Highly sensitive biomolecular interaction detection method using optical bound/free separation with grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS)

Abstract

Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS) with optical bound/free (B/F) separation technique was developed by employing a highly directional fluorescence with polarization of surface plasmon-coupled emission (SPCE) to realize highly sensitive immunoassay regardless of the ligand affinity. A highly sensitive immunoassay system with GC-SPFS was constructed using a plastic sensor chip reproducibly fabricated in-house by nanoimprinting and applied to the quantitative detection of an anti-lysozyme single-domain antibody (sdAb), to compare conventional washing B/F separation with optical B/F separation. Differences in the affinity of the anti-lysozyme sdAb, induced by artificial mutation of only one amino acid residue in the variable domain were attributed to higher sensitivity than that of the conventional Biacore surface plasmon resonance (SPR) system. The detection limit (LOD; means of six replicates of the zero standard plus three standard deviations) of the GC-SPFS immunoassay with optical B/F separation, was estimated to be 1.2 ng/ml with the low-affinity ligand (mutant sdAb Y52A: K D level was of the order of 10 −7 ~ 10 −6 M) and was clearly improved as compared to that (LOD: 9.4 ng/ml) obtained with the conventional washing B/F separation. These results indicate that GC-SPFS with the optical B/F separation technique offers opportunities to re-evaluate low-affinity biomaterials that are neither fully utilized nor widespread, and could facilitate the creation of novel and innovative methods in drug and diagnostic development.

Surface Plasmon-Assisted Fluorescence Enhancing and Quenching: From Theory to Application

“…Besides the RhB-doped PMMA film, the QD is adopted as a coupling object in the PMMA in SPCEi . The single-molecule super-resolution imaging can be achieved under strict conditions based on this diffraction grating SPCEi , grating . It utilized an inexpensively fabricated grating in combination with a scalable glancing angle deposition (GLAD).…”

“…174 The single-molecule super-resolution imaging can be achieved under strict conditions based on this diffraction grating SPCEi 175,176 grating. 176 It utilized an inexpensively fabricated grating in combination with a scalable glancing angle deposition (GLAD). The antigen-specific CD 4+ T cells in a blood sample are sensitively detected relying on a diffraction grating-induced SPCEi.…”