Oligonucleotide and Polymer Functionalized Nanoparticles for

May 21, 2012 - In contrast, amplification-free nucleic acid testing can circumvent these limitations ... Citation data is made available by participan...
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Oligonucleotide and Polymer Functionalized Nanoparticles for Amplification-Free Detection of DNA David A. C. Thomson,†,‡ Ernest H. L. Tee,† Nguyen T. D. Tran,‡ Michael J. Monteiro,‡ and Matthew A. Cooper*,† †

Institute for Molecular Bioscience and ‡Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia S Supporting Information *

ABSTRACT: Sensitive and quantitative nucleic acid testing from complex biological samples is now an important component of clinical diagnostics. Whereas nucleic acid amplification represents the gold standard, its utility in resource-limited and point-of-care settings can be problematic due to assay interferants, assay time, engineering constraints, and costs associated with both wetware and hardware. In contrast, amplification-free nucleic acid testing can circumvent these limitations by enabling direct target hybridization within complex sample matrices. In this work, we grew random copolymer brushes from the surface of silica-coated magnetic nanoparticles using azide-modified and hydroxyl oligo ethylene glycol methacrylate (OEGMA) monomers. The azide-functionalized polymer brush was first conjugated, via copper-catalyzed azide/alkyne cycloaddition (CuAAC), with herpes simplex virus (HSV)-specific oligonucleotides and then with alkyne-substituted polyethylene glycol to eliminate all residual azide groups. Our methodology enabled control over brush thickness and probe density and enabled multiple consecutive coupling reactions on the particle grafted brush. Brush- and probe-modified particles were then combined in a 20 min hybridization with fluorescent polystyrene nanoparticles modified with HSV-specific reporter probes. Following magnetic capture and washing, the particles were analyzed with an aggregate fluorescence measurement, which yielded a limit of detection of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Adoption of brush- and probe-modified particles into a particle counting assay will result in the development of diagnostic assays with significant improvements in sensitivity.

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INTRODUCTION Quantitative detection of nucleic acids from clinical samples has greatly aided the diagnosis and management of a number of human pathologies, including viral infections.1 Whereas serology (i.e., the identification of antibodies in serum) represents the classical route for diagnosis of viral infection, analysis of nucleic acids has vastly improved the pathologist’s ability to define infectious agents and thereby make informed decisions on disease etiology. Amplification methods, based on either temperature-cycled (e.g., polymerase chain reaction (PCR)) or isothermal (e.g., NASBA,2 LAMP3) methods can provide exquisite sensitivity while maintaining tight specificity. However, the integration of modules required for amplification into point-of-care integrated devices and instruments remains challenging, with only a handful of systems approaching the market.1b,4 Nanoparticles have found a number of applications in diagnostics that exploit their unique optical, electrical, and physical characteristics. This is largely due to their enhanced properties for rapid target molecule capture while delivering signal amplification for improved sensitivity. Methods for their reliable synthesis, control over elemental composition, and flexibility in surface chemistries have enabled creative development of assay architectures. Magnetic nanoparticles offer © 2012 American Chemical Society

specific advantages due to both their large distributed surface and ease of extraction and buffer exchange.5 Nanoparticles encompassing more than one modality, such as fluorescent and magnetic composites, have broad utility in biomedical applications including cancer and tumor cell imaging, chemisensors, and biosensors.6 Paramagnetic nanoparticles have been used to isolate and preconcentrate DNA from environmental samples,7 coated with gold to enable sufaceenhanced Raman scattering detection of DNA hybridization,8 and coupled to chemiluminescent readouts with DNAzyme amplification.9 To the best of our knowledge, there are very few reports of amplification-free methods for detection of nucleic acids. A biobar code nanoparticle complex has been reported for identification of nucleic acids using an electrochemiluminescent readout,10 and progress in the use of nanoparticles toward single-molecule sensitivity has been recently reviewed.11 These methods all involve some form of coupled amplification, albeit not involving amplification of the target nucleic acid. In an alternative approach, the direct hybridization of target nucleic Received: May 9, 2012 Revised: May 20, 2012 Published: May 21, 2012 1981

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Figure 1. Scheme for magnetic particle polymer modification and subsequent coupling of DNA oligonucleotides. (A) 500 nm diameter silica-coated magnetic nanoparticles were coated in polymerization initiator 6-CMS-BMP (6-(chlorodimethylsilyl)hexyl-2-bromo-2-methylpropanoate) (reagents: tetrahydrofuran (THF)); then, a copolymer of OEGMA-N3 and OEGMA-OH (reagents: poly(ethylene oxide) monomethyl ether pentynoate (MePEO-P), tris-hydroxylpropyl triazolylamine (THPTA), copper(II) bromide (CuBr2), tris[2-(dimethylamino)ethyl]amine (Me6TREN), and 2bromo-2-methylpropionic acid (BMPA)) was grafted with single electron transfer living radical polymerization (SET-LRP). (B) Alkyne-substituted herpes simplex virus (HSV) capture probes were then covalently grafted to the polymer brush via copper-catalyzed azide/alkyne cycloaddition (CuAAC) coupling reaction. Any remaining surface azides were “clicked” with an alkyne-substituted polyethylene glycol. (C) 200 nm carboxylicacid-functionalized fluorescent polystyrene particles were modified with amine-substituted HSV reporter probes via carbodiimide chemistry. (D) Magnetic and fluorescent particles were then combined in a hybridization reaction to generate a dose response to surrogate HSV viral DNA. (E) Capture and reporter probe complex to target consensus DNA sequence.

