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.

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

R. A. Langmuir 2001. F.. J. H. Pluen.23 All reagents were used without further purification.16 surface densities. A.. Elghanian. B... R. C. 2004. (19) Castelino. 5763-5765. after which the displaced fluorescence was seen not to increase further. Bedekar. A. Majumdar. M. where n is the number of nucleotides (nt’s)). Chakraborty. A. H. The concentration of the displaced probes in (15) Hagan.. We utilize a recently developed 2-dimensional microcantilever array.16 who worked with a hexagonally compressed array of doublestranded DNA. Furthermore. as shown in Figure 2. Viswanadham. S.. Microelectromech. and provide further insight regarding the key parameters that govern the chemomechanics of such hybrid structures. Syst. predicting an exponential increase in cantilever deflection with the density of double-stranded molecules. 290-299. where the 5′ end is thiolated (HS(CH2)6) and the 3′ end is fluorescently labeled (FAM). 5535-5541. Lin. where both ends are unmodified. Phys. Sundaram. K. 2000. A. 30. (Skokie. V... 2 of 12 cantilevers were broken during fabrication. Table 1.. B. 59. Chem. Mirkin... Surface Density Measurement Procedure. and (iv) hydration forces between DNA strands. 22. G. 795-799. D.17. Majumdar. Downloaded by READING UNIV on September 11. solution was measured using a fluorescence microscope and a CCD camera. In this paper. R. The resulting duplexes were displaced from the gold surface using β-mercaptoethanol. Persegian.. Kannan. H. We utilized a recently developed 2-dimensional microcantilver array. E. Jenkins... 22. single-stranded (ss) sequences immobilized on cantilever surfaces and used as probes to measure the hybridization density on gold surfaces (SS-n). J. 2004. L. thiolated probe oligonucleotides were first immobilized (SS-n) and then hybridized with fluorescently labeled complementary target strands of equal length (HD-n).. No. A... (21) Tinland. where the 5′ end is thiolated HS(CH2)6) and the 3′ end is unmodified. where the 5′ end is fluorescently labeled (FAM) and the 3′ end is unmodified. Gold substrates were incubated in the mercaptoethanol solution for 24 h. 1. Podgornik. 13. (Top) Cantilever array chip. S. A. SS-n. R... Gadegaard. Cui. Vol. Hagan et al.20 Results are interpreted according to published studies of molecular interaction potentials. complementary (C) sequences used for hybridization on cantilever surfaces (C-n). based on the empirical potentials developed by Strey et al.. Sturm. ReV. 271. Demers et al. M. Microcantilever Array Chip. A. have shown this procedure to be highly efficient in displacement of thiolated oligonucleotides from gold surfaces. 72. and sequences used to hybridize probes during hybridization density (HD) measurement (HD-n). N. M.20 To find the hybridization density. C-n. they highlighted the importance of DNA surface densities.. 10163-10173.15 provided the first theoretical framework for the chemomechanics in such hybrid structures. each of which contains multiple microcantilever sensors.. The effects of these parameter variations are correlated to changes in surface density through a previously published fluorescent measurement technique.. M. R. 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. Majumdar. 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. J. Methods Reagent Processing. Majumdar. N. Larsen.19 and persistence length21. A. 17. 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. Thiolated DNA molecules were treated with 100 mM DTT (dithiothreitol) immediately before use to break any disulfide bonds present... Anal. Table 1 lists the probe and target oligonucleotide sequences used in the experiments (GD-n. In the figure. Science 1996. J. A. C.18 Surface densities are modulated through variations in probe/ target length and ionic strength during probe immobilization.. B. Reynolds. 1457-1460. 2005 | doi: 10. Dedrick. Y.19. Biosyst. and the concentration of these displaced duplexes was again determined by measuring the fluorescence intensity. E 1999.15 compared the relative importance of various intermolecular forces: (i) electrostatics interactions between neighboring strands. Mech. (23) Papra. A. Bustamante. Langmuir 2005. B 2002. Oligonucleotide surface densities were measured using fluorescence-based techniques on gold-coated substrates (not on cantilever arrays). All oligonucleotide sequences were purified using HPLC and were obtained from Integrated DNA Technologies Inc. which functions to create a rigid reflective structure for cantilever tracking. and the oligonucleotides were immediately frozen to prevent the disulfide linkages from re-forming. C. (16) Strey. Chem. IL).17. Using DNA as a model molecule. This study predicted that hydration forces and osmotic pressure dominate interstrand mechanics. Phys. Mucic.org Publication Date (Web): November 17. The fabrication process for the microcantilever array chip and the design of optical multiplexing are described in detail in previous papers. Satyanarayana. (Bottom) An individually addressable well of the cantilever array chip containing up to 12 sensors and an injection port. Letsinger. (20) Demers. Thiolated oligonucleotides were synthesized with a 6-mercaptohexyl linker to facilitate adhesion to gold surfaces through gold-thiol bonding. Stachowiak. and HD-n). Chem. K. G.22 and compared to theoretical predictions.. (18) Yue. 2009 | http://pubs. 221-220. where n is the number of nucleotides (nt’s). L.264 Langmuir. as described in Castelino et al. 1. (17) Yue. Weill. we experimentally examine these predictions. J. DTT was removed by running the solution through commercially available size exclusion columns.19 Briefly. Hagan et al. S. (ii) the conformation entropy of DNA.18 The array contains 90 (15 × 6) individually addressable fluidic . Figure 2. 2006 Stachowiak et al. 999-1088.. W.acs. (22) Smith. 19561961.. S. Macromolecules 1997. B. Each cantilever in the well has a ridge structure around the square paddle at its end. (iii) the osmotic pressure of counterions. which contains individually addressable microfluidic wells. 106.1021/la0521645 the reaction kinetics to a Langmuir isotherm.

