Collaborative Research: Phage-based nanobiosensors for the rapid detection of food and waterborne pathogens

在大量的多种类型的细菌（通常相似但非致病）中准确检测低水平的致病细菌是具有挑战性的，并且将在该项目中通过设计一种纳米生物传感器来解决，该传感器能够从复杂的样品中分离出少量的坏细菌，并将其浓缩成小体积，最终检测致病细菌的存在。 拟议的工作包括第一代纳米机器人，由基因工程和磁化病毒（噬菌体）组成，可以特异性结合，分离，浓缩和感染液体样品中的目标细菌。该噬菌体对大肠杆菌具有特异性。大肠杆菌，并将基因工程产生大量的报告酶（磷酸酶）在1小时的感染。感染后，E.大肠杆菌被自然裂解以释放复制的病毒，从而也释放高浓度的报道酶。然后可以使用纳米结构电极上的电化学灵敏地检测工程酶。提出噬菌体可以与磁性纳米颗粒和纳米结构电极组合使用，以促进以实用的方式进行快速识别、分离和检测。本论文的主要研究内容如下：1）设计噬菌体T4，使其携带磷酸酶基因，并与磁性纳米颗粒靶向结合; 2）利用磁性纳米探针分离和富集大肠杆菌。3）电化学检测纳米结构电极上的磷酸酶，将安培信号与初始细菌浓度相关联。作为这项提案的一部分，PI？研究小组将开发高清晰度计算机生成的教学工具，使学生能够直观地了解遗传工程的概念和网页。教育录像将以纪录片的形式制作，并将包括逼真的动画。这些视频将通过YouTube和PI网站免费下载。

Accurately detecting low levels of disease-causing bacteria in a large population of many types of (often similar but non-disease causing) bacteria is challenging, and will be addressed in this project by designing a nanobiosensor that is able to separate low numbers of the bad bacteria from complex samples, and concentrate it into a small volume and finally detect the presence of the disease-causing bacteria. The proposed work consists of first generation nanobot consisting of a genetically engineered and magnetized virus (phage) which can specifically bind, separate, concentrate, and infect target bacteria in liquid samples. The phage is specific to E. coli bacteria and will be genetically engineered to produce large quantities of a reporter enzyme (phosphatase) during the 1-hour infection. Following infection, the E. coli is naturally lysed to release replicated viruses, thereby also releasing the high concentration of reporter enzyme. The engineered enzymes can then be sensitively detected using electrochemistry on a nanostructured electrode. It is proposed that phage can be utilized in combination with magnetic nanoparticles and nanostructured electrodes to promote the rapid recognition, separation, and detection in a pragmatic manner. The following research aims will be pursued: 1) Engineer the phage T4 to carry a gene for an engineered phosphatase enzyme and allow targeted attachment onto magnetic nanoparticles, 2) Use the magnetic nanoprobes to separate and concentrate E. coli from liquid samples, and 3) Electrochemically detect the phosphatase on a nanostructured electrode correlating amperometric signal to initial bacterial concentration. As part of this proposal, the PIs? research groups will develop high-definition computer-generated teaching tools which will allow students to visualize the concepts of genetic engineering and pages. The educational videos will be produced documentary-style and will include photo-realistic animations. The videos will be freely available for download via YouTube and the PI websites.