with low-fouling polymer brushes and functionalized with viral specific oligonucleotides. To date, many polymers have been synthesized for low binding surfaces, but only those based on oligo ethylene glycol methacrylate (OEGMA) and carboxybetaine acrylamide (CBAA) monomers have demonstrated low binding in undiluted blood samples.16 With this precedent and with well-developed techniques for its polymerization,17 OEGMA was selected in our work for the development of reagents that would enable sensitive and rapid hybridization directly within complex biological samples. A body of literature18 has demonstrated the effectiveness of CuAAC coupling techniques in polymer chemistry; for instance, Pokorski et al demonstrated that the ATRP of azide-modified OEGMA on viral nanoparticles could be in conjugation with fluorescent probes and gadolinium complexes.19 In this work, we utilized the rapid and highly efficient single electron transfer living radical polymerization (SET-LRP)20 for the copolymerization of an azide-modified and a hydroxyl OEGMA monomers on magnetic nanoparticles. (See Figure 1.) This provided the nanoparticles with a low fouling and

acid offers the possibility to achieve clinically relevant sensitivity without amplification.12 We have developed in our laboratory a nanoparticle-based hybridization assay that detects a singlestranded DNA molecule derived from the UL30 DNA polymerase gene of herpes simplex virus (HSV). The target molecule links probe-modified superparamagnetic nanoparticles with probe-modified fluorescent nanoparticles in a sequence specific manner. Magnetic extraction and washing steps removed the unbound fluorescent particles following the hybridization. The number of remaining fluorescent nanoparticles therefore provides a measurement of the concentration of the target nucleic acid in the sample. In our previous work, nonspecific interactions between particles gave some contribution in determining the sensitivity floor of the assay.13 Limiting nonspecific interactions is critical for biosensor performance,14 as particularly evident in label-free detection15 and single-molecule techniques,14b where any background signal can lead to poor performance. It is especially true when conducting hybridization in complex clinical samples (e.g., blood or plasma as desired within our research). We therefore set out to synthesize magnetic nanoparticles coated 1982

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glovebox with final resuspension into 2 mL. 6-(Chlorodimethylsilyl)hexyl-2-bromo-2-methylpropanoate, the polymerization initiator, was added (∼67 μmol, 60 μL), after which the particles were sealed in a 10 mL round-bottomed flask and mixed with a rotary shaker for 18 h after sonication for 20 s. Particles were then stored in THF until use. POEGMA-N3-co-POEGMA-OH Brush Synthesis. Immediately prior to polymerization the initiator-coated particles were magnetically exchanged five times into 3:7 methanol/water. Particles were suspended in a final volume of 8.75 mL within a Schlenk flask. Copper(II) bromine (5.12 mg, 22.9 μmol) was suspended in 1.28 mL of 3:7 methanol/water mixture (to achieve a concentration of 18 mM). Tris[2-(dimethylamino)ethyl]amine (Me6TREN, 6.1 μL, 22.9 μmol) was then added, after which 50 μL (900 nmol) of this combined Cu(II)Br2/Me6TREN mixture was added to the nanoparticle suspension. 2-Bromo-2-methylpropionic acid (14.18 mg, 85 μmol) was dissolved in 2.83 mL of 3:7 methanol/water (to achieve a 30 mM solution), after which 100 μL (3 μmol) was added to the nanoparticle suspension. OEGMA-N3 (200 mg, 180 μL, ∼360 μmol, av. 551 g/ mol) and OEGMA-OH (95 mg, 86 μL, 180 μmol, av. 526 g/mol) were added to the nanoparticle suspension. The nanoparticle suspension was then subjected to four freeze−thaw−pump cycles. The tube was then transferred into a sonication bath at 30 °C. Copper(I) chloride (3.67 mg, 36 μmol) was added to 1.22 mL of 3:7 methanol/water solution in a glass vial (to achieve a 30 mM solution), after which Me6TREN (9.8 μL, 36 μmol) was added. To assist disproportionation, the solution was sonicated for 20 s. Under argon, 100 μL (3 μmol) of the Cu(I)Cl/Me6TREN solution (100 μL) was added to the nanoparticle suspension. Final concentrations and molar ratios were OEGMA-N3 40 mM, OEGMA-OH 20 mM, BMPA 333 μM, CuCl 333 μM, CuBr2 100 μM, and Me6TREN 433 μM with ratios of 134/66/1/1/0.3/1.3, respectively. The reaction proceeded for 40 min with constant argon bubbling. The samples were manually swilled every minute for the first 5 min. Samples were collected under argon at 0, 5, 15, 30, and 40 min for dynamic light scattering (DLS). After 40 min the reaction was terminated by adding 5 mL of a 2 mM solution of Cu(II)Br2/Me6TREN representing a 3.3 fold molar excess over Cu(I). The particles were then repeatedly washed with water. CuAAC Oligonucleotide Immobilization. 3.6 × 1010 particles were washed three times with and transferred into 1 mL of degassed PBS/0.5% Tween- 20, pH 7.8. Degassing was conducted by bubbling nitrogen for at least 30 min prior to the conjugation. Copper sulfate (4 mg, 25 μmol) and THPTA (34 mg, 125 μmol) were combined in 157 μL of water to make a 500 mM THPTA, 100 mM CuSO4 stock solution. This was diluted 100 fold immediately prior to coupling and then diluted again to yield a reaction concentration of 100 and 500 μM for copper sulfate and THPTA respectively. Immediately prior to conjugation sodium L-ascorbate (10 mg, 50 μmol) was added to 1 mL of PBS/0.5% Tween-20. This was diluted to yield a reaction concentration of 5 mM. In the CuAAC reactions with Alk-DNA-665, 1.8 × 109 (equivalent with 50 μL stock) were combined with 300 pmol of fluorescent AlkDNA-665 oligonucleotide to yield 1.3 × 1013 probes/cm2 in a 300 μL reaction (6 × 109 particles/ml). Following 3 h of incubation particles were washed three times with Post-Hyb buffer and then transferred to a black 96-well microplate (BD Falcon ref 353241). Fluorescent intensity was determined using a Biotek Synergy 4 microplate reader (monochromators 9 nm setting, ex 640 nm, em 680 nm). Couplings with Alk-HSVp1 were conducted at 6 × 1013 probes/cm2 with 1.8 × 1010 particles on a 1 mL scale. AlkHSVp1 (14 nmol) was added (18.2 μL of the 770 μM stock). Following conjugation of AlkHSVp1, the particles were washed with conjugation buffer, and then the coupling was repeated under identical conditions using 700 nmol of the Alk-PEG reagent. Polymer Brush Characterization. DLS was conducted with a Malvern Zetasizer, ZS Nano. Particles extracted from the polymerization reaction (200 μL) were washed with a 400 μM CuBr2/ Me6TREN 3:7 methanol/water solution to terminate the reaction. Samples were then magnetically extracted and washed twice into water. DLS measurements are taken as the average of three polymerization reactions conducted in parallel. Each point was

