Langmuir 2006, 22, 263-268

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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.

Downloaded by READING UNIV on September 11, 2009 | http://pubs.acs.org Publication Date (Web): November 17, 2005 | doi: 10.1021/la0521645

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: majumdar@me.berkeley.edu. † 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.; Guntherodt, 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.; Guntherodt, 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.; Guntherodt, 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

. 271. have shown this procedure to be highly efficient in displacement of thiolated oligonucleotides from gold surfaces.. 1457-1460. where both ends are unmodified. A. This study predicted that hydration forces and osmotic pressure dominate interstrand mechanics.. L. The resulting duplexes were displaced from the gold surface using β-mercaptoethanol. and the oligonucleotides were immediately frozen to prevent the disulfide linkages from re-forming. 59. A. L. C. S. The concentration of the displaced probes in (15) Hagan... C. Mirkin. 72. E. Thiolated oligonucleotides were synthesized with a 6-mercaptohexyl linker to facilitate adhesion to gold surfaces through gold-thiol bonding. 5763-5765.. Persegian. Demers et al. Downloaded by READING UNIV on September 11. 2006 Stachowiak et al.20 Results are interpreted according to published studies of molecular interaction potentials. C-n. A.. S.. G. Science 1996. 17. Oligonucleotide surface densities were measured using fluorescence-based techniques on gold-coated substrates (not on cantilever arrays). W. Table 1 lists the probe and target oligonucleotide sequences used in the experiments (GD-n. No. 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... Each cantilever in the well has a ridge structure around the square paddle at its end. Syst. and sequences used to hybridize probes during hybridization density (HD) measurement (HD-n). The fabrication process for the microcantilever array chip and the design of optical multiplexing are described in detail in previous papers. A. N.. C. Using DNA as a model molecule.org Publication Date (Web): November 17.. 2009 | http://pubs. K. B. Majumdar.18 The array contains 90 (15 × 6) individually addressable fluidic . 106. after which the displaced fluorescence was seen not to increase further. 2004. Dedrick. A. Table 1. Viswanadham. where n is the number of nucleotides (nt’s)). 2004. B 2002. R. they highlighted the importance of DNA surface densities. Y. 1.15 compared the relative importance of various intermolecular forces: (i) electrostatics interactions between neighboring strands. We utilize a recently developed 2-dimensional microcantilever array. which functions to create a rigid reflective structure for cantilever tracking. A.15 provided the first theoretical framework for the chemomechanics in such hybrid structures.. Majumdar. Jenkins. (ii) the conformation entropy of DNA.. (Bottom) An individually addressable well of the cantilever array chip containing up to 12 sensors and an injection port. Phys. J.23 All reagents were used without further purification. Weill. A.. 30. Phys.17. 2000. based on the empirical potentials developed by Strey et al. Vol. and provide further insight regarding the key parameters that govern the chemomechanics of such hybrid structures. H. J. Bustamante. M. G.. Biosyst. (23) Papra. Surface Density Measurement Procedure. The effects of these parameter variations are correlated to changes in surface density through a previously published fluorescent measurement technique. ReV. Letsinger. 795-799. Chakraborty. Elghanian. which contains individually addressable microfluidic wells. each of which contains multiple microcantilever sensors. Methods Reagent Processing. M. 19561961.. as shown in Figure 2. K.. H. where the 5′ end is thiolated HS(CH2)6) and the 3′ end is unmodified. predicting an exponential increase in cantilever deflection with the density of double-stranded molecules.. 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. solution was measured using a fluorescence microscope and a CCD camera. as described in Castelino et al. Sundaram.. In the figure. R. (16) Strey. where the 5′ end is fluorescently labeled (FAM) and the 3′ end is unmodified. R. Majumdar.19. M. H. single-stranded (ss) sequences immobilized on cantilever surfaces and used as probes to measure the hybridization density on gold surfaces (SS-n). Chem. Microcantilever Array Chip. (18) Yue. Langmuir 2005. 