PMMA Microspheres with Embedded Lanthanide Nanoparticles by

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PMMA Microspheres with Embedded Lanthanide Nanoparticles by Photoinitiated Dispersion Polymerization with a Carboxy-Functional Macro-RAFT Agent Jianbo Tan,†,‡ Guangyao Zhao,† Zhaohua Zeng,*,‡ and Mitchell A. Winnik*,† †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto M5S 3H6, Ontario Canada Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, and Key Laboratory of Designed Synthesis and Application of Polymer Material, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China

‡

S Supporting Information *

ABSTRACT: Functional poly(methyl methacrylate) (PMMA) microbeads with a very narrow size distribution were synthesized by photoinitiated RAFT dispersion polymerization in aqueous ethanol using an acrylic acid−oligo(ethylene glycol) copolymer as a macro-RAFT agent. These particles are a prototype for multiparameter bead-based assays employing mass cytometry, a technique in which metalencoded beads are injected into the plasma torch of an inductively coupled plasma mass spectrometer (ICP-MS), and the metal ions generated are detected by time-of-flight mass spectrometry. To label the beads, the polymerization reaction was carried out in the presence of various types of small (ca. 5 nm) lanthanide fluoride (LnF3) nanoparticles (e.g., LaF3, CeF3, and TbF3) with polymerizable methacrylate groups on their surface. The type of metal ion and the metal content of the PMMA microbeads could be varied by changing the composition of the reaction medium. An important feature of these microbeads is that acrylic acid groups in the corona are available for covalent attachment of biomolecules. As a proof of concept, FITC−streptavidin (FITC-SAv) was covalently coupled to the surface of a Ln-encoded microbead sample. The number of FITC-SAv binding sites on the beads was determined through three parallel assays involving biotin derivatives. Interaction of the beads with a biotin−tetramethylrhodamine derivative was monitored by fluorescence, whereas interaction of the beads with a biotin-DOTA-Lu derivative was monitored both by ICP-MS and by mass cytometry. Each measurement detected an average of ca. 5 × 104 biotins per microsphere. Control experiments with beads covalently labeled with FITC−bovine serum albumin (FITC-BSA) showed only very low levels of nonspecific binding.

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of simultaneous detection is possible than with fluorescence detection. The lanthanide (Ln) elements are particularly interesting as encoding species. They have low natural abundance and thus low background signals. Meanwhile, they have similar chemistry, and there are in principle 54 natural nonradioactive isotopes available for multiplexed bioassays. To be used for mass cytometry-based bioassays, polymer microspheres encoded with lanthanide ions need to meet three requirements. First, the microspheres should have appropriate diameters within the range of 0.8−5 μm and with a very narrow size distribution. Smaller microspheres do not contain sufficient lanthanide ions for mass cytometry measurements, and larger microspheres may not pyrolyze completely in the ICP torch. Second, the microspheres should carry a sufficient and controlled number of metal ions to generate a strong mass cytometry signal. If the number of metal ions/microbead is to

INTRODUCTION Bead-based bioassays have been used in various bioanalytical fields, e.g., antibody profiling, blood pathogen detection, early cancer diagnostics, and characterizing immune responses to vaccination. Conventional bead-based flow cytometric assays employ fluorescent dye-encoded polymer microbeads.1 While this approach is powerful, the spectral overlap of fluorescent dyes limits the number of parameters that one can monitor. We are interested in multiplexed bead-based assays based on the relatively new technique of mass cytometry.2−5 In mass cytometry, cells or beads are injected individually but stochastically into the plasma torch of an inductively coupled plasma mass spectrometer (ICP-MS) equipped with time-offlight (TOF) detection.6,7 In the plasma at 7000 K, the beads are vaporized, atomized, and ionized to form an ion cloud for which 20−30 TOF mass spectra are taken. ICP-MS is a very powerful method for the quantitative detection of metals. It enables the use of metal atoms or individual isotopes as the encoding species. For instruments that are tuned to detect masses in the m/z range of 80−200, a much higher multiplicity © 2015 American Chemical Society

Received: April 2, 2015 Revised: May 8, 2015 Published: May 22, 2015 3629

DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640

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known to be effective at suppressing nonspecific protein adsorption and likely hinders protein conjugation to the carboxyl groups under the corona at the particle surface. Another promising approach to prepare uniform functional polymer particles with micrometer dimensions is photoinitiated RAFT dispersion polymerization of methyl methacrylate (MMA) in ethanol−water mixtures.12−14 In this reaction, the presence of the RAFT chain transfer agent is essential for obtaining particles uniform in diameter and appears to operate by modulating the rate of both the nucleation and growth stages of particle formation.12 This reaction can also be run using an acrylic acid (AA) copolymer macro-RAFT agent. In this way, one can obtain PMMA particles with a mean diameter d ≈ 1 μm with a narrow size distribution and with carboxylic acid groups in the corona surrounding the PMMA particle.13 There is a broad literature describing the synthesis via RAFTmediated dispersion polymerization, using macro-RAFT agents as the stabilizer, of colloidal polymer nanomaterials with different morphologies (e.g., worm-like and vesicle-like).15−28 Because of poor control of particle size distribution, however, the synthesis of uniform microspheres by RAFT dispersion polymerization is rare. Ki et al.29 reported a photo-RAFT dispersion polymerization of styrene at 70 °C using azodiisobutyronitrile (AIBN) as the initiator and poly(4vinylpyridine) as the macro-RAFT agent. They found a low particle yield (56%) after 12 h reaction, and only polydisperse PS particles were formed, with diameters (d) ranging from 130 to 310 nm. Wi et al.30 reported a similar RAFT dispersion polymerization of styrene by using poly(methacrylic acid) as the macro-RAFT agent and obtained PS particles with a broad particle size distribution. McKee et al.31 reported the dispersion polymerization of styrene in methanol with a poly(Nisopropylacrylamide)-based macro-RAFT agent as the stabilizer. Polystyrene particles with a bimodal size distribution were obtained. This project began with the hypothesis that −COOH groups in the corona of polymer microbeads will be much more accessible to biofunctionalization than −COOH groups on the particle surface buried under a PVP corona as in the particles synthesized by Abdelrahman et al.2 Thus, we synthesized a copolymer macro-RAFT agent containing pendant −COOH groups and examined whether it could be used for the synthesis of lanthanide-containing microbeads by dispersion polymerization of MMA in an ethanol−water mixture. Initial experiments were carried out in the batch mode by adding small amounts of AA and TbCl3 to the reaction medium, and this led to particles with a broad size distribution. Then we separated the reaction into two stages and added the AA and TbCl3 after the end of the nucleation stage. This approach led to very uniform particles but with no detectable Tb3+ ion content as measured by mass cytometry. As a last resort, we attempted to incorporate Ln ions in the form of LnF3 nanoparticles (NPs) prepared to contain polymerizable methacrylate groups in their surface ligands. Several years ago we showed that we could incorporate small LaF3 NPs (d ≈ 3−4 nm) with polymerizable groups into thermosensitive aqueous poly(N-isopropylacrylamide-co-N-vinylcaprolactam-co-methacrylic acid) nanogels.32 Since these nanogel particles were prepared by a type of precipitation polymerization, we had hopes that this approach might work for photoinitiated RAFT dispersion polymerization of MMA in ethanol−water.

