Institute of Information and Communication Technologies, Electronics and Applied Mathematics, UCLouvain, Place du Levant 1, 1348 Louvain-la-Neuve, Belgium.
Laboratory of Food and Environmental Microbiology, Earth and Life Institute, UCLouvain, Croix du Sud 2/12, 1348 Louvain-la-Neuve, Belgium.
ACS Appl Bio Mater. 2024 Nov 18;7(11):7292-7305. doi: 10.1021/acsabm.4c00965. Epub 2024 Nov 5.
Rapid and precise diagnostic techniques are essential for identifying foodborne pathogens, including (), which poses significant challenges to food safety. Traditional detection methods are limited by long incubation times and high costs. In this context, gold nanoparticle (AuNP)-based lateral flow assays (LFAs) are emerging as valuable tools for rapid screening. However, the use of antibodies in LFAs faces challenges, including complex production processes, ethical concerns, or variability. Here, we address these challenges by proposing an innovative approach using bacteriophage-derived proteins for pathogen detection on LFAs. We used the engineered endolysin cell-wall-binding domain (CBD) and distal tail proteins (Dit) from bacteriophages that specifically target . The protein-binding properties, essential for the formation of efficient capture and detection biointerfaces in LFAs, were extensively characterized from the microstructural to the LFA device level. Machine-learning models leverage knowledge of the protein sequence to predict advantageous protein orientations on the nitrocellulose membrane and AuNPs. The study of the biointerface binding quantified the degree of attachment of AuNPs to bacteria, providing, for the first time, a microscopic model of the number of AuNPs binding to bacteria. It highlighted the binding of up to one hundred 40 nm AuNPs per bacterium in conditions mimicking LFAs. Eventually, phage proteins were demonstrated as efficient bioreceptors in a straightforward LFA prototype combining the two proteins, providing a rapid colorimetric response within 15 min upon the detection of 10 cells. Recombinantly produced phage binding proteins present an opportunity to generate a customizable library of proteins with precise binding capabilities, offering a cost-effective and ethical alternative to antibodies. This study enhances our understanding of phage protein biointerfaces, laying the groundwork for their utilization as efficient bioreceptors in LFAs and rapid point-of-care diagnostic assays, thus potentially strengthening public health measures.
快速而准确的诊断技术对于识别食源性病原体至关重要，其中包括（），这给食品安全带来了重大挑战。传统的检测方法受到孵育时间长和成本高的限制。在这种情况下，基于金纳米颗粒（AuNP）的侧向流动分析（LFA）作为快速筛选的有价值工具正在出现。然而，LFA 中抗体的使用面临挑战，包括复杂的生产工艺、伦理问题或可变性。在这里，我们通过提出一种使用噬菌体衍生蛋白在 LFA 上进行病原体检测的创新方法来应对这些挑战。我们使用了针对的工程内切酶细胞壁结合域（CBD）和远位尾部蛋白（Dit）。这些噬菌体蛋白的结合特性对于在 LFA 中形成高效的捕获和检测生物界面至关重要，从微观结构到 LFA 设备水平都进行了广泛的表征。机器学习模型利用蛋白质序列的知识来预测在硝酸纤维素膜和 AuNP 上的有利蛋白质取向。生物界面结合的研究量化了 AuNP 与细菌的附着程度，首次提供了 AuNP 与细菌结合数量的微观模型。它突出了在模拟 LFA 的条件下，每个细菌可结合多达一百个 40nm 的 AuNP。最终，噬菌体蛋白被证明是一种高效的生物受体，在一个简单的 LFA 原型中结合了两种蛋白，在检测到 10 个细胞后 15 分钟内提供了快速的比色响应。重组产生的噬菌体结合蛋白提供了一个机会，可以生成具有精确结合能力的定制化蛋白文库，为抗体提供了一种具有成本效益和伦理的替代方案。本研究增强了我们对噬菌体蛋白生物界面的理解，为它们在 LFA 和快速即时诊断检测中的作为高效生物受体的应用奠定了基础，从而有可能加强公共卫生措施。
