*2.2. Biomolecules and Reagents*

Target cancer markers were purchased from companies. We used native human PSA protein (ab78528, Abcam, Cambridge, UK) and CEA (CEA15-N-100, Alpha Diagnostic, San Antonio, TX, USA) as the targets. In diluting the targets to specific concentrations, a sample diluent, NS buffer (ab193972, Abcam), was applied, consisting of phosphate-buffer saline (PBS) and bovine serum albumin. Human serum pool (12181201, Cosmo Bio, Tokyo, Japan), which was a mixture of 10-person serums free from specific diseases, was used for testing the robustness of the metasurface biosensors.

The sandwich complex of Ab—antigen—Ab was designed to have labels, such as biotin-Ab—antigen—Ab-FL. In accordance with this design, a FL label, HiLyte Flour 555 (HL555), was conjugated, in advance, to the Abs using a labeling kit (LK14, Dojindo Laboratories, Kumamoto, Japan). Additionally, biotin was conjugated using a labeling kit (LK03, Dojindo Laboratories). Abs for the PSA and CEA used in this study were mouse monoclonal, being reactive to human PSA and CEA, respectively. Anti-PSA Ab (8A9B8, GenScript, Nanjing, China) and anti-CEA Ab (ab4451, Abcam) were biotin-conjugated, and anti-PSA Ab (MAB6729, Abnova, Taipei, Taiwan) and anti-CEA Ab (10-2370, Fitzgerald Industries, Acton, MA, USA) were HL555-conjugated. After the conjugations, the Abs were collected nominally at 0.5 mg/mL, and then the concentrations of the labeled Abs were tested by light absorption measurement. The HL555-labeling ratios can be evaluated from the light absorbance; we found that HL555 molecules:anti-PSA Ab ≈ 4:1 and HL555 molecules:anti-CEA Ab ≈ 7:1. For the collection and dilutions of the labeled Abs, PBS at pH 7.4 (164-25511, FujiFilm Wako Pure Chemicals, Osaka, Japan) was used. To immobilize the sandwich complexes on the metasurfaces, Cys-streptavidin (Cys-SA, PRO1005, Click-Biosystems, Richardson, TX, USA) was employed, which can bind to the outermost surface of silicon nanopellets and effectively capture the biotin-labeled sandwich complexes.