be used as a variable to distinguish different classes of beads, then this requirement in conjunction with that for a narrow size distribution is necessary to keep the bead-to-bead metal ion content as similar as possible. Third, these microspheres must have functional groups on the surface for covalent attachment of biomolecules such as proteins or oligonucleotides. Previous experiments from our group have generated polymer microbeads that satisfied the first two requirements but suffered shortcomings in our ability to attach antibodies or other proteins to the particle surface.2 Dispersion polymerization is an obvious approach for synthesizing these microspheres. Under optimum conditions, such as the dispersion polymerization of styrene in ethanol, particles with diameters on the order of 1−5 μm and with an exceptionally narrow size distribution can be obtained.8,9 In a dispersion polymerization reaction, all the constituents are soluble in the polymerization medium at the start of the reactant, but monomer conversion leads to precipitation of the polymer formed. These reactions are carried out in the presence of a polymeric stabilizer such as poly(N-vinylpyrrolidone) (PVP). Graft copolymer formed in the reaction provides colloidal stability to the precipitated polymer, and at the end of the nucleation stage, the number of polymer particles is fixed. As the polymerization continues, these nuclei grow in parallel to form polymer particles with a narrow size distribution. For most dispersion polymerization reactions, particularly that of styrene in ethanol in the presence of PVP, the nucleation stage is particularly sensitive to the experimental conditions. The presence of functional comonomers, chain transfer agents, or cross-linking agents can interfere with and change the course of the reaction, often leading to particles with a very broad range of particle sizes. Many of these problems can be overcome if one delays the addition of these “problematic” comonomers until the nucleation stage is complete. We have referred to this reaction protocol as “twostage” dispersion polymerization and showed that in this way one can obtain cross-linked or functional PVP-stabilized polystyrene (PS) microspheres with the same narrow distribution of particle diameters as the reactions carried out in the absence of these problematic reagents.9−11 In order to synthesize lanthanide-containing PS microspheres with a narrow size distribution and uniform metal ion content, Abdelrahman et al.2 developed a multiple-stage dispersion polymerization protocol. In the first stage, they carried out dispersion polymerization of styrene in ethanol in the presence of PVP. After 30 min, well after the particle nucleation step was complete, they added acrylic acid (AA) and lanthanide chloride (LnCl3·6H2O) salts to the reaction mixture. Good results were obtained in this way. Even better results were obtained if they added the lanthanide salts in a third stage after the acrylic acid addition step was complete. With these protocols, they obtained a series of lanthanide-containing polystyrene (PS) microspheres with a very narrow size distribution and with control over the number and type of Ln ions incorporated into the particles. By titration, the microspheres were shown to contain a large number of surface carboxyl groups, which the authors imagined would be useful for covalently attaching antibodies to the particle surface. Unfortunately, typical coupling reactions were not effective, and only limited numbers of biomacromolecules could be coupled to the particle surface. They ascribed the difficulty to attach proteins to the surface of the PS microspheres to the presence of the PVP corona. PVP is 3630

DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640

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vinylpyrrolidone). In this case, silica sol coated with PVP acted as the stabilizer, and the incorporation efficiency of silica could reach 100%. The particle size could be varied from 200 to 1000 nm by varying the reaction conditions, e.g., monomer concentrations or stabilizer concentrations. Another general approach employs a macro-RAFT agent as an amphiphilic stabilizer for emulsion polymerization and leads to polymer particles with diameters on the order of 100 nm.40−42 For example, Nguyen et al.40 showed that a random copolymer based on acrylic acid (AA) and butyl acrylate (BA) synthesized by RAFT polymerization was able to interact with the surface of TiO2 NPs through the carboxylic acid groups, forming stabilized NP dispersions. By slowly feeding a mixture of hydrophobic monomers such as MMA and BA into this dispersion in the presence of a free radical initiator, hybrid particles formed that encapsulated the TiO2 NPs. In another example, Guimarães et al.41 reported emulsion polymerization of styrene or MMA by using a poly(ethylene oxide)-based trithiocarbonate macro-RAFT agent in the presence of laponite clay platelets. They found that the macro-RAFT agent had affinity for adsorption by the laponite, and the anchored RAFT groups were used to control the reaction leading, to in situ formation of amphiphilic block copolymers. Based on this method, multihollow laponite-armored polystyrene and P(MMA-co-BA) particles were prepared. Zgheib et al.42 provide a more detailed review of this approach and describe their attempts to encapsulate CeO2 (ceria) NPs into P(BA-co-MMA) particles using a macro-RAFT agent consisting of an oligo AABA copolymer. Several groups have examined more traditional emulsion polymerization using inorganic NPs with reactive or polymerizable functionality at the surface.43,44 For example, Zhang et al.43 used functional silica NPs as seeds for emulsion polymerization of styrene and obtained core−shell particles in which the thickness of the PS shell could be varied by adding different amounts of styrene in the reaction. Parole et al.44 reported emulsion polymerization of both MMA and styrene using a cationic initiator anchored on the surface of silica NPs. In this way they obtained anisotropic polymer particles (e.g., dissymmetric snowman- and dumbbell-like shapes). Returning to dispersion polymerization, we cite an example by Horák and Benedyk,45 who report the preparation of magnetic poly(glycidyl methacrylate) (PGMA) microspheres by dispersion polymerization in ethanol with the addition of an electrostatically stabilized ferrofluid. The Fe3O4 NPs formed the core of a core−shell structure, in which PGMA formed the shell. However, the particle size distribution was relatively broad, and the reaction was very sensitive to reaction conditions. In the following sections, we describe our results on the photoinduced RAFT dispersion polymerization of MMA in ethanol−water in the presence of various lanthanide NPs in which the NPs carry methacrylate groups at the surface. We show that reactions carried out in a batch process lead to polymer microbeads with a broad size distribution but that reactions carried out by a two-stage protocol lead to monodisperse polymer microbeads. These lanthanide-encoded particles are suitable for mass cytometry measurements as well as for the covalent attachment of bioaffinity agents. We end the paper with a proof-of-concept assay experiment involving attachment of streptavidin to the particles.

