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

17. V. 290-299. This study predicted that hydration forces and osmotic pressure dominate interstrand mechanics. and provide further insight regarding the key parameters that govern the chemomechanics of such hybrid structures. based on the empirical potentials developed by Strey et al.. (Skokie.. Langmuir 2005.. A. Biosyst. M. SS-n. Satyanarayana. The fabrication process for the microcantilever array chip and the design of optical multiplexing are described in detail in previous papers. C. M. Oligonucleotide surface densities were measured using fluorescence-based techniques on gold-coated substrates (not on cantilever arrays). Larsen.. (19) Castelino. and the concentration of these displaced duplexes was again determined by measuring the fluorescence intensity. The resulting duplexes were displaced from the gold surface using β-mercaptoethanol.23 All reagents were used without further purification. N. Table 1 lists the probe and target oligonucleotide sequences used in the experiments (GD-n. where n is the number of nucleotides (nt’s)). Thiolated DNA molecules were treated with 100 mM DTT (dithiothreitol) immediately before use to break any disulfide bonds present. L. 221-220.. 1457-1460. where both ends are unmodified. A.. B. and HD-n). G. Anal. 795-799. Chem.16 surface densities. we experimentally examine these predictions. they highlighted the importance of DNA surface densities. Table 1... Viswanadham. DTT was removed by running the solution through commercially available size exclusion columns. Hagan et al. R. All oligonucleotide sequences were purified using HPLC and were obtained from Integrated DNA Technologies Inc. A. Bedekar.22 and compared to theoretical predictions.19 and persistence length21. Sundaram.15 provided the first theoretical framework for the chemomechanics in such hybrid structures.17.18 Surface densities are modulated through variations in probe/ target length and ionic strength during probe immobilization. where the 5′ end is thiolated HS(CH2)6) and the 3′ end is unmodified. R.. 72. where the 5′ end is thiolated (HS(CH2)6) and the 3′ end is fluorescently labeled (FAM). Majumdar. Majumdar. Demers et al. S. Letsinger. Downloaded by READING UNIV on September 11. 5763-5765. 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. S. Each cantilever in the well has a ridge structure around the square paddle at its end.. E. each of which contains multiple microcantilever sensors.19 Briefly. Persegian. where the 5′ end is fluorescently labeled (FAM) and the 3′ end is unmodified. complementary (C) sequences used for hybridization on cantilever surfaces (C-n). Thiolated oligonucleotides were synthesized with a 6-mercaptohexyl linker to facilitate adhesion to gold surfaces through gold-thiol bonding.16 who worked with a hexagonally compressed array of doublestranded DNA. H. K. Gadegaard. (18) Yue... A. 10163-10173. Elghanian. 2 of 12 cantilevers were broken during fabrication... 2004. J.. In this paper... 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. No. Y. W.org Publication Date (Web): November 17. J. as shown in Figure 2. 1. (22) Smith. and sequences used to hybridize probes during hybridization density (HD) measurement (HD-n). Reynolds. and the oligonucleotides were immediately frozen to prevent the disulfide linkages from re-forming. Dedrick. A. C-n. 59. 2000. and (iv) hydration forces between DNA strands. 271. A. B. The effects of these parameter variations are correlated to changes in surface density through a previously published fluorescent measurement technique.. B 2002. S.1021/la0521645 the reaction kinetics to a Langmuir isotherm. 2006 Stachowiak et al. A. (20) Demers. (Top) Cantilever array chip. 2005 | doi: 10. Langmuir 2001. Phys. In the figure. A. 106. after which the displaced fluorescence was seen not to increase further. J. 2009 | http://pubs. Furthermore. 22. A. M. M. Kannan. (17) Yue.. 5535-5541.acs.. Syst. Sturm. Majumdar.. Microelectromech. Gold substrates were incubated in the mercaptoethanol solution for 24 h.18 The array contains 90 (15 × 6) individually addressable fluidic . Surface Density Measurement Procedure. Chem. Cui. G. Lin. C. IL). Majumdar. R. thiolated probe oligonucleotides were first immobilized (SS-n) and then hybridized with fluorescently labeled complementary target strands of equal length (HD-n). L. 22. 