A virulent phage named as PaP1 was isolated from hospital sewage based on a lambda phage isolation protocol. This phage showed a strong and highly specific binding ability to Pseudomonas aeruginosa (P. aeruginosa). Using this isolated phage as a recognition agent, a novel electrochemiluminescent (ECL) biosensor was developed for label-free detection of P. aeruginosa. The biosensor was fabricated through depositing phage-conjugated carboxyl graphene onto the surface of a glass carbon electrode. After specific binding of the host bacteria through the adsorption of P. aeruginosa cell wall by phage tail fibers and baseplate, the ECL signal of luminol suffered a decrease since the formed non-conductive biocomplex obstructed the interfacial electron transfer and blocked the diffusion of the ECL active molecules. The ECL emission declined linearly with P. aeruginosa concentration in the range of 1.4×102 -1.4×106CFUmL-1, with a very low detection limit of 56CFUmL-1. The whole detection process could be completed within 30min as a ready-for-use biosensor was adopted. This biosensor was successfully applied to quantitate P. aeruginosa in milk, glucose injection and human urine with acceptable recovery values ranging from 78.6% to 114.3%.
Keywords: Carboxyl graphene; Electrochemiluminescent biosensor; Luminol; Phage; Pseudomonas aeruginosa.
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