There are many reports describing the preparation of hybrid polymer latex incorporating inorganic NPs. For almost all of these examples, the polymer latex particles have diameters in the range of 100−500 nm. There are only rare examples of NP incorporation into uniform polymer microparticles. To put our work in context, we provide a short review of this literature. A common approach to the synthesis of hybrid polymer nanoparticles employs miniemulsion polymerization.33−35 Miniemulsions are typically surfactant-stabilized submicron dispersions of monomer droplets that contain an organic solute that acts to suppress Ostwald ripening. In miniemulsion polymerization, each monomer droplet can act like a tiny bulk reactor. NPs with a hydrophobic surface coating can be dispersed into the hydrophobic monomer phase with the intention that they remain in that phase as the monomer is polymerized. For example, Bechthold et al.33 reported the encapsulation of CaCO3 and carbon black NP pigments into PS latexes by miniemulsion polymerization. In the case of CaCO3, the nanoparticles were coated with stearic acid prior to dispersing the pigment into the styrene phase. In the case of carbon black, the choice of hydrophobe was crucial for the full encapsulation of carbon black into polystyrene latexes, and under optimum conditions, up to 8 wt % carbon black could be incorporated. Erdem et al.34 reported the encapsulation of both hydrophilic and hydrophobic titanium dioxide (TiO2) NPs into PS particles by miniemulsion polymerization. More recently, Fleischhaker et al.35 reported the preparation of PS/PMMA core−shell particles with CdS/ZnS/CdSe quantum dots (QDs) integrated into the core. For the synthesis of the core, they dispersed the oleate-coated CdS/ZnS/CdSe QDs in a styrene/ hexadecane miniemulsion prepared in the presence of SDS surfactant. The potassium persulfate initiated polymerization was carried out at 70 °C. These PS particles were then used as seeds for emulsion polymerization in which MMA was added slowly in the second stage, and in this way, particles with different shell thicknesses were obtained. Inorganic NPs, typically silica NPs, can serve as a (Pickering) stabilizer for emulsion droplets. When Pickering-stabilized monomer droplets are polymerized, one obtains polymer particles coated with a layer of the stabilizer NPs. For example, Tiarks et al.36 reported surfactant-free miniemulsion polymerization of styrene, MMA, and butyl acrylate (BA) by using 4vinylpridine-coated silica NPs as a Pickering stabilizer and examined the different hybrid morphologies that were obtained. They also examined the influence of adding different types and different amounts of surfactants into the reaction and found that the use of cationic surfactants led to an improvement in the incorporation of silica nanoparticles. Colver et al.37 reported the soap-free emulsion polymerization of MMA in the presence of 25 nm diameter silica NPs. These NPs adhered to the surface of the polymer colloids, and thus the polymer latexes were armored with a layer of NPs. In an application of this concept to dispersion polymerization, Schmid et al.38 report the synthesis of polystyrene− silica nanocomposite particles in methanol or in 2-propanol using commercial alcoholic silica sols (13−22 nm) as the stabilizing agent. The silica NPs covered the particle surfaces, and the nanocomposite particles exhibited a core−shell structure with diameters ranging from 300 to 1400 nm. In an analogous example, Zou and Armes39 synthesized colloidally stable poly(2-hydroxypropyl methacrylate)−silica nanocomposite particles by aqueous dispersion polymerization at 60 °C with the addition of ultrafine aqueous silica sol and poly(N3631

DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640

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Aldrich), sodium fluoride (NaF, Aldrich), and 2,2-dimethyl-2-phenylacetophenone (Darocur 1173, Aldrich) were used without further purification. Methyl methacrylate (MMA, Aldrich) was passed through a column of basic alumina (Aldrich) prior to storage under refrigeration at 4 °C. Bovine serum albumin (fluorescein conjugate (FITC-BSA)), streptavidin (fluorescein conjugate (FITC-SAv)), and 5-(and 6)-tetramethylrhodamine biocytin (biotin-TMR) were purchased from Life Technologies Inc. DOTA-biotin-sarcosine (C-100) (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was purchased from Macrocyclics Inc. Water was purified through a Milli-Q purification system. High purity nitrate acid was purchased from Seastar Chemical Inc. The synthesis of the macro-RAFT agent [poly(oligo(ethylene glycol) methyl ether acrylate-co-acrylic acid) trithiocarbonate (P(OEGA-co-AA)-TTC)] is described in the Supporting Information. Synthesis of Lanthanide Fluoride Nanoparticles (LnF3 NPs). To synthesize LnF3 NPs stabilized with both AEP and EGMAP, a solution of the ligands AEP (0.102 g, 0.746 mmol) and EGMAP (0.076 g, 0.36 mmol) (AEP/EGMAP = 3:1 mol/mol) in water (10 mL) was neutralized to pH 6.5 with 1.5 M aqueous NH3. Then a lanthanide salt Ln(NO3)3·6H2O or a mixture of salts (1.33 mmol) in water (10 mL) was added to the solution to form the Ln-AEP/ EGMAP complex at room temperature and stirred at 1000 rpm for 10 min. Then NaF (0.13 g, 3.00 mmol) in water (13 mL) was added dropwise at 0.2 mL/min via a feed pump. After 16 h stirring at 1000 rpm, the NPs in the clear solution were purified by dialysis at room temperature for 2−3 days using a dialysis bag (1 kDa Spectra/Pro 7 dialysis membranes). The concentrations based on the solids content of these NP solutions were LaF3 (3.7 mg/mL), TbF3 (5.3 mg/mL), CeF3 (5.2 mg/mL), PrF3 (4.3 mg/mL), and LaF3:Ce,Tb (5.2 mg/ mL). The synthesis of unreactive LaF3 NPs without double bonds on the surface was carried out in the same way but without the addition of EGMAP. The solids content of LaF3 capped with AEP was 6.1 mg/ mL. Two-Stage Photoinitiated RAFT Dispersion Polymerization of MMA in the Presence of TbCl3·6H2O and Acrylic Acid. A mixture of the monomer MMA (2.0 g, 10 wt % of the whole mixture), the macro-RAFT agent P(OEGA-co-AA)-TTC (0.3 g, 15 wt % relative to MMA), the RAFT agent DDMAT (5 mg, 0.25 wt % relative to MMA), and the photoinitiator Darocur 1173 (60 mg, 3 wt % relative to MMA) was dissolved in an ethanol/water mixture (7.2 g/10.8 g) to form a homogeneous solution. The mixture was gently purged with nitrogen for 15 min and then sealed. A LED UV lamp (λ = 365 nm, light intensity 16 mW/cm2) was employed to irradiate the reaction mixture from the top of the reaction cell. The reaction mixture turned turbid after 30 s of UV irradiation. After 15 min of irradiation, a degassed solution containing 1.0 g of MMA, 40 mg of AA, 30 mg of photoinitiator, 20 mg of TbCl3·6H2O, and 3.6 g/5.4 g of ethanol− water was added into the reaction. The reaction was further irradiated for another 45 min. These details are summarized in Table 1. The product was precipitated by centrifugation and washed three times with ethanol/water (40/60, w/w) mixtures. An aliquot of the dispersion was diluted with distilled water and then drop cast on a mica film for SEM measurements. One-Stage (Batch) Dispersion Polymerization of MMA in the Presence of LnF3 Nanoparticles. The recipe for this reaction is given in Table 1. A mixture in ethanol/water was prepared consisting of the monomer MMA, the macro-RAFT agent, the RAFT agent DDMAT, the photoinitiator Darocur 1173, and the LaF3 NP solution. It was gently purged with nitrogen for 15 min and sealed. After 1 h of UV irradiation, a stable dispersion was formed without any observable coagulum. The product was precipitated by centrifugation and washed three times with ethanol/water (40/60, w/w) mixtures. An aliquot of the dispersion was diluted with distilled water and then dropped on a mica film for SEM measurements. Two-Stage Dispersion Polymerization of MMA in the Presence of LnF3 Nanoparticles. The recipe for this reaction is given in Table 1. A mixture of the monomer MMA, the macro-RAFT agent, the RAFT agent DDMAT, and the photoinitiator Darocur 1173 was dissolved in the ethanol/water mixture, gently purged with

