Pseudomonas aeruginosa is one of the most common pathogenic Gram-negative bacteria associated with various infectious diseases, including burn wound infections, lung infections, ventilator-associated pneumonia (VAP), and sepsis [1]. The development of antimicrobial resistance (AMR) and the formation of biofilms make P. aeruginosa one of the significant pathogens contributing to outbreaks of severe healthcare-associated infections and community-acquired pneumonia [2], [3]. In 2017, P. aeruginosa was added to the World Health Organization's (WHO) priority pathogens list for research and development of new antibiotics [4]. Therefore, rapid and accurate detection technologies could provide personalized therapy to control P. aeruginosa infections.

Currently, traditional culture-based methods are still considered the gold standard for diagnosing bacterial infections. However, these procedures are laborious and time-consuming, resulting in unacceptable delays in obtaining results, especially in the treatment of severe infections [5]. Molecular techniques with high sensitivity and short turnaround time are commonly restricted by complicated procedures, the requirement for specialized personnel, and false-positive results [6]. To circumvent these limitations, various approaches have been developed for the rapid, accurate, and sensitive detection of P. aeruginosa, including immunoassays, and optical and electrochemical biosensors [7]. In addition to the choice of methodology, the accuracy and efficiency of identifying P. aeruginosa are significantly impacted by the biorecognition elements employed in the detection system. The biorecognition component ideal for bacterial detection should possess high stability and sensitivity, be cost-effective, and exhibit high specificity, i.e., the capability to identify a broad range of strains within the target species [8]. Due to the high specificity of antigen-antibody bindings, antibodies are the most common biorecognition elements applied in constructing immunoassays or biosensors. However, the cost of antibody production and their shorter shelf life are limitations for antibody-based detection methods [9]. Several alternatives to antibodies, such as aptamers [10], [11], antibiotics [12], and bacteriophage-based probes [13], [14], [15] have been successfully used in various detection platforms for P. aeruginosa diagnosis and have attracted more and more attention.

Lytic bacteriophages (or phages) are a class of bacterial viruses widely distributed in nature. The abundant sources and high natural specificity make phages potential biorecognition probes for detecting bacteria [16]. Nevertheless, due to the high strain specificity of most phages, the identification of broad-spectrum P. aeruginosa strains remains a tremendous challenge [17]. Generally, the host specificity of phages is determined by their receptor-binding proteins (RBPs), which may be tail fiber proteins (TFPs) or spike proteins. For Gram-negative bacteria, RBPs typically recognize lipopolysaccharides (LPS), outer membrane proteins, pili, and flagella on the surface of host bacteria [18], [19]. Compared to the highly specific phages, some RBPs have been reported to recognize strains not lysed by their originating phages [8], [16], [20]. Therefore, detection methods based on RBPs could reduce the potential false negative results caused by the extremely high specificity of phages. Furthermore, RBPs possess advantageous characteristics such as thermal and chemical stability, ease of production, and the ability to fuse with reporter enzymes or fluorescent proteins, making them potential biorecognition molecules [21]. Phage RBPs have been widely utilized as versatile tools for capturing, labeling, and detecting host bacterial cells. And a variety of RBP-based assays have already been successfully developed for the detection of pathogenic bacteria, including P. aeruginosa [12], [20], Bacillus anthracis [22], [23], Salmonella [24], [25], [26], Yersinia pestis [27], Klebsiella [8], Acinetobacter baumannii [16], [28], [29], Escherichia coli [30], Campylobacter [31], [32], Staphylococcus and Enterococcus [33]. For P. aeruginosa, a recombinant nonlytic TFP (P069) of the phage PaP1 has been used as a multifunctional recognition element for capturing or labeling P. aeruginosa in the bioluminescence or fluorescence assays [12], [20]. P069 was expressed in E.coli BL21 (DE3) in the form of inclusion bodies, although it can be renaturated and transformed into a soluble protein, the process of producing P069 is undoubtedly tedious [20].

Previously, a P. aeruginosa-targeting phage Henu5 (GenBank accession number: NC_073608.1) was isolated from sewage. Based on bioinformatics analysis, the protein Gp130 of phage Henu5 was predicted to be a TFP. This study reported a novel RBP (Gp130) that can be recombinant expressed in E. coli in a soluble form. The potential of Gp130 as a biorecognition molecule for identifying P. aeruginosa was assessed using a modified enzyme-linked phage receptor-binding protein assay (ELPRA). After enrichment of P. aeruginosa cells from complex samples by Gp130-coated solid-phase matrices, the modified ELPRA was conducted by linking the biotin-labeled Gp130 with a horseradish peroxidase-labeled streptavidin (HRP-SA), enabled rapid and colorimetric identification of P. aeruginosa.