dissolved in 200 mM sodium phosphate buffer. Further. 0. 392-399. We measured the change in surface stress resulting from DNA hybridization under various combinations of chain length and ionic strength. By measuring the motion of each spot. 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. Multiple injections of the hybridization buffer are used to ensure that the complementary strand is available to the cantilever sensor at its full concentration. The cantilevers were made of silicon-rich. Syst. Yue et al. Surface Density Measurements.25 Cantilever-Based Surface Stress Measurements. which required temperature control during biological experiments.1021/la0521645 Figure 3. in good agreement with theory. Yue et al. A micropipet was used to aspirate the buffer inside each well.19 showed using fluorescence measurements that the density of thiol-attached ssDNA could be controlled by changing the ion concentration from 0. Additional deflection occurs after the second injection. mitigate false readings. fully complementary to and of length equal to that of the immobilized probe DNA (C-n). the data in this figure are part of the set used to derive the results reported in Figure 5. Thermally induced deflections are calibrated in nanometer units using a white light interferometer (Veeco.18 A micropipet was used to inject 1 µL of 5 µM unlabeled. All biologically induced cantilever deflections are normalized against the cantilever deflection resulting from a unit temperature change. 22.24 To simultaneously image the entire cantilever array chip (100-500 cantilevers at a time).5 mJ/(m2 K). Langmuir. the probe solution was aspirated from each well. the cantilever tip deflections were monitored every 15-30 s until the baseline positions of all cantilevers appeared stable. 1. A. each of which contains multiple (4-12) cantilever sensors. Following the immobilization period. All cantilevers were hybridized with fully complementary strands (C20) at 5 µM concentration at a fixed ionic strength of 200 mM. low-stress silicon nitride and were 200-400 µm long. Woodbury. Greater than 50% of the cantilever response to hybridization was typically observed in the first 5-10 min following injection. After the deflection signals have reached stable values following the injection. 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. with the remainder of the response observed within 1/2 h following injection. Karnik. a laser beam was expanded and reflected off the cantilever array chip. defined as the fraction of probe ssDNA hybridized by complementary target ssDNA as a function of the grafting density.. 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. The hybridization solution consisted of 5 µM single-stranded. Once the cantilever array was mounted on the chip holder and aligned with the optical system. inset.18 Briefly. A third injection (not shown) resulted in no further deflection of the cantilevers. Cantilever deflections are set to zero at the time of the hybridization buffer injection. it was possible to detect the deflection of cantilever beams with 1-3 nm resolution. 14. After injection of the hybridization buffer (about 1 µL). each of which can be correlated to a specific grafting . S. J. which is about 2 cm2 in area. the cantilever response to DNA hybridization was optically monitored for at least 1 h following injection. thiolated probe sequences dissolved in sodium phosphate buffer (PB) (10 mM to 1 M) into individual microfluidic wells of the cantilever array. No. 500 mM.5 µm thick. R. After each injection the cantilevers are allowed to recover a stable position. Specifically. The Au-SiNx cantilever formed a thermal bimorph. Majumdar. A portion of the buffer within the wells (about 0. 2005. N. and no further injections are needed.acs. Figure 4B shows the density of hybridized DNA as a function of the ionic strength during attachment of probe ssDNA. 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.5-1 µL) is aspirated just prior to each injection to make room for the new injection. Three injections are typically necessary. unthiolated DNA. No further deflection typically results from a third injection. Microelectromech. indicating that the sensors have responded to the fully concentrated hybridization buffer. Most of the deflection occurs after the first injection. A typical set of deflection data from which the results presented in this paper were derived. 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. Results In all the experiments discussed below. was diluted by liquid remaining in the well after aspiration (just prior to the first injection). indicating that the first injection (24) Satyanarayana. 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.05 to 1 M (Figure 4A). 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. Vol. 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.Surface Stresses Induced by DNA Hybridization reaction wells.org Publication Date (Web): November 17. The variation in sensor response for each experiment is accurately reflected in the error bars of Figure 4.17 reported an experimentally observed thermomechanical sensitivity of 208 ( 14 nm/K for the 200 µm long cantilevers. 2006 265 Downloaded by READING UNIV on September 11. 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. Figure 4C plots the hybridization efficiency. The flat paddles at the end of the cantilevers produced spots on a CCD camera. Here two injections are visible. 1 M) is apparent. and the wells were washed three times in a buffer solution of concentration equal to that of the probe solution. the ionic strength of the phosphate buffer was varied only during immobilizing or grafting of the probe single-stranded DNA (ssDNA) using thiolAu chemistry. The cantilever tip deflection in response to a temperature change of 1 K was recorded to establish the thermomechanical sensitivity of each cantilever.18 demonstrated that cantilever sensors functionalized with end-thiolated. The response of multiple cantilevers in each well can be averaged to increase the accuracy of the measurements. 2009 | http://pubs. Multiple injections of the hybridization buffer are necessary to ensure that the complementary strand is available to the probe fully concentrated.. 2005 | doi: 10. Castelino et al. it was necessary to construct a ray-optics-based whole field illumination system. 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. and generally 30-40 µm wide. the offset is recorded for each cantilever. NY) to establish the sensitivity per unit of temperature change. These offsets are averaged for all the cantilevers in the population. During hybridization of the complementary strands.

