Spin-On End-Functional Diblock Copolymers for Quantitative DNA

Biomacromolecules 2008, 9, 2345–2352

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Spin-On End-Functional Diblock Copolymers for Quantitative DNA Immobilization Lu Chen,† Hernán R. Rengifo,† Cristian Grigoras,† Xiaoxu Li,†,‡ Zengmin Li,†,‡ Jingyue Ju,*,†,‡ and Jeffrey T. Koberstein*,† Columbia University Department of Chemical Engineering 500 West 120th Street, New York, New York 10027, and Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, New York 10032 Received March 10, 2008; Revised Manuscript Received June 17, 2008

We demonstrate a simple means to covalently bond DNA to both hard (i.e., glass and silicon wafers) and soft (i.e., polymeric) substrates that provides quantitative and precise control of the DNA areal density. The approach is based on spin coating an alkyne-end-functional diblock copolymer, R-alkyne-ω-Br-poly(tBA-b-MMA), that self-assembles on both types of substrates as an ordered monolayer and thereby directs alkyne groups to the surface. Azido-functionalized DNA is covalently linked to the alkyne functionalized substrates by means of a “click” reaction between azide and alkyne groups. The density of immobilized DNA can be quantitatively controlled by varying the parameters used for spin-coating the copolymer film, that is, solution concentration and rotational speed, or by varying the copolymer molecular weight. We find the yield of the DNA coupling reaction to be dependent on the nature of the polymer underlying the reactive alkyne functional groups, being higher for more hydrophilic polymers.

Introduction DNA microarrays have emerged as a powerful and universal tool for analysis of gene expression and polymorphism, for DNA sequencing and for gene discovery.1,2 DNA microarrays are solid substrates (e.g., glass) bearing different DNA probes immobilized at discrete locations. They are most frequently used to examine DNA hybridization3 and can generally be produced by two methods: direct on-chip synthesis of nucleic acids4 or attachment of presynthesized oligonucleotides that are chemically modified to allow for surface immobilization.5–8 The procedures for substrate activation and DNA attachment are well-established for glass,9–22 silicon wafers23–26 and gold surfaces.3,7,21,27–39 Polymeric substrates41–51 are an attractive alternative to glass and metal surfaces due to their low cost6,40 and their versatile chemical, physical, and surface properties. A number of factors have been identified that influence the DNA hybridization process in microarrays: nonspecific physisorption, probe density, probe orientation and layer structure, ionic strength and concentration of target solution, and temperature.3,7,8,14,29,31,35,38,52–61 Probe density, the focus of this publication, plays a crucial role in the extent of target hybridization and is a key parameter in hybridization kinetics.35,53,60,62 Typically, DNA probe films are characterized by an areal density of between 1012 and 1013 probes/cm2.31,54 Experiments indicate that surface hybridization is suppressed when the probe areal density is too high,14,29,31,35,54,58,59 a phenomenon that has been attributed to steric and electrostatic hindrances between closepacked DNA molecules. Optimal probe densities have been reported as 2 × 1012 ∼ 5 × 1012 probes/cm2 for gold substrates3,29,31,35,36,52–54,58–60,63,64 and 1.2 × 1011 to 2 × 1013 probes/cm2 for glass or polymeric substrates.5,6,9,12,14,15,24,53,59,61,65 The diversity of reported behavior reflects the large variety of * To whom correspondence should be addressed. E-mail: jk1191@ columbia.edu (J.T.K.); [email protected] (J.J.). † Department of Chemical Engineering. ‡ Columbia Genome Center.