EXPERIMENTAL SECTION

Instrumentation. A Hitachi S-5200 field emission scanning electron microscope was used at operating voltage of 1 kV to measure particle sizes and size polydispersities. The particles were diluted with distilled water and a drop was placed on a mica film. Images were analyzed by ImageJ software (National Institutes of Health). The values of the mean diameter d and CVd are defined by the equations n

d=

∑ nidi/n

(1)

i=1 n

CVd =

∑i = 1 (di − d)2 n−1

/d

(2)

Transmission electron microscope (TEM) images were obtained with a Hitachi H-7000 TEM at an accelerating voltage of 100 kV. Microspheres dispersed in ethanol−water mixtures were directly dropcast onto hydrophobic Formvar-carbon-coated copper TEM grids. For ion release and some biotin binding experiments, the content of lanthanide ions in the supernatant was measured with an ELAN 9000 inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer SCIEX). Details are provided in the Supporting Information. Confocal fluorescence microscopy measurements were performed on a Leica TCS SP2 microscope. The fluorescein chromophore of FITC−streptavidin was excited at 488 nm and examined with the green channel (490−540 nm) of the instrument. Fluorescence measurements were carried out with a SPEX Fluorolog-3 spectrometer (Jobin Yvon/SPEX, Edison, NJ). Tetramethylrhodamine (TMR) derivatives were excited at λex = 530 nm and monitored at λem = 580 nm. The molecular weight and the polydispersity of the macro-RAFT agent were measured with a Viscotek size-exclusion chromatograph (SEC) equipped with a Viscotek VE 3580 refractive index detector and Viscotek ViscoGEL G4000PWXL and G2500PWXL columns (kept at 30 °C). The flow rate was maintained at 1.0 mL/min using a Viscotek VE 1122 solvent delivery system and VE7510 degasser. An eluent of 0.2 mol/L KNO3, 200 ppm of NaN3, and 25 mmol/L, pH = 8.5, phosphate buffer was used. The system was calibrated with poly(methacrylic acid) standards. 1 H NMR spectra were recorded in CDCl3 or D2O using a Varian VNMRS 400 or a Bruker Avance III 400 MHz NMR spectrometer at a temperature of 25 °C. The bead-by-bead lanthanide ion content of each PMMA microparticle was measured by mass cytometry, using a CyTOF instrument from DVS Science Inc. (now Fluidigm Canada, Markham, ON). The instrument employs dual counting, a combination of digital counting and analogue modes of ion detection, allowing simultaneous detection of very small and very large signals. The transmission efficiency of mass cytometry is on the order of 10−4, which means that about 1 in every 104 ions generated in the plasma reaches the detector. The mean number of metal ions per particle can be calculated from the intensity value by the mass cytometry detector according to the equation N = I/T, where I is the mean intensity measured by the mass cytometry detector and T is the transmission coefficient of the detector (calculated from the turning solution). Materials. Ethylene glycol methacrylate phosphate (EGMAP) was kindly donated by Esstech, Inc., and used without further purification. Absolute ethanol, S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DDMAT, Aldrich), 4,4′-azobis(4-cyanovaleric acid) (ACVA, Aldrich), N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC, Aldrich), N-hydroxysuccinimide (NHS, Aldrich), oligo(ethylene glycol) methyl ether acrylate (OEGA, Mn = 475 g/mol, Aldrich), acrylic acid (AA, Aldrich), 2-aminoethyl dihydrogen phosphate (AEP, Aldrich), lanthanum(III) nitrate hexahydrate (La(NO3)3·6H2O, Aldrich), terbium(III) nitrate hexahydrate (Tb(NO3)3·6H2O, Aldrich), cerium(III) nitrate hexahydrate (Ce(NO3)3·6H2O, Aldrich), praseodymium(III) nitrate hexahydrate (Pr(NO3)3·6H2O, Aldrich), terbium(III) chloride hexahydrate (TbCl3· 6H2O, Aldrich), lutetium(III) chloride hexahydrate (LuCl3·6H2O, 3632

DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640

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smaller amount (60 μL, solids content 10 wt %) of the same PMMA microsphere dispersion was treated as described above. The carboxyl groups were activated with a solution of EDC and NHS in MES buffer (200 μL, containing 16 mg of EDC and 44 mg of NHS) as described above, washed by sedimentation−redispersion and redispersed in 100 μL of PBS buffer, which contained 60 μL of FITC-SAv (2 mg/mL in PBS), and incubated at room temperature overnight with gentle vortexing. The samples were washed by sedimentation−redispersion, redispersed in 300 μL of PBS, and then treated with an aqueous solution of Lu-DOTA-biotin (2 μL, 2.4 mM) for 90 min under gentle vortexing. The sample was sedimented at 8000g. The supernatant was collected and examined by ICP-MS after 50× dilution. Prior to analysis of this sample by mass cytometry, the microspheres were washed twice with 500 μL of PBS and finally redispersed in 500 μL of PBS. As a negative control, a sample of FITC-BSA-modified microspheres was treated with the same amount of Lu-DOTA-biotin.

Table 1. Recipes for the Photoinitiated RAFT Dispersion Polymerization of MMA in the Presence of Lanthanide Salts or LnF3 Nanoparticles in an Ethanol/Water Mixture materials MMA AA Darocur 1173 DDMAT P(OEGA-coAA)-TTC ethanol/water LnF3 NP solution TbCl3·6H2O

1st stagea,b 2.0 g 60 mg 5 mg 300 mg 7.2 g/ 10.8 g

2nd stagea 1.0 g 40 mg 30 mg

3.6 g/ 5.4 g

batchc

1st staged

2nd staged

2.0 g

2.0 g

1.5 g

60 mg 5 mg 300 mg

60 mg 5 mg 300 mg

60 mg

7.2 g/ 9.8 g 1.0 g

7.2 g/ 10.8 g

5.4 g/ 7.1 g 1.0 ge

■

20 mg

RESULTS AND DISCUSSION In our experimental design, PMMA microbeads would be synthesized by photoinitiated RAFT dispersion polymerization, using a functional macro-RAFT agent as the polymeric stabilizer. For this purpose, we synthesized a copolymer of oligoethylene glycol monomethyl ether acrylate (OEGA, Mn = 475 g/mol) and acrylic acid (AA) by RAFT polymerization. Details are provided in the Supporting Information. The role of the OEGA component was to provide a barrier to minimize nonspecific interaction with proteins, whereas the AA groups provide −COOH functionality for covalent attachment of proteins. The structure of the polymer synthesized is shown in Scheme 1. Its 1H NMR spectrum is presented in Figure S1, and

