Immunocapture of Escherichia coli in a fluoropolymer microcapillary array

Abstract

This study presents novel experimental insights into the direct quantitation and immunocapture of bacteria cells in a fluoropolymer microcapillary array, using Escherichia coli as work model, a pathogen responsible for around 80% of urinary tract infections (UTIs). In spite of the current clinical demand for sensitive tests for rapid identification and quantitation of pathogens in human samples, portable diagnostic tests developed to date lack the specificity, limit of detection and speed for effective implementation in bacteria detection at point-of-care. The ‘open microfluidic’ approach presented in this work directly addresses those challenges. We report for the first time evidence of immunocapture of bacteria using polyclonal antibodies immobilized on the inner surface of an inexpensive 10-bore, 200 μm internal diameter FEP-Teflon® MicroCapillary Film, with a limit of detection (LoD) of at just 1 colony forming unit (CFU). In capillaries coated with less than a full monolayer of capture antibody, we observed a first order equilibrium, with bacteria captured (in CFUs/ml) linearly proportional to the CFU/ml in the incubated sample. We captured up to 100% of E. coli cells, with clear evidence of immunospecificity as demonstrated by testing with a different bacteria specie (in this case Bacillus subtillis). We noticed gravity settling of bacteria within the capillaries created a gradient of concentration which on the overall enhanced the capturing of cells up to 6 orders of magnitude beyond the theoretic full monolayer (∼4.5 × 104 CFUs/ml), with washings having an unnoticeable effect. Our data particularly highlights quantitatively the relevance of interrogation volume in respect to the miniaturisation of bacteria quantitation, which cannot be solved with the most sophisticated imaging equipment. A further set of continuous flow experiments at a flow rate of just ∼1 μl/min (corresponding to a wall shear rate of ∼101 s−1 and superficial flow velocity ∼53 μm/s) showed a degree of flow focusing, yet the mobility, antibody affinity capturing and gravity settling of bacteria cells enabled successful capturing in the microcapillaries. These results will inform the future development of effective microfluidic approaches for rapid point-of-care quantitation of bacterial pathogens and in particular rule-in of E. coli in UTIs.