We acquired the single-point Raman spectrum with an XPLORA (HORIBA, Jobin Yvon, France) Raman microscope system. Raman mapping was performed in air using the 633-nm laser and a 60× S Plan Fluor air objective (NA, 0.7; Nikon). The laser power after the objective was measured to be 10.7 mW, and the laser power we used was reduced to 50% to protect the Raman dye. Raman mapping was performed at a step of 1.5 μm × 1.5 μm, and the acquisition time was 10 s per point. To obtain the pure Raman signal, the combined background of Au and SYBR Green I was subtracted from the spectrum.

We performed the reference Raman measurements of SYBR Green I in bulk solution at 2000× concentration. The corresponding concentration of commercial SYBR Green I was assumed to be 3.92 mM according to Zipper et al. (35). This dye can bind to DNA strands nonspecifically and can therefore randomly distribute on the whole surface of the tetrameric metamolecule (37). The SERS EF was calculated by comparing the signals measured from a single tetrameric metamolecule with the intensity of the Raman signal from the bulk solution. The EF was estimated from the excitation power P, the Raman intensity I, and the amount of molecules N contributing to the signal for the bulk SYBR Green I solution and for the tetrameric metamolecules, respectivelyEF=ISERS×Pbulk×NbulkIbulk×PSERS×NSERS

ISERS is the peak intensity of SYBR Green I at 1340 cm−1 in the tetrameric metamolecules, and Ibulk is the peak intensity of SYBR Green I in the solution. Pbulk is the same with PSERS. Nbulk is the estimated number of SYBR Green I molecules in the volume of the laser spot in the bulk measurement (~1.8 × 107). It has been reported that the DNA molecule has a 13-nm2 footprint for the 80-nm L-AuNPs (38). Therefore, NSERS was calculated, as the SYBR Green I molecules were absorbed on the surface of the L-AuNPs in the plasmonic hot spot (~6). The size of the plasmonic hot spot was estimated according to FDTD calculation results from fig. S9.

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