immobilization protocols used in the construction and application of DNA microarrays. It is clear from these experiments, however, that the surface density of immobilized DNA must be precisely controlled in order to optimize microarray performance. A variety of strategies have been reported to prepare functional polymer surfaces for quantitative DNA immobilization: surface-initiated atom transfer radical polymerization (ATRP) of functional polymer brushes,42,66 pulsed-plasma polymerization of amino-derivatized polymers,67 and poly(L-lysine)graft-poly(ethylene glycol) [PLL-g-PEG] coated onto glass.68 The areal density of immobilized DNA is a function of the areal density of surface functional groups as well as the efficiency of coupling/bioconjugation reactions to those groups. Traditional nucleophilic-electrophilic reactions used to immobilize oligonucleotides are susceptible to side reactions that both reduce the yields and can render the yields irreproducible.69 Recently, a chemoselective approach using 1,3-dipolar azide-alkyne cycloaddition chemistry (also termed Sharpless “click” chemistry)70 has been applied to the construction of DNA probe surfaces.69,71,72 This strategy is highly predictable, rapid, and highly selective due to the intrinsic properties of “click” reactions. In this report, we demonstrate a simple means for covalent immobilization of DNA onto solid and polymeric substrates that provides quantitative control of the DNA areal density. The approach is based upon synthesis of a block copolymer terminated with alkyne groups capable of coupling azidefunctional DNA by surface “click” reactions. The block copolymer self-assembles to form a bilayer on the substrate and thereby directs reactive alkyne groups to the surface as depicted in Figure 1. The areal density of reactive alkyne groups, σ is linearly dependent on the copolymer film thickness, t (σ ) FtNa/ M, where Na is Avogadro’s number, and F and M are the density and molecular weight of the copolymer, respectively), which is controlled by adjusting the solution concentration, c, or

10.1021/bm800258g CCC: $40.75  2008 American Chemical Society Published on Web 08/05/2008

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Figure 1. Schematic of the bilayer structure of (1) spin-on alkyne-end-functional diblock copolymers and (2) the immobilization of azido-functionalized DNA on alkyne-functionalized surfaces via “click” chemistry.

rotational speed, ω, used in spin coating the block copolymer thin film (t ∼ ω-1/2η1/3c, where η is the viscosity of the solution).73,74 The areal density of reactive sites for DNA immobilization can therefore be controlled by either varying the spin coating conditions or the copolymer molecular weight. The block copolymer contains a photoactive sequence adjacent to the alkyne group that allows us to examine the influence of surface hydrophilicity or polarity on the efficiency of the DNA immobilization reaction. When exposed to UV radiation the photoactive polymer is converted from a hydrophobic to a hydrophilic material.75 The effects of surface hydrophilicity on nonspecific adsorption and the signal-to-noise ratio of fluorescence-based DNA microarray sensors are also reported.

Scheme 1. Synthesis of R-Alkyne-ω-Br-poly(tBA-b-MMA) by a Two-Step Metal-Catalyzed Living Radical Polymerization (LRP)

Experimental Section Materials. All materials were purchased from Aldrich and used as received, unless otherwise noted. tert-Butyl acrylate (tBA) and methyl methacrylate (MMA; 99+ % purity) monomers were passed through a basic Al2O3 chromatographic column (flash) to remove inhibitor. Toluene was purchased from Acros (99.8%) and used as received. Diblock Copolymer Synthesis. A diblock copolymer comprising a Pt BA sequence, a PMMA sequence, one terminal bromine and one terminal alkyne, R-alkyne-ω-Br-poly(tert-BA-b-MMA), was synthesized by metal-catalyzed living radical polymerization (LRP).76 The copolymer synthesis required three steps: preparation of the alkynefunctional (alkyne-Br) LRP initiator; polymerization of the poly(tBA) block to form R-alkyne-ω-Br-poly(tBA); and polymerization of the poly(MMA) block to generate R-alkyne-ω-Br-poly(tBA-b-MMA). The initiator, propanoic acid, 2-bromo-2-methyl-2-propynyl ester, was prepared following a reported procedure.77 The procedure for R-alkyneω-Br-poly(tBA) synthesis was as follows: monomer (tBA, 2.0 g, 15.60 mmol), solvent (benzene, 1.5 mL), initiator (propanoic acid, 2-bromo2-methyl-2-propynyl ester, 32.0 mg, 0.016 mmol), catalyst (CuBr, 2.5 mg, 0.016 mmol), and ligand (N, N, N′, N′, N′′- pentamethyldiethylenetriamine [PMDETA], 99%, 42.1 mg, 0.03 mmol) were weighed directly into a 25 mL Schlenk tube. After three freeze-pump-thaw cycles, the tube was filled with argon, and the reaction mixture was heated at 70 °C in an oil bath. The sidearm of the tube was purged with argon for at least 5 min before it was opened for samples to be removed at predetermined times with an airtight syringe. Samples were dissolved in CDCl3, and the conversion was measured by 1H NMR spectroscopy. The number and weight average molecular weights were measured by GPC relative to PS standards. Once the desired conversion was achieved, the Schlenk tube was removed from the oil bath, was allowed to reach room temperature, and the polymerization mixture was diluted with CH2Cl2. This solution was passed through a basic alumina flash column and the catalyst-free mixture was collected. Solvent was removed under reduced pressure using a rotary evaporator. Polymer was recovered via filtration after precipitation of concentrated