19561961. Microcantilever Array Chip. C... S. R. F. N. (Bottom) An individually addressable well of the cantilever array chip containing up to 12 sensors and an injection port. B. Methods Reagent Processing. ReV.264 Langmuir.. J. have shown this procedure to be highly efficient in displacement of thiolated oligonucleotides from gold surfaces. We utilized a recently developed 2-dimensional microcantilver array.. H. Podgornik. H. Using DNA as a model molecule. K. solution was measured using a fluorescence microscope and a CCD camera. (21) Tinland. 1. Mirkin. predicting an exponential increase in cantilever deflection with the density of double-stranded molecules. Science 1996. Figure 2.19. Pluen. (ii) the conformation entropy of DNA. Bustamante.15 compared the relative importance of various intermolecular forces: (i) electrostatics interactions between neighboring strands. as described in Castelino et al. We utilize a recently developed 2-dimensional microcantilever array. A. 30. Stachowiak. A. J. Jenkins. (16) Strey.. 13. (23) Papra. D. 999-1088. 2004. Mucic. Weill. which functions to create a rigid reflective structure for cantilever tracking. (iii) the osmotic pressure of counterions. Phys.20 Results are interpreted according to published studies of molecular interaction potentials. Macromolecules 1997. where n is the number of nucleotides (nt’s). Vol. single-stranded (ss) sequences immobilized on cantilever surfaces and used as probes to measure the hybridization density on gold surfaces (SS-n). which contains individually addressable microfluidic wells. Chem. B. Mech. R.20 To find the hybridization density. 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. E 1999. 17. The concentration of the displaced probes in (15) Hagan. Chakraborty...

Yue et al. and linear drift trends are removed (Figure 3). Each cantilever has a rigid paddle structure at its end to provide a flat reflecting surface for optical diagnostics. The hybridization solution consisted of 5 µM single-stranded.18 demonstrated that cantilever sensors functionalized with end-thiolated. Results In all the experiments discussed below. each of which can be correlated to a specific grafting .5 mJ/(m2 K). which represent the first standard deviation. a laser beam was expanded and reflected off the cantilever array chip.Surface Stresses Induced by DNA Hybridization reaction wells.. 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.1021/la0521645 Figure 3. 2005 | doi: 10. it was possible to detect the deflection of cantilever beams with 1-3 nm resolution. Woodbury. The flat paddles at the end of the cantilevers produced spots on a CCD camera. The cantilevers were made of silicon-rich. dissolved in 200 mM sodium phosphate buffer. Once the cantilever array was mounted on the chip holder and aligned with the optical system. 14. A typical set of deflection data from which the results presented in this paper were derived. Additional deflection occurs after the second injection. Castelino et al. the ionic strength was fixed at 200 mM. and the wells were washed three times in a buffer solution of concentration equal to that of the probe solution. which required temperature control during biological experiments. 2009 | http://pubs. All biologically induced cantilever deflections are normalized against the cantilever deflection resulting from a unit temperature change. Vol. 1 M) is apparent. it was necessary to construct a ray-optics-based whole field illumination system. The Au-SiNx cantilever formed a thermal bimorph.19 showed using fluorescence measurements that the density of thiol-attached ssDNA could be controlled by changing the ion concentration from 0. Cantilever deflections are set to zero at the time of the hybridization buffer injection. indicating that the sensors have responded to the fully concentrated hybridization buffer. thiolated probe sequences dissolved in sodium phosphate buffer (PB) (10 mM to 1 M) into individual microfluidic wells of the cantilever array. 2005.18 Briefly.18 A micropipet was used to inject 1 µL of 5 µM unlabeled.25 Cantilever-Based Surface Stress Measurements. The variation in sensor response for each experiment is accurately reflected in the error bars of Figure 4. and fresh buffer of the same type and concentration was injected to establish that no significant signal resulted from the injection process alone. 