Linker-protein G mediated functionalization of polystyrene-encapsulated upconversion nanoparticles for rapid gene assay using convective PCR

The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 10³ copies μL⁻¹. The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 10⁷ and 10³ copies of DNA.