Chemomechanics of Surface Stresses Induced by DNA Hybridization

Jeanne C. Stachowiak,† Min Yue,†,‡ Kenneth Castelino,† Arup Chakraborty,§,| ... and Materials Sciences DiVision, Lawrence Berkeley National L...
0 downloads 0 Views 468KB Size
Download PDF
Langmuir 2006, 22, 263-268

263

Chemomechanics of Surface Stresses Induced by DNA Hybridization Jeanne C. Stachowiak,† Min Yue,†,‡ Kenneth Castelino,† Arup Chakraborty,§,| and Arun Majumdar*,†,| Departments of Mechanical Engineering, Chemical Engineering, and Chemistry, UniVersity of California, and Materials Sciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed August 9, 2005. In Final Form: October 14, 2005 When biomolecular reactions occur on one surface of a microcantilever beam, changes in intermolecular forces create surface stresses that bend the cantilever. While this phenomenon has been exploited to create label-free biosensors and biomolecular actuators, the mechanisms through which chemical free energy is transduced to mechanical work in such hybrid systems are not fully understood. To gain insight into these mechanisms, we use DNA hybridization as a model reaction system. We first show that the surface grafting density of single-stranded probe DNA (sspDNA) on a surface is strongly correlated to its radius of gyration in solution, which in turn depends on its persistence length and the DNA chain length. Since the persistence length depends on ionic strength, the grafting density of sspDNA can be controlled by changing a solution’s ionic strength. The surface stresses produced by the reaction of complementary single-stranded target DNA (sstDNA) to sspDNA depend on the length of DNA, the grafting density, and the hybridization efficiency. We, however, observe a remarkable trend: regardless of the length and grafting density of sspDNA, the surface stress follows an exponential scaling relation with the density of hybridized sspDNA.

Introduction Biology is replete with molecules such as molecular motors that convert the free energy of chemical reactions into mechanical work, which is critical in many life processes. Such molecules use the interplay between chemical reactions and thermal fluctuations to produce motion in processes that are relatively well understood,1-4 although the details of the effect of molecular structure are still being resolved. Recently, Fritz et al.5 showed that reaction-induced motion can be generated in synthetic mechanical systems as well. They demonstrated that when biomolecular reactions occur on one surface of a cantilever beam, the cantilever bends due to generation of a mechanical surface stress (see Figure 1). This phenomenon has been observed for DNA hybridization,6-8 antigen-antibody binding,9,5,10 and also many nonbiological reactions in vapor.11-13 While it has formed * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Mechanical Engineering, University of California. ‡ Current address: Applied Biosystems, Inc., Belmont, CA 94002. § Departments of Chemical Engineering and Chemistry, University of California. | Lawrence Berkeley National Laboratory. (1) Dimroth, P.; Wang, H.; Grabe, M.; Oster, G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4924-4929. (2) Wang, H.; Oster, G. Appl. Phys. A 2002, 75, 315-323. (3) Bustamante, C.; Keller, D.; Oster, G. Acc. Chem. Res. 2001, 34, 412-420. (4) Berg, H. C. Annu. ReV. Biochem. 2001, 72, 19-54. (5) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Gu¨ntherodt, H. J.; Gimzewski, J. K. Science 2000, 288, 316-318. (6) Hansen, K.; Ji, H.; Wu, G. H.; Datar, R.; Cote, R.; Majumdar, A. Anal. Chem. 2001, 73, 1567-1571. (7) Wu, G. H.; Ji, H. F.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1564. (8) McKendry, R.; Zhang, J. Y.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Gu¨ntherodt, H. J.; Gerber, C. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 9783-9788. (9) Thundat, T.; Oden, P. I.; Warmack, R. J. Microscale Thermophys. Eng. 1997, 1, 185-199. (10) Wu, G. H.; Datar, R.; Hansen, K.; Thundat, T.; Cote, R.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (11) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894-2896. (12) Berger, R.; Delmarche, E.; Lang, H.; Gerber, C.; Gimzewski, J. K.; Meyer, E.; Gu¨ntherodt, H. J. Science 1997, 276, 2022-2024.

