**4. Conclusions**

In conclusion, we proposed a simplified E-beam lithography method to fabricate SERS-active substrates. By eliminating the lift-o ff process, it is possible to make large-area, SERS-active substrates with high e fficiency. The fabricated nanograting substrates were highly uniform and thus exhibited reproducible and robust SERS signals. We observed the highest SERS intensity on nanogratings of periods 200–400 nm. The enhancement had an obvious periodicity dependence. By investigating the fluorescence enhancement on the lift-o ff and nonlift-o ff nanogratings, we found the enhancement on the two structures is due to distinct plasmonic e ffects. The excitation of SPP was responsible for the large enhancement and SERS e ffect on the substrate. Our numerical simulations agreed nicely with the experimental results and indicated that further increasing of SERS e fficiency is possible by the coupling of SPP and LSPR. We have demonstrated using this substrate for the detection of protein-specific binding. Our method has the potential to fabricate large-area SERS-active substrates based on the relatively matured lithographic technique in a shorter period. The proposed SERS-active substrate can be readily employed as a routine molecular sensing element for a wide range of applications, such as environmental pollutant surveillance and immunoassays.

**Author Contributions:** Conceptualization, Y.-C.C.; Methodology, Y.-C.C.; Experiment, Y.-C.C. and B.-H.H.; Simulation, Y.-C.C. and T.-H.L.; Validation, Y.-C.C.; Formal analysis, Y.-C.C.; Investigation, Y.-C.C.; Writing, Y.-C.C.; Visualization, Y.-C.C.; Supervision, Y.-C.C.; Project administration, Y.-C.C.; Funding acquisition, Y.-C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Science and Technology of Taiwan, gran<sup>t</sup> numbers MOST 106-2221-E-018-021, MOST 107-2221-E-018-006, and MOST 108-2221-E-018-016.

**Acknowledgments:** The authors would like to express their gratitude to the support from the Ministry of Science and Technology of Taiwan and the technical assistance from the nanofabrication facility of National Chang-Hua University of Education. Y.-C.C. would also like to appreciate the many fruitful discussions with Yu-Ju Hung, Kuo-Ping Chen, Kerwin Wang and Yang-Wei Lin and their technical assistances.

**Conflicts of Interest:** The authors declare no conflict of interest.
