Multifunctional Carbon Dot–Infused Agarose Hydrogel Film as an Adsorptive Platform for Efficient and Biosafe Endotoxin Clearance

ABSTRACT The effective removal of bacterial lipopolysaccharide (LPS) endotoxins from biological fluids remains a challenging task in bioprocessing and biomedical applications. This study presents a simple and cost‐effective approach for endotoxin separation using a poly‐L‐lysine functionalized carbon dots (PLL‐CDs) embedded within an agarose (AGR) hydrogel film (AGR/PLL‐CDs), fabricated via a microwave‐assisted process without chemical cross‐linkers. The resulting AGR/PLL‐CDs hydrogel film exhibited unique characteristic features, including structural stability, thermal resistance, rapid swelling, balanced mechanical strength and well‐defined morphology, contributing to its robust performance in endotoxin removal. The hydrogel film achieved 92% LPS removal within 120 min at 37°C, maintaining efficiency across a wide concentration range (10 pM–100 nM), physiological pH 7.4, and varying ionic strengths (0.1–1 M NaCl). The high removal efficiency of the hydrogel film was attributed to the specific LPS binding onto PLL‐CDs, rather than mere physical adsorption within the hydrogel matrix. The hydrogel film retained its LPS removal efficiency even in the presence of various potentially interfering substances, such as surfactants, proteins, lipids, chelating agents, divalent cations, and saccharides. This demonstrated its excellent selectivity and operational robustness. The adsorbent could be regenerated and reused over five successive cycles with minimal loss in efficacy. MTT cytotoxicity assays using human triple‐negative breast cancer cells demonstrated excellent cell viability, meeting the ISO 10993‐5 standards for biocompatibility. Thus, the AGR/PLL‐CDs hydrogel film combines the binding specificity, stability, and biosafety required for a cost‐effective, reusable platform for efficient endotoxin purification.