polymer solution (CH2Cl2) with a MeOH/H2O mixture (7:3 v/v). The procedure for R-alkyne-ω-Br-poly(tBA-b-MMA) synthesis was as follows: monomer (MMA, 1.78 g, 17.77 mmol), solvent (toluene, 3.0 mL), initiator (R-alkyne-ω-Br-Pt BA polymer, 0.49 g, 0.04 mmol), catalyst (CuBr, 10 mg, 0.06 mmol), and ligand (PMDETA, 78.0 mg, 0.54 mmol) were weighed directly into a 25 mL Schlenk tube. After three freeze-pump-thaw cycles, the tube was filled with argon, and the reaction mixture was heated in an oil bath kept at 90 ( 2 °C. Under an argon atmosphere, polymerization samples were removed, dissolved in CDCl3, and the conversion was measured by 1H NMR spectroscopy. A part of the solution was reserved for GPC analysis. Once the desired conversion was achieved, the reaction was quenched by cooling followed by dilution with CH2Cl2. This solution was passed through a basic alumina flash column and the catalyst-free mixture was collected and solvent was removed under reduced pressure, using a rotary evaporator. Polymer was recovered via filtration after precipitation of concentrated polymer solution (CH2Cl2) in hexanes. The overall synthesis procedure is shown in Scheme 1. Molecular weights and compositions of the Pt BA block and the overall block copolymer are shown in Table 1. DNA Synthesis. The azido-functional, FAM-labeled 20mer DNA used in this study, 5′-azido-(CH2)5-CONH-(CH2)6-CGCGTCAGTCATATGCACGT-FAM-3′ (N3-DNA-FAM) was prepared according to the following procedures: 5′-amino-modified, 3′-FAM-labeled 20mer DNA (5′-amino-(CH2)6-CGCGTCAGTCATATGCACGT-FAM-3′) was synthesized on a DNA synthesizer (Expedite 8909, Applied Biosystems,

Copolymers for Quantitative DNA Immobilization


Table 1. Molecular Weights and Compositions of End-Functional Pt BA and End-Functional Diblock Copolymer material

Mn (GPC)

Mn (NMR)

Mw/Mn (GPC)

φPt BA

R-alkyne-ω-Br-poly(t BA) R-alkyne-ω-Br-poly(t BA-b-MMA)

12000 62000

11100 n.a.

1.42 1.20

1.0 0.21

Scheme 2. Photochemical Deprotection of R-Alkyne-ω-Br-poly(t BA-b-MMA) under UV Light in the Presence of a PAG

