胞，其吸收吨之间的小储液器他从泵的机械噪声。由于光检测器的敏感性质，悬臂式测量系统最好在一个封闭的设置，以阻止任何杂散光干扰的光学读出系统操作。此外，感测层的质量显着降低，如果大量的洗涤​​步骤的传感器芯片的寿命限制的悬臂上执行。 掌握这一技术后的替代或未来的应用与终止在氨基或羧基的自组装膜涂覆的悬臂可以用作pH传感器。质子化或去质子化的官能端基取决于溶液的pH值，可以产生表面电荷，导致悬臂弯曲24。由于悬臂式传感器，可以跟踪与生命细菌的细胞壁1,6,7不稳定的药物靶相互作用，因此，它们将帮助搜索，并为开发新一代抗生素，以打击耐药菌感染。在未来的悬臂将用作微量天平测量的质量和单细胞的生长率26。的的悬臂技术将细胞反应，不同的生长因子或药物的研究证明是有益的。它也将快速检测多种生物标志物，里面有直接相关的医疗和护理点的应用提供了一种新的工具。由于悬臂式传感器的通用性，体积小，和鲁棒性，它们将形成一个敏感的监视器的有害环境的影响。他们已证明自己的灵敏度，检测有毒有害气体，可以逃离的实验室和工业生产单元到环境中，如氢氟酸27或氰化氢28。

分子识别范式的基础大片生物学和医学。药理学，例如，我们的目标是针对如transglycoslyation或催化细菌细胞壁的肽交联转肽的生化过程的主要参与者干扰与病理通路。因此，降低药物的设计来确定一个合适的分子针对特定细菌停靠站点，以诱使完美契合。仿效这种做法成功的一个典型的例子是万古霉素（VAN），它的目标肽聚糖的细胞壁，细菌一个保守的结构特征。在新生的革兰氏阳性菌的肽聚糖的基体由终止序列中的赖氨酸-D-丙氨酸-D-丙氨酸，拴通过C55脂质链接器8-10在这里称为D-丙氨酸的肽。这些暴露的肽肽前体为根本机械保护的细菌细胞对恶劣环境的力量，重要的是，都没有发现人类细胞中，使他们的理想目标抗生素药物。 凡特异性结合的细菌细胞壁肽形成中等强度的范肽的C-末端在图1中所示的复杂。 范和暴露的肽块的动作的transpeptidases和transglycosylases的，能催化交联的细胞壁1，有效地停止从交联，因此nanomechanically的减弱导致其死亡的细菌细胞破裂1,6之间的这种相互作用， 7。 出现耐万古霉素肠球菌 （VRE）是一个日益增加的医疗问题，因为产生细菌耐药性肠球菌由于酰胺林微妙的变化卡格酯键8，这种看似简单的结构变化，在细菌的表面上删除范的现场对接，从一个单一的氢键的结合口袋的后续修改。重要的是，这个过程中改变终端丙氨酸五肽存在于万古霉素敏感肠球菌 （VSE）到一个赖氨酸-D-丙氨酸-D-乳酸10,12，在这里被称为D-Lac的范-D-Lac的结合亲和力，产生了显着的减少因此，三个订单治疗无效的反对肠球菌感染，抗生素耐药性细菌的惊人增长幅度渲染范推动发展，创新的办法来加快和恢复抗菌剂的抗菌活性或发现更强大的药物对多药耐药细菌感染。 <p类=“jove_content”&gt;的灵感来自于我们的实验使用独立的悬臂系统，如利用化学能转化为运动细胞。悬臂式传感器，把化学能转化为机械运动的能力使它们独特的探针实时监测机械干扰细菌细胞壁的目标具有相当的优势，为。悬臂式传感器需要没有记者的标签，并在单步反应快速检测生物分子。他们可以筛选多种药物的生理环境下，如缓冲解决方案和全血清平行。此外，悬臂技术在原位引用使用差分测量，使他们能够特异性地检测药物靶相互作用，凭借其通过标准的半导体工艺路线制造，它们是适合于大批量生产和并行高通量筛选十万的每小时的药物。 particular多个悬臂梁传感器阵列研究对万古霉素的抗生素耐药性是有用的工具-因为耐万古霉素本质上是一个机械问题1,6,7。反应的模型细菌细胞壁的目标和 Van在溶液中可以检测到通过监视药物靶结合的相互作用引起的，在临床相关的应力的变化。体现在以悬臂弯曲信号从表面反应所产生的应力光学分析用激光束照亮传感器。此外，通过剪裁细菌悬臂梁传感器的顶部表面上的目标分子接收涂层，附近的一个未有限数目的分析物（车 ），特定的生化相互作用的生物力学模型细菌细胞壁基质进行实时监控。除了在分子识别事件，有几个因素会导致画质的悬臂传感器中选择，其中包括 - 温度变化，非特异性结合，或溶液的折射率的变化。为了解释的非特异性信号， 在测量和参考悬臂梁的弯曲持续监控，以分析的特异性相互作用进行原位的差分测量。此外，通过调节表面密度p（其中p占用的捕获分子的整个顶表面面积的悬臂的表面面积的比率被定义为依赖于表面的化学组成和几何形状的悬臂式传感器的检测灵敏度可提高所涵盖的X射线光电子能谱（XPS）所确定的总人口的分子。 这个协议的细节如何万古霉素或任何其他抗生素结合细菌细胞壁的前体的类似物（mucopeptides）在临床相关浓度的缓冲溶液中的一个第二血血清中可检测到的通过使用标签悬臂技术。新鲜的金表面自组装单分子膜（SAMs）的使用，因为往往容易形成稳定干净的金层13,14。自组装膜通常由短的硫醇基团的分子的共价键连接在金表面上，而在另一端所需的捕获单元被允许自由互动与被分析物在溶液中的目标。硫醇形成一个灵活的系统，特别是考虑到许多不同化学端基的巯基化合物是市售还是很容易在实验室中合成。但是，应注意，以确保与硫醇基团的分子必须具有正确的末端基团，例如在蛋白质或肽的共轭，氨基应该面对向内拴上的硫醇基团与羧基的反应表面发生。在这里，硫醇从b直接结合的三肽模拟物的细菌的脂质II固相方法合成的目标acterial细胞壁市售预装王-D-Ala和王-D-紫胶树脂和标准FMOC保护基化学15。虽然此说明的重点是对万古霉素，显然可以扩展到其他抗生素确实邀请来自不同的领域，特别是在生物化学，药理学，材料科学，轻松地通过此协议为自己的实验，研究人员进一步研究。