chemically functional (through the azide moiety for coupling and immobilization of oligonucleotides) brush coating. The efficient CuAAC “click” cross-coupling reaction allowed alkyneterminated DNA probes to be immobilized to the polymer brush in water with significantly greater efficiency compared with previous coupling techniques, which utilized cross-linking reagents in anhydrous solvents. The remaining azides on the surface were quenched by coupling alkyne PEG (Figure 1). The combination of SET-LRP and CuAAC offers the possibility for novel polymer probe architectures, such as multilayer brushes, to be grafted from the nanoparticle surface. We see these multistructured brushes as offering the potential for future improvements in sensitivity and the potential for clinically viable amplification-free DNA detection.

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MATERIALS AND METHODS

Full materials and methods is available in Supporting Information Synthesis of Hex-5-en-1-yl 2-bromo-2-methylpropanoate. Synthesis was based on procedures reported in Barbey and Klok,21 whereas the purification step followed procedures outlined by Ohno and coworkers.22 1H NMR (400 MHz, CDCl3) δ = 5.75−5.85 (m, H), 4.96−5.05 (m, 2H), 4.16−4.19 (t, 2H, J = 6.5 Hz), 2.07−2.13 (m, 2H), 1.93 (s, 6H), 1.68−1.74 (m, 2H), 1.46−1.54 (m, 2H). 13C NMR (100 MHz, CDCl3) δ = 171.9, 138.4, 115, 66.1, 56.1, 33.3, 30.9, 27.9, 25.2. Synthesis of 6-(Chlorodimethylsilyl)hexyl-2-bromo-2-methylpropanoate. The synthesis was performed based on modification of procedures reported in Barbey and Klok21 and Theato et al.23 1H NMR (400 MHz, CDCl3): δ = 4.15−4.18 (t, 2H, J = 6.6 Hz), 1.93 (s, 6H), 1.64−1.72 (m, 2H), 1.29−1.47 (m, 6H), 0.80−0.83 (m, 1H), 0.40 (s, 6H). 13C NMR (100 MHz, CDCl3): δ = 171.9, 66.2, 56.1, 32.6, 30.9, 28.4, 25.6, 23.0, 19.0, 1.8. Synthesis OEGMA-N3. Synthesis of OEGMA-N3 was adapted from the procedures reported by Pokorski et al.19 1H NMR (400 MHz, CDCl3): 6.13 (s, 1H), 5.57 (t, 1H), 4.3 (t, 2H), 3.62 − 3.78 (m, 24H), 3.38 (t, 1H), 1.95 (s, 3H). Poly(ethylene oxide) Monomethyl Ether Pentynoate (MePEO-P). The synthesis of the alkyne-PEG quenching reagent was adapted from the literature24 with N,N′-diisopropylcarbodiimide used instead of dicyclohexylcarbodiimide. 1H NMR (400 MHz, CDCl3): δ = 4.26 (t, 1H), 3.8−3.84 (m, 3H), 3.7 (t, 1H), 3.64−3.66 (m, 52H), 3.55 (m, 1H), 3.38 (s, 1H), 2.5−2.6 (m, 1H), 1.7 (s, 3H), 1.12 (d, 13H). MS-TOF 834, 861, 878, 905, 922, 949, 966, 993, 1010, 1037. Tris-hydroxylpropyl triazolylamine (THPTA). The synthesis of THPTA was adapted from the literature.25 1H NMR (400 MHz, CD3OD) δ = 7.98 (s, 3H), 4.5 (t, J = 7.2, 6H), 3.75 (s, 6H). 3.56 (t, J = 6, 6H), 2.11 (q, 6H), MS m/z calculated, 435.25; measured, 435.2. Fluorescent Nanoparticle Reporter Probe Immobilization. The protocol was adapted from our previous work.13 In brief, 500 μL of 200 nm yellow green Fluospheres (Invitrogen, F8811, LOT 759339, 4.5 × 1012 mL−1) was washed with 60 mM MES buffer and then combined with 30 mM EDC and 46 nmol of amine-substituted HSVp2 yielding an effective coupling concentration of 1013 probes/ cm2. After 90 min the coupling was repeated with methoxypolyethylene glycol (750 g/mol, 5 mM) and then ethanolamine (10 mM) for 30 min each. Magnetic Nanoparticle ATRP Initiator Grafting. All magnetic extractions took place within glass test tubes (ThermoFisher CSE73500-16100) using permanent magnets and use of sonication bath for resuspension (Thermoline Scientific, WUC-D06H, CSK Climatek). Batch sizes where done at 1 mL (3.6 × 1010) of as-received particles from Ademtech. Particles were washed with water to remove the biocide Proclin300 and resuspended into 500 μL of water. They were then cleaned and hydroxyl-activated with two cycles of piranha for 15 min (120 °C, 2 mL of 3:1 conc. H2SO4/30% H2O2). Particles were then first exchanged into water, then three times into THF, and finally three times into anhydrous THF within a nitrogen-purged 1983

