Liposome-Doped Nanocomposites as Artificial-Cell-Based Biosensors: Detection of Listeriolysin O

Abstract

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.

Fabrication, characterisation and performance of hydrophilic and super-hydrophilic silica as cell culture surfaces

“…4 The sol-gel method has also been used to produce bioactive porous glasses for bone regeneration and to fabricate silica films on glass surfaces for cell-growth. 5,6 It has been observed that biological entities such as proteins and liposomes, 7,8 in addition to mammalian and prokaryotic cells, remain active on sol-gel derived surfaces after immobilisation or adhesion and can demonstrate increased stability and growth.…”

Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors

“…On the other hand, single moleculebased biosensors may be more stable, but sometimes less functional compared to the whole cell counterpart (Zhao et al 2006). Synthetic materials are likely to be a feature of these two extremes, as extensively demonstrated by the latest bibliography on artificial biosensing systems, which mimic cells in their detection capabilities, showing also improved features in terms of robustness and sensitivity.…”

“…Moreover, conventional methods for identification of L. monocytogenes in food is a culture-based procedure that takes 5-7 days, consequently increasing the probabilities to commercialise a contaminated food product before the problem has been identified. For these reasons, the materials implemented by Zhao et al (2006) could be considered a good candidate to detect LLO, showing fast response (30 minutes) and high stability (at least 5 months in ambient conditions).…”

Biosensor: an emerging safety tool for meat industry

“…In a study to detect Listeriolysin O (LLO), the liposomes were encapsulated in porous silica using alcohol-free sol-gel synthesis methods. The immobilized liposomes act as a cellular compartment containing the fluorescent dyes and the dye release due to the pore formation by LLO indicated the presence of the toxin (Zhao et al 2006).…”