Figure 1. Specific, surface-bound biomolecular interactions between target and probe oligonucleotide strands can produce a sufficiently large surface stress to bend a cantilever beam measurably.

the basis for label-free detection of a variety of technologically important biomolecular and chemical reactions,14 the mechanism by which chemical reactions produce surface stresses is yet to be fully understood. A fundamental understanding of the chemomechanics of synthetic systems could lead not only to insight regarding intermolecular forces between biomolecules but also to a variety of novel hybrid mechanical devices. Since much is known about the structure of DNA and the chemistry of DNA reactions, DNA hybridization forms a simple model reaction that can be used for understanding the chemomechanical transduction mechanisms. Hansen et al.6 demonstrated the detection of single nucleotide mismatches using cantilever sensors. McKendry et al.8 detected hybridization at nanomolar concentrations and examined the effect of target and ionic strengths on the signal magnitude during hybridization, fitting (13) Pinnaduwage, L. A.; Gehl, A.; Hedden, D. L.; Muralidharan, G.; Thundat, T.; Lareau, R. T.; Sulchek, T.; Manning, L.; Rogers, B.; Jones, M.; Adams, J. D. Nature 2003, 425, 6957. (14) Thundat, T.; Majumdar, A. In Sensors and Sensing in Biology and Engineering; Barth, F. G., Humphrey, J. A. C., Seecomb, T. W., Eds.; Springer: New York, 2003.

10.1021/la0521645 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/17/2005

264 Langmuir, Vol. 22, No. 1, 2006

the reaction kinetics to a Langmuir isotherm. Using DNA as a model molecule, Hagan et al.15 provided the first theoretical framework for the chemomechanics in such hybrid structures, based on the empirical potentials developed by Strey et al.,16 who worked with a hexagonally compressed array of doublestranded DNA. Hagan et al.15 compared the relative importance of various intermolecular forces: (i) electrostatics interactions between neighboring strands, (ii) the conformation entropy of DNA, (iii) the osmotic pressure of counterions, and (iv) hydration forces between DNA strands. This study predicted that hydration forces and osmotic pressure dominate interstrand mechanics. Furthermore, they highlighted the importance of DNA surface densities, predicting an exponential increase in cantilever deflection with the density of double-stranded molecules. In this paper, we experimentally examine these predictions, and provide further insight regarding the key parameters that govern the chemomechanics of such hybrid structures. We utilize a recently developed 2-dimensional microcantilever array, which contains individually addressable microfluidic wells, each of which contains multiple microcantilever sensors.17,18 Surface densities are modulated through variations in probe/ target length and ionic strength during probe immobilization. The effects of these parameter variations are correlated to changes in surface density through a previously published fluorescent measurement technique.19,20 Results are interpreted according to published studies of molecular interaction potentials,16 surface densities,19 and persistence length21,22 and compared to theoretical predictions.

Stachowiak et al. Table 1. Oligonucleotide Nomenclature and Sequencesa name

sequence (5′ to 3′)

GD-10 HS(CH2)6 5′-TCT CAC CTT C-3′ FAM GD-20 HS(CH2)6 5′-GTG GTA GAT GAA GGT GAG AG-3′ FAM GD-30 HS(CH2)6 5′-GGA GGT GGA CAG ATA ATG GTA GAA GAT AGG-3′ FAM GD-40 HS(CH2)6 5′-CAC TAC GAG TCA AGC TCA CAC ATT CAG GAT TCG GCA TGA T-3′ FAM SS-10 HS(CH2)6 5′-TCT CAC CTT C-3′ SS-20 HS(CH2)6 5′-GTG GTA GAT GAA GGT GAG AG-3′ SS-30 HS(CH2)6 5′-GGA GGT GGA CAG ATA ATG GTA GAA GAT AGG-3′ C-10 5′-GAA GGT GAG A-3′ C-20 5′-CTC TCA CCT TCA TCT ACC AC-3′ C-30 5′-CCT ATC TTC TAC CAT TAT CTG TCC ACC TCC-3′ HD-10 FAM 5′-GAA GGT GAG A-3′ HD-20 FAM 5′-CTC TCA CCT TCA TCT ACC AC-3′ HD-30 FAM 5′-CCT ATC TTC TAC CAT TAT CTG TCC ACC TCC-3′ a Sequences used to measure the grafting density (GD) on gold surfaces (GD-n, where n is the number of nucleotides (nt’s)), where the 5′ end is thiolated (HS(CH2)6) and the 3′ end is fluorescently labeled (FAM), single-stranded (ss) sequences immobilized on cantilever surfaces and used as probes to measure the hybridization density on gold surfaces (SS-n), where the 5′ end is thiolated HS(CH2)6) and the 3′ end is unmodified, complementary (C) sequences used for hybridization on cantilever surfaces (C-n), where both ends are unmodified, and sequences used to hybridize probes during hybridization density (HD) measurement (HD-n), where the 5′ end is fluorescently labeled (FAM) and the 3′ end is unmodified.