22.264 Langmuir. N. Sturm. A. Kannan. and (iv) hydration forces between DNA strands.. Majumdar. Figure 2. Cui. Macromolecules 1997. thiolated probe oligonucleotides were first immobilized (SS-n) and then hybridized with fluorescently labeled complementary target strands of equal length (HD-n). B. complementary (C) sequences used for hybridization on cantilever surfaces (C-n). Reynolds. All oligonucleotide sequences were purified using HPLC and were obtained from Integrated DNA Technologies Inc. J. 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. Hagan et al. D.17. 13. Thiolated DNA molecules were treated with 100 mM DTT (dithiothreitol) immediately before use to break any disulfide bonds present.19 and persistence length21.acs. (20) Demers... 221-220. (iii) the osmotic pressure of counterions. Anal. R. Gadegaard. 290-299. DTT was removed by running the solution through commercially available size exclusion columns. 2005 | doi: 10. Podgornik. where the 5′ end is thiolated (HS(CH2)6) and the 3′ end is fluorescently labeled (FAM). Lin. Gold substrates were incubated in the mercaptoethanol solution for 24 h. Mech.1021/la0521645 the reaction kinetics to a Langmuir isotherm. V. 1. In this paper. 999-1088. Microelectromech. M.22 and compared to theoretical predictions.16 surface densities.. Larsen. 22.16 who worked with a hexagonally compressed array of doublestranded DNA. Satyanarayana. A. Pluen. S. R. A..20 To find the hybridization density. Mucic. (19) Castelino. 2 of 12 cantilevers were broken during fabrication. (22) Smith. (21) Tinland.. SS-n. 5535-5541. (Skokie.19 Briefly. Chem. where n is the number of nucleotides (nt’s). IL). Bedekar.18 Surface densities are modulated through variations in probe/ target length and ionic strength during probe immobilization. we experimentally examine these predictions. S. B. B. Langmuir 2001. 10163-10173. Stachowiak. Hagan et al... F. and HD-n). Furthermore. (17) Yue. E 1999. J. Chem. We utilized a recently developed 2-dimensional microcantilver array. and the concentration of these displaced duplexes was again determined by measuring the fluorescence intensity. A.. J. (Top) Cantilever array chip.

Cantilever deflections are set to zero at the time of the hybridization buffer injection. Figure 4C plots the hybridization efficiency. 22. A portion of the buffer within the wells (about 0. We measured the change in surface stress resulting from DNA hybridization under various combinations of chain length and ionic strength. a laser beam was expanded and reflected off the cantilever array chip. the offset is recorded for each cantilever. Following the immobilization period. The flat paddles at the end of the cantilevers produced spots on a CCD camera. 2005. dissolved in 200 mM sodium phosphate buffer. 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. which represent the first standard deviation. and no further injections are needed. 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. The hybridization solution consisted of 5 µM single-stranded. Figure 4B shows the density of hybridized DNA as a function of the ionic strength during attachment of probe ssDNA. All cantilevers were hybridized with fully complementary strands (C20) at 5 µM concentration at a fixed ionic strength of 200 mM. The response of multiple cantilevers in each well can be averaged to increase the accuracy of the measurements. thiolated probe sequences dissolved in sodium phosphate buffer (PB) (10 mM to 1 M) into individual microfluidic wells of the cantilever array. The cantilever tip deflection in response to a temperature change of 1 K was recorded to establish the thermomechanical sensitivity of each cantilever. J. 1. R. 1 M) is apparent. A typical set of deflection data from which the results presented in this paper were derived. the probe solution was aspirated from each well. it was possible to detect the deflection of cantilever beams with 1-3 nm resolution. Cantilevers in each well were incubated in the probe solution (SS-n) (thiolated DNA in sodium phosphate buffer) for at least 3 h. and show statistical variations. Greater than 50% of the cantilever response to hybridization was typically observed in the first 5-10 min following injection. each of which can be correlated to a specific grafting . The cantilevers were made of silicon-rich. During hybridization of the complementary strands. After the deflection signals have reached stable values following the injection. it was necessary to construct a ray-optics-based whole field illumination system. Karnik. Vol. A.5 µm thick. 0. After injection of the hybridization buffer (about 1 µL). NY) to establish the sensitivity per unit of temperature change. 