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. Majumdar.24 To simultaneously image the entire cantilever array chip (100-500 cantilevers at a time).. 1. each of which contains multiple (4-12) cantilever sensors. in good agreement with theory.5 µm thick. the ionic strength of the phosphate buffer was varied only during immobilizing or grafting of the probe single-stranded DNA (ssDNA) using thiolAu chemistry. N. 2006 265 Downloaded by READING UNIV on September 11. unthiolated DNA. During hybridization of the complementary strands. Karnik.org Publication Date (Web): November 17. 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. Three injections are typically necessary. Here two injections are visible. 392-399. Langmuir. These offsets are averaged for all the cantilevers in the population. The response of multiple cantilevers in each well can be averaged to increase the accuracy of the measurements. 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. mitigate false readings. 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. 22. defined as the fraction of probe ssDNA hybridized by complementary target ssDNA as a function of the grafting density. NY) to establish the sensitivity per unit of temperature change. and no further injections are needed. was diluted by liquid remaining in the well after aspiration (just prior to the first injection). fully complementary to and of length equal to that of the immobilized probe DNA (C-n). 0. inset. Multiple injections of the hybridization buffer are used to ensure that the complementary strand is available to the cantilever sensor at its full concentration. with the remainder of the response observed within 1/2 h following injection. No further deflection typically results from a third injection. Further. and show statistical variations. By measuring the motion of each spot.5-1 µL) is aspirated just prior to each injection to make room for the new injection. Figure 4C plots the hybridization efficiency.17 reported an experimentally observed thermomechanical sensitivity of 208 ( 14 nm/K for the 200 µm long cantilevers.05 to 1 M (Figure 4A). Figure 4B shows the density of hybridized DNA as a function of the ionic strength during attachment of probe ssDNA. the data in this figure are part of the set used to derive the results reported in Figure 5. After the deflection signals have reached stable values following the injection. After injection of the hybridization buffer (about 1 µL). the offset is recorded for each cantilever. A micropipet was used to aspirate the buffer inside each well. J. Following the immobilization period. Most of the deflection occurs after the first injection. the probe solution was aspirated from each well.acs. Specifically. No. which is about 2 cm2 in area. indicating that the first injection (24) Satyanarayana. the cantilever tip deflections were monitored every 15-30 s until the baseline positions of all cantilevers appeared stable. Surface Density Measurements. We measured the change in surface stress resulting from DNA hybridization under various combinations of chain length and ionic strength. Thermally induced deflections are calibrated in nanometer units using a white light interferometer (Veeco. Greater than 50% of the cantilever response to hybridization was typically observed in the first 5-10 min following injection. and generally 30-40 µm wide. the cantilever response to DNA hybridization was optically monitored for at least 1 h following injection. 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. S. All cantilevers were hybridized with fully complementary strands (C20) at 5 µM concentration at a fixed ionic strength of 200 mM. A third injection (not shown) resulted in no further deflection of the cantilevers. The cantilever tip deflection in response to a temperature change of 1 K was recorded to establish the thermomechanical sensitivity of each cantilever. 500 mM. R. Cantilevers in each well were incubated in the probe solution (SS-n) (thiolated DNA in sodium phosphate buffer) for at least 3 h. Multiple injections of the hybridization buffer are necessary to ensure that the complementary strand is available to the probe fully concentrated. A portion of the buffer within the wells (about 0. 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. low-stress silicon nitride and were 200-400 µm long. A. Microelectromech. Yue et al. Syst. After each injection the cantilevers are allowed to recover a stable position.