CA) by phosphoramidite chemistry with a set of dAbz, dGdmf, dCbz, and dT-CE phosphoramidite nucleosides (Glen Research, Sterling, VA). The resulting amino-modified, FAM-labeled DNA was coupled with 6-azidohexanoic acid succinimidyl ester in DMSO, 0.1 M Na2CO3/ NaHCO3 buffer (pH 8.7) for 6 h at room temperature to afford 5′azido-modified, 3′-FAM-labeled DNA. The resulting azido-modified, FAM-labeled DNA was purified by size-exclusion chromatography and desalted with an oligonucleotide purification cartridge, followed by vacuum drying. The dried crude DNA product was further purified with reverse-phase HPLC using a C-18 reverse column (Xterra MS C18, 4.6 mm × 50 mm). The fraction containing the desired product was collected and evaporated to dryness under vacuum. The final product was characterized by MALDI-TOF MS: calcd, 6978; found, 6979 [(M + 1), m/z]. Spin-On Film Preparation. Glass (glass slide, 75 mm × 25 mm) and silicon (silicon wafer) substrates were cleaned by soaking in piranha solution (1:3 30% H2O2/H2SO4) for 30 min, followed by an extensive rinse with deionized water. Caution: “Piranha” is a Very strong oxidizer and reacts Violently with many organic materials. Cleaned substrates were stored in water and dried with a stream of nitrogen before use. Silicon substrates were further cleaned by exposure to UV/ozone for 15 min before use. R-alkyne-ω-Br-poly(tBA-b-MMA) was dissolved in toluene to make a series of solutions with different concentrations (in the range of 0.1∼0.8 g polymer/100 g solution). Two control polymers Pt BA (Mn 14400, Mw/Mn 1.06, Polymer Sources, Inc.) and PMMA (Mn 15000, Mw/Mn 1.16, Polymer Sources, Inc.) were dissolved in toluene to make a solution of 1.0 g polymer/100 g solution. The solutions were spin-coated onto glass slides (for contact angle, XPS and fluorescence analyses) or silicon wafers (for XPS, ellipsometry and AFM analyses) at 2500 rpm for 1 min. The spin-coated films were annealed in a vacuum oven at 130 °C for 8 h. Surface Photochemical Deprotection. The photochemical deprotection of R-alkyne-ω-Br-poly(tBA-b-MMA)72 is shown in Scheme 2. The Pt BA copolymer sequence is hydrolyzed or deprotected to poly(acrylic acid) (PAA) by exposure to UV light in the presence of a photoacid generator (PAG).72 A layer of PAG (BOC-methoxy phenyldiphenyl sulfonium triflate, λmax ) 255 nm, purchased from Aldrich) was spin-coated (1000 rpm, 1 min) on top of R-alkyne-ω-Br-poly(tBAb-MMA) spin-coated films on glass substrates from a solution of PAG in ethanol (1.5% w/w). The PAG/copolymer/glass sample was placed under a hand-held UV lamp and exposed to deep UV light (254 nm wavelength) for 20 min. If necessary, the UV-exposed sample was postbaked at 90 °C for 30 s to a few minutes to facilitate diffusion of

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the generated acid and thereby increase the degree of tert-butyl ester deprotection. Excess PAG was washed away by ethanol after which the sample was dried with nitrogen. DNA Immobilization via “Click” Reaction and DNA Nonspecific Physisorption. 1,3-Dipolar azide-alkyne cycloaddition chemistry was used to immobilize N3-DNA-FAM onto either the hydrophobic R-alkyne-ω-Br-poly(tBA-b-MMA) coated glass surface or the hydrophilic R-alkyne-ω-Br-poly(AA-b-MMA) coated surface which was obtained by the surface photochemical deprotection of R-alkyne-ωBr-poly(tBA-b-MMA), as illustrated in Scheme 2. A coupling solution was prepared by mixing tetrakis-(acetonitrile) copper (I) hexafluorophosphate [CuI(CH3CN)4PF6] (2 mM in DMSO), tris-(benzyltriazolylmethyl) amine (TBTA; 2 mM in DMSO), sodium ascorbate (2.6 mM in H2O) and N3-DNA-FAM (50 µM in H2O) with a volume ratio of 2:2:2:3. The solution was then spotted onto hydrophobic or hydrophilic alkyne-functionalized surfaces in the form of 9 µL drops (with a spot size of about 3 mm in diameter). The spotted surface was incubated in a humid chamber at room temperature for 1 h in the absence of light. The surface was rinsed with copious amounts of deionized water and DMSO to remove unbound DNA and residual coupling agents and was finally dried with dry nitrogen before immediate fluorescence measurement. Control experiments were always conducted to monitor the nonspecific DNA physisorption on the same surface on which DNA was immobilized via “click reaction”. The coupling solution for the control experiments was the same as that for “click” reactions except that the catalyst, CuI(CH3CN)4PF6, was not included. DNA Quantification. The method for DNA quantification based on fluorescence measurements has been reported in the literature.6 Azido-functionalized, FAM-labeled 20mer DNA (N3-DNA-FAM) was diluted in H2O to a dilution series ranging from 0.005 to 2 µM DNA. A total of 10 replicas of each dilution in the form of a 9 µL drop were spin-coated on cleaned glass slides (75 mm × 25 mm) at 500∼1000 rpm for 1∼2 min. The fluorescence intensity of the spots was measured by fluorescence scanner and quantified using ScanArray Express software to obtain a standard curve for the DNA probes. After DNA immobilization via “click” reaction, the fluorescence signal of the spot was measured and the areal density of the immobilized DNA was extracted from the calibration curve. For each experimental condition, the experiment was repeated 3∼5 times and the immobilization data presented are the average of these repetitions. Instrumental Techniques. 1H NMR (400 MHz) spectra were recorded on Bruker-DRX400 spectrometer at 20 °C in CDCl3 with tetramethylsilane (TMS) as internal standard. Gel permeation chromatography (GPC) analysis was performed on a Shimadzu LC-10AT high pressure liquid chromatograph equipped with: a CTO-10 Å column oven (40 °C) hosting two Waters gel columns (Styragel HT2 and Styragel HT4), a Shimadzu RID-10A RI detector, and a SPD-10A UV-vis detector (254 nm). HPLC was performed on a Waters system consisting of a Rheodyne 7725i injector, 600 controller, and a 996 photodiode array detector calibrated with polystyrene standards. Mass measurement of DNA was made on a Voyager DE matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Applied Biosystems). Sessile drop contact angle measurement was carried out at room temperature with a model 100-00 contact angle goniometer (Rame-Hart, Inc.). The contact angles were recorded immediately after dispensing a 5 µL water or ethylene glycol droplet with a pipet onto the surface. Measurements were performed three times in different locations on the surface of at least three samples and averaged. Film thickness was measured with a Beaglehole ellipsometer (Beaglehole Instruments, Wellington, New Zealand) in variable angle mode. A refractive index of 1.473 was used for all polymer samples. Measurements were performed three times in different locations on the surface of at least three samples and averaged. Angle-dependent X-ray photoelectron spectroscopy (ADXPS) spectra were recorded with a PHI 5500 spectrometer equipped with a hemispherical electron energy analyzer, a multichannel detector, and an Al KR monochromator X-ray source run at 15 kV and 23.3 mA. The working pressure of the

