New Capability for Bacterial Testing in the Food Production Environment

这项后续基金研究计划涉及开发一种创新的生物纳米传感器，该传感器具有优异的性能，可用于冷藏食品生产中，以灵敏，定量和多路的方式检测细菌病原体。这涉及进一步开发基于纳米颗粒的李斯特菌检测分析技术，特别是单核细胞生成李斯特菌的检测，这是继之前BBSRC资助（BB/M018652/1, BB/R00899X/1）之后的工作。目前检测细菌的方法非常耗时（在选择性培养基上培养细菌需要1-2天，但实际上，在实验室不在现场的食品生产设施中需要7天），而且成本昂贵，需要专门的人员和设备。因此，迫切需要能够在食品生产环境中进行更快、更简单、更可靠的细菌性病原体的分离和检测。为了满足这一需求，我们将开发一种简单的便携式原型设备，能够在使用点（POU）检测细菌，特别是李斯特菌。这项研究涉及使用一种称为拉曼散射的光学检测技术，该技术将用于细菌病原体的POU检测。如果特定波长的光被射向一个分子，那么一些散射光就会改变波长。这种波长的变化与分子的结构有关，并提供了可用于确定鉴定的分子指纹。然而，拉曼散射是一个微弱的过程，如果分子被着色并吸附在粗糙的金属表面上（表面增强共振拉曼），信号可以大大增强。在这种情况下，我们将使用金纳米颗粒，因为金金属表面会放大表面分子的拉曼散射，从而产生分子独有的指纹，从而无需分离步骤即可轻松识别混合物的组成。将开发一种新型生物纳米传感器，用于一次检测多种李斯特菌病原体，并结合增强的拉曼检测。这将使用磁性纳米颗粒，其表面有一个生物分子，将与细菌表面结合。这将允许在施加磁场的情况下从周围介质中隔离和分离细菌。此外，还将添加带有彩色分子或标签的功能化金纳米粒子，从而产生强烈的表面增强拉曼信号，以及一种能够特异性结合特定细菌菌株的抗体。当正确的细菌存在时，结合将发生，导致细菌与基质的磁隔离和浓缩。通过对每种细菌使用不同的标签，将产生一个独特的光谱，从而可以同时检测到多种细菌。便携式拉曼光谱仪将用于检测POU中存在的细菌。我们将与Samworth兄弟集团（拥有12个生产基地的家族食品制造企业，年营业额约为10亿英镑）和Wasatch Photonics公司合作，进一步发展我们对原型开发的初步研究。这将使该技术沿着转化途径进一步发展，以期将基于实验室的方法发展为可直接用于粮食生产领域的POU检测。在成功演示POU检测之前，我们的未来目标是成立一家分拆公司，将该技术商业化，并通过我们的合作伙伴Samworth兄弟及其客户网络以及市场评估报告建立了明确的需求。

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.