Methods Reagent Processing. All oligonucleotide sequences were purified using HPLC and were obtained from Integrated DNA Technologies Inc. (Skokie, IL). Thiolated oligonucleotides were synthesized with a 6-mercaptohexyl linker to facilitate adhesion to gold surfaces through gold-thiol bonding. Thiolated DNA molecules were treated with 100 mM DTT (dithiothreitol) immediately before use to break any disulfide bonds present. DTT was removed by running the solution through commercially available size exclusion columns, and the oligonucleotides were immediately frozen to prevent the disulfide linkages from re-forming. Table 1 lists the probe and target oligonucleotide sequences used in the experiments (GD-n, where n is the number of nucleotides (nt’s), SS-n, C-n, and HD-n). Nonspecific attachment of oligonucleotides to the uncoated silicon nitride side of the cantilevers was prevented by self-assembly of a poly(ethylene glycol) (PEG) layer using silane chemistry.23 All reagents were used without further purification. Surface Density Measurement Procedure. Oligonucleotide surface densities were measured using fluorescence-based techniques on gold-coated substrates (not on cantilever arrays), as described in Castelino et al.19 Briefly, fluorescently labeled oligonucleotide probes were first attached on the gold substrate using thiol chemistry (GDn) and then displaced into solution by competitive binding of 12 mM β-mercaptoethanol. The concentration of the displaced probes in (15) Hagan, M. F.; Majumdar, A.; Chakraborty, A. K. J. Phys. Chem. B 2002, 106, 10163-10173. (16) Strey, H. H.; Persegian, V. A.; Podgornik, R. Phys. ReV. E 1999, 59, 999-1088. (17) Yue, M.; Lin, H.; Dedrick, D. E.; Satyanarayana, S.; Majumdar, A.; Bedekar, A. S.; Jenkins, J. W.; Sundaram, S. J. Microelectromech. Syst. 2004, 13, 290-299. (18) Yue, M.; Stachowiak, J.; Majumdar, A. Mech. Chem. Biosyst. 2004, 1, 221-220. (19) Castelino, K.; Kannan, B.; Majumdar, A. Langmuir 2005, 22, 19561961. (20) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535-5541. (21) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Macromolecules 1997, 30, 5763-5765. (22) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795-799. (23) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 1457-1460.

Figure 2. (Top) Cantilever array chip. (Bottom) An individually addressable well of the cantilever array chip containing up to 12 sensors and an injection port. Each cantilever in the well has a ridge structure around the square paddle at its end, which functions to create a rigid reflective structure for cantilever tracking. In the figure, 2 of 12 cantilevers were broken during fabrication. solution was measured using a fluorescence microscope and a CCD camera. Gold substrates were incubated in the mercaptoethanol solution for 24 h, after which the displaced fluorescence was seen not to increase further. Demers et al. have shown this procedure to be highly efficient in displacement of thiolated oligonucleotides from gold surfaces.20 To find the hybridization density, thiolated probe oligonucleotides were first immobilized (SS-n) and then hybridized with fluorescently labeled complementary target strands of equal length (HD-n). The resulting duplexes were displaced from the gold surface using β-mercaptoethanol, and the concentration of these displaced duplexes was again determined by measuring the fluorescence intensity. Microcantilever Array Chip. We utilized a recently developed 2-dimensional microcantilver array, as shown in Figure 2. The fabrication process for the microcantilever array chip and the design of optical multiplexing are described in detail in previous papers.17,18 The array contains 90 (15 × 6) individually addressable fluidic

