Optical Detection of Listeria in the Chilled Food Environment using Bionanosensors

这项研究计划涉及开发一种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，用于冷冻食品生产。这将涉及开发基于纳米颗粒的分析技术，以检测与冷冻食品行业的食品污染有关的多种细菌病原体(单核细胞增生性乳杆菌和肠杆菌科)。目前的细菌检测方法耗时(在选择性培养基上培养细菌需要1-2天，但实际上实验室位于异地的食品生产设施需要7天的周转时间)、昂贵且需要专门的人员和设备。因此，对能够在生产环境中进行的细菌病原体的更快、更简单和可靠的分离和检测有着强烈的需求。为了满足这一需求，我们将开发一个简单、便携的检测平台，能够对细菌进行使用点(POU)检测。成功地检测出食品生产中的细菌对公众的健康至关重要。在冷冻食品部门，李斯特菌特别令人担忧，因为它是一种异常有弹性的细菌，可以在4至42摄氏度的温度下生存。单核细胞增多性李斯特菌是最严重的食源性感染形式之一，总死亡率为30%，在脆弱的个人中上升至40%。感染这种疾病的风险最大的是孕妇、老年人和免疫功能低下的人，在某些情况下，如果血液受到感染，李斯特菌病可能会发展为脑膜炎和败血症。然而，由于对食品安全的严格控制，李斯特菌的爆发很少见，但我们的工业合作伙伴，生产三明治和沙拉的Bradgate Bakery，需要能够定位、检测和控制食品生产线上的任何污染。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POU细菌病原体的检测。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，将采取金属纳米颗粒的形式。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。一种新型的生物传感器将与增强拉曼检测相结合，用于同时检测多种细菌病原体，即单核细胞增生性乳杆菌和肠杆菌科细菌。这将使用磁性纳米颗粒，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，导致细菌从基质中磁隔离和浓缩。通过为每种细菌使用不同的标签，将为每种细菌实现独特的光谱，从而允许同时检测多种细菌。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 an innovative bionanosensor with superior performance for the detection of bacterial pathogens in a sensitive, quantitative and multiplexed manner for use in chilled food production. This will involve developing nanoparticle based analytical technology for the detection of multiple bacterial pathogens (L. monocytogenesis and enterobacteriaceae) associated with food contamination in the chilled food industry. Current methods for detecting bacteria are time consuming (1-2 days in the case of bacteria culturing on selective media, but in reality 7 day turnaround for food production facilities where laboratories are located offsite), expensive and require specialised personnel and equipment. Therefore, there is a strong need for faster, simpler and reliable isolation and detection of bacterial pathogens that can be carried out in the production environment. To address this need we will develop a simple, portable detection platform capable of point of use (POU) detection for bacteria.Successful bacteria detection in food production is crucial for the health of the general public. Within the chilled food sector, Listeria is of particular concern since it is an unusually resilient bacterium which can survive in temperatures from 4 to 42 degrees C. Listeria monocytogenes causes one of the most severe forms of foodborne infection with an overall mortality rate of 30 %, rising to 40 % in vulnerable individuals. Those most at risk of acquiring the disease are pregnant women, the elderly and the immuno-compromised and in some cases listeriosis can develop into meningitis and septicaemia if infection of the blood occurs. However, outbreaks of Listeria are rare due to strict controls on food safety however our industrial partner, Bradgate Bakery, who manufacture sandwiches and salads, require the ability to locate, detect and contain any contamination within food production lines.The research involves the use of an optical detection technique called Raman scattering which will be developed for the POU 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 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 bionanosensor for the detection of multiple bacterial pathogens, namely L. monocytogenesis and enterobacteriaceae in one assay combined with enhanced Raman detection will be developed. This will use magnetic nanoparticles which have a biomolecule on the surface known as a lectin which will bind to the surface of bacteria. 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 and concentration of the bacteria from the matrix. By using a different label for each bacteria, a unique spectrum will be achieved for each bacteria allowing multiple to be detected simultaneously. A portable Raman spectrometer will then be used to detect the bacteria present.