Characterization and quantification of nanoparticle-antibody conjugates on cells using C60 ToF SIMS in the event-by-event bombardment/detection mode

Cluster C-60 ToF-SIMS (time-of-night 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 and (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 similar to 10 nm 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-o). In the negative ion mass spectrum, the palmitate (C16H31O2-) and oletate (C18H33O2-) 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 similar to 23% of the cell surfaces while the AuNPs was found to be similar to 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. (C) 2011 Elsevier B.V. All rights reserved.