Characterization and quantification of nanoparticle–antibody conjugates on cells using C 60 ToF SIMS in the event-by-event bombardment/detection mode

Abstract

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 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 ∼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 (C 16H 31O 2 −) and oletate (C 18H 33O 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.