MEASURING PARTICLE AGGREGATION and AGGLUTINATION WITH ARCHIMEDES
Forces at the micro- and nano-scale make the aggregation of particles an almost universal phenomenon. Factors such as pH, temperature, surface treatment, molecular affinity, zeta potential, and contamination can have an enormous effect on the degree of aggregation. In some cases, aggregation is highly undesirable: when pigments cluster and clog inkjet nozzles, for example, or when therapeutic proteins aggregate and induce unwanted immune responses. Aggregation can also be put to use, as in immunoassays in which antibody-coated beads agglutinate in the presence of an analyte.
ARCHIMEDES measures aggregation with a degree of accuracy and detail that is unparalleled, with better resolution than light scattering, Brownian motion tracking, or field flow fractionation. Because ARCHIMEDES is responsive to mass, its output is directly proportional to the order of aggregation, i.e., dimers give twice the response of a primary particle. Each particle or aggregate is measured and counted separately, giving a quantitative picture of the sample content that cannot be attained with ensemble methods. In addition, aggregates can be measured in their native environments, without the need for re-suspension or dilution that can alter the aggregation.
In many applications, primary particles are of uniform size and are sufficiently massive to be measured individually by ARCHIMEDES. As an example, the buoyant mass distributions of 1 µm polystyrene beads (Duke 3900A) were measured after being exposed to Immunoglobulin G protein at a high concentration (~1 mg/ml). The proteins coat the beads and cause them to agglutinate. As shown in the frequency measurements (right), monomers, dimers, and trimers are clearly distinguished since each is an integer multiple of the monomer mass. The buoyant mass of several thousand particles and aggregates were monitored automatically with ARCHIMEDES over several hours by periodic sampling and continuous measurement. The time course of the agglutination process is seen in the histograms of buoyant mass (below).
The graph above shows quantitatively the formation of the various aggregate orders (monomers, dimers, and trimers) vs. time. Similar measurements enable mass-based readout for immunoassays in which the multimer populations reflect the concentration of an analyte that catalyzes the agglutination.1 Inverting this method, the analysis of dimer content (say) could provide a simple quality check for the uniformity and activity of commercially-available beads functionalized for use in a range of immunoassays and separation technologies.2
Continuing the above example, we can also extract detailed information about the coating on the beads. The plot at right compares the buoyant mass of the beads before exposure to IgG and after the agglutination is complete. During the process, the mean buoyant mass increased from 26.0 fg to 30.4 fg. Assuming this increase is caused by the accumulation of IgG (molecular weight 150 kD), it would form a coating 14 nm thick containing approximately 1010 proteins per bead.
Aggregation analysis of nanoparticles can also be carried out with ARCHIMEDES. The plot at right shows buoyant mass profiles of 57 nm gold nanoparticles (NIST Standard Reference Material 80133). Different samples of these nanoparticles were left untreated, exposed to bovine serum albumin (BSA), and exposed to ammonium acetate.
The histogram shows how the dimer fraction depends on the exposure. Exposing the beads to BSA, which is often used to minimize non-specific binding, results in the least aggregation. The ammonium acetate, which affects the ionic strength of the suspension, shows the most aggregation. The plot at right shows the quantitative results for the dimer fractions, and confirms that ARCHIMEDES is capable of measuring even subtle effects of nanoparticle functionalization.
ARCHIMEDES can also analyze aggregates containing very large numbers of primary particles. An important example is the aggregation of proteins, which has significant implications in protein therapeutics. The mass of a typical protein is 100 kD (~10-19 g), about 1000 times below ARCHIMEDES' mass limit of detection. The histograms below show the distributions of IgG aggregates contained in 4 µl of native sample. Because the underlying measurements are of mass, the distribution can be cast as the aggregate order, i.e., the number of base proteins that make up each aggregate, as is shown in the right plot.
An analysis of inkjet inks shows how ARCHIMEDES can analyze both primary particles and high-order aggregates. Samples of ink were first diluted in ethyl acetate by about 1000:1 so that the suspension was nearly transparent. This allowed the "primary" pigment particles to be measured, with good agreement in size of 130 nm for four different samples (below, left).
The same samples were then measured in their native, undiluted (and totally opaque) state. In this case the primary constituents are so concentrated (> 1011/ml) that several hundred are present in the sensor at any time, and so cannot be resolved individually. ARCHIMEDES was instead configured to detect only particles >1 µm to look for aggregates caused by instabilities in the suspension that cause flocculation. The results show a significant difference in number concentration of these larger aggregates between the ink samples (above, right). Larger aggregate concentrations correlate with longer shelf life, and with ink samples that caused clogging problems with inkjet nozzles (below).
1 Mass-based readout for agglutination assays, R.Chunara, M. Godin, S. M. Knudsen, and S.R. Manalis, APPLIED PHYSICS LETTERS 91 (2007) 193902.
2 For example, Dynabeads,® manufactured by Invitrogen.