A high-sensitivity lanthanide nanoparticle reporter for mass cytometry: tests on microgels as a proxy for cells.

This paper addresses the question of whether one can use lanthanide nanoparticles (e.g., NaHoF4) to detect surface biomarkers expressed at low levels by mass cytometry. To avoid many of the complications of experiments on live or fixed cells, we carried out proof-of-concept experiments using aqueous microgels with a diameter on the order of 700 nm as a proxy for cells. These microgels were used to test whether nanoparticle (NP) reagents would allow the detection of as few as 100 proteins per "cell" in cell-by-cell assays. Streptavidin (SAv), which served as the model biomarker, was attached to the microgel in two different ways. Covalent coupling to surface carboxyls of the microgel led to large numbers (>10(4)) of proteins per microgel, whereas biotinylation of the microgel followed by exposure to SAv led to much smaller numbers of SAv per microgel. Using mass cytometry, we compared two biotin-containing reagents, which recognized and bound to the SAvs on the microgel. One was a metal chelating polymer (MCP), a biotin end-capped polyaspartamide containing 50 Tb(3+) ions per probe. The other was a biotinylated NaHoF4 NP containing 15 000 Ho atoms per probe. Nonspecific binding was determined with bovine serum albumin (BSA) conjugated microgels. The MCP was effective at detecting and quantifying SAvs on the microgel with covalently bound SAv (20 000 SAvs per microgel) but was unable to give a meaningful signal above that of the BSA-coated microgel for the samples with low levels of SAv. Here the NP reagent gave a signal 2 orders of magnitude stronger than that of the MCP and allowed detection of NPs ranging from 100 to 500 per microgel. Sensitivity was limited by the level of nonspecific adsorption. This proof of concept experiment demonstrates the enhanced sensitivity possible with NP reagents in cell-by-cell assays by mass cytometry.

Synthesis and mass cytometric analysis of lanthanide-encoded polyelectrolyte microgels.

This article describes the synthesis and characterization of two series of functional polyelectrolyte copolymer microgels intended for bioassays based upon mass cytometry, a technique that detects metals by inductively coupled plasma mass spectrometry (ICP-MS). The microgels were loaded with Eu(III) ions, which were then converted in situ to EuF(3) nanoparticles (NPs). Both types of microgels are based upon copolymers of N-isopropylacrylamide (NIPAm) and methacrylic acid (MAA), poly(NIPAm/VCL/MAA) (VCL = N-vinylcaprolactam, V series), and poly(NIPAm/MAA/PEGMA) (PEGMA = poly(ethylene glycol)methacrylate, PG series). Very specific conditions (full neutralization of the MAA groups) were required to confine the EuF(3) NPs to the core of the microgels. We used mass cytometry to measure the number and the particle-to-particle variation of Eu ions per microgel. By controlling the amount of EuCl(3) added to the neutralized microgels. we could vary the atomic content of individual microgels from ca. 10(6) to 10(7) Eu atoms, either in the form of Eu(3+) ions or EuF(3) NPs. Leaching profiles of Eu ions from the hybrid microgels were measured by traditional ICP-MS.

Metal-Containing Polystyrene Beads as Standards for Mass Cytometry.

We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.

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

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.

Lanthanide-containing polymer microspheres by multiple-stage dispersion polymerization for highly multiplexed bioassays.

We describe the synthesis and characterization of metal-encoded polystyrene microspheres by multiple-stage dispersion polymerization with diameters on the order of 2 mum and a very narrow size distribution. Different lanthanides were loaded into these microspheres through the addition of a mixture of lanthanide salts (LnCl(3)) and excess acrylic acid (AA) or acetoacetylethyl methacrylate (AAEM) dissolved in ethanol to the reaction after about 10% conversion of styrene, that is, well after the particle nucleation stage was complete. Individual microspheres contain ca. 10(6)-10(8) chelated lanthanide ions, of either a single element or a mixture of elements. These microspheres were characterized one-by-one utilizing a novel mass cytometer with an inductively coupled plasma (ICP) ionization source and time-of-flight (TOF) mass spectrometry detection. Microspheres containing a range of different metals at different levels of concentration were synthesized to meet the requirements of binary encoding and enumeration encoding protocols. With four different metals at five levels of concentration, we could achieve a variability of 624, and the strategy we report should allow one to obtain much larger variability. To demonstrate the usefulness of element-encoded beads for highly multiplexed immunoassays, we carried out a proof-of-principle model bioassay involving conjugation of mouse IgG to the surface of La and Tm containing particles and its detection by an antimouse IgG bearing a metal-chelating polymer with Pr.