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obtained from DLS runs of 10 s in triplicate. The supernatant of polymerization reaction was rotary evaporated to remove the methanol and then freeze-dried to remove the water. These samples were then diluted into THF and analyzed by GPC. Particles were freeze-dried prior to analysis with X-ray photoemission spectroscopy (XPS) and thermogravimetric analysis (TGA). XPS analysis was conducted on a Kratos Axis Ultra using Al Kα (1253.6 eV) X-rays. Survey scans were conducted over an area of 0.7 mm × 0.3 mm with a pass energy of 160 eV, with 1 eV steps and 100 ms delay. High-resolution scans were conducted with three sweeps with pass energy of 20 eV and 250 ms dwell times for each step. Results were analyzed with Casa XPS data processing, exported, and plotted using GraphPad Prism 5. TGA) was conducted using a Mettler Toledo TGA/DSC1 Star system under nitrogen at 10 °C/min from 25 to 800 °C. Hybridization Reactions. Hybridization reactions took place within assay plates (BD Falcon V-bottom polypropylene cat. 353263). All liquid handling was conduction on an Agilent Bravo Robotic Pipettor station with tips (Axygen VT-250-L-R) treated by pipetting 150 μL of blocking buffer for 20 s. The assay plate was first incubated at 30 °C for 15 min with 120 μL of blocking buffer, after which it was replaced with 80 μL of hybridization buffer or 30 μL of hybridization buffer and 50 μL of fetal bovine serum. The concentration of HSVTar (683 μM) and VZVTar (312 μM) was first determined with a Thermoscientific Nanodrop 1000. Target molecules were then diluted into 5 mL of hybridization buffer to achieve a 1 μM stock concentration. Dilution was then conducted into a second tube to achieve a 50 nM working solution. Target DNA was then loaded into a 12 column plate and robotically serially diluted to achieve desired assay concentrations (5000, 500, 50, 5, 0.5, 0 pM) within the 100 μL final hybridization volume. Magnetic particles were diluted to 3 × 109 particles/mL (0.125 mL stock equivalent) and combined with 1.9 × 1011 fluorescent particles (41.6 μL stock equivalent) in 1.5 mL of PostHyb Buffer. Ten μL of this combined particle containing buffer was added to each well to yield final reaction concentrations of 3 × 108 and 1.9 × 1010 particles/mL, respectively. The assay plate was shaken (rpm: 1200) on a Velocity11 magnetic shaker every 2 min to keep the particles suspended and placed on a hot plate station at 30 °C otherwise. After a 20 min hybridization the plate was transferred to a 96-position magnetic rack (Agencourt Bioscience Corporation, AGN #32782 SPRIplate); then, after 2 min the supernatant was removed with particles resuspended by shaking in 100 μL of Post Hyb Buffer. This was repeated four times with finally resuspension into 80 μL and injected into a black 96-well microplate. The plate was fluorescently analyzed using a BMG Labtech PolarSTAR (ex 485/10 nm, em 510/10 nm, gain 1500). Buffers. Blocking buffer: (5× SPPE [750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA], 2× Denhardt’s (400 mg/L each of Ficoll, PVP, and BSA), 200 μg/mL sheared salmon sperm DNA, 0.1% Tween 20). Hybridization buffer (5× SPPE (5× SSPE = 750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA), 1× Denhardt’s (200 mg/L of Ficoll, PVP and BSA), 100 ug/mL sheared salmon sperm DNA, 0.1% Tween 20). Post-Hyb buffer (0.3× SPPE (0.3× SSPE = 45 mM NaCl, 3 mM NaH2PO4, 0.3 mM EDTA), 0.1% Tween 20. All buffers were prepared with sterilized and filtered using 0.2 μm filters. Conjugation buffer: 1× PBS, 0.05% v/v Tween 20.