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

for 10 bp DNA hybridization. Thus. in agreement with Smith et al.22 we examine the radii of gyration for the molecules of interest in this work. This finding is in agreement with the model by Hagan et al. R.3 Å for single-stranded DNA. Ser. 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 large screening length consequently results in lower probe densities at low ionic strengths. the hybridization efficiency in this regime is high. we conclude that the key parameter controlling surface stress is the hybridization density of DNA molecules. (Inset) Ratio of separations predicted by a model and measurement. arising from intrastrand sterics at high ionic strengths.5 Å. the surface stress increases monotonically with increasing grafting density (decreasing chain length).acs.. which suggests that the surface stress is in some way related to either the grafting (probe) or hybridization (target) densities. and follows an exponential relation. However.2 nm. and followed an exponential fit reasonably well (Figure 7).. are dominant and determine the spacing between probes. or both. are caused by perturbation of the hydrogen-bonding network of water surrounding DNA molecules.27 The results for the hybridization density and efficiency are shown in Figure 4.26-28 However. if the Tinland model is used and the molecules are presumed to pack in a dense cubic array on the sensor surface. At low ionic strength conditions. p is the persistence length in angstroms. the Debye length is much smaller than the hydration length and. Acad. the Debye length is large and the osmotic forces. C.B and shown in Figure 7.19 That is.and 30-nucleotide singlestranded probes. it is well-known that when alkanethiols bind to one surface of a cantilever beam. as seen for all the lengths of probes considered.org Publication Date (Web): November 17. the surface stress (a linear function of the cantilever deflection) is a strong function of the hybridization density. the experimentally measured mean center-to-center molecular spacing of singlestranded DNA probes is roughly equal to their radii of gyration in solution. D. Antigen-antibody binding and various other protein-protein interactions have been observed to produce (26) Kelley.Surface Stresses Induced by DNA Hybridization Langmuir. 2912-2919. the grafting density is relatively constant in this regime. Strey et al.. hence. 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. Schorr. However. where N is the number of nucleotides. where the decay length of the interactions is about 0. Argillier. we may consider the surface of the cantilever not as a set of isolated molecules but as a semicontinuous film. (28) Pincus. Frisbie. the immobilization density of probe ssDNA is high and. 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. beyond a certain ionic strength.. and b0 is the proportionality constant between the strand contour length and N ) 4. which creates surface stress to bend the cantilever beam. To examine this relationship further.13 When this SAM is exposed to gas molecules. a separate trend exists for each ionic strength during immobilization. because the length of the 10-nucleotide probes is comparable to the minimum persistence lengths. T. 2009 | http://pubs. there are few steric constraints for the target molecules to hybridize with the probe ssDNA. No.3 nm. they produce a self-assembled monolayer (SAM). For example.15 which predicted an exponential increase in cantilever deflection with the density of DNA duplexes. However. 2005 | doi: 10. Hence. hydration forces determine the probe spacing. In Figure 6 we observe that. What we found remarkable was that when the surface stress was plotted against the hybridization density.28 That is. the surface stress data in Figure 5 are plotted against the grafting (probe) and hybridization (target) densities of Figure 4A. Under these conditions. Sci. Hence. then depending on the binding affinity adsorption of gas molecules also produces further surface stress. Under high ionic strength conditions. the actual number density of hybridized probes on the surface ends up being much larger as compared to that of the low-concentration case. 22. which has been used as a model molecular system. . C. 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. Tirrell. which reduces interprobe repulsion and leads to a higher grafting density in this intermediate regime. When plotted against the grafting density of probe ssDNA (Figure 7B. 31. the density of which is controlled by the radius of gyration. Johnson. 1. which have a much longer range. Macromolecules 1991. inset). D. 1163-1169.. 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. K. Increasing the ionic strength reduces the Debye length. On the basis of this hypothesis.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. J. P. 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. The above discussion has focused on DNA hybridization. M. P. since the original number density of immobilized probes is low. Figure 7A shows that. Tinland et al. or about 0. 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 of ionic strength on their formation have been well studied. Macromolecules 1998. hence. the decrease in hybridization efficiency is not large enough to account for the higher immobilization density of the probe ssDNA. P. IV 2000. However.. We apply this analysis only to 20. The results in Figure 5 show that the cantilever mechanical response increases with ionic strength. W. the hybridization densities achieved are still quite low in this regime. Vol. Using this result and imposing an intrinsic minimum persistence length of 7.. 24. F. 4297-4300. 2006 267 Downloaded by READING UNIV on September 11.1 chains/nm2.16 showed that hydration forces dominate interstrand dynamics at separations below 3. M. A. The lowest ionic strengths produce the minimum grafting density. (27) Guenoun. the distribution of counterions is not uniform in the coordinate direction parallel to the surface.. as seen in our experiments.1021/la0521645 Figure 6.. 9. Tirrell. However.

