A fluorescence-based synthetic LPS sensor.

Abstract

For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.