Precisely Controlled Smart Polymer Scaffold for Nanoscale

Nov 23, 2012 - ABSTRACT: We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA...
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Precisely Controlled Smart Polymer Scaffold for Nanoscale Manipulation of Biomolecules Philipp S. Spuhler,† Laura Sola,§ Xirui Zhang,† Margo R. Monroe,† Joseph T. Greenspun,‡ Marcella Chiari,§ and M. Selim Ü nlü*,†,‡ †

Department of Biomedical Engineering, ‡Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts 02215, United States § Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milan, Italy S Supporting Information *

ABSTRACT: We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA) and the conformation of a polymer scaffold through variation in buffer pH and ionic strength. An amphoteric poly(dimethylacrylamide) based coating containing weak acrylamido acids and bases, which are copolymerized together with the neutral monomer, is covalently bound to the surface. The coating can be made to contain any desired amount of buffering and titrant ionogenic monomers, allowing control of the surface charge when the surface is bathed in a given buffer pH. Spectral self-interference fluorescence microscopy (SSFM) is utilized to precisely quantify both the DNA orientation and the polymer conformation with subnanometer resolution. It is possible to utilize the polymer scaffold to functionalize a variety of common materials used in microfabrication, making it a general purpose building block for the next generation of nanomachines and biosensors.

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These ionogenic monomers make it possible to design the coating to achieve a controlled surface charge for a given buffer pH. A third monomer, N-acryloyloxysuccinimide, is added to the polymer to allow the covalent binding of end amino modified dsDNA molecules to the polymer backbone. We utilize spectral self-interference fluorescence microscopy (SSFM), which permits axial localization of fluorophore tags on DNA probes with subnanometer resolution13,14 to precisely quantify both the DNA orientation and the polymer conformation. The basis of the system is an SSFM chip consisting of a transparent optical spacer on a reflecting surface (in this case, 17.5 μm thermally grown silicon dioxide on a polished silicon substrate), which is functionalized with the smart polymer scaffold. The monomer’s structure and the schematic representation of the polymer network are depicted in Figure 1. The acidic Immobiline used in this work contains a weak carboxyl group with a pKa value of 3.6 while the basic Immobiline contains a tertiary amino group with a pKa value of 8.5, and ionization and protonation of Immobilines result in a pH dependent net charge of the dimethylacrylamide coating, Figure 1B. The pH dependent polymer charge was characterized by measuring electro-osmotic flow (EOF) in a capillary coated by a polymer with identical composition. EOF, which depends on the surface charge density, was found to be negligible around pH 6, indicating that, at this pH

ynthetic molecular motors, and especially DNA motors, have great potential in various applications including molecular sensing and intelligent drug delivery.1The basic mechanisms by which many DNA nanomachines induce motion is through conformation changes in DNA in response to environmental changes in pH2−4 or ionic strength5 or through the binding of signaling molecules.6 Such nanomachines were previously used to induce bending of cantilevers7 or as gates for synthetic nanopores.8 Other platforms utilize orientation changes of surface immobilized DNA, termed “DNA switching”, which are induced by the modulation of an electric potential to control the surface charge of the sensor.8 The characteristic behavior of the DNA switching, such as the switching amplitude (the difference between upright and horizontal orientations) or switching dynamics are influenced by buffer pH, ionic strength, and temperature.9,10 Additionally, the switching amplitude is influenced by conformation changes of the immobilized DNA, making it possible to detect DNA hybridization or denaturation11 or to utilize the platform for high-throughput characterization of the DNA sequence dependence on conformation changes that are induced by DNA binding proteins such as transcription factors.12 We present a molecular motor, which consists of short double stranded DNA (dsDNA) probes anchored on a smart polymer surface. Both the dsDNA orientation and the conformation of the polymer scaffold are precisely controlled through the variation in the buffer pH and ionic strength. The pH dependence is achieved through synthesis of the surface coating through copolymerization of dimethyl acrylamide, with weak acrylamido acids and bases that go under the trade name of Immobilines. © 2012 American Chemical Society

Received: July 1, 2012 Accepted: November 23, 2012 Published: November 23, 2012 10593

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Figure 1. (A) The acidic Immobiline (pKa 3.6) contains a weak carboxyl group and the basic Immobiline (pKa 8.5) contains a tertiary amino group. Ionization and protonation of Immobilines results in a pH dependent net charge. (B) These ionizable groups are incorporated in a smart dimethyl acrylamide scaffold. (C) At low pH, the tertiary amino groups are protonated and the polymer is positively charged; an increase in the pH results in ionization of the carboxyl groups and the net polymer charge decreases; at high pH, tertiary amino groups are deprotonated and the polymer charge becomes increasingly negative.

