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

Abstract

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 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, 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.

Bacteriophage Based Biosensors: Trends, Outcomes and Challenges

“…While, this DNA in the host genome is called a prophage, the lysogenic cycle can continue indefinitely, except in some cases, such as adverse conditions of the environment and bacteria exposure to stress [14]. Consequently, the bacteriophage used like bio-probe in biosensor devises offer several advantages, such as (1) specificity to host bacteria, consequently efficient bacteria screening [15], (2) easy to generate mass quantities of progeny phages, due to their short replication time, (3) ability to tolerate critical conditions, such as organic solvents and large range of pH and temperature [16]. It is important to understand the characteristics of phages, such as the physical and chemical properties, in order to design the biosensor platform able to bind the phage without losing their ability to recognize the bacteria target.…”

Applications of Phage-Based Biosensors in the Diagnosis of Infectious Diseases, Food Safety, and Environmental Monitoring

“…The increased sensitivity enabled E. coli detection of 10 3 CFU/mL in broth while 100 CFU/100 mL of E. coli in inoculated river water. Such combination of phage-based diagnosis on paper fluidics offer new platforms to establish innovative detection techniques owing to sensitivity, robustness, and specificity and are personal friendly [47]. Additional improvement in the sensitivity of this method was reported by using fluorescent quantum dots (QDs) for phage labeling.…”

Principle and Development of Phage-Based Biosensors

“…As mentioned earlier, biosensor involves some biological recognition elements like bacteriophages [17], enzyme [18], whole cell [19], nucleic acid [20], and antibody [21], etc. These common bio-probe are briefed in the following sections:…”

“…A phage as a bio-recognition probe offers numerous benefits in rapid bacterial sensing [17] as they are: (•) extremely specific to their host [26], (•) ability of producing extraordinary titers of descendant phages, (•) tolerant to extreme environmental conditions like ultrahigh temperatures, organic solvents and wide-ranging pH compared to Abs, [27], (•) safe handling, (•) discriminating among dead and live bacteria as they proliferate only in live bacterial cells [28], (•) production in bulk are artless and economical. These compensations make phages as leading bio-recognition probes to develop biosensors for bacterial screening [15].…”