**6. Patents**

Some of the contents in this article were filed in a Japanese patent (JP2022175611).

**Funding:** This study was partially supported by M-Cube Project and the fourth mid-term project in NIMS.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The author thanks Takashi Hironaka for his contribution to the experiment in this study and Naoki Ikeda for the nanofabrication of the metasurfaces.

**Conflicts of Interest:** The author declares no conflict of interest.

## **Appendix A**

Figure A1 shows further results of FL detection for CEA using a metasurface biosensor and of CEA detection using a commercial ELISA kit (ab264604, Abcam). Figure A1a shows a lower CEA-concentration range than that is Figure 3a, and indicates that CEA was actually detected at the concentrations of 0.008 ng/mL (or 8 pg/mL). The zero level of the FL measurement is shown with an arrow. Thus, it is confirmed that the dynamic range of the metasurface biosensors for CEA covers from ∼1 pg/mL to 25 ng/mL.

**Figure A1.** Detection of CEA by the metasurface biosensor and ELISA. (**a**) CEA detection curve in a low concentration range down to 8 pg/mL, measured by the metasurface biosensor. Measured data are shown using orange closed circles with error bars. Dashed curve is a fitted curve by the Hill equation (Equation (1)). (**b**) Detection results for three CEAs (black closed circle: ab264604, Abcam, blue triangle: 15-N-100, Alpha Diagnostic, green open circle: ab742, Abcam) by ELISA, which was conducted using a commercial kit. The vertical axis represents optical density at 450 nm. Three horizontal bars indicate zero levels for the three CEAs; the colors match up the measured data.

Figure A1b shows a set of ELISA results using the commercial kit, which claims high throughput (i.e., 1.5 h running time) and high sensitivity. We used three CEA targets: block closed circles correspond to CEA, ab264604, Abcam, blue triangles to CEA, CEA15-N-100, Alpha Diagnostic, and green open circles to CEA, ab742, Abcam. The detection profiles of the black closed circle and the blue triangle are similar; the dynamic ranges are estimated to be from 0.63 to 50 ng/mL, based on intersection of the two profiles. In contrast, another profile of the green open circle exhibited a lower response than the others, and the dynamic range covers from 1.25 to 200 ng/mL. These results suggest that the enzyme reaction in ELISA can change for detection targets. Practically, we can conclude that the ELISA kit is effective at 1–50 ng/mL for a given CEA, and that the sub-ng/mL ranges are unreliable.

Additionally, we note that the actual running time for the ELISA was approximately 2 h, due to additional procedures (e.g., washing of a microplate) are inevitable; the runtime falls within a short-time category among various ELISA kits. The metasurface biosensors completed the runtime within 2 h and, moreover, exhibited the one-digit pg/mL detection for CEA in a dynamical manner, which is 100-times more effective than the ELISA kit.

#### **References**


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