However, this surface coating can be formulated to control the pH value at which the surface charge transitions. This design parameter is executed through an adjustment in the concentration and ratio of the buffering and titrating monomers.

value, the positive and negative charges are balanced and the surface is neutral. In order to observe polymer conformation and to demonstrate controlled orientation of the dsDNA, dsDNA probes containing fluorophore tags on the surface distal or the surface proximal ends were end grafted onto the smart polymer scaffold. This was achieved through covalent binding between the amine modification on the 60 base pair long dsDNA probes and the N-hydroxysuccimde esters (NHS-ester) in the polymer. The dsDNA probe orientation is controlled through pH dependent charging of the polymer scaffold, which can be made to contain any desired amount of buffering and titrating monomers. The chemistry of these monomers is well-known,15,16 as these compounds have been used for electrophoretic separations in the technique called “isoelectric focusing in immobilized pH gradients”.17 The polymer behaves like a buffer with pH and buffering capacity defined by the concentration and ratio of buffering and titrating acrylamide monomers. When the polymer scaffold is in plain water, an excess of positive or negative charges on the polymer is neutralized by the excess of hydroxyl ions or protons in the solution, thus determining the pH of the hydrogel solution. When a buffer that overcomes its buffering capacity surrounds the polymer scaffold, the scaffold acquires a net negative or positive charge, depending on the buffer pH. Negatively charged polymer (buffer pH > pIpolymer) repels dsDNA, and the positively charged polymer (buffer pH < pIpolymer) attracts dsDNA to induce an upright or horizontal probe orientation, respectively. The surface coating presented in this paper transitions from a positive to a negative surface charge at neutral pH.

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EXPERIMENTAL SECTION SSFM Measurement Setup. The principle of operation, the experimental setup for data acquisition, and the analysis of spectra for subnanometer localization of fluorophore heights by SSFM were described previously.18 Briefly, a continuous wave laser (HeNe 633 nm) is focused on the sample by a 0.13 NA microscope objective, and the fluorophore emissions are collected through the same objective and coupled to a spectrometer (spectral resolution of 3 cm−1 at 700 nm). The spectral data are then fitted using custom software, which gives the distance between the fluorophores and the Si/SiO2 interface. The SSFM samples were purchased from Silicon Valley Microelectronics Inc. and consist of single side polished silicon with a top layer of 17.5 μm thick thermally grown silicon dioxide. An overview of the SSFM measurements is shown in Figure 2. Short 60 base pair long dsDNA are immobilized on the SSFM chip. Half the probes are modified with Atto647N fluorophores on the surface distal end, and the remaining probes are modified with the fluorophores on the surface proximal end. The ensemble fluorophore heights are initially measured in a dry environment. Under these conditions, it is known that the polymer collapses on the sensor and the DNA assume a horizontal orientation.19 The axial positions of the fluorophores are then measured in a wet environment for various buffers, and the fluorophore heights are 10594

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the capillary, followed by the application of an electric field. Since an uncharged molecule has zero electrophoretic mobility, the velocity, at which this specie moves from the inlet to the UV detector, corresponds to the electro-osmotic velocity of the buffer.20 The EOF mobility was calculated as μ = (LD/tr)(Lt/V), where LD is the distance from the inlet reservoir to the detector, V is the voltage difference applied across the length of the capillary (Lt), and tr is the migration time. A P/ACETM MDQ capillary electrophoresis system from Beckman Coulter Inc. was used for EOF measurements. The fused silica capillary had an internal diameter of 75 μm and a length of 32 cm. The column temperature was 25 °C, the detection wavelength was 214 nm, the injection pressure was 0.3 psi for a duration of 2 s, and the applied voltage during separation was 15 kV with a measurement time of 60 min. The running buffers were diluted to achieve a final concentration of 25 mM, and the buffer composition is given in the previous section. The EOF measurements, as shown in Figure 3, show a negligible value at pH 6. Figure 2. The sensor surface is functionalized with an amphoteric 3D polymeric binding scaffold. Amine functionalized dsDNA probes are end-grafted to the polymer. (A) The ensemble height of fluorophore tags on short, 60 base pair long, surface anchored double stranded oligonucleotides (dsDNA) is first measured in a dry environment, with subnanometer resolution, using spectral self-interference fluorescence microscopy (SSFM). (B) The ensemble fluorophore heights are then measured in a wet environment. The surface becomes negatively charged for buffer with pH > 6.5, and dsDNA are repelled by the polymer to assume an upright orientation. The polymer itself is repelled by the negative charge residing on the oxide surface so that the mean height of the DNA anchor points is elevated. (C) The surface becomes positively charged for pH < 6.5, and the dsDNA are attracted to the polymer to assume a horizontal orientation. Under these conditions, the positively charged polymer collapses on the oxide surface.

