(394j) Transport of Filamentous Viruses As Reporters in Lateral Flow Assays

Lateral-flow assays (LFAs) are a simple and rapid point-of-care format to detect biological analytes such as proteins, viruses, and bacteria. In LFAs, reporter particles bearing antibodies to a given analyte are transported by capillary action through a fibrous affinity membrane; particles bind to the analytes during flow and are in turn captured on the membrane, thereby generating a visual signal. While LFAs are particularly well-suited for use in economically challenged areas with limited laboratory infrastructure, their lack of sensitivity limits their use in scenarios requiring early detection. Previously, we showed that filamentous viruses can be employed as viral nanoparticle reporters in a quantitative LFA whose limit of detection is up to two orders of magnitude less than that of existing methods, but the reasons underlying this enhanced sensitivity remain poorly understood.

Here, we test the idea that the transport properties of filamentous viruses in LFA membranes gives rise to the increased sensitivity of viral nanoparticle LFAs. High-aspect-ratio rodlike M13 bacteriophage (M13) displaying biotin on the M13 tail protein p3 are conjugated with fluorescent labels and suspended in solutions of high-molecular-weight polymers, which mimic viscoelastic biofluids. We functionalize glass fiber LFA membranes with antibodies to M13, which primarily capture M13 on the p8 coat proteins on the lateral phage surface, and with avidin, which captures M13 at the biotinylated tail. Using microscopy and image analysis, we characterize the flow and binding of viruses bearing side- or tip-binding recognition elements in solutions of various polymer concentration. The number of M13 captured on the membrane is greater for side-binding than for tip-binding, as expected from the number of recognition elements, and in both cases depends on the flux of M13. We show that most binding events require the rodlike viruses to reorient prior to capture on the membrane. This result is consistent with the idea that the elongated M13 shape couples with the local flow field in an open and disordered fibrous LFA membrane to increase capture and hence LFA sensitivity.