Responsive Hydrogel Binding Matrix for Dual Signal Amplification in Fluorescence Affinity Biosensors and Peptide Microarrays

Abstract

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.

Rapid Actuation of Thermo-Responsive Polymer Networks: Investigation of the Transition Kinetics

“…This value translates to the swelling ratio change of a flat film of 7.5, which is in the range of the one measured previously for the same type of polymer and cross-linking. 30 It is worth of noting that the simulated LSPR spectra qualitatively agree with the measured ones, but the LSPR wavelengths are red-shifted with respect to the measured ones by about 150 nm. This discrepancy can be ascribed to the over-estimation of Au nanoparticle short- and long-axis lengths D ⊥ and D ∥ due to the convolution of tip geometry of the used AFM tip with the particle geometry.…”

“…By raising temperature above LCST to 40 °C, the thickness decreases by a factor of 4.7 and the refractive index increases to n h = 1.42. 30 …”

“…Their considering may be important for systems researched to perform non-reciprocal morphing as a prerequisite for locomotion of soft micro-swimmers, 12 time-dependent switching between the hydrophobic and hydrophilic state can be utilized for construction of micro-crawlers, 6 or in design of responsive biointerfaces that allow rapid compacting of molecular species at plasmonic hotspots for subsequent sensitive plasmonically enhanced fluorescence spectroscopy detection. 30 …”

Plasmonic nanomaterials with responsive polymer hydrogels for sensing and actuation

“…By studying this effect in more detail, it was observed that the temperature-induced collapse of similar pNIPAAm layers led to a decrease in the swelling ratio Q v by a factor of 4.7, but after its postmodification with IgG this ratio dropped to only 1.6. 167 This work suggests that a possible route to circumvent this problem is using ligands with lower molar mass imposing smaller changes to the thermoresponsive characteristics of the pNIPAAm binding matrix. Fig.…”

The Bioanalytical and Biomedical Applications of Polymer Modified Substrates

“…The finding opens up possibilities for discovering lectin biomarkers toward various biomedical applications, including cancer diagnosis and therapy. Recently, Hageneder et al developed a responsive hydrogel binding matrix for dual signal amplification in fluorescence affinity biosensors and peptide microarrays by using a terpolymer modified metallic sensor surface [148]. The terpolymer, derived from poly(Nisopropylacrylamide) (pNIPAAm), was arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein−Barr virus (EBV) in diluted human plasma by using a set of peptide ligands.…”