Detection of Gram (-) endotoxins: a study on the biorecognition-target interaction by means of Neutron Reflectometry

Gram(-) bacteria are pathogenic microorganisms whose outer membrane of the external envelope is composed of complex molecules, such as lipopolysaccharides (LPS), consisting of three structural domains: lipid A, the core oligosaccharide and the O antigen. They are usually considered as endotoxins responsible of many infections induced by bacterial pathogens, so representing a suitable target for a selective detection of their presence in aqueous environment. Selective detection of this analyte can be achieved through specifically designed biosensors, exploiting the biological response deriving from the biorecognition molecule and the analyte into a measurable signal. Among biorecognition molecules, aptamers are very appealing. They are single-stranded DNA or RNA with high affinity and specificity towards specific analytes. Recently, a small aptamer, named LA27, has been identified for a selective recognition of LPS. Actually, the LPS portion interacting with LA27 is not well-understood yet, although preliminary studies seem to suggest a direct affinity with lipid A as well as a capability of this aptamer to interact with LPS deriving from different strains of Gram(-) bacteria. In this context, we performed a biophysical investigation on the interaction of LA27 biorecognition molecule with asymmetric Supported Lipid Bilayers (SLBs) prepared by the Langmuir-Blodgett method: the outer leaflet was composed by the deuterated di-palmitoyl-phosphocholine (d-DPPC), while three LPS extracted from three different Gram(-) strains, such as Akkermansia, Flavobacterium and Paenalcaligenes hominis respectively, were alternatively used to form the outer leaflet of the biomimicking bacterial membranes. For each of these samples, the interaction study was carried out at 25 °C and 38 °C in the presence of various solvent contrasts by performing Neutron Reflectometry experiments. This analysis gave us information about the aptamer positioning within the bilayer regions, so that we shed light on the binding affinity of LA27 with a specific portion of LPS