We acknowledge the funding through the DARPA Simbiosys program. and small molecules that bind to membrane-bound proteins and receptors on a cell surface. though it must be taken into account that the dynamics of flexible cell membranes are very different from those of rigid surfaces. Acknowledgment. the NCI IMAT program. produces surface stresses on micromechanical structures. where hydration forces dominate the response. 500 mM. 2009 | http://pubs. In particular. J. acknowledges funding from the ARCS Foundation and the NSF through a graduate fellowship. Downloaded by READING UNIV on September 11. the osmotic pressure of counterions dominates the intermolecular forces while. a model biomolecular reaction. Cote. We acknowledge helpful suggestions and discussions with Richard J. LA0521645 Conclusions In this paper. The surface stress response to the grafting density appears to be influenced by the chain length. Jerry Hu. during which much of this work was performed.org Publication Date (Web): November 17. 2005 | doi: 10. No. The cantilever response increased monotonically with an increase in the ionic strength used during immobilization. 2006 Stachowiak et al. the grafting density is independent of the ionic strength and hydration interactions dominate.S. we observe that the surface stresses collapse onto a single curve and can be described with a single-exponentially increasing trend. We show that. density and efficiency are also found to depend on the grafting density and the length of the DNA. and Henry Lin of the Department of Pathology at the Keck School of Medicine of the University of Southern California and with Thomas Thundat.M. (B. Immobilization took place in 50 mM. inset) is scaled by the hybridization efficiency to produce a single trend that is less influenced by the DNA chain length. 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. at low ionic strength. and it would be worth investigating how the surface stress depends on the density of the binding events. and Karolyn Hansen of Oak Ridge National Laboratories. the implications of our observations on the mechanics of cell membranes are also worth investigation. inset) Cantilever response during DNA hybridization as a function of the grafting density. we investigate how DNA hybridization. The lessons learned from this work could perhaps be useful in providing insight into the chemomechanics of cell membranes.10 Finally. which depends on the persistence length. surface stress. when the surface stress is plotted against the hybridization density. 1. at higher ionic strength. main) Cantilever response during DNA hybridization as a function of the hybridization density. The surface stress response to the grafting density (B. and the DOE Basic Energy Sciences. Ram Datar. the density is governed by the radius of gyration of ssDNA. Two aspects of this investigation include (i) identifying the role of various intermolecular forces and (ii) finding a key parameter that controls mechanical response. (A) Cantilever response to hybridization of 10-nucleotide DNA in 200 mM sodium phosphate buffer as a function of the hybridization density. Our measurements of the grafting density of probe single-stranded DNA suggest that. However.1021/la0521645 Figure 7.9. (B. 100 mM. Life Sciences Division.C. The facilities provided by the Berkeley Microfabrication Laboratory are much appreciated. 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. there are many proteins. A. Vol. which depends on the chain length. peptides. which in turn depends on the ionic strength. and 1 M sodium phosphate buffer. The hybridization .268 Langmuir. 22. at low ionic strength.5. 200 mM.acs. acknowledges the support of the Miller Institute through a professorship. Measurement of surface stresses generated from DNA hybridization reactions suggests that surface stress is a strong function of the grafting density of probe molecules.

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