Optical Detection of Foodborne Bacterial Pathogens using Nanosensors

这项研究计划涉及开发一种新的工具，该工具基于使用创新的生物纳米传感器，具有上级性能，以灵敏，定量和多重方式检测细菌病原体。这将涉及开发基于纳米颗粒的分析技术，用于同时检测与食物中毒相关的多种细菌病原体。目前用于检测细菌的方法是耗时的（在选择性培养基上培养细菌的情况下为1-2天）、昂贵的并且需要专门的人员和设备。因此，强烈需要更快、更简单和更可靠的细菌病原体的分离和检测，其可以在现场进行，并且可以在单个测试中同时检测多种细菌。因此，开发一种简单、便携的检测平台，可以进行多重床旁检测（POC）。成功的病原体检测对公众的健康至关重要，因为细菌对抗菌药物的耐药性导致传染病的威胁急剧增加。大肠杆菌等细菌感染是人类健康的主要威胁。大肠杆菌的感染导致迫切需要开发用于分离和检测与食品安全、医学诊断、水质和反恐有关的微生物的高效策略。E.大肠杆菌可引致肠胃炎、泌尿道感染及新生儿脑膜炎，而沙门氏菌则会侵袭胃壁及肠道，严重时可引致血液中毒。这项研究采用一种名为拉曼散射的光学检测技术，该技术将发展用于细菌病原体的POC检测。如果特定波长的光被引导到分子上，那么一些散射光将改变波长。这种波长的变化与分子的结构有关，并提供了可用于最终鉴定的分子指纹。然而，拉曼散射本质上是弱过程，并且如果分子被着色并被吸附到粗糙的金属表面上，则信号可以大大增强（表面增强共振拉曼）。金属可以被认为基本上放大了来自表面上的分子的拉曼散射，并且在这种情况下，金属将采取金属纳米颗粒的形式。由于产生了分子特有的指纹，混合物的组成可以很容易地识别而无需分离。将开发一种新的诊断工具，用于检测多种细菌病原体，即大肠杆菌，鼠伤寒沙门氏菌和空肠弯曲杆菌在一个单一的测定结合增强拉曼检测。然而，这项技术将不限于这些生物体，并可以很容易地应用于其他病原体。这将涉及使用磁性纳米颗粒，其表面上具有称为凝集素的生物分子，该生物分子将结合到细菌细胞的表面。这将允许在施加磁性时从周围介质中分离和分离细菌。此外，将添加用有色分子或标记官能化的银纳米颗粒，导致强烈的表面增强拉曼信号，以及将特异性结合特定细菌菌株的生物分子（抗体或适体）。当正确的细菌存在时，将发生结合，导致细菌从基质中的磁性分离，以及它现在具有Sers响应。通过对每种生物标志物使用不同的标记，将为每种生物标志物实现独特的光谱，从而允许同时检测多种生物标志物。然后将使用便携式拉曼光谱仪检测存在的细菌。

This programme of research involves the development of a new tool based on the use of innovative bionanosensors with superior performance for the detection of bacterial pathogens in a sensitive, quantitative and multiplexed manner. This will involve developing nanoparticle based analytical technology for the simultaneous detection of multiple bacterial pathogens associated with food poisoning. Current methods for detecting bacteria are time consuming (1-2 days in the case of bacteria culturing on selective media), expensive and require specialised personnel and equipment. Therefore, there is a strong need for faster, simpler and more reliable isolation and detection of bacterial pathogens that can be carried out in the field and can simultaneously detect multiple bacteria within a single test. Therefore, development of a simple, portable detection platform is proposed which can carry out multiplexed point of care (POC) detection.Successful pathogen detection is crucial for the health of the general public as the threat of infectious disease is dramatically increasing as a result of bacteria developing resistance to antimicrobial drugs. Major threats to human health from bacterial infections such as E. coli have led to urgent demands to develop highly efficient strategies for isolating and detecting microorganisms in connection with food safety, medical diagnostics, water quality, and counter terrorism. Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis and Salmonella attacks the stomach lining and intestines and in severe cases can result in blood poisoning.The research involves the use of an optical detection technique called Raman scattering which will be developed for the POC detection of bacterial pathogens. If light of a particular wavelength is directed onto a molecule then some of the scattered light will change wavelength. This change in wavelength is related to the structure of the molecules and provides a molecular fingerprint that can be used for definitive identification. However Raman scattering is an intrinsically weak process and the signal can be greatly enhanced if the molecule is coloured and is adsorbed onto a roughened metal surface (surface enhanced resonance Raman). The metal can be thought of as essentially amplifying the Raman scattering from a molecule on the surface and in this case the metal will take the form of metal nanoparticles. Since a fingerprint unique to the molecule is produced, the composition of mixtures can easily be identified without separation. A novel diagnostic tool will be developed for the detection of multiple bacterial pathogens, namely Escherichia coli, Salmonella typhimurium and Campylobacter jejunii in a single assay combined with enhanced Raman detection. However, this technology will not be limited to these organisms and can readily be applied to other pathogens. This will involve using magnetic nanoparticles which have a biomolecule on the surface known as a lectin which will bind to the surface of bacterial cells. This will allow isolation and separation of bacteria from the surrounding medium upon application of a magnetic. Additionally, silver nanoparticles which are functionalised with a coloured molecule or label, resulting in intense surface enhanced Raman signals, and a biomolecule which will bind specifically to a particular strain of bacteria (antibody or aptamer) will be added. When the correct bacteria are present binding will occur resulting in magnetic isolation of the bacteria from the matrix as well as it now having a SERS response. By using a different label for each biomarker, a unique spectrum will be achieved for each biomarker allowing multiple biomarkers to be detected simultaneously. A portable Raman spectrometer will then be used to detect the bacteria present.