A combined approach to signal enhancement in fluorescence affinity biosensors and assays is reported. It is based on the compaction of specifically captured target molecules at the sensor surface followed by optical probing with a tightly confined surface plasmon (SP) field. This concept is utilized by using a thermoresponsive hydrogel (HG) binding matrix that is prepared from a terpolymer derived from poly(N-isopropylacrylamide) (pNIPAAm) and attached to a metallic sensor surface. Epi-illumination fluorescence and SP-enhanced total internal reflection fluorescence readouts of affinity binding events are performed to spatially interrogate the fluorescent signal in the direction parallel and perpendicular to the sensor surface. The pNIPAAm-based HG binding matrix is arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein-Barr virus (EBV). The detection is performed in diluted human plasma or with isolated human IgG by using a set of peptide ligands mapping the epitope of the EBV nuclear antigen. Alkyne-terminated peptides were covalently coupled to the pNIPAAm-based HG carrying azide moieties. Importantly, using such low-molecular-weight ligands allowed preserving the thermoresponsive properties of the pNIPAAm-based architecture, which was not possible for amine coupling of regular antibodies that have a higher molecular weight.

Biosensor Technologies AIT Austrian Institute of Technology GmbH Konrad Lorenz Straße 24 Tulln an der Donau 3430 Austria

FZU Institute of Physics Czech Academy of Sciences Na Slovance 2 Prague 182 21 Czech Republic

Ginolis Ltd Automaatiotie 1 Oulunsalo 90460 Finland

Macromolecular Chemistry Department Chemistry Biology University of Siegen Adolf Reichwein Strasse 2 Siegen 57076 Germany

Molecular Diagnostics Health and Environment AIT Austrian Institute of Technology GmbH Giefinggasse 4 Vienna 1210 Austria

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