from the assay work-flow. This more convenient assay format would simplify the design and manufacturing complexity of both disposable fluidic cartridges and instruments. With these considerations in mind, the development of magnetic nanoparticle reagents that would enable specific and rapid hybridization of target nucleic acids while avoiding nonspecific interactions was the focus of this work. The synthetic steps for synthesis of the probe-modified core−shell particles was as follows: (a) immobilization of the organosilane polymerization initiator 6-(chlorodimethylsilyl)hexyl-2-bromo2-methylpropanoate through siloxane bonds onto silica modified magnetic nanoparticles; (b) random copolymerization of the monomers OEGMA-OH and OEGMA-N3 from the nanoparticle surface using SET-LRP; (c) conjugating HSV specific capture probes with alkyne substituents (Alk-HSVp1) to the polymer brush via CuAAC, and (d) quenching all remaining azides on the surface with a 750 Da PEG also possessing alkyne substituents. The magnetic particles were then combined with reporter probe-modified fluorescent nanoparticles and used within an assay to detect surrogate HSV DNA. (See Figure 1.) This assay architecture was based on our previous work and utilized magnetic capture for purification of target-molecules-linked fluorescent nanoparticles.13 The fluorescent nanoparticles were quantified in an aggregate fluorescence detection method providing a dose response. Pinhole-free polymer brushes are important in the development of surfaces that exhibit low protein binding properties. Therefore, because dense grafting of the polymerization initiator was desired, adventitious carbon was first removed from nanoparticle surface using a piranha treatment. The particles “as received” from the supplier were analyzed by XPS. The complete absence of signal originating from elemental iron confirmed conformal silica coating. (See Figure 1 in the Supporting Information.) The XPS also demonstrated carbon (∼16%) on the particle surface, assumed to be remnants from the proprietary emulsion synthesis process. Studies revealed that two successive piranha treatments conducted at 120 °C for 15 min with a 30 s sonication step between piranha treatments with washes into copious (3 × 10 mL) amounts of water were able to reduce this down to 11% carbon. Following transfer of the magnetic particles into water and then into anhydrous tetrahydrofuran (THF), the polymerization initiator was immobilized onto the nanoparticle surface. Initiator immobilization was confirmed by XPS, with supporting evidence from carbon, silicon, and bromine regions. The appearance of the ester carbon species at 289.3 eV (Figure 2A), the addition peak at 101.4 eV in the silicon spectrum attributed to silicon bound with carbon (Figure 2B), and the appearance of bromine at 70.4 to 71.5 eV (Figure 2C) provide evidence of initiator immobilization. The strong bromine doublet with a binding energy difference of 1.12 eV agrees with previous reports of bromine functionality.26 With the initiator successfully immobilized on the nanoparticle surface, polymerization was conducted using both the hydroxyl and azide forms of the oligoethylene glycol methacrylate monomer. Removal of the bismethacrylate impurities, conducted here via silica flash chromatography,19 was important for reproducible and controlled polymerization. It was believed that the bismethacrylate species contributed to cross-linking reactions with resultant gel formation. The polymer brush synthesized using SET-LRP at varying mole ratios of the monomers OEGMA-N3 and OEGMA-OH

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RESULTS AND DISCUSSION Rapid and sensitive quantification of viral nucleic acid sequences from clinical samples is an important technology. Whereas nucleic acid amplification offers excellent performance, its transfer into integrated devices as required for point of care diagnostics is challenging. In an alternative and more attractive approach, sequence-specific hybridization, as represented in Figure 1, can be conducted directly in minimally processed clinical samples, such as serum of ideally blood treated to lyse the viral capsid. If the clinically required sensitivity and specificity can be achieved, then both the amplification and nucleic acid purification step can be omitted 1984

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content in the SET-LRP, then the particles were not colloidally stable under aqueous conditions. The CuAAC of Alk-HSVp1 capture probes followed by CuAAC of a 750 Da PEG-alkyne afforded improved colloidal stability. However, to generate a methodology that would afford reliable grafted probe density, we decided to randomly copolymerize OEGMA-N3 with the hydroxyl terminated OEGMA-OH to afford colloidal stability (Figure 2 in the Supporting Information). We suggest that this is due to hydrogen bonding with water by the ω-terminal hydroxyl group. All work reported here was conducted with particles polymerized with 67% OEGMA-N3 and 33% OEGMA-OH. XPS was used to confirm grafting of the polymer brush to the nanoparticle surface. Figure 3A presents the survey scan showing O 1s, N 1s, C 1s, and Si 2p peaks. The silicon appears from the underlying particle. High-resolution spectra of nitrogen and carbon binding energies are presented in Figure 4B,C, respectively. The nitrogen region demonstrates a signal

Figure 4. Thermogravimetric analysis of (a) “as received” magnetic particles, (b) initiator-coated particles, and (c) POEGMA-N3-coPOEGMA-OH grafted particles.

characteristic of azides, as previously reported.27 The azide signal contains two peaks at 400.9 and 404.5 eV with a binding energy split of 3.6 eV. Peak areas are in the ratio of 2.6:1, as expected from the structure of the azide with internal nitrogen surrounded by two nitrogen atoms. The carbon components are assigned from the reference C−C component at 285 eV and yielded C−O at 286.5 eV and the ester carbon CO at 289 eV. The C−C is assigned to the alkane backbone of the polymer, whereas the C−O signal originates from the ethylene oxide side chain. In addition to the initiators immobilized on the magnetic nanoparticle surface, the polymerization also contained the

Figure 2. High-resolution XPS of (A) carbon, (B) silicon, and (C) bromine regions of magnetic particles functionalized with polymerization initiator.

