**1. Introduction**

The detections of biomolecules is a crucial part of clinical diagnosis and health monitoring. To detect target biomolecules in the practical situations, we need not only high sensitivity, but also robustness against other biomolecules present in abundance. It is widely known that human blood usually comprises 55% blood cells, such as red corpuscles and platelets, and 45% plasma, which has proteins, such as immunoglobulin G (IgG) and albumin, other glycerin, and inorganic salts. Normally, concentrations of globulin and albumin in blood are 20–35 and 35–45 g/L, respectively [1]. For medical diagnosis, cancer markers, such as prostate specific antigen (PSA) and carcinoembryonic antigen (CEA), are examined in a range around 5 ng/mL [2]. Thus, biomarker molecules are much rarer than abundant biomolecules; for example, IgG concentration, which is normally 8.61–17.47 mg/mL [2], is 106-fold higher than the clinical criteria of PSA and CEA. Therefore, robustness is inevitably a requirement for practical biosensors.

Human serums are most commonly examined for cancer markers in clinical examinations [3–5]. Accordingly, biosensors applicable to cancer markers were often tested for their robustness using human serums [6–17]. All-dielectric metasurface biosensors [18–21] and plasmon–photon hybrid metasurface biosensors [22] were recently reported as efficient fluorescence (FL) biosensors. Figure 1a,b illustrates two situations where sandwich complexes of antibody (Ab)—antigen—Ab are formed. Complexes in the former are in a buffer suitable for proteins, whereas complexes in the latter are in a human serum that contains abundant biomolecules, such as albumin and IgG. For immobilization of the complexes, one of the Abs is labeled with biotin, and for FL detection, the other Ab is labeled with FL molecules. The biotin-labeled Abs serve as capture Abs and the FL-labeled Abs work

**Citation:** Iwanaga, M. Robust Detection of Cancer Markers in Humans Serum Using All-Dielectric Metasurface Biosensors. *Biosensors* **2023**, *13*, 377. https://doi.org/ 10.3390/bios13030377

Received: 27 February 2023 Revised: 9 March 2023 Accepted: 10 March 2023 Published: 13 March 2023

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

as detection Abs in an ordinary immunoassay scheme, e.g., enzyme-linked immunosorbent assay (ELISA). Here, we report a series of proof-of-concept experiments using the all-dielectric metasurface biosensors for the detection of cancer markers that coexist with other actual proteins, such as albumin, IgG, and different cancer markers, thereby clarifying the robustness of metasurface biosensors.

**Figure 1.** Key concepts of this study. (**a**) Target antigens in a purified condition. (**b**) Targets in a human serum where abundant impeding molecules, exist together with a small number of targets. Antibody (Ab) labeled with fluorescence (FL) molecule (magenta dot), antigen (purple), Ab labeled with biotin (black dot), and bovine serum albumin (BSA, light brown) are shown. Additionally, albumin (brown) and immunoglobulin G (IgG, Y-shaped) are schematically illustrated. (**c**) Photograph (color) of an alldielectric metasurface substrate and top-view scanning-electron-microscopy image (gray scale), providing a magnified view. The metasurface was a 300-nm periodic array of silicon nanopellets. Six metasurface areas of small rectangular shapes (2.1 <sup>×</sup> 0.7 mm2 each) in the photo were designed to correspond to six microfluidic channels. White and black scale bars indicate 10 mm and 500 nm, respectively.

A color photograph of an all-dielectric metasurface substrate is shown in Figure 1c, where six metasurface areas of small rectangular shapes appear, exhibiting diffraction colors. A white scale bar indicates 10 mm. An scanning-electron-microscopy (SEM) image, which magnifies a metasurface area, is shown on a gray scale. The SEM image was taken in a top-view manner, presenting a periodic array of circular silicon nanopellets of 200 nm height. The periodic length was 300 nm and the diameter of the silicon nanopellets was 224 ± 4 nm. A black scale bar represents 500 nm.