Surface Stresses Induced by DNA Hybridization reaction wells, each of which contains multiple (4-12) cantilever sensors. The response of multiple cantilevers in each well can be averaged to increase the accuracy of the measurements, mitigate false readings, and show statistical variations. Each cantilever has a rigid paddle structure at its end to provide a flat reflecting surface for optical diagnostics. Each reaction well was physically separated from its neighboring wells by adhesive bonding of a Pyrex wafer containing an array of wells wet-etched to the cantilever array.24 To simultaneously image the entire cantilever array chip (100-500 cantilevers at a time), which is about 2 cm2 in area, it was necessary to construct a ray-optics-based whole field illumination system.18 Briefly, a laser beam was expanded and reflected off the cantilever array chip. The flat paddles at the end of the cantilevers produced spots on a CCD camera. By measuring the motion of each spot, it was possible to detect the deflection of cantilever beams with 1-3 nm resolution. The cantilevers were made of silicon-rich, low-stress silicon nitride and were 200-400 µm long, 0.5 µm thick, and generally 30-40 µm wide. One surface of the cantilevers was coated with a 25 nm thick Au film that was deposited on SiNx with a 5 nm thick Cr adhesion layer. The Au-SiNx cantilever formed a thermal bimorph, which required temperature control during biological experiments. All biologically induced cantilever deflections are normalized against the cantilever deflection resulting from a unit temperature change. Thermally induced deflections are calibrated in nanometer units using a white light interferometer (Veeco, Woodbury, NY) to establish the sensitivity per unit of temperature change. Yue et al.17 reported an experimentally observed thermomechanical sensitivity of 208 ( 14 nm/K for the 200 µm long cantilevers, in good agreement with theory. Further, the surface stress sensitivity for the devices used in this study has been related to the thermomechanical sensitivity through a mechanical model based on Stoney’s formula and estimated at 24.5 mJ/(m2 K).18 A micropipet was used to inject 1 µL of 5 µM unlabeled, thiolated probe sequences dissolved in sodium phosphate buffer (PB) (10 mM to 1 M) into individual microfluidic wells of the cantilever array. Cantilevers in each well were incubated in the probe solution (SS-n) (thiolated DNA in sodium phosphate buffer) for at least 3 h. Following the immobilization period, the probe solution was aspirated from each well, and the wells were washed three times in a buffer solution of concentration equal to that of the probe solution. The entire cantilever array was then immersed in 200 mM sodium phosphate buffer for at least 10 min prior to being mounted on the temperature-controlled chip holder. Once the cantilever array was mounted on the chip holder and aligned with the optical system, the cantilever tip deflections were monitored every 15-30 s until the baseline positions of all cantilevers appeared stable. The cantilever tip deflection in response to a temperature change of 1 K was recorded to establish the thermomechanical sensitivity of each cantilever. A micropipet was used to aspirate the buffer inside each well, and fresh buffer of the same type and concentration was injected to establish that no significant signal resulted from the injection process alone. The hybridization solution consisted of 5 µM single-stranded, unthiolated DNA, fully complementary to and of length equal to that of the immobilized probe DNA (C-n), dissolved in 200 mM sodium phosphate buffer. After injection of the hybridization buffer (about 1 µL), the cantilever response to DNA hybridization was optically monitored for at least 1 h following injection. Greater than 50% of the cantilever response to hybridization was typically observed in the first 5-10 min following injection, with the remainder of the response observed within 1/2 h following injection. Multiple injections of the hybridization buffer are used to ensure that the complementary strand is available to the cantilever sensor at its full concentration. After each injection the cantilevers are allowed to recover a stable position. Three injections are typically necessary. A portion of the buffer within the wells (about 0.5-1 µL) is aspirated just prior to each injection to make room for the new injection. Most of the deflection occurs after the first injection. Additional deflection occurs after the second injection, indicating that the first injection (24) Satyanarayana, S.; Karnik, R. N.; Majumdar, A. J. Microelectromech. Syst. 2005, 14, 392-399.