allowed fine-tuning of the concentration of azide moieties within the brush. A sufficiently dense azide functionality was required to enable efficient grafting of HSV-specific probes; if the ratio of OEGMA-N3 was too high (>∼66%), then colloidal stability of the particles was compromised. For example, if the OEGMA-N3 monomer represented the entire monomer

Figure 3. XPS spectra of OEGMA-N3-co-OEGMA-OH coated particles. (A) Survey scan of polymer-coated particles, (B) high-resolution nitrogen region with clearly defined azide moiety, and (C) high-resolution carbon region showing species characteristic of POEGMA polymers. 1985

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Figure 5. (A) CuAAC coupling scheme for immobilization of fluorescent oligonucleotide. (B) Aggregate fluorescent signal obtained following CuAAC conjugation of Alk-DNA-TYE665 onto OEGMA-N3-co-OEGMA-OH modified particles (n = 3).

particles (2 g/cm3), Vparticle is the volume of the particle calculated from a radius of 500 nm, NA is Avogadro’s number, Mpolymer is the molecular weight of the polymer calculated from GPC (12 000 g/mol), and Sparticle is the surface area of the particles. Grafting density was estimated to be 0.8 chains/nm2. With the grafting of the POEGMA-N3-co-POEGMA-OH brush from the nanoparticle surface confirmed, the next step was to immobilize the brush covalently with HSV oligonucleotides. Several methods have been reported in the literature for coupling biomolecules to POEGMA and structurally related polymer brushes. The Klok group demonstrated the activation of terminal hydroxyl moieties with p-nitrophenyl chloroformate (NPC) in anhydrous THF with subsequent amine nucleophilic attack.30 More recently, N,N′-disuccinimidyl carbonate (DSC) was selected from a number of cross-linking reagents examined for biomolecule conjugation onto surface-grafted POEGMA brushes,31 whereas several other reports have demonstrated the use of DSC for biomolecule coupling.32 Utilization of the activated ester approach can be experimentally challenging for nanoparticle manipulations due to the hydrolytic susceptibility of the activated ester with its resultant requirement for anhydrous techniques. The avidin family of proteins was avoided because these would putatively create additional moieties, in particular, basic amino acids, which may increase nonspecific interactions. Additionally, a covalent conjugation technique was preferred because it would enable the use of particles within highly denaturing viral lysis conditions. These challenges, and the desire to obtain an optimized capture probe density for rapid and reliable hybridization, compelled the investigation of an alternative approach. In contrast with the activated ester approach, the CuAAC reaction can be performed in aqueous solvents, which simplifies the modification process. The CuAAC is one of a family of “click reactions” that provide high yields, minimum sidereactions, and orthogonal specificity and has inspired its application throughout the materials sciences,33 polymer engineering,18a,34 and biomolecule coupling.35 Typically,

sacrificial initiator 2-bromo-2-methylpropionic acid (BMPA) in the water phase. Its inclusion in the reaction mixture followed the work of Ohno28 and served two purposes. First, the sacrificial initiator enhanced control over the polymerization by increasing the concentration of Cu(II) deactivating species. Second, the molecular weight of the polymer polymerized from the sacrificial initiators enabled an estimation of the grafted polymer molecule weight. Gel permeation chromatography (GPC) reported a molecular weight (Mn) of 12 100 g/mol with a polydispersity index (PDI) of 1.35. At regular intervals during the polymerization an aliquot (∼2.2% of the reaction, 200 μL) of the reaction was removed and analyzed by DLS to determine the particle diameter. The results provided strong evidence of particle modification with the polymer brush with a significant change in particle diameter from 500 to 1000 nm. However, the DLS PDI of brushmodified particles was typically between 0.4 and 0.6, and thus the accuracy of hydrodynamic particle size from the DLS gave qualitative size information (Figure 2 of the Supporting Information). In a third characterization method, TGA was conducted to quantify the mass of polymer grafted from the nanoparticle surface and thus the grafting density. The particles “as-received” from the supplier retained 91.8% of their weight, the initiatorcoated particles retained 87.8%, and the polymer coated particles retaining 78.1% of their weight (Figure 4). Taking the difference between the polymer and initiator-coated particles leaves 9% of weight loss due to grafted OEGMA-N3-coOEGMA-OH. The grafting density was calculated using the formula29 ⎛ Wpolymer ⎞ ⎜ ⎟ρ V N ⎛ chains ⎞ ⎝ 100 − Wpolymer ⎠ particle A ⎟ = surface density⎜ ⎝ nm 2 ⎠ M polymerSparticle

where Wpolymer is the percent weight loss due to the decomposition of grafted polymer, ρ is the density of the 1986

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Figure 6. Dose response data for 20 min hybridization reactions conducted with magnetic particles modified with Alk-HSVp1 and those with both Alk-HSVp1 and Alk-PEG. (A) Hybridization conducted in 100% hybridization buffer with HSV target nucleic acid and negative control VZV. (B) Hybridization conducted in 50% fetal bovine serum/50% hybridization buffer. Consecutive CuAAC with Alk-PEG improves sensitivity from a LoD of 500 pM to 60 pM, n = 3. Arrow indicates improvement in LoD due to alk-PEG conjugation.

