Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria

• 3 schemes for phage-based lateral flow assays were designed to detect E. coli.
• 2 schemes utilized genetically engineered bacteriophages.
• Alkaline phosphatase and maltose binding protein was expressed during the phage infection.
• Genetic engineering allowed a 100x improvement in LOD.
• CFU/100 mL of E. coli were detected in environmental water samples.

Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages’ ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 103 CFU/mL of Escherichia coli in broth after 7 h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100 CFU/100 mL in inoculated river water after 9 h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.