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instrument was maintained below 2 × 10-9 Torr. Survey and highresolution spectra were acquired with pass energies of 93.9 and 23.5 eV, respectively. No beam damage was induced on the sample surface. At least three samples were analyzed and the results were averaged. The scanned fluorescence images were recorded on ScanArray Express scanner (Perkin-Elmer Life Sciences). Each microarray was scanned at 5 µm resolution using the laser with the excitation wavelength of 488 nm and the emission filter centered at 522 nm.

Results and Discussion Our strategy for quantitative DNA immobilization requires four steps: (1) design and synthesis of a diblock copolymer comprising a surface-active copolymer sequence that is also photoactive and is terminated with a reactive alkyne group; (2) delivery of the reactive functional groups to solid substrates by spin-coating a thin block copolymer film; (3) covalent immobilization of DNA onto the copolymer modified surfaces; and in some cases, (4) photochemical deprotection of the surface-active copolymer block underlying the functional groups to change that block from hydrophobic to hydrophilic (performed before DNA immobilization). Design of the block copolymer is straightforward.75 The first block must adhere strongly to glass or silicon substrates or interpenetrate with polymeric substrates to provide an anchoring function. The second block is surface-active and causes the copolymer to self-assemble at the surface presenting a surface layer of the surface-active block. Since the second block is terminated with the functional group, self-assembly delivers the functional groups to the surface as illustrated in Figure 1. It is desirable that the anchor block have a glass transition temperature above 100 °C to survive the rigors involved with applications of DNA arrays.40 Poly(methyl methacrylate) (PMMA; Tg, 105 °C; surface tension, 44 mN/m)79 was chosen as the anchor block because it has a high Tg, high surface tension, and a strong interaction with glass or silicon substrates. Because a PMMA anchor block is used, the copolymer can also be used to modify the surfaces of PMMA homopolymer, a promising polymeric substrate that has been successfully used for the immobilization of enzymes,80 peptides, and DNA.6,81,82 Poly(tert butyl acrylate) (Pt BA; Tg, 40 °C; surface tension, 30 mN/m)75,79 was chosen as the surfaceactive block in our design because it has a low surface tension, low Tg, and is photoactive. Pt BA can be converted to poly(acrylic acid), PAA, by exposure to light in the presence of a PAG to allow for the control of surface hydrophilicity.72 The block copolymer concept is very versatile. Other polymers containing “protected” functional groups could also be used as the surface-active block because the protecting groups commonly used, such as trimethylsilane and tert-butoxy, render the polymers to be of low surface tension. The surface tension of alkyne-end-functional groups, CHtCsCH2sOsC(dO)sC[(CH3)2]sCH2s, was calculated by the group contribution method (see Supporting Information) as 26.0 mN/m. Because the alkyne groups have a lower surface tension than that of the Pt BA backbone, they are expected to be enriched in the topmost surface region78 and to be readily accessible for the surface click reaction with azido-functionalized DNA for immobilization. The synthesis of R-alkyne-ω-Brpoly(tBA-b-MMA) block copolymer is shown in Scheme 1. Molecular weights (Mn) of Pt BA and PMMA blocks were measured by GPC as 12k and 50k, respectively; the volume fraction of Pt BA in the block copolymer, φPt BA, was calculated to be 0.21, as shown in Table 1.