maintained a 30 times higher fluorescent intensity than the fluorescent silica particles, and thus their use was continued (Figure 3, Supporting Information). Future studies will investigate grafting of POEGAM-N3-co-POEGMA-OH from the surface of the fluospheres to minimize nonspecific binding and enable reporter probe grafting. To achieve this, all processing steps (initiator grafting, polymerization, and probe immobilization) must be conducted under aqueous conditions, as fluospheres are not stable in solvents other than water. Once the CuAAC of alkyne-substituted fluorescent oligonucleotides had been demonstrated, OEGMA-N3-co-OEGMAOH modified particles were modified with alkyne-substituted HSVp1 capture probes, Alk-HSVp1. Hybridization rates for solution-phase target nucleic acids and surface-immobilized probes have been shown to be strongly dependent on the probe density.37 If probe density is too low, then slow hybridization results, while at higher concentrations steric interference, electrostatic repulsion, and base stacking, can impede target entry to the probe surface, causing an inefficient hybridization. In preliminary studies, a titration of probe concentration and nanoparticle surface area within the CuAAC coupling reaction was made to determine optimum concentration for use in the amplification free detection assay. From these studies, 6.6 × 1013 probes/cm2 was found to be the optimum for the coupling reaction. Polymers containing azide moieties in combination with CuAAC have enabled consecutive coupling reactions to be conducted.24a Here we utilized this concept to quench azides remaining following immobilization of Alk-HSVp1 with an alkyne-substituted poly(ethylene oxide). MS-TOF demonstrated this Alk-PEG to have, on average, 16 monomer units. There were a number of functional objectives in conducting this quenching step. The first was to convert ω-terminal hydrophobic azide groups into terminal hydroxyl groups to impart further colloidal stability upon the particles. Second, the AlkPEG contributed to the density of the polymer brush by “backfilling” gaps in the polymer brush. The consequence of the quenching step with Alk-PEG on the limit of detection was investigated. A 20 min hybridization reaction was conducted with particles modified with Alk-HSVp1 and those that had been modified with HSVp1 and Alk-PEG. In 100% hybridization buffer, the level of detection (LoD) was not significantly affected with the conjugation of the Alk-PEG (Figure 6A). However, in serum the LoD improved from 400 pM to ∼60 pM (Figure 6B). The strong dose response evident in Figure 6 demonstrates that the CuAAC and SET-LRP were successful in

CuAAC biomolecule coupling reactions are conducted with copper sulfate and sodium ascorbate as the reducing reagent to yield the Cu(I) active catalyst. However, the copper-mediated generation of oxygen radical species can cause undesired oxidative damage to biomolecules. This effect can be minimized with the appropriate ligand and thus the water-soluble polytriazole, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), as reported by Hong et al.,25b was synthesized and used throughout this study. For added precaution, the conjugation buffer was degassed by bubbling nitrogen prior to the CuAAC reaction. To first establish coupling conditions required for the immobilization of oligonucleotides to the azide-modified polymer brush, the CuAAC reaction was first used to couple an alkyne-substituted 11-mer oligonucleotide fluorescent probe, Alk-DNA-665, (Figure 5A). Following a 3 h coupling reaction conducted with 300 pmol of Alk-DNA-665 the particles were washed and then analyzed for fluorescence intensity (Figure 5B). The strong fluorescent signal (17,636, s.d. 472) from the particles provided strong evidence that the conjugation was effective in immobilizing the fluorescent oligonucleotide. The conjugation reaction containing particles and fluorescent dye without the copper or ligand species showed a small signal (534, s.d 142) that was attributed to background binding, whereas particles and copper and ligand alone showed a signal of virtually zero (4, s.d. 2.3). Once the probe immobilization reaction had been established, particles were conjugated with alkyne-substituted capture probes specific against HSV and then used in dose response assay. The assay format of the amplification free nucleic acid assay was based on our previous work.13 The assay was based on target nucleic-acid-dependent linkage between polymer brush and capture-probe-modified 500 nm magnetic nanoparticles and 200 nm reporter-probe-modified fluorescent polystyrene nanoparticles (Fluospheres). Fluorescent particles were modified with amine-substituted HSVp2 oligonucleotides using carbodiimide chemistry (EDC). The fluospheres contain on average 10 000 encapsulated fluorescent dye molecules and provide an effective method for signal amplification of target hybridization. However, they have been reported to yield high background in a number of assay formats,36 and thus in preliminary studies the fluorescent intensity of the Fluosphere was compared with 200 nm fluorescent silica particles (42-00202, Micromod Partikeltechnologie, Germany), which would enable initiator immobilization in anhydrous solvent, followed by brush grafting and probe conjugation. The fluospheres 1987