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. Yue et al. Once the cantilever array was mounted on the chip holder and aligned with the optical system. indicating that the first injection (24) Satyanarayana. unthiolated DNA. the ionic strength was fixed at 200 mM. and fresh buffer of the same type and concentration was injected to establish that no significant signal resulted from the injection process alone. Further. the cantilever response to DNA hybridization was optically monitored for at least 1 h following injection. the cantilever tip deflections were monitored every 15-30 s until the baseline positions of all cantilevers appeared stable. Multiple injections of the hybridization buffer are necessary to ensure that the complementary strand is available to the probe fully concentrated. By measuring the motion of each spot. Castelino et al. No further deflection typically results from a third injection. in good agreement with theory.18 A micropipet was used to inject 1 µL of 5 µM unlabeled. Yue et al. 2009 | http://pubs. Additional deflection occurs after the second injection. inset.25 Cantilever-Based Surface Stress Measurements. Langmuir. Specifically. defined as the fraction of probe ssDNA hybridized by complementary target ssDNA as a function of the grafting density.acs. These offsets are averaged for all the cantilevers in the population. which is about 2 cm2 in area. with the remainder of the response observed within 1/2 h following injection. 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. 392-399. N. Most of the deflection occurs after the first injection. indicating that the sensors have responded to the fully concentrated hybridization buffer. the data in this figure are part of the set used to derive the results reported in Figure 5. Syst. S. 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. and linear drift trends are removed (Figure 3).18 Briefly. 2006 265 Downloaded by READING UNIV on September 11. the ionic strength of the phosphate buffer was varied only during immobilizing or grafting of the probe single-stranded DNA (ssDNA) using thiolAu chemistry. Results In all the experiments discussed below. 14.org Publication Date (Web): November 17. mitigate false readings. and generally 30-40 µm wide. Thermally induced deflections are calibrated in nanometer units using a white light interferometer (Veeco.19 showed using fluorescence measurements that the density of thiol-attached ssDNA could be controlled by changing the ion concentration from 0. each of which contains multiple (4-12) cantilever sensors. low-stress silicon nitride and were 200-400 µm long. A third injection (not shown) resulted in no further deflection of the cantilevers. and the wells were washed three times in a buffer solution of concentration equal to that of the probe solution. Each cantilever has a rigid paddle structure at its end to provide a flat reflecting surface for optical diagnostics. All biologically induced cantilever deflections are normalized against the cantilever deflection resulting from a unit temperature change.Surface Stresses Induced by DNA Hybridization reaction wells. fully complementary to and of length equal to that of the immobilized probe DNA (C-n).5 mJ/(m2 K).. 500 mM.24 To simultaneously image the entire cantilever array chip (100-500 cantilevers at a time). The variation in sensor response for each experiment is accurately reflected in the error bars of Figure 4. No.17 reported an experimentally observed thermomechanical sensitivity of 208 ( 14 nm/K for the 200 µm long cantilevers. Multiple injections of the hybridization buffer are used to ensure that the complementary strand is available to the cantilever sensor at its full concentration. Microelectromech. which required temperature control during biological experiments.05 to 1 M (Figure 4A). was diluted by liquid remaining in the well after aspiration (just prior to the first injection). A micropipet was used to aspirate the buffer inside each well. After each injection the cantilevers are allowed to recover a stable position.18 demonstrated that cantilever sensors functionalized with end-thiolated. Surface Density Measurements. Three injections are typically necessary. Woodbury. Here two injections are visible. 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.1021/la0521645 Figure 3. 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. Majumdar. The Au-SiNx cantilever formed a thermal bimorph.5-1 µL) is aspirated just prior to each injection to make room for the new injection. 2005 | doi: 10..

