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 (
Functionalized SERS Nanoparticle Conjugation
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Corresponding Organization :
Other organizations : Stony Brook University, University of Toronto, Princess Margaret Cancer Centre
Protocol cited in 6 other protocols
Variable analysis
- Three "flavors" of NPs (S420, S421, and S440) with characteristic Raman spectra
- Cyto 647-maleimide at 4.5 × 10^5 molar equivalents per NP
- Three different monoclonal antibodies (anti-EGFR "panitumumab", anti-HER2, or an isotype control mAb) at 500 molar equivalents per NP
- Heterobifunctional PEG cross linker SM(PEG)^12 at 1.5 × 10^4 molar equivalents per NP
- MM(PEG)^12 at 6 × 10^5 molar equivalents per NP
- 2-mercaptoethanesulfonate at 9 × 10^5 molar equivalents per NP
- Conjugation of Cyto 647 fluorophores to the NP surface
- Conjugation of monoclonal antibodies to the NP surface
- Final concentration of the conjugated NPs
- Gold-core diameters of ~60 nm and overall dimension of ~120 nm
- Silica surface of the NPs functionalized with thiols
- Stock concentrations of SERS NPs (800 pM in water) diluted in 10 mM MOPS buffer, pH 7.25, at a volume ratio of 1:1
- All reagents degassed with dry argon gas prior to use and all reactions conducted under anhydrous conditions
- Reactions conducted in light-protected amber vials on a vortex mixer at 800 rpm
- Purification of conjugated NPs via centrifugation (1000 g for 10 min) and replacement with storage buffer (20 mM MOPS at pH 7.5 with 0.1% BSA and 0.05% sodium azide)
- Storage of conjugated NPs at 4°C and protected from light
- None explicitly mentioned
- Isotype control mAb
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