calculated by taking the axial position of the fluorophores relative to the position in a dry environment. DNA Probe Immobilization. The dsDNA probes consist of a 60 base pair long oligonucleotide (purchased from IBA DNA Gmbh, Germany) with an Amine-C6 modification on the 5′ end hybridized to a complementary oligonucleotide with a Atto647N fluorophore modification on the 3′ end for the proximal dsDNA probes and a Atto647N fluorophore modification on the 5′ end for the distal dsDNA probes. The amine modified DNA sequence is: 5′ TCA TCG GTC AGG TGC AAC AAA TTG ATA AGC AAT GCT TTT TTG GCC CTA TCT TCT AAC AGC 3′. The probes were hybridized (30 μM dsDNA in 150 mM Na2HPO4/NaH2PO4, pH 8.5) and spotted (15 μM dsDNA in 150 mM NaPO4, pH 8.5) using a robotic spotter, immobilized for 3 h in 65% humidity at room temperature, blocked in ethanolamine buffer for 15 min at 50 °C (50 mM ethanolamine, 20 mM Tris, 100 mM NaCl), washed (twice in 2× SSC for 5 min at 50 °C, once in 0.2× SSC for 5 min), dried with argon, and left under vacuum overnight, prior to making dry measurements. SSC buffer is saline-sodium citrate buffer, which is commonly used in wash protocols for DNA microarrays. Electro-Osmotic Flow Measurements. The pH dependent polymer charge was characterized by measuring electroosmotic flow (EOF) velocity in a capillary coated with the same polymer used for the remaining experiments in the paper. EOF velocity was experimentally measured by injecting, hydrodynamically, a neutral marker (5 mM acrylamide in water) into

Figure 3. The electro-osmotic flow measurements indicate an isoelectric point between pH 5−6 for a fused silica surface coated with the smart polymer.

Polymer Synthesis. The basic structure of the polymeric scaffold is obtained by radical copolymerization of N,N-dimethylacrylamide with N-acryloyloxysuccinimide. This copolymer was modified by incorporating acidic (pKa 3.6) and basic (pKa 8.5) Immobilines during polymer synthesis. Immobilines are ionogenic monomers with the general formula CH2CH−CO−NH−R, where R denotes one of several ionizable groups with pKa in the 3.6−10.3 range, and these ionizable groups provide a net charge in the polymer that is dependent on the buffer pH.21 An overview for the reaction steps for polymerization of DMANAS-Immobiline polymer is shown in Figure 4. SSFM samples were first silanized with methacryloxypropyl-trimetoxysilane (MAPS). Following silanization of SSFM samples with MAPS, the samples are immersed in a solution consisting of 100 mL of N,N-dimethylformamide (DMF), 9.58 mL of dimethyl acrylamide (DMA; filtered on aluminum oxide to eliminate the inhibitor), 331 mg of NAS, 9.75 mL from a 200 mM stock solution of Immobiline, pKa 8.5, in isopropanol and 126 mg of Immobiline, pKa 3.6. The solution is degassed with argon, and (azobisisobutyronitrile) AIBN is added. This solution is then heated overnight at 65 °C. Each sample is then washed with 10595

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Figure 4. Reaction steps for polymerization of p-DMA-NAS-Immobiline (pKa 8.5) and Immobiline (pKa 3.6) amphoteric polymer.

