1) Functional Metal-Chelating Polymers for Mass Cytometry Bioassays

The focus of this project is the synthesis, characterization, and application of metal-chelating polymers intended for mass cytometric bioassays. Polymers were synthesized from an inexpensive poly(tert-butyl acrylate) backbone using RAFT (reversible addition-fragmentation chain transfer) polymerization. At this stage, thiol end-functionality was converted to a protected disulfide through aminolysis and air oxidation. Subsequent polymer modifications yielded polymers with a DTPA (diethylenetriaminepentaacetic acid) ligand on every repeat unit, Mw/Mn ≤ 1.2, and a maleimide as an orthogonal functional group for conjugation to antibodies. Thermogravimetric analysis, ITC (isothermal titration calorimetry), and conventional ICP-MS analysis were performed to show that a polymer-antibody conjugate, comprised of the polymer and GAM (goat anti-mouse) antibody, contained an average of 2.4 ▒ 0.3 polymer chains per antibody. Eleven monoclonal primary antibodies were labeled with different lanthanide isotopes using the same labeling methodology. Single cell analysis of whole umbilical cord blood stained with a mixture of 11 metal-tagged antibodies was performed by mass cytometry. Future goals of this project include the following: (A) Increased multiplexity through the investigation of different metals such as palladium and platinum, as well as new ligand systems to fully sequester these metals. (B) Dual-purpose polymer tags with both fluorescent dyes and metal-chelating groups for use in both FACS (fluorescence-activated cell sorting) and mass cytometry. (C) Polymer-antibody conjugation chemistries alternative to the Michael addition method currently in use. This method involves partial reduction of the hinge region disulfide groups of an antibody, which can deactivate some antibodies. Alternative chemistries, such as those that utilize lysine side-chain amino groups, are desirable.

2) Lanthanide Encoded Polymer Microspheres for Biological Assays

The objective of this project is the development of an advanced element (metal) encoding system and methodology for functionalized microspheres, which in combination with an elemental detector will provide researchers and clinicians with massively multiplexed analytical capabilities. The focus is on the development of a large variety of element-tagged functionalized polymeric beads employing a novel encoding system which can be used in two assay formats: individual (cytometric) bead analysis at high throughput (employing Flow Cytometer-Mass Spectrometer (mass cytometry)), and homogeneous assays (using Inductively Coupled Plasma Mass Spectrometer (ICP-MS)). Our specific aims include 1) synthesis of polystyrene microspheres uniquely imbibed with elements; 2) appropriate surface functionalization of the encoded bead for attachment of affinity reagents (antibodies, oligonucleotides, peptides); 3) development of bio-analytical methods using these reagents in a multiplex format and validation against existing fluorochrome based technologies. Microspheres are an attractive option for supporting surface chemistries for immunoassays and oligonucleotide hybridization assays. One of the advantages of microspheres is the ability to increase the reaction surface area per volume of the reaction mixture, which provides a reliable means of increasing the capacity and dynamic range potential of an assay, as well as miniaturizing the reaction. We expect that our team will be able to produce many thousands of distinguishable analytical beads by the incorporation of various concentrations and ratios of metals. We hope that this integrated massively multiplex reagent support/encoding system will simplify and enhance the diagnostic, prognostic and therapeutic efficacy available to physicians and their patients, increasing the effectiveness of healthcare while substantially reducing the human and financial costs of modern personalized treatment.

3) Lanthanide Nanoparticles for Higher Sensitivity Mass Cyometry Immunoassays

The overall goal of this project is to develop a new generation of reagents for mass cytometry immunoassays that can detect antigen present at low copy number on cell surfaces or within cells. A meaningful target is 100 or fewer copies per cell. At its current state of development, mass cytometry detects 1 ion for every 104 ions created in the plasma, and the ion cloud from a single cell is sampled ca. 20 times in 250 Ás. While the background signal is very low (1 to 2 counts per s), so that 1 count per cell is statistically significant, we target 5 counts per cell as lowest level of abundance for quantitative data. Thus we require reagents that carry at least 5000 atoms of a given isotope, so that staining low copy number biomarkers with these reagents will lead to a minimum of 50,000 copies of a given isotope per cell. Our hypothesis is that we can achieve the necessary level of labeling by attaching to a monoclonal antibody a nanoparticle of the appropriate diameter. The characteristic size of a monoclonal antibody is comparable to the diameter of a 10-nm nanoparticle (for IgG dh=9.7▒0.1 nm). Rare earth ions offer several advantages as element labels: low natural abundance, similar chemistry, and the availability of a variety of elements and multiple isotopes available as isotopically enriched samples. For mass cytometry applications, two key requirements are colloidal dispersability in water and a narrow size distribution, so that antibodies and other bioaffinity reagents carry similar numbers of Ln ions. In this section we consider reactions that can provide spherical or nearly spherical polyhedral nanoparticles with a narrow size distribution. For those reactions that lead to hydrophobic surface ligands, we will propose strategies for transferring the nanoparticles into water.