Langmuir, Vol. 22, No. 1, 2006 265

Figure 3. A typical set of deflection data from which the results presented in this paper were derived. Specifically, the data in this figure are part of the set used to derive the results reported in Figure 5, inset. Here the difference in cantilever deflection for hybridization of 20-nt probes (SS-20) at a 5 µM concentration immobilized at various ionic strengths (10 mM, 500 mM, 1 M) is apparent. All cantilevers were hybridized with fully complementary strands (C20) at 5 µM concentration at a fixed ionic strength of 200 mM. Multiple injections of the hybridization buffer are necessary to ensure that the complementary strand is available to the probe fully concentrated. Here two injections are visible. A third injection (not shown) resulted in no further deflection of the cantilevers. was diluted by liquid remaining in the well after aspiration (just prior to the first injection). No further deflection typically results from a third injection, indicating that the sensors have responded to the fully concentrated hybridization buffer, and no further injections are needed. Cantilever deflections are set to zero at the time of the hybridization buffer injection, and linear drift trends are removed (Figure 3). After the deflection signals have reached stable values following the injection, the offset is recorded for each cantilever. These offsets are averaged for all the cantilevers in the population. The variation in sensor response for each experiment is accurately reflected in the error bars of Figure 4, which represent the first standard deviation. Yue et al.18 demonstrated that cantilever sensors functionalized with end-thiolated, single-stranded DNA do not deflect measurably when exposed to noncomplementary targets that only contain 1-3 consecutive complementary nucleotides over a length of 10-30 nucleotides.

Results In all the experiments discussed below, the ionic strength of the phosphate buffer was varied only during immobilizing or grafting of the probe single-stranded DNA (ssDNA) using thiolAu chemistry. During hybridization of the complementary strands, the ionic strength was fixed at 200 mM. Surface Density Measurements. Castelino et al.19 showed using fluorescence measurements that the density of thiol-attached ssDNA could be controlled by changing the ion concentration from 0.05 to 1 M (Figure 4A). The probe ssDNA (SS-n) of known grafting density was then hybridized using fluorescently labeled complementary target ssDNA (C-n) at 200 mM ionic strength. Figure 4B shows the density of hybridized DNA as a function of the ionic strength during attachment of probe ssDNA. Figure 4C plots the hybridization efficiency, defined as the fraction of probe ssDNA hybridized by complementary target ssDNA as a function of the grafting density.25 Cantilever-Based Surface Stress Measurements. We measured the change in surface stress resulting from DNA hybridization under various combinations of chain length and ionic strength, each of which can be correlated to a specific grafting

266 Langmuir, Vol. 22, No. 1, 2006

Stachowiak et al.

Figure 4. (A) Grafting density of single-stranded, thiolated probe DNA as a function of the probe length and ionic strength. (B) Hybridization density of fully complementary DNA as a function of the chain length and ionic strength at which the probe molecules were immobilized. (C) Hybridization efficiency, defined as the fraction of probe ssDNA hybridized by complementary target ssDNA, at various ionic strengths. Hybridization took place at 200 mM ionic strength for all targets. In all figures error bars represent the first standard deviation from measurements on multiple gold substrates and are generally about 5%.

