Electrochemical aptasensor for monitoring of bacterial endotoxins in mammalian cell cultures

Mammalian cells, particularly human cell culture models, are essential for studying disease pathophysiology and producing cell-based therapeutic products. Monitoring and controlling cell culture conditions accurately is essential for optimal cell growth and health, as even minor variations can significantly influence cell behavior. The presence of viruses, bacteria, and their by-products are key indicators of cell culture contamination. Conventional assays for quantifying cellular health and microbial contaminants such as endotoxins are end-point assays that are often laborious, require specialized equipment, and typically detect contamination only at advanced stages. For example, the chromogenic Limulus amoebocyte lysate assay, used for quantifying endotoxin, a bacte rial by-product, is often susceptible to interference from serum proteins in the culture medium. In this work, we present a simple and sensitive aptamer-based biosensor designed to detect bacterial-secreted endotoxins in various complex cell culture media. As a proof of concept, human induced pluripotent stem cells (hiPSCs) were deliberately contaminated with Escherichia coli (E. coli), and the biosensor's response to endotoxins released by the bacteria was monitored over a 24-h period. The biosensor demonstrated a reliable linear response with a detection limit of 0.33 ± 0.06 pg/mL in DMEM and 0.142 ± 0.025 pg/mL in StemFlex medium. Its performance in complex sample matrices suggests the potential for integration with industrial-scale cell culture systems for real-time contamination detection, providing a cost-effective, efficient, and timely method to monitor cell health and ensure sterile conditions for therapeutic cell cultivation.