Rapid Detection of Endotoxin Based on G-quadruplex/Thioflavin T Label-Free Fluorescent Aptamer Sensor

Endotoxin (lipopolysaccharide, LPS), a major toxic component of Gram-negative bacteria. It shows high stability and resistance to conventional sterilization. Therefore, rapid and effective detection of LPS is crucial to mitigate its threats to human health. This study developed a sensitive, simple, and rapid LPS detection method. It uses a label-free fluorescent aptamer sensor based on an LPS-specific aptamer and the commercial fluorescent probe Thioflavin T (ThT). A novel LPS-detecting aptamer (G4-LA47) was synthesized by attaching guanine-rich DNA sequences to both ends of the LPS-specific aptamer LA27. The G4-LA47 aptamer retained high affinity for LPS, while its terminal guanine-rich sequences formed a G-quadruplex structure. Upon binding with ThT, the G-quadruplex/ThT complex exhibited strong fluorescence. The presence of LPS unfolds of the G-quadruplex, releases free ThT and reduces the fluorescence intensity. LPS was quantitative detected by monitoring fluorescence intensity changes. The linear range was 10–80 ng/mL. The LOD and LOQ were 3.26 ng/mL and 10.86 ng/mL, respectively. The sensor was successfully applied to juice and drinking water samples, with recoveries ranging from 93.34% to 113.29%. This label-free fluorescent aptamer sensor demonstrates high sensitivity, specificity, and simplicity, showing promising potential for practical applications.