Figure 2. Ellipsometry measurements of overall thickness of R-alkyneω-Br-poly(t BA-b-MMA) block copolymer as a function of solution concentration.

Figure 3. Water contact angle (filled circles) and ethylene glycol contact angle (open circles) of R-alkyne-ω-Br-poly(t BA-b-MMA) block copolymer as a function of spin coating solution concentration.

The second step of our strategy for quantitative DNA immobilization involves the delivery of alkyne-end-functional groups to solid substrates and the subsequent control of their areal density. Spin-coating was chosen to fabricate block copolymer thin films because it allows for precise control of the film thickness and therefore the surface areal density of alkyne groups.73,74 The ability to precisely control the overall thickness of the R-alkyne-ω-Br-poly(tBA-b-MMA) block copolymer is illustrated in Figure 2, where the overall copolymer thickness measured by ellipsometry is shown as a function of the concentration of solution used for spin-coating. The overall block copolymer thickness increased linearly with the solution concentration, as predicted by the relationship:73 t ∼ ω-1/2η1/3c, where t is the film thickness, ω, η, and c are rotational speed, solution viscosity and solution concentration, respectively. Preferential segregation of the low-surface-tension Pt BA block to the surface is confirmed by the water and ethylene glycol contact angle data shown in Figure 3. The measured water contact angles fall between those of the PMMA (72°) and Pt BA (90°) controls as do those for ethylene glycol (55° and 73° for PMMA and PtBA controls, respectively). As the polymer concentration increases, the water contact angle increases until it reaches a plateau value close to that of pure Pt BA. This result signals the formation of a pure Pt BA surface layer at a concentration of about 0.3%. The spatially averaged volume fraction of Pt BA at the surface, φS, Pt BA, was calculated from ADXPS measurements



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Table 2. ADXPS Measurements of the Elemental Atomic Percentagea of R-Alkyne-ω-Br-poly(t BA-b-MMA) Copolymers, Pt BA, and PMMA Homopolymers polymer sample copolymer

solution concentration

take-off angle, θ

C

O

Si

φS,Pt BA

0.1%

10° 45° 75° 10° 45° 75° 10° 45° 75° 45° 45°

76.5% 66.6% 57.2% 77.2% 72.3% 69.1% 77.7% 74.2% 73.3% 77.7% 71.6%

23.5% 26.2%b 27.7%b 22.8% 25.6%b 26.2%b 22.3% 25.8% 26.7% 22.3% 28.4%

ND 8.1% 15.1% ND 2.0% 4.7% ND ND ND ND ND

0.82

0.3% 0.5% pure Pt BAd pure PMMAd

1.0% 1.0%

0.92 0.42c 0.99 0.47 0.33 1 0

a The error in the elemental composition is less than (5%. Si signals are from the silicon substrate. ND: not detected. b Oxygen concentration includes contributions from both the copolymer and the oxidized layer of the silicon substrate. c φS,Pt BA is slightly skewed by the contribution of a small amount of oxygen in the substrate. d Pt BA and PMMA are control samples.

