(521e) Single Particle ICP-MS: An Emerging Technique for Quantifying Size and Aggregation of Inorganic Nanomaterials for Biomedical Applications

The efficacy, toxicity, and fate of engineered nanomaterials depends on size and colloidal stability. Conventional techniques to characterize the size and aggregation of nanomaterials are limited to batch-based measurements or require harsh conditions to obtain single nanoparticle resolution. Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) is an emerging technique that offers high-throughput in situ nanoparticle size analysis with single nanoparticle resolution.

To accomplish this, SP-ICP-MS utilizes a microfluidics sample introduction system to introduce nanoparticles—one at a time—into an argon plasma. In the argon plasma, individual nanoparticles are atomized and ionized to generate an ion cloud. This discrete ion cloud then travels through a quadrupole mass spectrometer. Once detected, the measured intensity of a singular ion cloud is used to calculate the size of individual nanoparticles. This process occurs at a rate of +200 nanoparticles per minute for a given element.

We show that SP-ICP-MS accurately determined size distributions of different sizes of gold nanoparticles (AuNPs) that we synthesized in house at a rate of ≥ 200 particles/minute. Dynamic light scattering and transmission electron microscopy corroborated our SP-ICP-MS results. After successfully determining the mass and size distributions of AuNPs with different sizes, we used SP-ICP-MS to differentiate between nanoparticles of different sizes in mixtures. Our results confirmed that SP-ICP-MS can simultaneously and precisely measure AuNP mass distributions of multiple nanoparticle subpopulations in a nanoparticle mixture with single particle resolution. To further validate SP-ICP-MS, we demonstrated that SP-ICP-MS can accurately determine the mass distribution of non-spherical anisotropic nanoparticles.

Since SP-ICP-MS differentiated subpopulations of nanoparticles in mixtures and quantified the mass distribution of anisotropic nanoparticles, we hypothesized that SP-ICP-MS could quantify nanoparticle aggregation with single particle (i.e., single aggregate) resolution. To generate AuNP aggregates, we exposed citrate-coated 16-nm AuNPs with a narrow size distribution to physiologically relevant saline concentrations. Given that the mean mass of our 16-nm AuNPs was 50 attograms, we assumed that 100 attograms corresponded to an aggregate consisting of 2 AuNPs, 150 attograms corresponded to an aggregate consisting of 3 nanoparticles, and so forth.

SP-ICP-MS detected nanoparticle aggregation within 5 minutes of saline exposure. After 60 minutes of saline exposure, the number of individual single AuNPs decreased by ~70% when compared with AuNPs in the initial measurement without saline. At this time point, more than half (52%) of the aggregates were comprised of 2–5 AuNPs per aggregate while only ~ 21% of the detected aggregates consisted of 5 or more AuNPs per aggregate.

Our SP-ICP-MS experiments confirmed the aggregation data obtained from dynamic light scattering and UV-Vis spectrophotometry which showed rapid progression of salt-induced AuNP aggregation. However, in stark contrast to batch-based methods, we quantified the individual masses of AuNP aggregates. When compared with light scattering techniques, SP-ICP-MS results were not skewed by larger nanoparticles or by the extra layer of hydration, as we only measure gold ions from the AuNPs. When compared to electron microscopy, SP-ICP-MS circumvents harsh sample preparation conditions that may introduce artifacts and the need for subsequent image analysis. Ultimately, SP-ICP-MS is an unbiased approach for quantifying nanoparticle aggregation in situ.

To mitigate nanoparticle aggregation, we hypothesized that PEGylation of AuNPs, —the decoration of nanoparticle surfaces with polyethylene glycol (PEG) polymers—could be adopted for our study. We prepared 16-nm AuNPs modified with various surface densities of thiol-PEG5kDa-methoxy. Upon exposure to saline, SP-ICP-MS results confirmed that PEGylation of nanoparticles physically hinders the rapid onset of salt-induced aggregation in a PEG surface density–dependent fashion. After 60 min in saline, more than 90% of all detected nanoparticle events were detected as individual nanoparticles for each PEG surface density compared with the 27% of individual nanoparticles observed for citrate coated AuNPs. SP-ICP-MS results show that the formation of larger aggregates (3 or more nanoparticles per aggregate) was completely inhibited by PEG. Our SP-ICP-MS findings confirmed as little as 0.010 PEG/nm² was sufficient for reducing the time-dependent effects of aggregation for monodisperse 16-nm AuNPs.

In summary, we show that SP-ICP-MS accurately analyzed the size of engineered AuNPs with varying shapes and sizes. Furthermore, we applied SP-ICP-MS as an in situ technique to quantify aggregation of AuNPs in physiologically relevant saline conditions and showed the mass distributions of AuNP aggregates with single aggregate resolution. We applied surface engineering strategies to demonstrate that increasing PEG surface densities on AuNPs could block aggregation.

SP-ICP-MS analysis of inorganic nanomaterials has far reaching implications for researchers who seek to understand how engineered nanoparticles change over time in biologically relevant environments and how surface modifications impact nanoparticle transformations.