SERS nanotags were prepared by functionalizing AuNPs with antibodies and Raman reporters and stabilizing with bovine serum albumin (BSA; Life Technologies Australia Pty Ltd.) coatings. Briefly, 60-nm AuNPs were synthesized by citrate reduction of HAuCl4 (39). Ten microliters of 1 mM Raman reporters in ethanol (either MBA, TFMBA, DTNB, or MPY) and subsequently 2 μl of 1 mM dithiobis(succinimidyl propionate) (Thermo Fisher Scientific) in dimethyl sulfoxide were added into 1 ml of AuNP solutions and incubated for 5 hours at RT to form a complete self-assembled monolayer. For the functionalization of MPY Raman reporters, 20 μl of 0.1 M NaOH was first added to adjust AuNP solutions to pH = 8. After incubation, the mixture was centrifuged at 7600 rpm for 10 min to remove the residual reactants. The mixture was then resuspended in 200 μl of 0.1 mM PBS and incubated with 2 μg of primary antibodies against either MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367), calnexin (Abcam, ab112995), or CD45 (BioLegend, 368502) for 30 min at RT. The mixture was then centrifuged at 600g at 4°C for 10 min to remove free antibodies and resuspended in 200 μl of 0.1% (w/v) BSA for 0.5 hour at RT to block nonspecific binding sites and stabilize SERS nanotags. The SERS nanotags were stored at 4°C and were stable for months.

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