Figure 5. Proposed schematic structures for poly(t BA-b-MMA) diblock copolymer films prepared from different concentrations of spin-coating solutions (Pt BA, solid lines in boxes; PMMA, dotted lines). Figure 4. ADXPS (filled circles) and ellipsometry (open circles) estimation of the apparent thickness of Pt BA as a function of spin coating solution concentration of R-alkyne-ω-Br-poly(t BA-b-MMA) copolymer.

of the C1s to O1s atomic ratio according to eq 1, where 128 is the molecular weight of tBA repeat unit, 100 is the molecular weight of MMA repeat unit, 1 is the density of Pt BA and 1.17 is the density of PMMA.79

φS,PtBA × 1 (1 - φS,PtBA) × 1.17 ×7+ ×5 C 128 100 ) O XPS φS,PtBA × 1 (1 - φS,PtBA) × 1.17 ×2+ ×2 128 100

()

(1)

φS, Pt BA represents the average volume fraction over the sampling depth. Table 2 contains a summary of the ADXPS results. The Si signal from the silicon substrate was detected on thinner films (at the polymer concentration of 0.1 and 0.3%) but not on thicker films (at the concentration of 0.5%). At the same takeoff angle, φs,Pt BA is higher in a thicker film. ADXPS was also applied to determine the apparent thickness of Pt BA and to confirm the formation of a layered structure. The apparent thickness of Pt BA estimated from the ADXPS substrate-overlayer model is shown in Figure 4 (filled circles). The development of the ADXPS substrate-overlayer model is described in detail in the Supporting Information and elsewhere.75,83 The apparent thickness of Pt BA can also be estimated from ellipsometry measurements because the volume fraction of Pt BA is known to be 0.21. Figure 4 shows that the Pt BA thicknesses estimated by both methods are in good agreement over the concentration range of 0-0.5%, but that a strong discrepancy is observed for higher concentrations. This behavior indicates that a saturated monomolecular film of block copolymer forms at about 0.5%.

When the polymer concentration exceeds 0.5%, the block copolymer forms a multilayered structure and neither the ADXPS substrate-overlayer model nor the ellipsometry calculations of the tBA thicknesses are valid. While the data above 0.5% are not accurate, they do reflect the change in surface layer morphology. The thicknesses estimated by ADXPS depend on the value of the inelastic mean free path adopted for analysis of the C1s spectra. A value of 3.1 nm84 was employed in our calculation. Within the range 0.4-0.5%, the ADXPS-estimated thickness of Pt BA is about 2.4-3.1 nm, close to the radius gyration of the Pt BA block (2.7 nm).75 This result indicates that Pt BA sequences are configured as wet polymer brushes in the saturated monomolecular layer. Figure 5 gives schematic representations of the layered structures we believe are manifest for different spin coating concentrations. The third step of our strategy for quantitative DNA immobilization is the covalent coupling of DNA onto the surface. Azido-terminated, FAM-labeled 20mer DNA (N3-20mer-FAM) was immobilized to R-alkyne-ω-Br-poly(tBA-b-MMA) block copolymer surfaces via a “click” reaction. Figure 6 shows the surface areal density of immobilized DNA quantified by fluorescence measurements (filled circles) and the surface areal density of alkyne groups calculated from ellipsometry measurements (open circles) as a function of the spin-coating solution concentration. The surface areal density of alkyne groups (σ) are calculated from the overall thickness of the copolymer film (t, measured by ellipsometry) through the relationship σ ) FtNa/ M, where Na is Avogadro’s number and F and M are the respective density and molecular weight of the block copolymer.74 Both alkyne and DNA surface areal densities increase with solution concentration from 0.2 to 0.5%. The linear regression equations are σ [10-12 × molecules/cm2] ) 40.3c for the alkyne surface density (R ) 0.9916) and σ [10-12 × molecules/cm2] ) 20.6c for immobilized DNA (R ) 0.9675).

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Figure 6. Areal density of covalently bound DNA (filled circles) and areal density of alkyne groups (open circles) as a function of spincoating solution concentration of R-alkyne-ω-Br-poly(t BA-b-MMA) block copolymer. The lines represent the result of linear regression for data points up to 0.5%. Table 3. Water Contact Angle (WCA) Measurements of Pt BA, PAA, and R-Alkyne-ω-Br-poly(t BA-b-MMA) Block Copolymer Before and After Photochemical Deprotection R-alkyne-ω-Br-poly(t BA-b-MMA)a Pt BA

before deprotection

after deprotection

PAA

90 ( 1°

88 ( 3°

38 ( 3°

35 ( 3°

a

The spin-coating solution concentration is 0.5%.