Figure 5C shows the height changes of the fluorophore tags that result due to hydration of the platform. The mean height change observed in the surface proximal fluorophore tags, which are adjacent to the functional amine groups on the dsDNA probes, is a measure of the polymer conformation because the amine groups are covalently bound to the functional NHS-ester groups, which are distributed throughout the polymer. Similarly, the probe orientation under hydrated conditions is characterized by the ensemble height changes of both the surface proximal and distal fluorophores. As seen in Figure 5C, the height change of surface distal fluorophores significantly exceeds that of the surface proximal fluorophore tags and the mean probe orientation of the ensemble, Figure 5D, is approximated for each spot as θProbe = sin−1[(ΔhDistal − ΔhProximal)/(R2)1/2], where (R2)1/2 is the root-mean-square end-to-end length of the dsDNA probes. An analysis of the error introduced by this approximation is given in the Supporting Information. The variation in the polymer charge results in controlled changes in the polymer conformation. The polymer charge, in turn, is controlled by the solution pH, and the solution’s ionic strength is the dominant parameter affecting the electric-field screening of the polymer charge. Figure 6 shows the conformation changes in the polymer and probe orientation for buffer pH ranging from 4.4 to 9.0, and ionic strengths of 40 and 300 mM. The spontaneous ionization of the silicon dioxide in solution leads to a surface charge density of −0.1 to −0.2 μC/cm2,24 which causes the positively charged polymer to collapse at low buffer pH and results in the repulsion of the negatively charged polymer at buffer pH values above the polymer pI. For a buffer ionic strength of 40 mM, the surface proximal fluorophore heights are seen to vary between 1.5 and 8.0 nm, corresponding to a significant elevation in the dsDNA probe binding sites. An increase in the buffer ionic strength, to 300 mM, results in counterion condensation and reduces the strength of electrostatic interactions between the fixed charges on the polymer and the oxide. Consequently, the degree of variation between the surface proximal fluorophore heights on the positively and negatively charged polymer was reduced to only 4 to 6 nm for the same range of buffer pH. The buffer pH and ionic strength is also used to control the orientation of immobilized dsDNA probes on the polymer scaffold, as seen in Figure 6. As expected, the effect of the polymer charge on the dsDNA orientation is most apparent at low ionic

DMF and tetrahydrofuran (THF) and dried under nitrogen flow. Finally, the samples are dried in a vacuum oven. Calculation of dsDNA Probe Orientation. The persistent or worm-like model is used to characterize the flexibility of the double stranded DNA probes.22 The root-mean-square end-toend length of the probe is calculated as: ⟨R2⟩ =

2l 2[(L /l) − 1 + e(−L / l)]

(1)

where l = 50 nm and is the persistence length of double stranded DNA, and the contour length of the probe, L, is 0.34 nm per nucleotide. The root-mean-square (rms) end-to-end length is the taken as the probe length to calculate the mean probe orientation based on the measured height difference between the surface distal and surface proximal fluorophores.The model by Dobryn23 is used to correct for the effect of ionic strength on the dsDNA persistence length, for the calculations of both the empirical and theoretical values of the DNA orientation. It is known that the axial height of surface distal and surface proximal fluorophores are equivalent in a dry environment,19 and the measured fluorophore heights are taken relative to the measured axial distance in a dry environment: Δh = zwet − zDry. This is done to account for the slope in the thickness of the silicon dioxide spacer layer.

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RESULTS AND DISCUSSION An overview of the approach for precise control and measurement of the polymer scaffold conformation and dsDNA probe orientation is shown in Figure 5. An array of 504 spots of 60 base pair long amine functionalized dsDNA probes were end-grafted to active NHS-ester groups on the polymer functionalized silicon dioxide chip, as shown in Figure 5A. To account for the effect of slope in the oxide thickness (approximately 1 nm/mm), we spot an array of surface proximal and surface distal fluorophore tagged dsDNA and take the difference between the mean axial silicon− fluorophore distances in four adjacent spots, as shown in Figure 5B. It was previously shown that the polymer collapses on the surface in a dry environment,19 and the difference between the surface proximal and distal fluorophore heights, measured to be 0.4 nm, indicate a horizontal orientation of the dsDNA probes. 10596

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Figure 5. (A) An array of 18 × 28, 60 base pair long dsDNA is spotted on a functionalized silicon dioxide chip. Spots of surface distal and surface proximal fluorophore tagged dsDNA probes are spotted in a checkered pattern to eliminate the effects of variation in the silicon dioxide thickness. (B) The mean height difference between surface distal and surface proximal fluorophores is 0.4 nm, indicating that the oligonucleotides assume a horizontal orientation on the surface in a dry environment. (C) The change in fluorophore−silicon height is measured for all spots in pH 7.6 buffer (10 mM Tris/ TES/NaOH, 50 mM NaCl) relative to the axial position under dry conditions. A histogram of the fluorophore height differences in a wet versus dry environment, Δh = zWet − zDry, is plotted for surface distal and surface proximal fluorophores. (D) The mean angle assumed for dsDNA probes in wet conditions is calculated on the basis of the height differences between surface proximal and distal fluorophore heights.