<table border="1">
	<tbody>
		<tr>
			<td>Items</td>
			<td>Catalog Number </td>
			<td>Company</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Scentris</td>
			<td></td>
			<td>Scentris, Veeco Instruments, Inc</td>
			<td>Not in production</td>
		</tr>
		<tr>
			<td>Six-way valve</td>
			<td></td>
			<td>MVP, Hamilton, Reno, NV</td>
			<td>Enables multiple concentration injection</td>
		</tr>
		<tr>
			<td>EB Evaporator</td>
			<td></td>
			<td>BOC Edwards Auto 500, UK</td>
			<td>Electron beam is preferable because of deformation of cantilevers caused by thermal heating problems</td>
		</tr>
		<tr>
			<td>Storage vessel</td>
			<td></td>
			<td>Agar Scientific, UK</td>
			<td>Keeps gold coated films fresh</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td></td>
			<td>King Precision Glass, Claremont, CA, USA</td>
			<td>For capillary functionalization of the cantilever arrays</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td></td>
			<td>Model Genie Plus, Kent Scientific, Torrington, CT, USA</td>
			<td>Good for controlled flow rate of liquid samples in microfluidic channels</td>
		</tr>
		<tr>
			<td>0.2-4.5 &#956;m Filters</td>
			<td>1511940001</td>
			<td>Merck Millipore</td>
			<td>For sample filtering</td>
		</tr>
		<tr>
			<td>Cantilever chips</td>
			<td></td>
			<td>London Centre for Nanotechnology (LCN)</td>
			<td>Highly sensitive</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>10049-21-5</td>
			<td>Sigma-Aldrich, UK</td>
			<td>For making buffer solutions</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>7558-79-4</td>
			<td>Sigma-Aldrich, UK</td>
			<td>For making buffer solutions</td>
		</tr>
		<tr>
			<td>Polysorbate 80 (PS80) or Tween 80</td>
			<td>9005-65-6</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used in buffer solutions to prevent nonspecific interactions on glassware</td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td>Millipore Co., Billerica, MA, USA</td>
			<td>Used for making solutions</td>
		</tr>
		<tr>
			<td>Whole serum</td>
			<td>GEM-700-110-H</td>
			<td>Sera Laboratories International Ltd, UK</td>
			<td>Used in measurements that mimic physiological conditions</td>
		</tr>
		<tr>
			<td>Vancomycin (Van)</td>
			<td>1404-93-9</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Drug used</td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>7664-93-9</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used for glassware cleaning procedures</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>7722-84-1</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used for glassware cleaning procedures</td>
		</tr>
		<tr>
			<td>D-Ala and D-Lac</td>
			<td></td>
			<td>London Centre for Nanotechnology (LCN)</td>
			<td>Surface receptors coated on the cantilever for detecting drug-target interactions</td>
		</tr>
		<tr>
			<td>LabView</td>
			<td></td>
			<td>National Instruments Co., Austin, TX, USA</td>
			<td>Software used for instrumental interface to allow controlled measurements</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