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

T. The lowest ionic strengths produce the minimum grafting density. are caused by perturbation of the hydrogen-bonding network of water surrounding DNA molecules. IV 2000. K.. for 10 bp DNA hybridization. it is well-known that when alkanethiols bind to one surface of a cantilever beam. then depending on the binding affinity adsorption of gas molecules also produces further surface stress.19 That is. 4297-4300. We apply this analysis only to 20. P. What we found remarkable was that when the surface stress was plotted against the hybridization density. where N is the number of nucleotides. the grafting density is relatively constant in this regime.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. because the length of the 10-nucleotide probes is comparable to the minimum persistence lengths. or both. the surface stress data in Figure 5 are plotted against the grafting (probe) and hybridization (target) densities of Figure 4A.. The above discussion has focused on DNA hybridization. Under these conditions. To examine this relationship further.and 30-nucleotide singlestranded probes. D. However.acs.. Ser. p is the persistence length in angstroms. However. which creates surface stress to bend the cantilever beam. (Inset) Ratio of separations predicted by a model and measurement. 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. we may consider the surface of the cantilever not as a set of isolated molecules but as a semicontinuous film. the surface stress (a linear function of the cantilever deflection) is a strong function of the hybridization density. R. The large screening length consequently results in lower probe densities at low ionic strengths.13 When this SAM is exposed to gas molecules.1021/la0521645 Figure 6. . they produce a self-assembled monolayer (SAM).. the immobilization density of probe ssDNA is high and. Schorr. M. Macromolecules 1991.Surface Stresses Induced by DNA Hybridization Langmuir. and follows an exponential relation.1 chains/nm2.. where the decay length of the interactions is about 0.2 nm. the hybridization efficiency in this regime is high. Hence. (27) Guenoun. 31.3 nm. Tirrell. hydration forces determine the probe spacing. the distribution of counterions is not uniform in the coordinate direction parallel to the surface.. the surface stress increases monotonically with increasing grafting density (decreasing chain length).. Argillier. 2912-2919.26-28 However. 2006 267 Downloaded by READING UNIV on September 11. 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. 22. For example. in agreement with Smith et al. the density of which is controlled by the radius of gyration. beyond a certain ionic strength. 2009 | http://pubs. Under high ionic strength conditions. C. We note that the theory of planar electrolyte brushes and the impact of ionic strength on their formation have been well studied. Johnson. the actual number density of hybridized probes on the surface ends up being much larger as compared to that of the low-concentration case. and followed an exponential fit reasonably well (Figure 7). A. 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.org Publication Date (Web): November 17. Tinland et al. since the original number density of immobilized probes is low. arising from intrastrand sterics at high ionic strengths. Vol. Tirrell. the Debye length is large and the osmotic forces. Strey et al.28 That is. the Debye length is much smaller than the hydration length and. P. the hybridization efficiency is lowered due to steric and electrostatic hindrances experienced by target molecules. which suggests that the surface stress is in some way related to either the grafting (probe) or hybridization (target) densities. inset). Increasing the ionic strength reduces the Debye length. as seen for all the lengths of probes considered. Macromolecules 1998. F. the decrease in hybridization efficiency is not large enough to account for the higher immobilization density of the probe ssDNA.16 showed that hydration forces dominate interstrand dynamics at separations below 3. 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. 1. a separate trend exists for each ionic strength during immobilization. the experimentally measured mean center-to-center molecular spacing of singlestranded DNA probes is roughly equal to their radii of gyration in solution. D. Acad. No. which is influenced by the molecular length and ionic strength (through the persistence length). or about 0..3 Å for single-stranded DNA. 9. we conclude that the key parameter controlling surface stress is the hybridization density of DNA molecules. Sci. M. 1163-1169. C. In Figure 6 we observe that. Figure 7A shows that. which reduces interprobe repulsion and leads to a higher grafting density in this intermediate regime. At low ionic strength conditions. W. and b0 is the proportionality constant between the strand contour length and N ) 4. Since the hydration forces do not depend strongly on the ionic strength. The results in Figure 5 show that the cantilever mechanical response increases with ionic strength. 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. hence. However.5 Å. Hence. Antigen-antibody binding and various other protein-protein interactions have been observed to produce (26) Kelley.22 we examine the radii of gyration for the molecules of interest in this work. 24.. are dominant and determine the spacing between probes.B and shown in Figure 7. the hybridization densities achieved are still quite low in this regime. which have a much longer range. P. which has been used as a model molecular system. Frisbie. 2005 | doi: 10. This finding is in agreement with the model by Hagan et al. hence. (28) Pincus. When plotted against the grafting density of probe ssDNA (Figure 7B. there are few steric constraints for the target molecules to hybridize with the probe ssDNA. as seen in our experiments. J.15 which predicted an exponential increase in cantilever deflection with the density of DNA duplexes. Thus. if the Tinland model is used and the molecules are presumed to pack in a dense cubic array on the sensor surface. On the basis of this hypothesis. Using this result and imposing an intrinsic minimum persistence length of 7. However. However.27 The results for the hybridization density and efficiency are shown in Figure 4.

