Ultrasensitive Detection of Viable Enterobacter sakazakii by a Continual Cascade Nanozyme Biosensor

Recent outbreaks of life-threatening neonatal infections linked to Enterobacter sakazakii (ES) heightened the need to develop rapid and ultrasensitive detection strategies, especially those capable of determining the viable cells. This study introduced a continual cascade nanozyme biosensor for the detection of viable ES based on propidium monoazide (PMA), loop-mediated isothermal amplification (LAMP), and Nanozyme strip. The ompA gene of ES was determined using FITC-modified and BIO-modified primers in the LAMP process. LAMP combined with PMA treatment was applied for distinguishing the viable from the dead state of ES. Then, using Fe₃O₄ magnetic nanoparticles as a nanozyme probe, a magnetic nanoparticle (MNP)-based immunochromatographic strip (Nanozyme strip) was further employed for amplifying signal to allow visual detection and quantification by a strip reader. The LAMP products were sandwiched between the anti-FITC and the anti-BIO, and the accumulation of the Fe₃O₄ magnetic nanoparticles enabled the visual detection of ES. The detection limit of the nanozyme biosensor was improved by 10 CFU/mL compared with previously reported techniques, and the whole manipulation process was much faster (within 1 h) and simpler (without specialist facilities). Hence, the developed continual cascade nanozyme biosensor has provided a rapid, ultrasensitive, and simple tool for on-site detection of viable ES.