Liquid Nebulization-Ion Mobility Spectrometry Based Quantification of Nanoparticle-Protein Conjugate Formation

Despite the importance of examining the formation of nanoparticle-protein conjugates, there is a dearth of routine techniques for nanoparticle-protein conjugate characterization. The most prominent change to a nanoparticle population upon conjugate formation is a shift in the nanoparticle size distribution function. However, commonly employed dynamic light scattering based approaches for size distribution characterization are ineffective for nonmonodisperse samples, and further they are relatively insensitive to size shifts of only several nanometers, which are common during conjugate formation. Conversely, gas phase ion mobility spectrometry (IMS) techniques can be used to reliably examine polydisperse samples, and are sensitive to ∼1 nm size distribution function shifts; the challenge with IMS is to convert nanoparticle-protein conjugates to aerosol particles without bringing about nonspecific aggregation or conjugate formation. Except in limited circumstances, electrospray based aerosolization has proven difficult to apply for this purpose. Here we show that via liquid nebulization (LN) with online, high-flow-rate dilution (with dilution factors up to 10 000) it is possible to aerosolize nanoparticle-protein conjugates, enabling IMS measurements of their conjugate size distribution functions. We specifically employ the LN-IMS system to examine bovine serum albumin binding to gold nanoparticles. Inferred maximum protein surface coverages (∼0.025 nm^-2) from measurements are shown to be in excellent agreement with reported values for gold from quartz crystal microbalance measurements. It is also shown that LN-IMS measurements can be used to detect size distribution function shifts on the order of 1 nm, even in circumstances where the size distribution function itself has a standard deviation of ∼5 nm. In total, the reported measurements suggest that LN-IMS is a potentially simple and robust technique for nanoparticle-protein conjugate characterization.