Characterization and Quantification of Nanoparticle-Antibody Conjugates on Cells Using C(60) ToF SIMS in the Event-By-Event Bombardment/Detection Mode.

Cluster C(60) ToF-SIMS (time-of-flight secondary ion mass spectrometry) operated in the event-by-event bombardment-detection method has been applied to: a) quantify the binding density of Au nanoparticles (AuNPs)-antiCD4 conjugates on the cell surface; b) identify the binding sites between AuNPs and antibody. Briefly, our method consists of recording the secondary ions, SIs, individually emitted from a single C(60) (1,2+) impact. From the cumulative mass spectral data we selected events where a specific SI was detected. The selected records revealed the SIs co-ejected from the nanovolume impacted by an individual C(60) with an emission area of ~ 10nm in diameter as an emission depth of 5-10 nm. The fractional coverage is obtained as the ratio of the effective number of projectile impacts on a specified sampling area (N(e)) to the total number of impacts (N(0)). In the negative ion mass spectrum, the palmitate (C(16)H(31)O(2) (-)) and oletate (C(18)H(33)O(2) (-)) fatty acid ions present signals from lipid membrane of the cells. The signals at m/z 197 (Au(-)) and 223 (AuCN(-)) originate from the AuNPs labeled antibodies (antiCD4) bound to the cell surface antigens. The characteristic amino acid ions validate the presence of antiCD4. A coincidence mass spectrum extracted with ion at m/z 223 (AuCN(-)) reveals the presence of cysteine at m/z 120, documenting the closeness of cysteine and the AuNP. Their proximity suggests that the binding site for AuNP on the antibody is the sulfur-terminal cysteine. The fractional coverage of membrane lipid was determined to be ~23% of the cell surfaces while the AuNPs was found to be ~21%. The novel method can be implemented on smaller size NPs, it should thus be applicable for studies on size dependent binding of NP-antibody conjugates.