Two-stage synthesis using AA + TbCl3·6H2O in the second stage. bA one-stage (batch) reaction also containing AA (40 mg) and TbCl3 (20 mg) led to a polydisperse mixture of polymer particles (results not shown). cOne-stage (batch) reaction using 1.0 g of LaF3 NP solution (3.7 mg/mL). dTwo-stage synthesis adding 1.0 g of LnF3 NP solution in the second stage along with additional monomer and photoinitiator. e LaF3 NP solution (3.7 mg/mL), TbF3 NP solution (5.3 mg/mL), CeF3 NP solution (5.2 mg/mL), LaF3:Ce,Tb NP solution (5.2 mg/ mL). a

nitrogen for 15 min, and then irradiated for 45 min. A stable dispersion was formed without any observable coagulum. Then a degassed solution in ethanol/water containing MMA, the photoinitiator Darocur 1173, and the LnF3 nanoparticle solution was added to the reaction mixture. The UV lamp was turned on, and the mixture was irradiated for another 45 min. The product was precipitated by centrifugation and washed three times with an ethanol/water (40/60, w/w) mixture. Preparation of FITC-SAv-Coated PMMA Microspheres and Determination of Biotin Binding Capacity by Fluorescence Titration. An aliquot of a carboxyl-functional PMMA microsphere dispersion in water (160 μL, solids content 10 wt %, prepared by the two-stage method with LaF3 NPs as described above) was washed twice with 500 μL of MES buffer (pH 5.5, 100 mM) in a 2.0 mL Eppendorf tube with a slanted bottom, and the microspheres were redispersed in 400 μL of MES buffer. A solution of EDC and NHS in MES buffer (400 μL, containing 32 mg of EDC and 88 mg of NHS) was added to the vial with gentle vortexing for 25 min, and then the microspheres were sedimented by centrifugation at 8000g and washed twice with 500 μL of PBS. The activated microspheres were redispersed in 100 μL of PBS buffer, which contained 160 μL of FITC-SAv (2 mg/mL in PBS). The samples were incubated overnight at room temperature with gentle vortexing, then sedimented at 8000g, and resuspended in PBS. This washing process was repeated twice more, and the supernatant was discarded. An aliquot of the FITC-SAvlabeled microspheres was examined by confocal microscopy. For biotin titration experiments, monitored by fluorescence, a washed sample of the FITC-SAv-labeled microspheres was redispersed in 1800 μL of PBS and split into nine centrifuge tubes. Then different amounts of biotin-TMR were added to the tubes and incubated for 90 min under gentle vortexing. The samples were sedimented by centrifugation at 8000g; the supernatants were collected, and the intensity of the TMR fluorescence was measured. As a negative control, we also attached FITC-BSA to the microspheres in the same way and then treated these samples with different amounts of biotin-TMR. The supernatants were collected; these samples were excited at 530 nm, and the TMR emission was measured at 580 nm. Preparation of FITC-SAv-Coated PMMA Microspheres and Determination of Biotin Binding Capacity by ICP-MS and CyTOF. A separate sample of FITC-SAv-labeled PMMA microspheres was prepared for titration experiments with Lu-DOTA-biotin. Here a

Scheme 1. Structure of the Macro-RAFT Agent P(OEGA-coAA)-TTC) Used for Photoinitiated Dispersion Polymerization

its SEC trace is shown in Figure S2, yielding Đ = 1.4. We refer to it as P(OEGA131-co-AA113)-TTC, where the subscripts refer to the number-average degree of polymerization and TTC refers to the trithiocarbonate end group. The recipes for the dispersion polymerization reactions are collected in Table 1. The first set of reactions built on the approach used by Abdelrahman et al. for the synthesis of lanthanide-encoded PS microbeads, namely a two-stage polymerization in which acrylic acid (AA) and TbCl3 were added to the reaction in a second stage.2 More specifically, we began the photoirradiation prior to the addition of AA and the lanthanide salt. After 15 min (partial monomer conversion), we removed the sample from the light source, added AA and TbCl3·6H2O dissolved in an ethanol/water mixture, and then continued the irradiation. Figure 1 shows the SEM image and diameter distribution histogram of PMMA particles prepared in this way. The particles have a narrow size distribution (CVd = 2.9%) with a mean diameter d = 0.98 μm. Unfortunately, when the particles were examined by mass cytometry, we could not detect any signal from Tb ions. This result indicates the combination of AA and TbCl3·6H2O did not incorporate lanthanide ions into the particles. 3633

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Figure 1. SEM image and diameter distribution histogram of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with P(OEGA-co-AA)-TTC as stabilizer in the presence of 2 wt % AA and 1 wt % TbCl3·6H2O (relative to MMA). d = 0.98 μm and CVd = 2.9%.

The lack of incorporation of Ln ions into the particles must reflect important differences between this reaction and the dispersion copolymerization of styrene + acrylic acid in ethanol in the presence of Ln salts. One difference might be related to the kinetics of the incorporation of AA into the copolymers. Another is the reaction medium. The lanthanide chloride salts are likely to be more soluble in an ethanol−water mixture than in ethanol. A third difference is the reaction temperature: 70 °C for the styrene + acrylic acid system and room temperature for the photoinitiated polymerization in the methyl methacrylate + acrylic acid system. In an attempt to overcome these problems, we turned to an alternative strategy for Ln ion incorporation. We took advantage of the fact that treatment of La3+ salts in water with NaF in the presence of a mixture of aminoethylphosphate (AEP) and ethylene glycol methacrylate phosphate (EGMAP) produces small nanoparticles with both amino groups and methacrylate groups on the surface. The polymerizable surface groups enable incorporation of the NPs into aqueous nanogel particles and may also be effective for encapsulating them in PMMA particles produced by photoinitiated RAFT dispersion polymerization. LnF3 Nanoparticle Synthesis. Several years ago, when we first examined the synthesis of LaF3 NPs in the presence of AEP/EGMAP, we found that we could obtain relatively uniform NPs with diameters in the range of 4−6 nm.32 This reaction did not work well for higher lanthanides, although it was possible to prepare mixed NPs containing primarily LaF3 doped with other lanthanides.46 Here, when we repeated this preparation, using a 3:1 mole ratio of AEP/EGMAP, we obtained the NPs shown in the TEM image in Figure 2. As the TEM images in Figure 2 show, NPs were obtained, but they were not particularly monodisperse. Similar information is provided by dynamic light scattering studies (Figure S3). These show that the NPs prepared in this way consist of mixtures of small NPs and their aggregates. XRD spectra of the nanoparticles are shown in Figure S4. To proceed, we imagined that NPs with a narrow size distribution might not be important in terms of their incorporation into PMMA microbeads. If the dispersion polymerization was successful, sufficiently large numbers of these NPs would be encapsulated into each microbead that the size of the individual NPs themselves might not matter. We also prepared a sample of “unreactive” LaF3 NP in the presence of AEP but no EGMAP. These NPs do not have polymerizable methacrylate groups on their surface. Photoinitiated RAFT Dispersion Polymerization in the Presence of LnF3 NPs. In our first attempt at photoinitiated

Figure 2. TEM images of LnF3 NPs. (A) LaF3 NPs capped with EGMAP and AEP, (B) LaF3 NPs capped with AEP only, (C) CeF3 NPs, (D) PrF3 NPs, (E) TbF3 NPs, and (F) LaF3:Ce,Tb NPs. Scale bar: 100 nm.