density in Castelino et al.19 and to a hybridization density and efficiency in Figure 4. Single-stranded, end-thiolated probe DNA of three lengths (10, 20, and 30 nucleotides) was immobilized on cantilever surfaces at three ionic strengths (without saltsin deionized water, with moderate salts100 mM PB, and with excess salts1 M PB) for a total of nine experiments, each performed in an individually addressable microfluidic well containing multiple cantilevers. Following incubation in the probe solution, sensors were washed, transferred to 200 mM PB, and mounted on the optical detection system. The hybridization buffer contained 5 µM single-stranded DNA of length equal to that of and fully complementary to the immobilized probe in 200 mM PB. Figure 5 plots the steady-state surface stress changes for different probe/ target lengths and ionic strengths used during grafting of probe ssDNA. For a given ionic strength during probe immobilization, the cantilever response was observed to decrease with increasing probe/target length. For a given probe/target length, the cantilever response was observed to increase with increasing ionic strength during immobilization. These trends appear to follow those in the surface density measurements, suggesting, as is discussed in the next section, that surface densities significantly influence the cantilever response. To better resolve the relationship between surface stress change during hybridization and ionic strength during immobilization, we immobilized 10-nucleotide probes at 6 PB concentrations (10 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1 M) and monitored the surface stress response resulting from their hybridization in 200 mM PB (Figure 5, inset). The 10-nucleotide DNA was chosen because it grafts at a higher density, resulting in a greater cantilever response, permitting the resolution of a greater number of response levels over the range of ionic strengths considered. The results from this experiment are presented relative (25) We attempted to measure the hybridization density of probe surfaces immobilized at ionic strengths below 50 mM, which are expected to yield densities lower than those previously measured by Castelino et al.,19 but found that difficulties in the experimental procedure prevented the collection of reliable data. Owing to these difficulties, the surface stress measurements for hybridization of probes immobilized in DI water are in all cases correlated to the measurements of the hybridization density and efficiency of probes immobilized in 50 mM PB. On the basis of the results of Castelino et al., we expect slight decreases in hybridization density and slight increases in efficiency below 50 mM. We do not expect these small changes to significantly affect the trends observed when the two data sets are compared in the Discussion. We note that this measurement technique does not exclude partially hybridized targets or targets that partially hybridize more than one probe, but counts them equally with fully hybridized targets. That is, any of the fluorescently labeled target strands that remain attached to displaced probes at the time of the fluorescence measurement will be “counted” by the measurement technique whether they are fully hybridized to the probes or not. The density of partially hybridized strands, if present, is expected to increase with increasing ionic strength. The effectiveness of the washing procedures toward removal of incompletely hybridized targets is not known.

Figure 5. Cantilever response to hybridization of 10-, 20-, and 30-nucleotide strands in 200 mM sodium phosphate buffer. Immobilization of probe strands occurred in DI water, 100 mM sodium phosphate buffer, and 1 M phosphate buffer. Numbers in parentheses are the numbers of cantilever measurements contributing to the 1 standard deviation error bar. (Inset) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer. Immobilization took place in 10 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1 M sodium phosphate buffer.

to the largest surface stress change (immobilization in 1 M PB). We present these data to show qualitatively the trends observed in the previously described experiment in greater detail.

Discussion The most important observation of this work is that, for the range of ionic strengths and DNA chain lengths considered, the surface stress is dominated by the density of hybridization events, as demonstrated by the discovery of a single-exponential scaling relation between surface stress and the density of hybridized DNA strands, regardless of chain length. This conclusion arises from correlating the surface density and surface stress as a function of ionic strength and DNA chain length, as discussed below. In Figure 4A, the transition from a strong dependence of the grafting density on ionic strengths (at low ionic strengths) to a weaker dependence (at higher ionic strengths) suggests a transition from the dominance of osmotic forces to dominance of hydration forces. Osmotic forces arise due to the entropic motion of the counterions that are localized around the DNA. For monovalent ions, these forces are repulsive in nature and are active on length scales comparable to the Debye screening length. Hydration forces

Surface Stresses Induced by DNA Hybridization

Figure 6. Comparison of center-to-center separation distances of surface-grafted probes predicted by grafting density data and by persistence length data21 with the assumption that probe radii are in direct contact. (Inset) Ratio of separations predicted by a model and measurement.

