Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells.

There have been important advances in characterizing the surface coverage of ligands on colloidal inorganic nanoparticles (NPs), but our knowledge of ligand coverage on lanthanide NPs is much more limited. The assynthesized NPs are often coated with hydrophobic ligands that need to be replaced with hydrophilic ligands such as poly(ethylene glycol) (PEG) for biomedical applications. The two challenges in terms of characterizing ligand coverage on NPs are first to show that different analytical methods give consistent results and second to show how the sample preparation protocol affects ligand density. Here, we report a quantitative study of the native oleate content of as-synthesized NaYF 4 and NaTbF 4 NPs, as well as the surface coverage after ligand exchange with three methoxyPEG-monophosphates with M n = 750, 2000, and 5000 Da. For NaYF 4 , we obtained consistent results for both oleates and PEGs by three independent methods (TGA, 1 H NMR, and ICP-AES). The oleate coverage was very sensitive to the sample isolation/purification protocol, with a high surface coverage (5.5 to 8 nm −2 ) for ethanol/hexane sedimentation/ redispersion but only 2 nm −2 if THF was used in place of hexanes. The surface coverages PEG750 (∼1.1 nm −2 ), PEG2000 (∼1.7 nm −2 ), and PEG5000 (∼0.2 nm −2 ) suggest that corona repulsion limits the number of PEG5000 molecules that can graft to the surface. For NaTbF 4 NPs, we compared the surface coverage of PEG2000-monophosphate with a PEG2000-tetraphosphonate ligand shown to provide enhanced colloidal stability in PBS buffer. We found the surprising result that the footprints of these ligands were comparable, suggesting that there was insufficient room for all four phosphonate groups of the tetradentate ligand to bind simultaneously to the NP surface.

Over the past decade, there have been extensive developments in the field of lanthanide-based nanoparticles (NPs). Most studies have focused on the application of upconverting NaYF4-based NPs for deep tissue imaging and paramagnetic NaGdF4 NPs for MRI. Current applications for the remaining members of the lanthanide series are rather limited. Recently, a novel bioanalytical technique known as mass cytometry (MC) has been developed which can benefit from the entire lanthanide series of NPs. MC is a high-throughput multiparametric cell-by-cell analysis technique based on atomic mass spectrometry that uses antibodies labeled with metal isotopes for biomarker detection. NaLnF4 NPs offer the promise of high sensitivity coupled with multiparameter detection, provided that NPs can be synthesized with a narrow size distribution. Here we describe the synthesis of six members of this NP family (NaSmF4, NaEuF4, NaGdF4, NaTbF4, NaDyF4, NaHoF4) with the appropriate size (5–30 nm) and size distribution (CV < 5%) for MC. We employed the coprecipitation method developed by Li and Zhang [Nanotechnology 2008, 19, 345606], and for each member of this series, we examined the heating rate, final reaction temperature, and composition of the reaction mixture in an attempt to optimize the synthesis. For each of the six NaLnF4, in the range of the target sizes, we were able to identify “sweet spots” in the reaction conditions to obtain NPs with a narrow size distribution. In addition, we investigated the oleate surface coverage of the NPs and the effect of long-term storage (2 years) on the colloidal stability of the NPs. Finally, NaTbF4 NPs were rendered hydrophilic via lipid encapsulation and tested for nonspecific binding with KG1a and Ramos cells by mass cytometry.

Polydimethylsiloxane (PEP) is widely used in medical prostheses and therefore is in contact with plasma and secretory proteins. Two pair of globular proteins, lactoferrin (Lf) and transferrin (Trf), and bovine IgG1 and IgG2a, which differ substantially between pair members in their pl, were used to study the interaction of a PEP widely used in breast implants and soluble protein. Studies were done using iodinated proteins over a concentration range that resulted in an apparent protein monolayer. Secondary incubations with dilute protein solutions were needed to form the monolayer on PEP, possibly as a consequence of micro air bubbles trapped on its highly textured surface as shown by atomic force microscopy. Immunoassay quality polystyrene microtiter wells were used as controls. Adsorption studies were routinely performed at pH 4, 7 and 10 and at ionic strengths corresponding to 0.95, 9.5 and 90.0 mS. The protein capture capacity (PCC) of PEP for Lf and Trf was optimal at physiological pH and ionic strength and comparable under these conditions to that of Immulon 2 (Imm 2) microtiter wells. While increasing the ionic strength and pH further increases the PCC of Imm 2 for Lf and Trf, this markedly lowered the PCC of PEP for these proteins suggesting that initial polar interactions may precede subsequent hydrophobic bonding to PEP. This was tested using a hydrophilic variant of PEP, which when tested in a 90.0 mS buffer, showed a > five-fold lower PCC at neutral and alkaline pH. The greatly reduced PCC of the hydrophilic variant might also suggest that hydrophilic variants of silicone would be more biocompatible than those currently used. The PCC of PEP for the IgGs was less than that of Imm 2 but still optimal at physiological conditions. Consistent with the data on Lf/Trf, PCC progressively decreased with increasing ionic strength at alkaline pH. Differences in pl between the protein pairs had only a marginal effect on the PCC of PEP. Monolayer adsorption on both PEP and Imm 2 was slowly reversible and greater in the presence of free ligand (< 2% in 16 h) suggesting that the process follows Mass Law principles. However, even in the presence of non-ionic detergent and free ligand, 85-90% remained bound on either surface. Thus, desorption of proteins in the monolayer should not complicate subsequent immunochemical studies conducted on adsorbed monolayers.