RAFT dispersion polymerization in the presence of LaF3 NPs containing polymerizable surface functionality, we carried out the reaction in the batch mode (cf. Table 1) in which all the reactants were present at the start of the reaction. An SEM image of the particles obtained in this way is shown in Figure 3a. PMMA particles with a broad distribution of sizes were obtained. This result suggests that the presence of LaF3 NPs at the beginning of the reaction disturbed the particle nucleation stage of the reaction. We then tried a two-stage protocol with the same quantity of reactants, in which we added the LaF3 NPs in the second stage. We began by irradiating a mixture (cf. Table 1) containing photoinitiator, the macro-RAFT agent, and the MMA monomer in an ethanol−water (40/60, w/w) mixture for 45 min. In this way, we obtained seed particles. Then a degassed ethanol−water solution containing additional MMA and photoinitiator as well as the LaF3 NP solution was added to the reaction and irradiated for another 45 min. Figures 3b and 3c show the SEM image and diameter distribution histogram of PMMA microspheres obtained in this way. The particles have an average diameter of ca. 1.02 μm with a narrow particle size distribution (CVd = 2.9%). Figures 3d and 3e show TEM images of these microspheres. The high contrast and the presence of small particles at the edges of the microbeads (Figure 3e) suggest that a large number of nanoparticles are incorporated into the microspheres. The metal ion content of the microspheres was then measured by mass cytometry. Figure 3f shows the 139La distribution of the microspheres. The average content of the particles was 1.4 × 106 La atoms per particle with a coefficient of variation of the La content distribution (CVLa) of 42%. The main conclusion from these initial experiments was that a significant amount of La ions could be incorporated into PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization of MMA with LaF3 NPs as the lanthanide source. A control experiment was carried out to assess whether the double bonds on the surface of LaF3 NPs were essential for the incorporation of the NPs into the PMMA microspheres. For these experiments, we employed the sample of “unreactive” LaF3 NPs lacking methacrylate groups on the surface. Figure 4 shows SEM and TEM images of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the addition of the unreactive LaF3 NPs plus additional 3634

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Figure 3. (a) SEM image of PMMA microspheres prepared by batch (one-stage) photoinitiated RAFT dispersion polymerization in the presence of 1.0 g of LaF3 NP solution (3.7 mg/mL). (b) SEM image of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization in which the LaF3 NP solution plus additional monomer and photoinitiator were added in the second stage. (c) Diameter histogram of the PMMA microspheres prepared in the two-stage dispersion polymerization. (d, e) TEM images of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the LaF3 NP solution added in the second stage. (f) Analysis by mass cytometry of the La content distribution of the PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization.

To test the versatility of this two-stage method, we also carried out photoinitiated RAFT dispersion polymerization with the addition of different kinds of LnF3 NPs (TbF3, CeF3, and PrF3). SEM images for these PMMA microbead samples and corresponding metal ion mass cytometry signal intensity distributions are shown in Figure 5. Values of the mean particle diameters d and the coefficients of variation of the particle diameter (CVd) and of the lanthanide ion distribution (CVLn) are listed in the caption to Figure 5. These results show that various lanthanide-containing PMMA microspheres can be prepared by using two-stage photoinitiated RAFT dispersion polymerization with the addition of different LnF3 NPs. Recall that the macro-RAFT agent designed for this reaction consisted of a copolymer of OEGMA and acrylic acid. Thus, we anticipated that the particle surface would contain numerous −COOH groups. The mean number of carboxyl groups per particle for one sample (the LaF3−PMMA microbeads) was determined by titration (Figure S5) to be 6.8 × 106 −COOH groups per microbead. We assume that this result is typical for all of the PMMA microbeads reported here. This sample was also tested by ICP-MS for loss of Ln ions upon storage in aqueous buffer. These experiments are described in the Supporting Information (Figure S6). The PMMA microbeads containing LaF3 NPs showed no loss of La ions over 2 weeks in PBS buffer. In phosphate buffer, pH 7.4, the sample lost 1% of its La ions immediately, with no further loss seen. In MES buffer at pH 5.5, the sample lost about 3% of its La content over 2 weeks. These results indicate that the leakage of embedded Ln ions into the aqueous medium is unlikely to be a problem during bioconjugation or in subsequent assays. Synthesis of PMMA Microbeads Containing Two or More Types of Ln Atoms. Polymer particles for multiparametric bead-based immunoassays by mass cytometry should contain at least two different lanthanide elements. We examined two ways to incorporate two or more lanthanide

Figure 4. (a) SEM image, (b) TEM image, and (c) diameter distribution histogram of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the addition of a solution of “unreactive” LaF3 NP solution plus additional monomer and photoinitiator in the second stage. (d) Mass cytometry measurement of the La content of the PMMA microspheres.

monomer and photoinitiator in the second stage. The PMMA microspheres obtained had a narrow size distribution but a negligible LaF3 NP content. The mass cytometry result shows essentially no La ion signal above background (Figure 4d). These results demonstrate that double bonds on the NPs surfaces play an important role in the incorporation of LaF3 NPs into PMMA microspheres by photoinitiated RAFT dispersion polymerization. 3635

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Figure 5. SEM images, diameter distribution histogram, and lanthanide content distributions of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization in the presence of 15 wt % P(OEGA-co-AA)-TTC with the addition of (a) 1.0 g of TbF3 NPs solution, d = 1.06 μm, CVd = 3.1%, CVTb = 29%; (b) 1.0 g of CeF3 NPs solution, d = 1.03 μm, CVd = 2.9%, CVCe = 25%; and (c) 1.0 g of PrF3 NPs solution, d = 1.08 μm, CVd = 4.5%, CVPr = 27%.

elements into our PMMA microspheres. One approach used LnF3 NPs doped with cerium and terbium (LaF3:Ce,Tb).46 The other involved two-stage photoinitiated RAFT dispersion polymerization of MMA in the presence of a mixture of different LnF3 NPs. Figures 6a and 6b show an SEM image and the diameter distribution histogram of PMMA microspheres prepared with LaF3:Ce,Tb NPs added in the second stage. The particles have an average diameter of 0.92 μm with a narrow particle size distribution (CVd = 2.8%). The metal content was measured by mass cytometry. Mass cytometry data for both 140 Ce and 159Tb are presented as an isotopic “dot−dot” diagram in Figure 6c. The average Ce atom content of the sample was 4.5 × 105 per bead, and the coefficient of variation of the Ce content distribution (CVCe) was 45%, while the average per bead Tb atom content of the sample was 7.3 × 104 with CVTb equal to 50%. Figure 6d shows the 139La distribution for the same sample. The mean per bead La atom content of the sample was 4.7 × 105 with CVLa equal to 43%. These results indicate that lanthanide-encoded PMMA microspheres can be obtained by using lanthanide-doped LnF3 NPs as the lanthanide source. This strategy, however, also requires a time-consuming synthesis of different lanthanide-doped LaF3 NPs. We then tested the strategy of using a mixture of two different LnF3 NPs as the lanthanide source for the two-stage photoinitiated RAFT dispersion polymerization. In each case the dispersion polymerization reaction led to microspheres with diameters on the order of 1 μm and a narrow size distribution (CVd ≤ 4%). Figure 7 shows mass cytometry data for a sample of PMMA microspheres prepared using different TbF3/CeF3

Figure 6. (a) SEM image and (b) diameter distribution histogram of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the addition of 0.5 g of LaF3: Ce,Tb NP solution in the second stage; (c) 140Ce/159Tb bivariate plot of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the addition of 0.5 g of LaF3: Ce,Tb NP solution in the second stage; (d) La content of PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with 0.5 g of LaF3: Ce,Tb NP solution added in the second stage.