nanomechanics of drug-target interactions and antibacterial resistance detection

悬臂梁传感器，作为在细菌细胞壁的模型的基体固定在其表面上和在溶液中的抗生素药物反应之间的换能器，以前所未有的灵敏度和特异度1-5生化传感应用中表现出相当大的潜力。药物靶相互作用产生的表面应力，使悬臂弯曲，它是通过激光照射时，可以分析的信号的光学。表面应力测量纳米级精度的变化允许中断的生物力学模型细菌细胞壁的目标实时跟踪。尽管提供了相当大的优势，多悬臂梁传感器阵列从未被应用在量化药物靶结合的相互作用。 在这里，我们报告硫醇自组装单分子膜，模仿细菌细胞壁基质定量斯图涂的多个硅悬臂阵列DY抗生素结合的相互作用。为了理解万古霉素对细菌细胞壁的结构1,6,7的力学的影响。我们开发了一种新的模式，可以说是由两个独立的因素提出该悬臂弯曲;我）即一种化学因子，这是一个经典的Langmuir吸附等温，从中我们计算热力学平衡解离常数（K D） ⅱ）几何因素，本质上是一种细菌肽受体的悬臂的表面上分布的措施。肽受体（对）的表面分布是用来研究的几何形状和配位体加载的依赖性。结果表明，传感应用的p〜10％的阈值是至关重要的。下面的，有弯曲的信号是没有可检测到的，而高于此值时，弯曲信号几乎呈线性增加，揭示该应力是一个本地的化学结合因子的商品器和几何因子结合反应区域的机械连接和设计实力的代理商，以打击超级病菌感染提供了新的范式。

