Optical Detection of Foodborne Bacterial Pathogens using Nanosensors

这一研究方案涉及开发一种新的工具，其基础是使用性能优越的创新生物传感器，以灵敏、定量和多重的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，以同时检测与食物中毒有关的多种细菌病原体。目前检测细菌的方法耗时(在选择性培养基上培养细菌需要1-2天)，费用昂贵，并且需要专门的人员和设备。因此，迫切需要更快、更简单、更可靠的细菌病原体分离和检测，可以在现场进行，并可以在一次测试中同时检测多个细菌。因此，提出了一种简单、便携的检测平台，可以进行多路护理点(POC)检测。由于细菌对抗菌素产生耐药性，传染病的威胁急剧增加，成功的病原体检测对公众的健康至关重要。大肠杆菌等细菌感染对人类健康的重大威胁导致了迫切的需求，即在食品安全、医疗诊断、水质和反恐方面开发高效的微生物分离和检测策略。强毒的大肠杆菌可引起胃肠炎、尿路感染和新生儿脑膜炎，沙门氏菌攻击胃壁和肠道，严重时可导致血液中毒。研究涉及使用一种名为拉曼散射的光学检测技术，该技术将被开发用于细菌病原体的POC检测。如果特定波长的光照射到分子上，那么一些散射光就会改变波长。这种波长的变化与分子结构有关，并提供了可用于最终鉴定的分子指纹。然而，拉曼散射是一个本质上很弱的过程，如果分子着色并吸附到粗糙的金属表面上，信号可以大大增强(表面增强的共振拉曼)。金属可以被认为基本上放大了表面分子的拉曼散射，在这种情况下，金属将以金属纳米颗粒的形式存在。由于产生了分子特有的指纹，因此无需分离就可以很容易地识别混合物的组成。将开发一种新的诊断工具，与增强拉曼检测相结合，在单一检测中检测多种细菌病原体，即大肠杆菌、鼠伤寒沙门氏菌和Jejunii弯曲杆菌。然而，这项技术不会局限于这些生物，也可以很容易地应用于其他病原体。这将涉及到使用磁性纳米颗粒，这种纳米颗粒表面有一种被称为凝集素的生物分子，它将结合到细菌细胞的表面。这将允许在施加磁力时将细菌从周围的介质中分离出来。此外，还将添加与有色分子或标签功能化的银纳米颗粒，从而产生强烈的表面增强拉曼信号，以及将专门与特定菌株的细菌(抗体或适配子)结合的生物分子。当正确的细菌存在时，将发生结合，导致细菌与基质的磁性隔离，以及它现在具有SERS反应。通过对每个生物标记物使用不同的标签，将为每个生物标记物实现唯一的光谱，从而允许同时检测多个生物标记物。然后将使用便携式拉曼光谱仪来检测存在的细菌。

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.