The NPs (Cabot Security Materials Inc, Mountain View, CA) utilize a gold core and are encapsulated in silica. The gold-core diameters are ~60 nm and the overall dimension is ~120 nm. Additional details may be found in the literature9 (link). The silica surface of the NPs are functionalized with thiols to allow conjugation to a variety of targeting molecules (Fig. 6a). Three “flavors” of NPs were used, identified as S420, S421 and S440, each of which emits a characteristic Raman spectrum (Fig. 5b).
Stock concentrations of SERS NPs (800 pM in water) were diluted in 10 mM MOPS (Sigma-Aldrich, part No. M1254) buffer, pH 7.25, at a volume ratio of 1:1 (e.g. 200 μL buffer to 200 μL NPs in water), and then reacted with Cyto 647-maleimide (Cytodiagnostics Inc, part No. NF647-3-01), at 4.5 × 105 molar equivalents per NP, at room temperature for 1 h to conjugate Cyto 647 fluorophores to the NP surface (Fig. 6a). Three different monoclonal antibodies (mAb) were used for conjugation: anti-EGFR “panitumumab” (Vectibix, NDC 55513-954-01), anti-HER2 (Thermo Scientific, MS-229-PABX), or an isotype control mAb (Thermo Scientific, MA110407). These mAbs were first eluted over a desalting spin column (Thermo Scientific, 89882) and buffer-exchanged into the MOPS buffer to remove preservatives such as sodium azide. All mAbs were purchased free of protein stabilizers such as BSA or gelatin. Antibodies were added to fluorescent NPs at 500 molar equivalents per NP, along with the heterobifunctional PEG cross linker SM(PEG)12 (link) (Thermo Scientific, 22112), at 1.5 × 104 molar equivalents per NP, and incubated at room temperature for 3 h. Following the primary conjugation reaction, MM(PEG)12 (link) (Thermo Scientific, 22711), at 6 × 105 molar equivalents per NP, was then added to the NPs and reacted over night at 4°C to block residual thiols on the NPs. Note that all reagents were degassed with dry argon gas prior to use and all reactions were conducted under anhydrous conditions in light-protected amber vials (Fisher Scientific, 03-391-36&03-391-17) on a vortex mixer (Fisher Scientific™ Microplate Vortex Mixers, 02-216-101) set at 800 rpm. Finally, the NPs were reacted at room temperature with 2-mercaptoethanesulfonate (Sigma-Aldrich, M1511) for 30 min, at 9 × 105 molar equivalents per NP, and the reaction mixture was then diluted at a volume ratio of 1:1 with storage buffer (20 mM MOPS at pH 7.5 with 0.1% BSA (Jackson Immuno, 001-000-162) and 0.05% sodium azide (Sigma-Aldrich, S2002)). The diluted reaction mixture was purified four times via centrifugation (1000 g for 10 min), in which the supernatant was removed and replaced with storage buffer after each round of centrifugation. The conjugated NPs were stored at 4°C and protected from light. UV-VIS spectrophotometry was used to measure the final concentration of the conjugated NPs (Supplementary Fig. 2).