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Scheme 2. Bioconjugation Conditions Used To Attach FITC-SAv or FITC-BSA to the Surface of Microspheres

Figure 7. Bivariate dot−dot plots of mass cytometry results for PMMA microspheres prepared by two-stage photoinitiated RAFT dispersion polymerization with the addition of (a) 1.0 g of TbF3 NP solution and 0.2 g of CeF3 NP solution; (b) 1.0 g of TbF3 NP solution and 1.0 g of CeF3 NP solution; (c) 0.2 g of TbF3 NP solution and 1.8 g of CeF3 NP solution; and (d) mass cytometry data of a mixed sample with microspheres (a), (b), and (c) showing three separate populations.

NP ratios in the second stage of the reaction. Unsurprisingly, there is a tight correlation between the Tb and Ce content of each bead. We then examine a mixture of these three microspheres by mass cytometry. The results are shown in Figure 7d. The isotopic “dot−dot” diagrams show three wellseparated populations. This is a satisfying result that demonstrates that one can control the ratios of metal ion content in the PMMA microbeads using reactive LnF3 NPs for lanthanide incorporation in the bead synthesis reaction. FITC-SAv-Coated Lanthanide-Containing Microspheres. As a proof-of-concept experiment to test our ability to attach bioaffinity agents to the surface of microspheres, we examined fluorescein-labeled streptavidin (FITC-SAv) as a model protein. The fluorescein chromophore renders the microspheres fluorescent so that they can be detected by fluorescence microscopy and, if desired, by flow cytometry. Initially, we tested the noncovalently adsorption of FITCSAv to the microspheres. For these experiments we used the PMMA microspheres labeled with LaF3 NPs. An aliquot of a PMMA microsphere dispersion in water (20 μL, solids content 10 wt %) was diluted with phosphate buffer. Then a FITC-SAv solution (100 μL, 0.1 mg/mL, in PBS) was added to the vial. After 1 h of incubation, the sample was washed by five cycles of sedimentation−redispersion with 1 mL of PBS (Scheme 2). Figures 8a and 8b show an optical image and a confocal fluorescence microscopy image of the microspheres exposed to FITC-SAv labeled. No fluorescent signal was observed in the green channel (excited at 488 nm), which suggests that noncovalent adsorption of FITC-SAv to the microspheres is negligible. For bead-based bioassays, low noncovalent adsorption of proteins to particles is very important for highly specific detection. Normally, using BSA to block the bead surface is an effective method to decrease the noncovalent adsorption. In

Figure 8. Optical (a) and CFM (b) images of La-containing PMMA microspheres treated with FITC-Sav without activation. Optical (c) and CFM (d) images of La-containing PMMA microspheres activated with EDC first and then treated with FITC-SAv. Scale bar: 8 μm.

this case, where P(OEGA-co-AA)-TTC was used as the stabilizer in photoinitiated RAFT dispersion polymerization, the corona on the microspheres appears to be effective at preventing nonspecific adsorption of proteins such as streptavidin. To test whether these microspheres with carboxyl groups in the stabilizer have the ability to covalently bind FITC-SAv, we carried out a bioconjugation experiment with FITC-SAv. An aliquot of a PMMA microsphere dispersion in water (20 μL, solids content 10 wt %) was diluted with phosphate buffer and activated with EDC + NHS. Then a FITC-SAv solution (100 μL of FITC-SAv, 0.1 mg/mL in PBS) was added to the vial and incubated for 1 h. The sample was then washed by five cycles of sedimentation−redispersion with 1 mL of PBS. Figures 8c and 8d show an optical image and a fluorescent image of the particles treated in this way. Well-separated fluorescent microspheres were observed in the green channel of the confocal microscope. These results indicate that FITC-SAv was covalently attached to the microspheres. The microspheres prepared by photoinitiated dispersion polymerization with the P(OEGA-co-AA)-TTC macro-RAFT agent exhibit both a low 3637

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microsphere, similar to the number of TMR-biotins detected by fluorescence. The sample of FITC-SAv-labeled LaF3 NP encoded microspheres saturated with Lu-DOTA-biotin was then examined by mass cytometry. The results are shown in Figure 10 along with corresponding results for FITC-BSA-labeled microspheres treated in the same way with Lu-DOTA-biotin. The “dot−dot” in Figure 10a shows a strong correlation of 175 Lu and 139La, consistent with binding of Lu-DOTA-biotin to the FITC-SAv on the surface of the LaF3-encoded NPs. The signal for 139La from the microbeads (ca. 4.5 × 105 per bead) is about an order of magnitude stronger than that from the 175Lu bound to the PMMA particle surface. From the signal intensity for 175Lu, we calculate that ca. 6.6 × 104 Lu-DOTA-biotin are bound to each FITC-SAv-coated PMMA microsphere. The results of the control experiment in which the FITC-BSAcoated PMMA beads were treated with Lu-DOTA-biotin in shown in Figure 10c. Here the La signal for the beads is strong, but the Lu signal associated with these beads is very weak. Another insight into the biotin-SAv binding is given by the screen capture from the mass cytometry data shown in Figures 10b,d. In Figure 10b, for the FITC-SAv-coated LaF3/PMMA microspheres, signals for 139La are accompanied by signals for 175 Lu from the biotin derivative. In Figure 10d, for the FITCBSA-coated LaF3/PMMA microspheres treated with the same amount of Lu-DOTA-biotin, signals for 139La show no indication of a coincident signal from the 175Lu biotin derivative.

level of noncovalent protein absorption as well as a capacity for covalent protein attachment. Quantification of the Biotin Binding Capacity of the FITC-SAv-Coated PMMA Microspheres by Fluorescence Titration. The confocal microscopy results show that the microspheres have accessible functional groups in the corona to attach bioaffinity agents. In this section, we describe fluorescence titration experiments to measure the binding capacity of biotin-TMR to the streptavidin-coated PMMA microspheres (Scheme 2). PMMA microspheres covalently labeled with FITC-SAv were prepared as described in the Experimental Section. Then these microspheres were titrated with different amounts of biotin-TMR. After centrifugation of each sample, the supernatants were collected and their fluorescence was measured. As a negative control, we attached FITC-BSA to the particles and then titrated these samples in the same way with biotin-TMR. In both experiments, emission was monitored at 580 nm where only TMR emits. The FITCBSA-coated PMMA microspheres serve to monitor the extent of nonspecific biotin binding to the surface of the proteincoated microparticles relative to FITC-SAv. In Figure 9, we plot fluorescent intensities of the supernatant solutions against the amount of biotin-TMR added in the