orientation as a function of the ionic strength. In the model, dsDNA probes are treated as freely rotating rigid rods anchored on a charged surface. Each nucleotide is represented as a point charge separated by 0.34 nm and a value of −0.24e per point charge to account for the counterion condensation. The Gouy− Chapman theory is used to calculate the electrostatic potential as a function of distance from the surface; the electrostatic energy of the dsDNA is calculated as a function of their orientation, and the mean orientation is calculated using Boltzmann statistics.25 Figure 7A gives the calculated mean orientation for 60 bp long dsDNA as a function of monovalent salt concentration for a surface potential of −100 and 100 mV. The results are intuitive: at high ionic strength, the mean dsDNA orientation drops as the effects of the electric field become negligible and the orientation approaches that of randomly orientated dsDNA, while low ionic strengths result in efficient repulsion of the DNA probes to induce an upright orientation in excess of 70°. The dependency on the buffer ionic strength holds for buffers of salts with different valencies.25 The measured orientation for the negatively charged polymer (pH 7.6) matches well with the calculated results at low ionic strength, as the maximum orientation exceeds 70°(Figure 7B). At high ionic strength, the effects of negative charge on the polymer become negligible and the mean dsDNA orientation approaches 45°. The expected mean orientation is 33° when probes occupy available states between 0° and 90° with an equal

strengths, where a nearly horizontal probe orientation is observed at low pH, whereas the negatively charged polymer at high pH repels the probes to induce a standing orientation in excess of 70°. The controlled orientation of dsDNA on the charged polymer scaffold may be understood by considering the electrical DNA switching on a gold surface, which was studied extensively by Rant and co-workers.10 They demonstrated that the underlying mechanism by which dsDNA probes are oriented is due to the diffuse double layer of counterions, which accumulates at the interface of a charged surface and an electrolyte solution. A high gradient of the charge density at the buffer−polymer interface results in an intense electric field on the order of 100 kV/cm within a distance of approximately one Debye length from the charged surface, and this electric field imparts a large electrostatic energy to the charged nucleotides.11 However, the thickness of the double layer, which varies from 3 nm at low ionic strength (10 mM) to about 0.6 nm at high ionic strength (300 mM), does not span the entire length of the probes, and the electrostatic interactions are confined to the base of the dsDNA probes. Hence, the electric torque that is applied to the probes is greatest for buffers of low ionic strength, where the field extends far from the charged surface. A quantitative analysis of the dsDNA probe orientation over a wide range of salt concentrations gives further insight into the behavior of the system. A first order model, presented previously by Rant,25 is used to calculate the dependence of probe 10597

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Figure 7. (A) Calculated orientation of 60 bp dsDNA probes on a charged planar surface as a function of ionic strength. The surface potential is taken as −100 and 100 mV for the negatively and positively charged surfaces, respectively. (B) The measured orientation of 60 mer dsDNA probes for negatively charged (pH = 7.6) and positively charged (pH = 4.4) polymeric surfaces.

Figure 6. (A) The mean height change of surface proximal and surface distal fluorophore tags on 60 base pair long dsDNA probes upon surface hydration, as a function of the buffer pH, for ionic strengths of 40 and 300 mM. The cartoon shows the polymer swelling and change in probe orientation at an ionic strength of 40 mM. The surface proximal fluorophore tags are used to measure the polymer conformation: The positively charged polymer collapses onto the negatively charged oxide surface, and negatively charged polymer is repelled from the oxide surface. The surface distal and surface proximal fluorophore heights allow precise quantification of dsDNA orientation: The dsDNA is oriented in a lying position by the positively charged polymer and in a standing position by the negatively charged polymer. (B) The calculated probe orientation as a function of buffer pH at ionic strengths of 40 and 300 mM is shown. Low ionic strength allows the electric field to penetrate far from the charged polymer to effectively orient immobilized dsDNA.

charged surface as opposed to their repulsion from a negatively charged surface.26 When the probe orientation is switched from an upright to a lying position, it is known that their dynamic behavior is stochastic, as the attracting electric field does not exceed its thermal motions for the majority of orientational states available. The probes are only pulled down and captured when their orientation passes a threshold where the attracting electric field dominates over the thermal motion.26 For our steady state ensemble measurements, the observed orientation represents a weighted average of the captured and uncaptured probes. Hence, when the ionic strength decreases beneath the threshold value, where the size of the diffuse layer is large enough to effectively capture a majority of the probes, the observed ensemble of the probe orientation is seen to switch from 45° (probes are freely moving) to