Abstract
Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.
Highlights
Gram-negative bacteria are the most common pathogens, and severely affect food/environmental safety and threaten public health
The Limulus Amebocyte Lysate (LAL) extract reacts with LPS, and has a reported detection limit of around 10−9 g·mL−1 5,6. This is an extremely sensitive limit that is useful for quantitative analysis, but due to the limitation of horseshoe crab (Limulus polyphemus) resources, and results of samples that tested negative using the LAL test but that were associated with adverse events determined by the use of Mono-Mac 6 cells and TLR expressing cells[7], scientists are focusing their research on the development of complementary sensing methods for LPS detection
We describe the development and validation of a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells transformed by a red fluorescent protein gene under the transcriptional control of an nuclear factor κB (NF-κB) response element
Summary
Gram-negative bacteria are the most common pathogens, and severely affect food/environmental safety and threaten public health. The LAL extract reacts with LPS, and has a reported detection limit of around 10−9 g·mL−1 5,6 This is an extremely sensitive limit that is useful for quantitative analysis, but due to the limitation of horseshoe crab (Limulus polyphemus) resources, and results of samples that tested negative using the LAL test but that were associated with adverse events determined by the use of Mono-Mac 6 cells and TLR expressing cells[7], scientists are focusing their research on the development of complementary sensing methods for LPS detection. There is a strong correlation between chemical structure of LPS, especially the lipid A structure, and the immunological response via the TLR4 pathway[44,45] These functions are crucial components for the development of cell-based biosensors that use molecules with LPS affinity as an alternative for sensitive and accurate detection of LPS and evaluation of its inflammatory activity
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