Bio-Functional, Lanthanide-Labeled Polymer Particles by Seeded Emulsion Polymerization and their Characterization by Novel ICP-MS Detection

Abstract

We present the synthesis and characterization of monodisperse, sub-micron poly(styrene) (PS) particles loaded with up to and including 10(7) lanthanide (Ln) ions per particle. These particles have been synthesized by seeded emulsion polymerization with a mixture of monomer and a pre-formed Ln complex, and analyzed on a particle-by-particle basis by a unique inductively coupled plasma mass cytometer. Seed particles were prepared by surfactant-free emulsion polymerization (SFEP) to obtain large particle sizes in aqueous media. Extensive surface acid functionality was introduced using the acid-functional initiator ACVA, either during seed latex synthesis or in the second stage of polymerization. The loading of particles with three different Ln ions (Eu, Tb, and Ho) has proven to be close to 100 % efficient on an individual and combined basis. Covalent attachment of metal-tagged peptides and proteins such as Neutravidin to the particle surface was shown to be successful and the number of bound species can be readily determined. We believe these particles can serve as precursors for multiplexed, bead-based bio-assays utilizing mass cytometric detection.

Fig. 1

SEM image of seed latex st040.

Fig. 2

SEM image analysis of the seeded growth of st040 by Method 1 polymerization (KPS as initiator) for the attempted loading of Eu at 10⁶ ions per particle. Shown are (A) – polymerization in the absence of Me-β-CD and (B) – polymerization in the presence of Me-β-CD.

Fig. 3

Number PSD of sample ST124 (seeded emulsion polymerization of st109 via Method 2 to load Eu at a target of 1.17 × 10⁷ ions per particle. Shown in the PSD histogram is the original seed PSD (light grey) as well as the PSD after seeded growth (dark grey) prior to further centrifugation and washing. Note the population due to secondary nucleation at approximately 200 nm diameter. These are washed away during centrifugation.

Fig. 4

Amount of secondary nucleation (expressed as Φ_NEW, the volume fraction of new particles from the volume PSD for each sample) plotted as a function of theoretical Ln loading per particle. Shown are seeded emulsion polymerization systems initiated by persulfate (black squares, ‘Method 1’) and by ACVA (open circles, ‘Method 2’).

Fig. 5

Eu isotopic dot-dot diagram for sample ST162 (particles loaded with Eu with targeted value of 1.03 × 10⁷ per particle) as measured by mass cytometry. Two main peaks (circled) and a long tail are evident.

Fig. 6

Lanthanide content distribution (LnCD) for ¹⁵³Eu from sample ST162 (obtained from Figure 5). The two peaks shown here correspond to the two populations observed by mass cytometry. Note than the number of ions per particle is related to intensity by a factor of 2300.

Fig. 7

Lanthanide loading via seeded emulsion polymerization: comparison of theoretically predicted loading values with experimentally determined averages from mass cytometry. (A): Ln loading using ‘Method 1.’ (B): Ln loading using ‘Method 2.’ The dark colours correspond to the predicted value for each sample. Unless otherwise indicated, the Ln complex used is Eu(TNB)₃. Experimental average Ln values are obtained from the first peak in each LnCD from mass cytometry.

Fig. 8

LnCDs for A)¹⁶⁹Tm and B) ¹⁵¹Eu for the conjugation of labeled Neutravidin to acid-stabilized particles ST073. Key: Dashed line – commercially available PS-COOH particles; Solid line – ST073 labeled with Neutravidin with reduced X4 with 40 fold excess of SMCC.

Fig. 9

Mass cytometry analysis used for a model sandwich assay involving ST073 particles labeled with Tm-tagged Neutravidin, treated with biotin-labeled mouse IgG, in turn recognized with Tb-tagged anti-mouse IgG. The LnCD of the all three detected elements are shown.

Scheme 1

Cartoon schematic of the mass cytometer instrument. Metal-labeled particles are introduced into the ICP torch on an individual basis, whereby they are burned and the atomic composition of each particle is determined by TOF-MS. Mass spectra are recorded on the order of every 20 μs. The size of the ion cloud corresponding to each particle as it passes through the TOF chamber allows for 20 to 30 mass spectra to be collected per particle.

Scheme 2

Schematic of particle metal-labeled sandwich immunoassay for mass cytometry analysis.