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: KD 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.
Highlights
In recent years, various biomaterials for drug development, including cancer immunity drugs [1] and anti-drug antibodies (ADAs) [2,3,4], have rapidly gained importance in next-generation precision medicine [5]
All points were measured at three to five times using one same sensor chip for reproducibility, and the origin of the horizontal axis indicates that the polarizer is orthogonal to the diffraction grating on the Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-Surface plasmon field-enhanced fluorescence spectroscopy (SPFS)) sensor surface, i.e., the p-polarized position
We previously explored the angular distribution of the GC-SPFS fluorescence signal using the same optical setup and confirmed the narrow directionality of the fluorescence signal with Au and Ag as plasmonic materials [29]
Summary
Various biomaterials for drug development, including cancer immunity drugs [1] and anti-drug antibodies (ADAs) [2,3,4], have rapidly gained importance in next-generation precision medicine [5]. Sensitive immunoassay without washing process specific roles of these authors are articulated in the “author contributions” section
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