are caused by perturbation of the hydrogen-bonding network of water surrounding DNA molecules. Strey et al.16 showed that hydration forces dominate interstrand dynamics at separations below 3.2 nm, or about 0.1 chains/nm2, where the decay length of the interactions is about 0.3 nm. At low ionic strength conditions, the Debye length is large and the osmotic forces, which have a much longer range, are dominant and determine the spacing between probes. The large screening length consequently results in lower probe densities at low ionic strengths. Increasing the ionic strength reduces the Debye length, which reduces interprobe repulsion and leads to a higher grafting density in this intermediate regime. However, beyond a certain ionic strength, the Debye length is much smaller than the hydration length and, hence, hydration forces determine the probe spacing. Since the hydration forces do not depend strongly on the ionic strength, the grafting density is relatively constant in this regime. Tinland et al.21 have measured the effect of ionic strength on the persistence length of ssDNA and suggested that the radius of gyration of the ssDNA polymer strand can be correlated to the persistence length according to the relation Rg ) (b0Np/3)1/2 Å, where N is the number of nucleotides, p is the persistence length in angstroms, and b0 is the proportionality constant between the strand contour length and N ) 4.3 Å for single-stranded DNA. Using this result and imposing an intrinsic minimum persistence length of 7.5 Å, arising from intrastrand sterics at high ionic strengths, in agreement with Smith et al.,22 we examine the radii of gyration for the molecules of interest in this work. We apply this analysis only to 20- and 30-nucleotide singlestranded probes, because the length of the 10-nucleotide probes is comparable to the minimum persistence lengths. In Figure 6 we observe that, if the Tinland model is used and the molecules are presumed to pack in a dense cubic array on the sensor surface, the resulting predictions of mean center-to-center probe spacing are in good agreement with those arising from grafting density measurements by Castelino et al.19 That is, the experimentally measured mean center-to-center molecular spacing of singlestranded DNA probes is roughly equal to their radii of gyration in solution. On the basis of this hypothesis, we may consider the surface of the cantilever not as a set of isolated molecules but as a semicontinuous film, the density of which is controlled by the radius of gyration, which is influenced by the molecular length and ionic strength (through the persistence length). We note that the theory of planar electrolyte brushes and the impact

Langmuir, Vol. 22, No. 1, 2006 267

of ionic strength on their formation have been well studied.26-28 However, the mean spacing between probes observed in this work is significantly larger than the Debye length and thus does not fit the definition of a brush.28 That is, the distribution of counterions is not uniform in the coordinate direction parallel to the surface.27 The results for the hybridization density and efficiency are shown in Figure 4. The lowest ionic strengths produce the minimum grafting density. Under these conditions, there are few steric constraints for the target molecules to hybridize with the probe ssDNA. Hence, the hybridization efficiency in this regime is high, as seen for all the lengths of probes considered. However, since the original number density of immobilized probes is low, the hybridization densities achieved are still quite low in this regime. Under high ionic strength conditions, the immobilization density of probe ssDNA is high and, hence, the hybridization efficiency is lowered due to steric and electrostatic hindrances experienced by target molecules. However, as seen in our experiments, the decrease in hybridization efficiency is not large enough to account for the higher immobilization density of the probe ssDNA. Hence, the actual number density of hybridized probes on the surface ends up being much larger as compared to that of the low-concentration case. The results in Figure 5 show that the cantilever mechanical response increases with ionic strength, which suggests that the surface stress is in some way related to either the grafting (probe) or hybridization (target) densities, or both. To examine this relationship further, the surface stress data in Figure 5 are plotted against the grafting (probe) and hybridization (target) densities of Figure 4A,B and shown in Figure 7. Figure 7A shows that, for 10 bp DNA hybridization, the surface stress (a linear function of the cantilever deflection) is a strong function of the hybridization density, and follows an exponential relation. This finding is in agreement with the model by Hagan et al.,15 which predicted an exponential increase in cantilever deflection with the density of DNA duplexes. When plotted against the grafting density of probe ssDNA (Figure 7B, inset), the surface stress increases monotonically with increasing grafting density (decreasing chain length). However, a separate trend exists for each ionic strength during immobilization. What we found remarkable was that when the surface stress was plotted against the hybridization density, all the data points collapsed to yield a single relationship between the surface stress and hybridization density for all lengths of DNA and various ionic strengths, and followed an exponential fit reasonably well (Figure 7). Thus, we conclude that the key parameter controlling surface stress is the hybridization density of DNA molecules. The above discussion has focused on DNA hybridization, which has been used as a model molecular system. However, it would be intriguing to see if the principles learned from DNAsthat surface stress depends on the number of binding events per unit areashold true for other molecular reactions as well. For example, it is well-known that when alkanethiols bind to one surface of a cantilever beam, they produce a self-assembled monolayer (SAM), which creates surface stress to bend the cantilever beam.13 When this SAM is exposed to gas molecules, then depending on the binding affinity adsorption of gas molecules also produces further surface stress. Antigen-antibody binding and various other protein-protein interactions have been observed to produce (26) Kelley, T. W.; Schorr, P. A.; Johnson, K. D.; Tirrell, M.; Frisbie, C. D. Macromolecules 1998, 31, 4297-4300. (27) Guenoun, P.; Argillier, J. F.; Tirrell, M. C. R. Acad. Sci., Ser. IV 2000, 9, 1163-1169. (28) Pincus, P. Macromolecules 1991, 24, 2912-2919.

