Bifunctional phage@isoluminol microgels coupled with peroxidase-like activity phagomagnetic nanoparticles for ultrasensitive chemiluminescence detection and in-situ inactivation of Shewanella in seafood matrices

Abstract

Shewanella putrefaciens (S. putrefaciens), a bacterium commonly found in seafood, contributes to spoilage even in cold environments, making its rapid early detection and control essential. In this study, we developed a Phagomagnetic-Phagomicrogel Chemiluminescence (PhMS-PhMG-CL) system for the detection and control of S. putrefaciens. This system combines bifunctional phage@isoluminol microgels with catalytic activity phagomagnetic nanoparticles, which enable enhanced detection signals and in-situ inactivation of S. putrefaciens. First, we synthesized a bifunctional microgel complex, P(NIPAm-co-MAA)@Phage@Isoluminol (piMGs), a 206.55 ± 16.15 nm particle capable of forming clear plaques and emitting a peak at 378 nm. After 9 h of treatment with piMGs, bacterial counts decreased by 2.11 ± 0.15 Log₁₀ CFU/mL from an initial concentration of 3 Log₁₀ CFU/mL (>99 %). Additionally, by coupling phage SPX1 with Fe₃O₄ nanoparticles, a phage nanoconjugate (pMBs) with both pre-enrichment and peroxidase-like activity was obtained. By combining both complexes, the PhMS-PhMG-CL system was developed. This system could specifically detect S. putrefaciens in the range of 7.6 × 10¹ to 5.9 × 10⁶ CFU/mL within 25 min, with a detection limit as low as 8 CFU/mL. It was also successfully applied to seafood matrices, such as aquaculture water and shrimp meat. Following highly sensitive detection, the system demonstrated an inactivation rate of approximately 97 % within 3 h. This study employed multifunctional phage nanoconjugates to develop a highly sensitive and specific PhMS-PhMG-CL system. It demonstrated an excellent recovery yield ranging from 90.48 ± 6.74 % to 104.28 ± 0.69 % and showed good selectivity toward the target bacteria. The system is capable of detecting and in-situ inactivation of S. putrefaciens, providing significant potential for the detection and control of other bacterial pathogens