New Capability for Bacterial Testing in the Food Production Environment

后续基金的这项研究方案涉及开发一种性能卓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。这涉及进一步开发基于纳米颗粒的分析技术，以检测李斯特菌，特别是单核细胞增多性李斯特菌，这是在前一次BBSRC资助(BB/M018652/1，BB/R00899X/1)之后进行的。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，确实需要在食品生产环境中进行更快、更简单、更可靠的细菌病原体分离和检测。为了满足这一需求，我们将开发一种简单、便携式的原型设备，能够对细菌，特别是李斯特菌进行使用点(POU)检测。这项研究涉及到一种名为拉曼散射的光学检测技术的使用，该技术将被开发用于POU对细菌病原体的检测。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，拉曼散射是一个微弱的过程，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(表面增强共振拉曼)。在这种情况下，我们将使用金纳米颗粒，因为金金属表面将放大表面分子的拉曼散射，产生分子特有的指纹，从而无需分离步骤即可轻松识别混合物的组成。一种新型的生物传感器将与增强拉曼检测相结合，用于一次检测多个李斯特菌。这将使用磁性纳米颗粒，它的表面有一个生物分子，它将结合到细菌的表面。这将允许在施加磁场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金纳米颗粒，从而产生强烈的表面增强拉曼信号，以及将专门与特定细菌菌株结合的抗体。当存在正确的细菌时，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，将产生独特的光谱，从而同时检测多种物种。我们将使用便携式拉曼光谱仪检测POU中存在的细菌。我们将与Samworth Brothers Group(拥有12个生产基地的家族食品制造企业，年营业额约10亿GB)和Wasatch Photonics Inc.合作，进一步发展我们面向原型开发的初步研究。这将使这项技术沿着转化途径进一步发展，以期将以实验室为基础的方法发展成可直接用于食品生产领域的POU检测。在POU检测成功演示之前，我们的未来目标是组建一家衍生公司，通过我们的合作伙伴Samworth Brothers及其客户网络以及市场评估报告，已经确定了明确的需求，将该技术商业化。

This Follow on Fund 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 involves further development of a nanoparticle based analytical technology for the detection of Listeria, in particular L. monocytogenesis following on from previous BBSRC funding (BB/M018652/1, BB/R00899X/1). 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), is expensive and requires specialised personnel and equipment. Therefore, there is a real need for faster, simpler and reliable isolation and detection of bacterial pathogens that can be carried out in the food production environment. To address this need we will develop a simple, portable protype device capable of point of use (POU) detection of bacteria, specifically Listeria.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 a weak process and the signal can be greatly enhanced if the molecule is coloured and adsorbed onto a roughened metal surface (surface enhanced resonance Raman). In this case we will use gold nanoparticles as the gold metal surface will amplify the Raman scattering from a molecule on the surface producing a fingerprint unique to the molecule allowing the composition of mixtures to be easily identified without separation steps. A novel bionanosensor for the detection of multiple Listeria pathogens in one assay combined with enhanced Raman detection will be developed. This will use magnetic nanoparticles which have a biomolecule on the surface which will bind to the surface of the bacteria. This will allow isolation and separation of bacteria from the surrounding medium upon application of a magnetic field. Additionally, gold nanoparticles which are functionalised with a coloured molecule or label, resulting in intense surface enhanced Raman signals, and an antibody which will bind specifically to a particular strain of bacteria 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 produced allowing multiple species to be detected simultaneously. A portable Raman spectrometer will be used to detect the bacteria present at the POU.We will partner with The Samworth Brothers Group (family-owned food manufacturing business with 12 production sites, generating an annual turnover of c. £ 1 bn) and Wasatch Photonics Inc to further develop our initial research towards prototype development. This will allow the technology to be moved further along the translational pathway with a view to developing the laboratory based approach into POU detection that can be used directly in food production areas. Our future aim, pending successful demonstration of POU detection, is to form a spin out company to commercialise the technology having already established a clear need through our partners Samworth Brothers and their customer network, as well as a market assessment report.