Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10μl to >100μl are concentrated within a sub-50nl region at the PAM filter edge in the microchannel, thus concentrating them over 1000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0×2.0×0.3mm PAM filter, as few as 10 rickettsial organisms per 100µl of lysed blood sample can be analyzed within 60min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases. •Capturing bacteria via monolith in a micro-flow channel overcomes transport issues.•Bacteria from blood volumes up to 100μl were captured and stained within minutes.•Hydrophilic monolith offers low non-specific fouling, enhances detection sensitivity.•Packing captured bacteria into microscale imaging area enhances detection sensitivity.•Fewer than 10 Rickettsia typhi cells were detected in blood with ~100% capture rate.