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acid testing for infectious diseases. Trends Biotechnol. 2011, 29, 240− 250. (2) Gracias, K. S.; McKillip, J. L. Nucleic acid sequence-based amplification (NASBA) in molecular bacteriology: a procedural guide. J. Rapid Methods Autom. Microbiol. 2007, 15, 295−309. (3) Curtis, K. A.; Rudolph, D. L.; Owen, S. M. Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1. J. Med. Virol. 2009, 81, 966−972. (4) Tanriverdi, S.; Chen, L. J.; Chen, S. Q. A rapid and automated sample-to-result HIV load test for near-patient application. J. Infect. Dis. 2010, 201, S52−S58. (5) Haukanes, B. I.; Kvam, C. Application of magnetic beads in bioassays. Nat. Biotechnol. 1993, 11, 60−63. (6) (a) Corr, S.; Rakovich, Y.; Gun’ko, Y. Multifunctional magneticfluorescent nanocomposites for biomedical applications. Nanoscale Res. Lett. 2008, 3, 87−104. (b) Iwata, A.; Satoh, K.; Murata, M.; Hikata, M.; Hayakawa, T.; Yamaguchi, T. Virus concentration using sulfonated magnetic beads to improve sensitivity in nucleic acid amplification tests. Biol. Pharm. Bull. 2003, 26, 1065−1069. (c) Katz, E.; Willner, I. Integrated nanoparticle−biomolecule hybrid systems: synthesis, properties, and applications. Angew. Chem., Int. Ed. 2004, 43, 6042− 6108. (7) Galluzzi, L.; Bertozzini, E.; del Campo, A.; Penna, A.; Bruce, I. J.; Magnani, M. Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples. J. Appl. Microbiol. 2006, 101, 36−43. (8) (a) Zhang, H.; Harpster, M. H.; Wilson, W. C.; Johnson, P. A. Surface-enhanced Raman scattering detection of DNAs derived from virus genomes using Au-coated paramagnetic nanoparticles. Langmuir 2012, 28, 4030−7. (b) Zhang, H.; Harpster, M. H.; Park, H. J.; Johnson, P. A.; Wilson, W. C. Surface-enhanced Raman scattering detection of DNA derived from the west nile virus genome using magnetic capture of Raman-active gold nanoparticles. Anal. Chem. 2011, 83, 254−60. (9) Wang, C.; Wu, J.; Zong, C.; Ju, H.; Yan, F. Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification. Analyst 2011, 136, 4295−300. (10) Zhu, D.; Tang, Y.; Xing, D.; Chen, W. R. PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method. Anal. Chem. 2008, 80, 3566−71. (11) Zanoli, L. M.; D’Agata, R.; Spoto, G. Functionalized gold nanoparticles for ultrasensitive DNA detection. Anal. Bioanal. Chem. 2012, 402, 1759−71. (12) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes. J. Am. Chem. Soc. 1998, 120, 1959−1964. (13) Thomson, D. A. C.; Dimitrov, K.; Cooper, M. A. Amplification free detection of herpes simplex virus DNA. Analyst 2011, 136, 1599− 1607. (14) (a) Cooper, M. A. Biosensor profiling of molecular interactions in pharmacology. Curr. Opin. Pharmacol. 2003, 3, 557−562. (b) Walter, N. G.; Huang, C. Y.; Manzo, A. J.; Sobhy, M. A. Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat. Methods 2008, 5, 475−489. (15) Cooper, M. A. Non-optical screening platforms: the next wave in label-free screening? Drug Discovery Today 2006, 11, 1068−1074. (16) Rodriguez-Emmenegger, C.; Kylian, O.; Houska, M.; Brynda, E.; Artemenko, A.; Kousal, J.; Alles, A. B.; Biederman, H. Substrateindependent approach for the generation of functional protein resistant surfaces. Biomacromolecules 2011, 12, 1058−1066. (17) (a) Wang, X. S.; F. Lascelles, S.; A. Jackson, R.; P. Armes, S. Facile synthesis of well-defined water-soluble polymers via atom transfer radical polymerization in aqueous media at ambient temperature. Chem. Commun. 1999, 1817−1818. (b) Wang, X. S.; Armes, S. P. Facile atom transfer radical polymerization of methoxycapped oligo(ethylene glycol) methacrylate in aqueous media at ambient temperature. Macromolecules 2000, 33, 6640−6647. (c) Ma,

generating a oligonucleotide-modified polymer brush. To prove the specificity of the hybridization, we also conducted the dose response with a DNA sequence derived from Varicella Zoster Virus. The absence of the dose response from these reactions demonstrates the specificity of these reagents.

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CONCLUSIONS To our knowledge, this is the first work detailing the immobilization, via a CuAAC reaction, of virus-specific capture oligonucleotides to POEGMA-N3-co-OEGMA-OH polymer brushes. The combination of “living” radical polymerization and click chemistry provides a powerful toolset for developing and optimizing particle reagents for use in bioassays. The “living” radical polymerization methods enable the synthesis of dense polymer brushes, whereas the orthogonal and efficient nature of CuAAC enables a multitude of species to be covalently conjugated to the polymer brush. Consecutive click reactions enabled the tuning of both HSVp1 capture probe density and subsequent backfilling of the polymer brush to fill voids within the brush. The capture-probe-functionalized particles were combined with fluorescent nanoparticles and used within a 20 min amplification-free detection assay, which yielded an LoD for single-stranded DNA of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Whereas many architectures for low binding surfaces have been developed with OEGMA, there has been little work using OEGMA-N3 monomers. This work reports a reliable synthesis scheme for the synthesis of azide-modified polymer brushes and subsequent modification with oligonucleotides for use in hybridization assays. The versatility of the method provides the opportunity for the further development of core−shell particles for improved sensitivity. For instance, CuAAC of a second polymer initiator enables the development of multilayer polymer brushes that may provide a binding matrix with kinetics approaching those provided by solution-phase probes.

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ASSOCIATED CONTENT

* Supporting Information S

Chemicals, detailed materials and methods, and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.T. acknowledges the dynamic learning environment of the Cooper Group and is grateful for the support of its members. He is also very grateful for the support, assistance, and editorial advice of M.J.M. XPS was conducted at the Centre for Microscopy and Microanalysis. Funding Sources: This work was supported by NHMRC Australia Fellowship AF511105.

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