These trends appear to follow those in the surface density measurements. In Figure 4A. inset). 500 mM. Immobilization of probe strands occurred in DI water. we expect slight decreases in hybridization density and slight increases in efficiency below 50 mM. and mounted on the optical detection system.19 but found that difficulties in the experimental procedure prevented the collection of reliable data. and 1 M phosphate buffer. as demonstrated by the discovery of a single-exponential scaling relation between surface stress and the density of hybridized DNA strands. sensors were washed.266 Langmuir. at various ionic strengths. suggesting. For monovalent ions. (C) Hybridization efficiency. The effectiveness of the washing procedures toward removal of incompletely hybridized targets is not known.org Publication Date (Web): November 17. and 30 nucleotides) was immobilized on cantilever surfaces at three ionic strengths (without saltsin deionized water. these forces are repulsive in nature and are active on length scales comparable to the Debye screening length. Owing to these difficulties. permitting the resolution of a greater number of response levels over the range of ionic strengths considered. We present these data to show qualitatively the trends observed in the previously described experiment in greater detail.1021/la0521645 Figure 5. 20-. Discussion The most important observation of this work is that. 500 mM. (Inset) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer. and 30-nucleotide strands in 200 mM sodium phosphate buffer. transferred to 200 mM PB. each performed in an individually addressable microfluidic well containing multiple cantilevers.acs. The 10-nucleotide DNA was chosen because it grafts at a higher density. Hydration forces . That is. and 1 M sodium phosphate buffer. The density of partially hybridized strands. Cantilever response to hybridization of 10-. On the basis of the results of Castelino et al. Hybridization took place at 200 mM ionic strength for all targets. regardless of chain length. end-thiolated probe DNA of three lengths (10. that surface densities significantly influence the cantilever response. For a given ionic strength during probe immobilization. the surface stress is dominated by the density of hybridization events. Osmotic forces arise due to the entropic motion of the counterions that are localized around the DNA. To better resolve the relationship between surface stress change during hybridization and ionic strength during immobilization. 2005 | doi: 10. In all figures error bars represent the first standard deviation from measurements on multiple gold substrates and are generally about 5%. 100 mM. we immobilized 10-nucleotide probes at 6 PB concentrations (10 mM. 1. 22. This conclusion arises from correlating the surface density and surface stress as a function of ionic strength and DNA chain length. Following incubation in the probe solution. 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. as is discussed in the next section. We do not expect these small changes to significantly affect the trends observed when the two data sets are compared in the Discussion. 200 mM. 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. defined as the fraction of probe ssDNA hybridized by complementary target ssDNA. Downloaded by READING UNIV on September 11. 100 mM sodium phosphate buffer. for the range of ionic strengths and DNA chain lengths considered. Immobilization took place in 10 mM. the cantilever response was observed to increase with increasing ionic strength during immobilization. 20. density in Castelino et al. with moderate salts100 mM PB. but counts them equally with fully hybridized targets. We note that this measurement technique does not exclude partially hybridized targets or targets that partially hybridize more than one probe. Numbers in parentheses are the numbers of cantilever measurements contributing to the 1 standard deviation error bar. which are expected to yield densities lower than those previously measured by Castelino et al. 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. to the largest surface stress change (immobilization in 1 M PB). 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. the cantilever response was observed to decrease with increasing probe/target length. (B) Hybridization density of fully complementary DNA as a function of the chain length and ionic strength at which the probe molecules were immobilized. (A) Grafting density of single-stranded. 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. Vol. 100 mM. Figure 4. and with excess salts1 M PB) for a total of nine experiments... No. thiolated probe DNA as a function of the probe length and ionic strength. 2009 | http://pubs. is expected to increase with increasing ionic strength. For a given probe/target length.19 and to a hybridization density and efficiency in Figure 4. and 1 M) and monitored the surface stress response resulting from their hybridization in 200 mM PB (Figure 5. 50 mM. 200 mM. 2006 Stachowiak et al. 50 mM. Single-stranded. Figure 5 plots the steady-state surface stress changes for different probe/ target lengths and ionic strengths used during grafting of probe ssDNA. resulting in a greater cantilever response. if present. as discussed below.

