Zhao Jianxiu, Jedlicka Sabrina S, Lannu Josh D, Bhunia Arun K, Rickus Jenna L
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, USA.
Biotechnol Prog. 2006 Jan-Feb;22(1):32-7. doi: 10.1021/bp050154o.
Listeriolysin O (LLO) is a pore-forming hemolysin secreted by the foodborne pathogen Listeria monocytogenes and is required for bacterial virulence. Current detection methods for L. monocytogenes are time-consuming, labor-intensive, and expensive, which is impractical considering the limitations of food storage. To overcome these problems, we developed a liposome-doped silica nanocomposite as a simple, inexpensive, and highly stable biosensor material that mimics existing whole-cell assays for LLO. Small unilamellar liposomes containing fluorescent dyes were immobilized within porous silica using alcohol-free sol-gel synthesis methods. The immobilized liposomes served as cellular surrogates for membrane insertion and pore formation by LLO. The integrity of liposomes in the solid-state sol-gel glass was investigated by fluorescence quenching and leaching assays. The materials were stable for at least 5 months in ambient conditions. Both free and immobilized liposomes responded to LLO at pH 6.0 with concentration dependent kinetics. The pore formation of LLO in liposome-doped silica composites displayed similar kinetic curves as free liposomes but with slower rates. LLO insertion into the immobilized liposomes was pH dependent. No increase in membrane permeability was observed at pH 7.4 for the liposome-doped composites in the presence of LLO. Immobilized liposomes can detect LLO in approximately 1.5 h using a steady state calibration and within 30 min using a kinetic calibration. These liposome silica composites potentially could be used for the detection of hemolysin producing L. monocytogenes as well as the many other bacteria that produce pore-forming toxins.
李斯特菌溶血素O（LLO）是食源性病原体单核细胞增生李斯特菌分泌的一种形成孔道的溶血素，是细菌毒力所必需的。目前用于检测单核细胞增生李斯特菌的方法耗时、费力且昂贵，考虑到食品储存的局限性，这是不切实际的。为了克服这些问题，我们开发了一种掺杂脂质体的二氧化硅纳米复合材料，作为一种简单、廉价且高度稳定的生物传感器材料，可模拟现有的LLO全细胞检测方法。使用无醇溶胶-凝胶合成方法将含有荧光染料的小单层脂质体固定在多孔二氧化硅中。固定化的脂质体作为LLO进行膜插入和孔形成的细胞替代物。通过荧光猝灭和浸出试验研究了固态溶胶-凝胶玻璃中脂质体的完整性。这些材料在环境条件下至少稳定5个月。游离脂质体和固定化脂质体在pH 6.0时均对LLO有响应，且具有浓度依赖性动力学。LLO在掺杂脂质体的二氧化硅复合材料中的孔形成显示出与游离脂质体相似的动力学曲线，但速率较慢。LLO插入固定化脂质体中依赖于pH。在pH 7.4时，在LLO存在下，掺杂脂质体的复合材料未观察到膜通透性增加。使用稳态校准，固定化脂质体可在约1.5小时内检测LLO，使用动力学校准可在30分钟内检测到。这些脂质体二氧化硅复合材料有可能用于检测产生溶血素的单核细胞增生李斯特菌以及许多其他产生孔形成毒素的细菌。