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SUMMARY In this paper, we report a photoinitiated dispersion polymerization of MMA using a macroRAFT agent P(OEGA131-coAA113)-TTC, consisting of a copolymer of acrylic acid and oligo(ethylene glycol) methyl ether acrylate (OEGA, Mn = 475). Polymerizations were carried out in the presence of various small (d ∼ 5 nm) LnF3 NPs carrying polymerizable methacrylate groups on their surface. In this way, we obtained PMMA microspheres with d ∼ 1 μm and a very narrow size distribution. These particles were encoded with various levels of Ln ions (on the order of 105 per bead). In our design, the macro-RAFT agent would serve as the stabilizing polymer for the microbeads. The role of the POEGA in the stabilizer was to minimize nonspecific protein adsorption to the microparticles whereas the AA groups were to provide functionality for the covalent attachment of proteins. In this way, the microbeads would serve as classifier beads in bead-based assays by mass cytometry. In a proof-of-concept experiment, a sample of La-encoded PMMA microbeads (ca. 4.5 × 105 La atoms per bead) was covalently labeled with FITC-streptavidin (FITC-SAv). As a negative control, a sample of the same beads was covalently labeled with FITC-BSA. Confocal fluorescence microscopy measurements showed strong fluorescence in both the green (FITC) and red (TMR) channels for FITC-SAv-labeled beads treated with a biotin−tetramethylrhodamine (TMR) derivative. A corresponding experiment, in which FITC-BSA-labeled beads were treated with the same amount of biotin-TMR and then washed, showed fluorescence only in the green channel. We learn that there is sufficient SAv on the beads to detect a fluorescent biotin derivative. The biotin binding capacity of the beads was determined by three parallel experiments: (i) titration of the FITC-SAv-labeled beads with biotin-TMR,

Figure 9. Plots of fluorescent intensity of supernatants containing unbound biotin-TMR versus the amount of biotin-TMR added for FITC-BSA-coated PMMA microspheres and FITC-SAv-coated PMMA microspheres.

titration. For the FITC-BSA-coated PMMA microspheres, the intensity plotted in Figure 9 increases linearly with the amount of added biotin-TMR, and the line extrapolates to zero. This result suggests very little nonspecific binding. For the FITCSAv-coated PMMA microspheres, the fluorescence from the isolated supernatant is close to zero until a certain threshold is reached, and then it increases linearly. The crossover at ca. 0.25 nmol of added biotin-TMR corresponds to saturation of the biotin binding sites on the SAv-coated microspheres. From this value, we calculate about 5.6 × 104 biotin-TMR per microsphere. Quantification of the Biotin Binding Capacity of the FITC-SAv-Coated PMMA Microspheres by ICP-MS and Mass Cytometry. In this section we describe the use of ICPMS and mass cytometry to quantify the binding capacity of SAv-coated LaF3 NP-encoded PMMA microspheres using LuDOTA-biotin (1H NMR spectrum, Figure S7) as the probe. In one set of experiments, an aliquot of microbeads (6 mg beads in 300 μL of PBS, ca. 9.0 × 109 beads) was treated with 4.8 nmol of Lu-DOTA-biotin. After 90 min incubation, the beads were sedimented, and in the supernatant we detected 4.0 nmol of free Lu-DOTA-biotin by ICP-MS. On the basis of this result, we calculated approximately 5.3 × 104 Lu-DOTA-biotin per 3638

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Figure 10. (a) 139La/175Lu bivariate plot and (b) mass cytometry screen capture of FITC-SAv-coated PMMA microspheres treated with Lu-DOTAbiotin. (c) 139La/175Lu bivariate plot and (d) mass cytometry screen capture of FITC-BSA-coated PMMA microspheres treated with Lu-DOTAbiotin.

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ACKNOWLEDGMENTS The authors thank NSERC Canada and DVS Sciences for their financial support. J.T. thanks the Chinese Scholarship Council for a scholarship to come to the University of Toronto. J.T. and Z.Z. thank the National Natural Science Foundation of China (Grants 20974126 and 21174165). The authors thank Yijie Lu for the DLS measurements.

followed by measuring the residual biotin-TMR in the supernatant; (ii) treatment of the FITC-SAv-labeled beads with an excess of a biotin-DOTA-Lu derivative, followed by measuring the residual amount of 175Lu in the supernatant. Finally (iii), we measured the 139La and 175Lu content of the beads by mass cytometry. Each of these measurements indicated that at saturation an average of ca. 5 × 104 biotin were bound to each PMMA microsphere. Control experiments with beads covalently labeled with FITC−bovine serum albumin (FITC-BSA) showed only very low levels of nonspecific binding. These results are encouraging for the development of bead-based assays by mass cytometry.

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(1) Dunbar, S. A.; Vander Zee, C. A.; Oliver, K. G.; Karem, K. L.; Jacobson, J. W. J. Microbiol. Methods 2003, 53 (2), 245−252. (2) Abdelrahman, A. I.; Dai, S.; Thickett, S. C.; Ornatsky, O.; Bandura, D.; Baranov, V.; Winnik, M. A. J. Am. Chem. Soc. 2009, 131 (42), 15276−15283. (3) Abdelrahman, A. I.; Thickett, S. C.; Liang, Y.; Ornatsky, O.; Baranov, V.; Winnik, M. A. Macromolecules 2011, 44 (12), 4801−4813. (4) Closson, T. L. L.; Feng, C.; Halupa, A.; Winnik, M. A. Macromolecules 2013, 46 (7), 2523−2534. (5) Thickett, S. C.; Abdelrahman, A. I.; Ornatsky, O.; Bandura, D.; Baranov, V.; Winnik, M. A. J. Anal. At. Spectrom. 2010, 25 (3), 269− 281. (6) Bandura, D. R.; Baranov, V. I.; Ornatsky, O. I.; Antonov, A.; Kinach, R.; Lou, X.; Pavlov, S.; Vorobiev, S.; Dick, J. E.; Tanner, S. D. Anal. Chem. 2009, 81 (16), 6813−6822. (7) Ornatsky, O. I.; Lou, X.; Nitz, M.; Sheldrick, W. S.; Baranov, V. I.; Bandura, D. R.; Tanner, S. D. Anal. Chem. 2008, 80 (7), 2539−2547. (8) Lok, K. P; Ober, C. K. Can J. Chem. 1985, 63 (1), 209−216. (9) Song, J.-S.; Tronc, F.; Winnik, M. A. J. Am. Chem. Soc. 2004, 126 (21), 6562−6563. (10) Song, J.-S.; Winnik, M. A. Macromolecules 2006, 39 (24), 8318− 8325. (11) Song, J.-S.; Winnik, M. A. Macromolecules 2005, 38 (20), 8300− 8307. (12) Tan, J.; Rao, X.; Wu, X.; Deng, H.; Yang, J.; Zeng, Z. Macromolecules 2012, 45 (21), 8790−8795.

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details for synthesis and characterization of P(OEGA-co-AA)-TTC and Lu-DOTA-biotin, lanthanide ions release experiments, pH and conductometric titration of PMMA microspheres, quantification of the biotin binding capacity of FITC-SAv labeled PMMA microparticles by biotin-TMR or Lu-DOTA-biotin titration, DLS measurements, and XRD spectra of LnF3 NPs. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00688.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (Z.Z.). *E-mail [email protected] (M.A.W.). Author Contributions

J.T. and G.Z. contributed equally to this work. Notes

The authors declare no competing financial interest. 3639

DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640

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DOI: 10.1021/acs.macromol.5b00688 Macromolecules 2015, 48, 3629−3640