268 Langmuir, Vol. 22, No. 1, 2006

Stachowiak et al.

Figure 7. (A) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer as a function of the hybridization density. Immobilization took place in 50 mM, 100 mM, 200 mM, 500 mM, and 1 M sodium phosphate buffer. The cantilever response increased monotonically with an increase in the ionic strength used during immobilization. (B, inset) Cantilever response during DNA hybridization as a function of the grafting density. The surface stress response to the grafting density appears to be influenced by the chain length. (B, main) Cantilever response during DNA hybridization as a function of the hybridization density. The surface stress response to the grafting density (B, inset) is scaled by the hybridization efficiency to produce a single trend that is less influenced by the DNA chain length.

surface stress, and it would be worth investigating how the surface stress depends on the density of the binding events.9,5,10 Finally, the implications of our observations on the mechanics of cell membranes are also worth investigation. In particular, there are many proteins, peptides, and small molecules that bind to membrane-bound proteins and receptors on a cell surface. Mechanical interactions between these receptor molecules and their interplay with a membrane’s mechanical compliance play a critical role in many cell functions and responses. The lessons learned from this work could perhaps be useful in providing insight into the chemomechanics of cell membranes, though it must be taken into account that the dynamics of flexible cell membranes are very different from those of rigid surfaces.

density and efficiency are also found to depend on the grafting density and the length of the DNA. Measurement of surface stresses generated from DNA hybridization reactions suggests that surface stress is a strong function of the grafting density of probe molecules, which depends on the chain length. However, when the surface stress is plotted against the hybridization density, we observe that the surface stresses collapse onto a single curve and can be described with a single-exponentially increasing trend. This finding suggests that the hybridization density brings together the impact of the ionic strength and chain length as a controlling parameter in the creation of surface stress by DNA hybridization in the medium to high ionic strength regime, where hydration forces dominate the response.

Conclusions

Acknowledgment. We acknowledge the funding through the DARPA Simbiosys program, the NCI IMAT program, and the DOE Basic Energy Sciences. The facilities provided by the Berkeley Microfabrication Laboratory are much appreciated. We acknowledge helpful suggestions and discussions with Richard J. Cote, Ram Datar, and Henry Lin of the Department of Pathology at the Keck School of Medicine of the University of Southern California and with Thomas Thundat, Jerry Hu, and Karolyn Hansen of Oak Ridge National Laboratories, Life Sciences Division. J.C.S. acknowledges funding from the ARCS Foundation and the NSF through a graduate fellowship. A.M. acknowledges the support of the Miller Institute through a professorship, during which much of this work was performed.

In this paper, we investigate how DNA hybridization, a model biomolecular reaction, produces surface stresses on micromechanical structures. Two aspects of this investigation include (i) identifying the role of various intermolecular forces and (ii) finding a key parameter that controls mechanical response. Our measurements of the grafting density of probe single-stranded DNA suggest that, at low ionic strength, the density is governed by the radius of gyration of ssDNA, which depends on the persistence length, which in turn depends on the ionic strength. We show that, at low ionic strength, the osmotic pressure of counterions dominates the intermolecular forces while, at higher ionic strength, the grafting density is independent of the ionic strength and hydration interactions dominate. The hybridization

LA0521645