500 mM. though it must be taken into account that the dynamics of flexible cell membranes are very different from those of rigid surfaces. We acknowledge the funding through the DARPA Simbiosys program. main) Cantilever response during DNA hybridization as a function of the hybridization density. Acknowledgment.9. 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. we investigate how DNA hybridization. Downloaded by READING UNIV on September 11. and Karolyn Hansen of Oak Ridge National Laboratories. Ram Datar. inset) is scaled by the hybridization efficiency to produce a single trend that is less influenced by the DNA chain length.org Publication Date (Web): November 17. a model biomolecular reaction.acs. where hydration forces dominate the response. 2009 | http://pubs. the osmotic pressure of counterions dominates the intermolecular forces while. peptides.1021/la0521645 Figure 7. at higher ionic strength. surface stress. The hybridization . No. Measurement of surface stresses generated from DNA hybridization reactions suggests that surface stress is a strong function of the grafting density of probe molecules.S. and Henry Lin of the Department of Pathology at the Keck School of Medicine of the University of Southern California and with Thomas Thundat. and small molecules that bind to membrane-bound proteins and receptors on a cell surface. Our measurements of the grafting density of probe single-stranded DNA suggest that. inset) Cantilever response during DNA hybridization as a function of the grafting density. Vol. and 1 M sodium phosphate buffer. (B. We show that. We acknowledge helpful suggestions and discussions with Richard J. which depends on the chain length. which depends on the persistence length. The cantilever response increased monotonically with an increase in the ionic strength used during immobilization. Jerry Hu. and the DOE Basic Energy Sciences. at low ionic strength. (B. 1. The surface stress response to the grafting density (B. 200 mM. (A) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer as a function of the hybridization density. LA0521645 Conclusions In this paper. However. J. 100 mM. Cote. 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. 22. there are many proteins. The lessons learned from this work could perhaps be useful in providing insight into the chemomechanics of cell membranes. when the surface stress is plotted against the hybridization density. during which much of this work was performed. the density is governed by the radius of gyration of ssDNA. In particular. The surface stress response to the grafting density appears to be influenced by the chain length. 2005 | doi: 10. The facilities provided by the Berkeley Microfabrication Laboratory are much appreciated.268 Langmuir. we observe that the surface stresses collapse onto a single curve and can be described with a single-exponentially increasing trend. produces surface stresses on micromechanical structures. Immobilization took place in 50 mM. density and efficiency are also found to depend on the grafting density and the length of the DNA.M. which in turn depends on the ionic strength. the implications of our observations on the mechanics of cell membranes are also worth investigation. Life Sciences Division. A.10 Finally. Two aspects of this investigation include (i) identifying the role of various intermolecular forces and (ii) finding a key parameter that controls mechanical response. the grafting density is independent of the ionic strength and hydration interactions dominate. acknowledges funding from the ARCS Foundation and the NSF through a graduate fellowship. acknowledges the support of the Miller Institute through a professorship. at low ionic strength.C. the NCI IMAT program.5. and it would be worth investigating how the surface stress depends on the density of the binding events. 2006 Stachowiak et al.