Molecularly Imprinted Polymers as an Indirect Sensing Approach for Bacillus cereus Detection.

Bacterial endospores are a major challenge in food safety due to their high resistance and potential to cause spoilage and foodborne illness. Current detection methods can be labor-intensive and require specialized instrumentation. A low-cost, label-free thermal sensing platform for indirect detection of Bacillus cereus endospores using molecularly imprinted polymers (MIPs) targeting dipicolinic acid (DPA), a spore biomarker, was developed. The MIPs were synthesized by free-radical bulk polymerization, characterized by rebinding assays, and evaluated using the Heat Transfer Method (HTM). At 100 μM DPA, the MIPs exhibited an imprinting factor (IF) of 2.62 ± 0.18 (n = 3), demonstrating preferential DPA binding. Sensor performance was evaluated using aqueous DPA, structural analogues, and a complex food matrix. HTM measurements produced concentration-dependent responses, with limits of detection of 0.20 ± 0.15 μM in aqueous DPA solutions and 0.060 ± 0.0098 μM in unpretreated, spiked ultrahigh-temperature (UHT) milk. Terbium(III) fluorescence independently supported DPA retention by the MIPs. Real-time assays with germinated B. cereus suspensions at 105 colony-forming units (CFU) mL-1 enabled proof-of-concept monitoring of biologically released DPA under laboratory conditions. These findings highlight the potential of combining DPA-imprinted polymers with HTM for real-time and label-free indirect endospore detection.