posted on 2021-06-03, 23:44authored bySimone Hageneder, Vanessa Jungbluth, Regina Soldo, Christian Petri, Matthias Pertiller, Marjut Kreivi, Andreas Weinhäusel, Ulrich Jonas, Jakub Dostalek
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.