When the concentration exceeds 0.5%, the immobilized DNA density falls off, the disordered copolymer multilayers cannot present their alkyne groups at the surface. The data illustrate that a single block copolymer is capable of controlling DNA areal density over a range of ∼4-10 × 1012 molecules/cm2. This range corresponds well to that (i.e., 1011-1013) reported for optimal DNA probe densities and could be further broadened by varying the block copolymer molecular weight. The fourth and final step in our strategy for quantitative DNA immobilization involves the photochemical deprotection of the hydrophobic Pt BA to form a hydrophilic poly(acrylic acid) (PAA) polymer underlying the alkyne functional groups as shown in Scheme 2. The conversion of Pt BA to PAA was confirmed with the water contact angle measurements shown in Table 3. Before deprotection, the contact angle was 88 ( 3°, equivalent within error to that of pure Pt BA (90 ( 1°); after deprotection, the contact angle dropped to 38 ( 3°, equivalent within error to that of pure PAA (35 ( 3°). DNA (N3-20mer-FAM) was immobilized through “click” reactions to both the hydrophobic and the hydrophilic surfaces and the scanned fluorescence images are shown in Figure 7 (top left, bottom left). Control experiments are also shown (top right, bottom right). In control experiments, CuI(CH3CN)4PF6, the catalyst for “click” reactions, was not included in the coupling solution. The fluorescence signals detected for the unreactive controls can arise only from physisorbed DNA. The figure shows that DNA physisorption is low on both the hydrophobic and hydrophilic surfaces, but slightly lower for the hydrophilic surface (fluorescence intensity is 160 ( 30 on the hydrophilic surface and 190 ( 30 on the hydrophobic surface) which can take on a negative charge that can repel negatively charged DNA. The areal density of covalently bound DNA is higher for the hydrophilic surface (bottom left, with the fluorescence intensity of 9000 ( 1500, corresponding to (1.3 ( 0.2) × 1013 DNA strands/cm2) than for the hydrophobic surface (top left, with the fluorescence intensity of 7400 ( 1950,

Figure 7. Scanned fluorescence images of DNA (N3-20mer-FAM) immobilization on hydrophobic (top) and hydrophilic (bottom) surfaces via “click” reaction (left) and nonspecific physisorption (right). The spincoating solution concentration is 0.5%.

corresponding to (1.1 ( 0.3) × 1013 DNA strands/cm2). The immobilization efficiency increases by converting the hydrophobic Pt BA surface to a hydrophilic PAA surface. At present, we can only speculate as to the origin of this effect; clearly additional experiments are necessary to better understand its origins. We believe this to be the first data to illustrate that the nature of the material underlying surface functional groups can influence the yield of covalent DNA immobilization.

Conclusions Spin-coated films of an alkyne-end-functional block copolymer constitute an effective means to quantitatively control the immobilization density of DNA on glass, silicon, and polymeric substrates. The alkyne groups provide sites for covalent attachment of azide functional DNA by means of a surface “click” reaction. When a single block copolymer formulation was used, it was possible to quantitatively control the density of immobilized DNA over a range of 4.8 × 1012-1.3 × 1013 probes/ cm2, by simply varying the concentration of solutions used to prepare spin coated films of the block copolymer. The areal densities could also be controlled through variation of the block copolymer molecular weight or the rotational speed of the spin coater. The range of probe density available with the spin coating method matches well with the experimental range of 1.2 × 1011-2 × 1013 probes/cm2 reported for optimal hybridization of DNA (i.e., 20mers), that is, covalently immobilized on glass or polymeric supports. We also find that the efficiency of the DNA surface immobilization reaction is dependent on the nature of the polymer underlying the surface functional groups and is higher for higher polymer hydrophilicity. The ability to readily control the surface density of immobilized DNA provides a simple means to optimize the degree of target DNA hybridization and is generally useful for the quantitative design of other biological microarray sensors as well. Acknowledgment. This work was supported by NIH Grants P50 HG002806 and R01 HG003582. H.R.R. acknowledges partial support by a GEM fellowship. Supporting Information Available. Calculation of surface tension of alkyne-end-functional groups by the group contribution method and estimation of Pt BA surface layer thicknesses


by the ADXPS substrate-overlayer model. This material is available free of charge via the Internet at http://pubs.acs.org.

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Spin-On End-Functional Diblock Copolymers for Quantitative DNA

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