Investigating conjugated polymer nanoparticle formulations for lateral flow immunoassays

Lateral flow immunoassays (LFI) are valuable tools for point-of-care testing. However, their sensitivity is limited and can be further improved. Nanoparticles (NP) of conjugated polymers (CPNs), also known as Pdots, are reported to be highly sensitive fluorescent probes, but a direct comparison with conventional colloidal gold-based (Au-NP) LFI using the same antibody-antigen pair is missing to date. Furthermore, the influence of brightness and Stokes shift of CPs on the signal : background ratio (SBR) needs to be evaluated. In this study, we encapsulated two different CPs, poly-(9,9-di-n-octyl-fluorenyl-2,7-diyl) (PDOF) and poly-(2,5-di-hexyloxy-cyanoterephthalylidene) (CN-PPV) in silica shell-crosslinked Pluronic© micelles (Si-NP) and Pdots and investigated the NP brightness with respect to CP loading dose. The brightest formulation of each NP system was conjugated to rabbit IgG as a model antigen and the SBR was investigated in an ELISA-like microplate assay and LFI. Two reference particles, Au-NP and a polystyrene NP (PS-NP) loaded with a small-molecule fluorescent dye were conjugated to IgG and compared to the Si-NP and Pdots. The mass of Pdots required for detection in LFI was at least two orders of magnitude lower than that of Si-NP and the reference NP. The SBR of CN-PPV (moderate brightness, large Stokes shift) was two to three times higher than the SBR of PDOF (high brightness, small Stokes shift). To combine the favourable properties of both CPs, a polymer blend of PDOF and CN-PPV was encapsulated in Pdots, and resulted in further increase of SBR in the microplate assay and LFI. In summary, combining two CPs with different properties can lead to fluorescent signal-transducers for applications such as ELISA and LFIs, which can enhance the detection limit of the assay by 2-3 orders of magnitude.