Tirrell. 2005 | doi: 10. because the length of the 10-nucleotide probes is comparable to the minimum persistence lengths. In Figure 6 we observe that. F. Hence. P. and followed an exponential fit reasonably well (Figure 7).1 chains/nm2. No. Vol. since the original number density of immobilized probes is low. 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. where N is the number of nucleotides. 1. At low ionic strength conditions. Macromolecules 1991. 22. M.15 which predicted an exponential increase in cantilever deflection with the density of DNA duplexes. p is the persistence length in angstroms. they produce a self-assembled monolayer (SAM). there are few steric constraints for the target molecules to hybridize with the probe ssDNA.B and shown in Figure 7. for 10 bp DNA hybridization. which have a much longer range. However. IV 2000. 2009 | http://pubs. as seen in our experiments. Strey et al. Increasing the ionic strength reduces the Debye length. When plotted against the grafting density of probe ssDNA (Figure 7B.. Sci. On the basis of this hypothesis. (28) Pincus. Figure 7A shows that. However. or both. the surface stress increases monotonically with increasing grafting density (decreasing chain length). (Inset) Ratio of separations predicted by a model and measurement. Tinland et al.2 nm. Macromolecules 1998.and 30-nucleotide singlestranded probes. Schorr. as seen for all the lengths of probes considered. which suggests that the surface stress is in some way related to either the grafting (probe) or hybridization (target) densities.26-28 However. 9. M. the immobilization density of probe ssDNA is high and. we conclude that the key parameter controlling surface stress is the hybridization density of DNA molecules. The large screening length consequently results in lower probe densities at low ionic strengths.. D.13 When this SAM is exposed to gas molecules. the Debye length is large and the osmotic forces. the decrease in hybridization efficiency is not large enough to account for the higher immobilization density of the probe ssDNA. However. hydration forces determine the probe spacing. Ser. the actual number density of hybridized probes on the surface ends up being much larger as compared to that of the low-concentration case. where the decay length of the interactions is about 0. arising from intrastrand sterics at high ionic strengths. However. To examine this relationship further..Surface Stresses Induced by DNA Hybridization Langmuir. J.. W. which creates surface stress to bend the cantilever beam.. the Debye length is much smaller than the hydration length and. 1163-1169. Frisbie. if the Tinland model is used and the molecules are presumed to pack in a dense cubic array on the sensor surface. the experimentally measured mean center-to-center molecular spacing of singlestranded DNA probes is roughly equal to their radii of gyration in solution. Since the hydration forces do not depend strongly on the ionic strength. the hybridization efficiency is lowered due to steric and electrostatic hindrances experienced by target molecules. are dominant and determine the spacing between probes. then depending on the binding affinity adsorption of gas molecules also produces further surface stress. Johnson. inset). the density of which is controlled by the radius of gyration. Argillier. We apply this analysis only to 20.. C. the hybridization efficiency in this regime is high. . beyond a certain ionic strength. and follows an exponential relation. However. A. it is well-known that when alkanethiols bind to one surface of a cantilever beam. Tirrell.3 Å for single-stranded DNA.. R. K. the grafting density is relatively constant in this regime.acs. Antigen-antibody binding and various other protein-protein interactions have been observed to produce (26) Kelley.1021/la0521645 Figure 6.5 Å. hence. the hybridization densities achieved are still quite low in this regime. a separate trend exists for each ionic strength during immobilization. which reduces interprobe repulsion and leads to a higher grafting density in this intermediate regime.3 nm. We note that the theory of planar electrolyte brushes and the impact of ionic strength on their formation have been well studied. Under these conditions. Using this result and imposing an intrinsic minimum persistence length of 7. This finding is in agreement with the model by Hagan et al. Thus. P. 2912-2919.. which has been used as a model molecular system. the surface stress data in Figure 5 are plotted against the grafting (probe) and hybridization (target) densities of Figure 4A. C. we may consider the surface of the cantilever not as a set of isolated molecules but as a semicontinuous film. 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. The lowest ionic strengths produce the minimum grafting density. which is influenced by the molecular length and ionic strength (through the persistence length). or about 0. The results in Figure 5 show that the cantilever mechanical response increases with ionic strength. Acad.16 showed that hydration forces dominate interstrand dynamics at separations below 3.27 The results for the hybridization density and efficiency are shown in Figure 4. Hence. For example. Under high ionic strength conditions. The above discussion has focused on DNA hybridization. the surface stress (a linear function of the cantilever deflection) is a strong function of 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. What we found remarkable was that when the surface stress was plotted against the hybridization density. 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. (27) Guenoun..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. 31. hence. are caused by perturbation of the hydrogen-bonding network of water surrounding DNA molecules.org Publication Date (Web): November 17. 24. T.19 That is. P. 2006 267 Downloaded by READING UNIV on September 11. and b0 is the proportionality constant between the strand contour length and N ) 4. in agreement with Smith et al.22 we examine the radii of gyration for the molecules of interest in this work. 4297-4300.28 That is. the distribution of counterions is not uniform in the coordinate direction parallel to the surface. D. 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.