A microfluidic approach to chemically driven assembly of colloidal particles at gas-liquid interfaces.

Bubbling up: Dissolution of CO(2) bubbles in a suspension of colloidal particles chemically induces the assembly of particles on the surface of shrunken bubbles, and thus yields rapid continuous formation of a colloidal armor. This approach maintains the high colloidal stability of particles in bulk, has increased productivity, and allows the formation of bubbles with precisely controlled dimensions.

Fabrication and electrochemical application of three-dimensional gold nanoparticles: self-assembly.

Multilayers film of nanostructured citrate-stabilized gold particles (AuNPs) has been fabricated based on the layer-by-layer (LBL) technique using a self-assembled monolayer of 1,4-benzenedimethanethiol (BDMT). The formation of AuNPs and BDMT self-assemblies as alternative multilayers was confirmed by transmission electron microscopy (TEM), X-ray photoelectron spectroscope (XPS), and quartz crystal microbalance (QCM). The formation of uniform AuNP layers with an average monolayer thickness of 5-6 nm was obvious in the TEM images. The existence of BDMT molecules as cross linkers for the AuNPs' layers was proved by XPS measurements. The greater affinity of AuNPs' layers to bind BDMT molecules in comparison with the bare Au bulk electrode was revealed by QCM measurements. Electrochemically, the AuNPs' layers on the electrode surface did not only catalyze the reduction of oxygen (ca. 100-mV positive shift of the reduction peak potential compared with that at the bare Au bulk electrode) but also showed a fascinating nature of working as a renewed activated-electrode surface; a zigzag response was observed for oxygen reduction during alternative immobilization of BDMT and the AuNP layer. The self-assembly of a new AuNPs layer restored the catalytic activity that was entirely blocked by the preceding BDMT layer.

Structure-Tuned Lead Halide Perovskite Nanocrystals.

Colloidally stable suspensions of lead halide perovskite nanocrystals are prepared from high-quality lead halide nanocrystal seeds. Perovskite nanocrystals with different layered crystal structures are reported. These systems are well suited for investigations of the intrinsic photophysics and spectroscopy of organic-inorganic metal halide perovskites.

Surface Functionalization Methods to Enhance Bioconjugation in Metal-Labeled Polystyrene Particles.

Lanthanide-encoded polystyrene particles synthesized by dispersion polymerization are excellent candidates for mass cytometry based immunoassays, however they have previously lacked the ability to conjugate biomolecules to the particle surface. We present here three approaches to post-functionalize these particles, enabling the covalent attachment of proteins. Our first approach used partially hydrolyzed poly(N-vinylpyrrolidone) as a dispersion polymerization stabilizer to synthesize particles with high concentration of -COOH groups on the particle surface. In an alternative strategy to provide -COOH functionality to the lanthanide-encoded particles, we employed seeded emulsion polymerization to graft poly(methacrylic acid) (PMAA) chains onto the surface of these particles. However, these two approaches gave little to no improvement in the extent of bioconjugation. In our third approach, seeded emulsion polymerization was subsequently used as a method to grow a functional polymer shell (in this case, poly(glycidyl methacrylate) (PGMA)) onto the surface of these particles, which proved highly successful. The epoxide-rich PGMA shell permitted extensive surface bioconjugation of NeutrAvidin, as probed by an Lu-labeled biotin reporter (ca. 7 × 10(5) binding events per particle with a very low amount of non-specific binding) and analyzed by mass cytometry. It was shown that coupling agents such as EDC were not needed, such was the reactivity of the particle surface. These particles were stable and the addition of a polymeric shell was shown did not affect the narrow lanthanide ion distribution within the particle interior as analyzed by mass cytometry. These particles represent the most promising candidates for the development of a highly multiplexed bioassay based on lanthanide-labeled particles to date.