A microfluidic platform combined with bacteriophage receptor binding proteins for multiplex detection of Escherichia coli and Pseudomonas aeruginosa in blood

Bloodstream infections (BSIs) are triggered by the existence of pathogens in blood and are considered a major health burden worldwide, especially when they result in sepsis and septic shock. Common diagnostic methods are time-consuming, present low specificity, or suffer from interference of blood components, which hampers a timely and effective treatment of BSIs. In this work, a novel microfluidic assay was developed combining a bead-based chip and bacteriophage receptor binding proteins (RBPs) as extremely specific and sensitive recognition molecules for the multiplex concentration and detection of Escherichia coli and Pseudomonas aeruginosa, which are highly prevalent bacteria in BSIs. The device comprises a microcolumn in which antibody-functionalized agarose beads were packed allowing the entrapment of the target bacterium from blood, providing its concentration and separation. For bacterial detection, two recombinant RBPs (Gp54 and Gp17) were fused with different fluorescent proteins and used for the identification of P. aeruginosa and E. coli by the measurement of the distinct fluorescent signals obtained. The developed microfluidic-based assay enabled a fast (70 min) and highly specific multiplex detection of both pathogens in whole blood, achieving a detection limit of around 103 CFU, without requiring any time-consuming bacterial pre-enrichment step. Furthermore, it provided a quantitative assessment of bacterial loads present in blood. Noteworthy, this miniaturized and inexpensive device presents simple fabrication and operation, showing great potential to be fully automated, demonstrating to be ideal in point-of-care settings.