Cell Based-Green Fluorescent Biosensor Using Cytotoxic Pathway for Bacterial Lipopolysaccharide Recognition

Abstract

Lipopolysaccharide (LPS), a characteristic component of the outer membrane of Gram-negative bacteria, can be used as an effective biomarker to detect bacterial contamination. Here, we reported a 293/hTLR4A-MD2-CD14 cell-based fluorescent biosensor to detect and identify LPS, which is carried out in a 96-well microplate which is nondestructive, user-friendly, and highly efficient. The promoter sequence of the critical signaling pathway gene ZC3H12A (encoding MCPIP1 protein) and enhanced green fluorescence protein (EGFP) were combined to construct a recombinant plasmid, which was transferred into 293/hTLR4A-MD2-CD14 cells through lipid-mediated, DNA-transfection way. LPS was able to bind to TLR4 and coreceptors-induced signaling pathway could result in green fluorescent protein expression. Results show that stable transfected 293/hTLR4A-MD2-CD14 cells with LPS treatment could be directly and continually observed under a high content screening imaging system. The novel cell-based biosensor detects LPS at low concentration, along with the detection limit of 0.075 μg/mL. The cell-based biosensor was evaluated by differentiating Gram-negative and Gram-positive bacteria and detecting LPS in fruit juices as well. This proposed fluorescent biosensor has potential in sensing LPS optically in foodstuff and biological products, as well as bacteria identification, contributing to the control of foodborne diseases and ensurance of public food safety with its high throughput detection way.