Long range surface plasmon and hydrogel optical waveguide field-enhanced fluorescence biosensor with 3D hydrogel binding matrix: on the role of diffusion mass transfer.

An implementation of evanescent wave affinity biosensor with a large-capacity three-dimensional binding matrix for ultra-sensitive detection of molecular analytes is investigated. In the experimental part of the work, highly swollen carboxylated poly(N-isopropylacryamide) (NIPAAm) hydrogel with up to micrometer thickness was grafted to a sensor surface, functionalized with antibody recognition elements and employed for immunoassay-based detection of target molecules contained in a liquid sample. Molecular binding events were detected by long range surface plasmon (LRSP) and hydrogel optical waveguide (HOW) field-enhanced fluorescence spectroscopy. These novel methods allowed probing an extended three-dimensional biointerface with an evanescent field reaching up to several micrometers from the sensor surface. The resonant excitation of LRSP and HOW modes provided strong enhancement of intensity of electromagnetic field that is directly translated into an increased fluorescence signal associated with the binding of fluorophore-labeled molecules. Experimental observations were supported by numerical simulations of mass transfer and affinity binding of target molecules in the hydrogel. Through the optimization of the hydrogel thickness and profile of the probing evanescent wave, low femtomolar limit of detection was achieved.