使用纳米级精度测量，以前所未有的灵敏度到一个单一的氢键缺失的应力变化，利用跟踪干扰细菌细胞壁的生物力学模型的实时（ 图2a-D）。 凡药物检测灵敏度的限制通过连续稀释其浓度步骤从1,000μm≤10纳米，露出最低检出浓度上升到平均〜-9±10纳米（〜15毫微克/毫升） 2纳米的差分弯曲信号（ 图2c）。进一步研究血清中能够检测到在生理环境下的抗生素，临床相关浓度范围为3-27μM17。注射后7μM 范血清（90％胎牛血清加10％甲醇钠的磷酸盐缓冲液pH 7.4）在相同的条件下在所有的悬臂，所述差分信号达拉血清 105±4纳米，而为DLac涂层的悬臂，没有观察到弯曲（ 图3）。 DALA涂层（万古霉素易受）悬臂弯曲观测到的信号相当强的药物靶结合的相互作用所造成的。然而，对于DLAC涂层（耐万古霉素）悬臂，存在基态氧孤对万古霉素结合口袋10,12和减少NH键斥力的减少，从而削弱药物靶点结合的相互作用或无悬臂弯曲，特别是对万古霉素的浓度较低（ 图3）。然而，对于万古霉素浓度高，我们观察到可测量弯曲的DLAC信号。此外，我们的实验设计是非常重要的原位引用所有CA涂有以下ntilevers 达拉和DLAC的同时实时暴露于相同的分析物。 要了解化学和几何中的确切作用，我们设计了一个模型1，说明溶剂相互作用之间的相关性和表面力学。这将产生表面应力： <img alt="公式1" src="/files/ftp_upload/50719/50719eq1.jpg" /> P&gt; PC，否则为零（1） 式（1）中的第一项为Langmuir吸附等温线，占药物靶结合事件，第二项是功法的形式描述大型机械强调网络形成的后果。 常数对应的最大表面应力时，所有的访问的结合位点被占用，K d的表面平衡解离的利弊桑特在悬臂上。 图4中所示的分析结果为：全局拟合公式（1）测得的应力差信号，揭示叠加到的结果; 约 29.7±1.0或14.1±3.0 MN /米和Kð〜1.0±0.3 800±310 μMD-Ala或DLAC的肽，其中一个对应于表面应力的措施时，所有可访问的结合位点被占用的。通过系统不同的肽密度P中监测的应力数据作为p的函数，而抗生素的浓度固定为10，100，和250μM，分别（ 图5）进行了调整，增强灵敏度的悬臂。重复该过程应力的范浓度为一个函数，而该肽密度固定在p〜100％。 &lt;IMG ALT =“图1”FO：内容宽度=“5英寸的”SRC =“/ files/ftp_upload/50719/50719fig1.jpg”/&gt; 图1。悬臂检测抗生素表面结合的相互作用。一）的示意图，以悬臂传感器阵列和检测到的药物靶标相互作用的模式，通过该模式下nanomechanically。b）化学结合的药物分子（车 ）的细菌粘肽类似物的（D-丙氨酸或D-Lac的 ）之间的相互作用。 c）本机制的突变万古霉素敏感细菌（VSE）收购对万古霉素耐药删除单一氢键结合口袋。 <a href="https://www.jove.com/files/ftp_upload/50719/50719fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50719/50719fig2.jpg" /&gt; 图2。破坏细菌细胞壁的生物力学模型实时跟踪。 a）绝对信号的D-丙氨酸，D-Lac和磷酸盐缓冲液和250μM的万古霉素的聚乙二醇涂层悬臂弯曲。黑线所示的参考信号的差PEG b）差信号的D-丙氨酸和D-Lac的磷酸盐缓冲液和250μM万古霉素。）差分悬臂偏转以剂量依赖的信号作为时间的函数的涂层的悬臂弯曲万古霉素的浓度分别为10nM（黄线），100纳米（红色线）和1000纳米（暗红色线）的顺序的存在。 四）差分悬臂偏转信号为三D-丙氨酸涂层悬臂式传感器作为的存在下，10个时间函数在NM万古霉素<a href="https://www.jove.com/files/ftp_upload/50719/50719fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig3.jpg" /> 图3。跟踪模型细菌细胞壁的生物力学实时为一个D-丙氨酸和D-Lac的涂层7μM万古霉素胎牛血清存在下悬臂的破坏。 <img alt="图4" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig4.jpg" /> 图4。剧情显示测量差分表面应力响应D-丙氨酸 （黑圈）和D-LAC（红色圆圈）涂层悬臂作为万古霉素的函数调查药物靶相互作用的纳米力学。在溶液中的浓度[货车]。由方程（1）（实线）中描述的数据。 <img alt="图5" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig5.jpg" /> 图5。作为D-丙氨酸的表面覆盖的函数，对在存在固定万古霉素浓度在悬臂式传感器的测量差的表面应力响应优化的纳米机械检测灵敏度的受体-配体相互作用调查受体加载和通过混合单分子膜的几何形状的依赖关系。溶液在10μM（黑线），100μM（红线），和250μM（绿线）。

Watch this Scientific Journal Video about Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection at JoVE.com

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

收购抗生素的耐药性是一个重大的公共医疗问题，目前排名由世界卫生组织对人类生命的最大威胁之一。在这里，我们描述了使用悬臂技术，以量化的抗菌性，发现新颖和强大剂对多药耐药细菌的关键。

纳米力学的药物靶标相互作用及耐药性检测

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

nanomechanics-drug-target-interactions-antibacterial-resistance

Research

JoVE Journal

Immunology and Infection

14.1K 次观看.  University College London. 收购抗生素的耐药性是一个重大的公共医疗问题，目前排名由世界卫生组织对人类生命的最大威胁之一。在这里，我们描述了使用悬臂技术，以量化的抗菌性，发现新颖和强大剂对多药耐药细菌的关键。

视频: Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

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 ...

科研

免疫与感染

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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 ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

使用抗生素耐药性平台进行抗生素脱除

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.