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 10 μl to >100 μl are concentrated within a sub-50 nl 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 2.0×2.0×0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 µl of lysed blood sample can be analyzed within 60 min, 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.