A. inset) Cantilever response during DNA hybridization as a function of the grafting density. though it must be taken into account that the dynamics of flexible cell membranes are very different from those of rigid surfaces. produces surface stresses on micromechanical structures. the density is governed by the radius of gyration of ssDNA. surface stress. and Henry Lin of the Department of Pathology at the Keck School of Medicine of the University of Southern California and with Thomas Thundat. 500 mM. However. peptides. when the surface stress is plotted against the hybridization density. Vol.M. 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. main) Cantilever response during DNA hybridization as a function of the hybridization density. The hybridization . the osmotic pressure of counterions dominates the intermolecular forces while. Jerry Hu. Downloaded by READING UNIV on September 11. at low ionic strength. acknowledges the support of the Miller Institute through a professorship. 1. during which much of this work was performed.org Publication Date (Web): November 17. Two aspects of this investigation include (i) identifying the role of various intermolecular forces and (ii) finding a key parameter that controls mechanical response. LA0521645 Conclusions In this paper. and the DOE Basic Energy Sciences. at low ionic strength.acs. The surface stress response to the grafting density (B. where hydration forces dominate the response. inset) is scaled by the hybridization efficiency to produce a single trend that is less influenced by the DNA chain length. a model biomolecular reaction. Cote. we observe that the surface stresses collapse onto a single curve and can be described with a single-exponentially increasing trend. We acknowledge the funding through the DARPA Simbiosys program. acknowledges funding from the ARCS Foundation and the NSF through a graduate fellowship.S. 2009 | http://pubs. the NCI IMAT program. We show that. No. and small molecules that bind to membrane-bound proteins and receptors on a cell surface.10 Finally. Acknowledgment. The surface stress response to the grafting density appears to be influenced by the chain length. The lessons learned from this work could perhaps be useful in providing insight into the chemomechanics of cell membranes. J. Ram Datar. (B. there are many proteins. and Karolyn Hansen of Oak Ridge National Laboratories. at higher ionic strength. which depends on the persistence length. the implications of our observations on the mechanics of cell membranes are also worth investigation.1021/la0521645 Figure 7. which in turn depends on the ionic strength. (B. 100 mM. density and efficiency are also found to depend on the grafting density and the length of the DNA. Immobilization took place in 50 mM. We acknowledge helpful suggestions and discussions with Richard J. The cantilever response increased monotonically with an increase in the ionic strength used during immobilization. and it would be worth investigating how the surface stress depends on the density of the binding events. Life Sciences Division. 2006 Stachowiak et al.5.268 Langmuir. 22. we investigate how DNA hybridization. 200 mM. (A) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer as a function of the hybridization density. 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. and 1 M sodium phosphate buffer. the grafting density is independent of the ionic strength and hydration interactions dominate. 2005 | doi: 10. In particular. The facilities provided by the Berkeley Microfabrication Laboratory are much appreciated. Our measurements of the grafting density of probe single-stranded DNA suggest that. Measurement of surface stresses generated from DNA hybridization reactions suggests that surface stress is a strong function of the grafting density of probe molecules.C.9. which depends on the chain length.