SERS Study of Rotational Isomerization of Cysteamine Induced by

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SERS Study of Rotational Isomerization of Cysteamine Induced by Magnetic Pulling Force Takeyoshi Goto and Hitoshi Watarai* Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received September 25, 2009. Revised Manuscript Received January 15, 2010 The effects of unidirectional pulling forces on covalently bridged cysteamine between superparamagnetic particles and Ag nanoparticles (NPs) were studied with SERS spectroscopy. With an increase in the pulling force from 0 to 100 pN per magnetic particle, the ν(C-S)Trans/ν(C-S)Gauche intensity ratio was increased from 0.6 to 1.08, the Raman frequency of ν(C-S)Trans was shifted from 716 to 719 cm-1, and the Raman bands associated with the amide groups were diminished. From these observations, it was concluded that the magnetic forces induced the extension of distance between the magnetic particle surface and the Ag NP surface and the rotational isomerization equilibrium of SC-CN was shifted from the gauche to the trans conformation with the longer molecular length.

Introduction Mechanical forces can regulate molecular structures and their functions in various ways. Molecules sense the variations in their environmental fields and transduce those to the other molecular system as mechanical forces. A typical example is that cells sense the environmental stimuli through the molecular interactions of transmembrane proteins with the extracellular matrix and physically reorganize their cytoskeleton.1,2 External forces on molecular systems strain the Gibbs free energy landscape of molecules and result in the changes in their kinetic and equilibrium. The range of biologically relevant forces is from piconewtons (van der Waals interactions) to nanonewtons (covalent interactions) for a single bond.3,4 For a scale comparison, the thermal energy kBT at 300 K corresponds to the translational energy to move a molecule by 1 nm with the external force of 4 pN or to the vibrational energy to stretch the C-S bond by 0.06 A˚ with the force constant (k = 267 N/m). This leads to the idea that an external force pulling a molecule will have a similar effect with temperature to the Gibbs free energy on chemical reactions such as bond dissociation. The studies of a relationship between a mechanical strain and the resultant dynamic chemical processes at a molecular level are still progressive subjects. Dynamic force spectroscopy (DFS) has been developed recently, in which bond strengths and energy landscape are determined by measuring the loading rate (force/time) dependence on an external force. The DFS studies have been conducted possibly with the advents of new nanotools and specifically functionalized materials: atomic force microscopy (AFM),5,6 optical tweezers,7,8 *Corresponding author. E-mail: [email protected]. (1) Geroges, P. C.; Janmey, P. A. J. Appl. Physiol. 2005, 98, 1547–1553. (2) Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Nat. Rev. Mol. Cell Biol. 2001, 2, 793–805. (3) Vogel, V. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 459–488. (4) Evans, E. Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 105–28. (5) Zlanatova, J.; Lindsay, S. M.; Leuba, S. H. Prog. Biophys. Mol. Biol. 2000, 74, 37–61. (6) Willemsen, O. H.; Snel, M. M. E.; Cambi, A.; Greve, J.; Grooth, B. G.; Fidgor, C. G. Biophys. J. 2000, 79, 3267–3281. (7) Bustamante, C.; Smith, S. B.; Liphardt, J.; Smith, D. Curr. Opin. Struct. Biol. 2000, 10, 279–285. (8) Wang, M. D.; Yin, H.; Landlick, R.; Gelles, J.; Block, S. M. Biophys. J. 1997, 72, 1335–1346. (9) Danilowicz, C.; Greenfield, D.; Prentiss, M. Anal. Chem. 2005, 77, 3023– 3028.

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magnetic tweezers,9,10 and electromagnetophoresis.11,12 The subjects are covering a wide range from single molecular noncovalent interactions13 to cellular interactions.14 Raman spectroscopy is one of the feasible ways to study external force effects on a sample at the molecular level. A Raman spectrum directly reflects a bond energy landscape, as the force constant k = (δ2V/δx2)x=x0 with the harmonic-oscillator approximation. The changes in the slope of an activation energy barrier and the equilibrium states induced by an external force are directly observed from the shift of molecular vibrational frequency and the Raman intensity. Various studies on the effect of shear forces on bulk liquid and solid under high pressures (∼GPa)15-17 and on single molecules have been reported with Raman spectroscopy and tip-enhanced Raman (TER) technique.18-20 The detection of Raman scattering from a molecular monolayer is highly challenging, since its light path length is typically less than 10 nm. Surface-enhanced Raman scattering (SERS) spectroscopy is very effective for the analyses of a molecular monolayer or even a submonolayer adsorbed on a certain metal. The SERS technique can enhance the Raman scattering intensity of adsorbed molecules roughly 106 times by using an appropriate metal substrate and an excitation laser. The enhancement effect is based on electromagnetic field (EM) enhancement and charge transfer (CT) between the adsorbed molecules and the metal surface.21 (10) Gore, J.; Byrant, Z.; Stone, M. D.; Nollmann, M.; Cozzarelli, N. R.; Bustamante, C. Nature 2006, 439, 100–104. (11) Iiguni, Y.; Watarai, H. Anal. Sci. 2007, 23, 121–126. (12) Iignu, Y.; Watarai, H. J. Chromatogr. A 2005, 1073, 93–98. (13) Merkel, R.; Nassoy, P.; Leund, A.; Ritchie, K.; Evans, E. Nature 1999, 397, 50–53. (14) Lele, T. P.; Pendse, J.; Kumar, S.; Salanga, M.; Karavitis, J.; Ingber, D. E. J. Cell. Physiol. 2006, 207, 187–194. (15) Kavitha, G.; Narayana, C. J. Phys. Chem. B 2006, 110, 8777–8781. (16) Davydov, V. A.; Kashevarova, L. S.; Rakhmanina, A. V. Phys. Rev. B 2000, 61, 11936–11945. (17) Goryainov, S. V.; Boldyreva, E. V.; Kolesnik, E. N. Chem. Phys. Lett. 2006, 419, 496–500. (18) Spitsina, N. G.; Motyakin, M. V.; Bashkin, I. V.; Meletov, K. P. J. Phys.: Condens. Matter 2002, 14, 11089–11092. (19) Watanabe, H.; Ishida, Y.; Hayazawa, N.; Inouye, Y.; Kawata, S. Phys. Rev. B 2004, 69, 155418. (20) Saito, Y.; Motohashi, M.; Hayazawa, N. Appl. Phys. Lett. 2006, 88, 143109. (21) McCreery, R. L. In Raman spectroscopy for Chemical Analysis; Winefordner, J. D., Ed.; John Wiley & Sons: New York, 2000; Vol. 15, Chapter 13.

Published on Web 01/28/2010

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We have recently proposed a possibility of a new force-spectroscopic method, which analyzed the effects of pulling force on the molecular submonolayer by the combination of a magnetic force application and SERS spectroscopy.22 The most important advantage of this method in comparison with other forced methods such as an optical trapping method7,8 is the capability to directly obtain conformational and structural information on the forced molecules from the vibrational spectra (SERS). In the present work, we will show the dependence of the rotational isomerization equilibrium of cysteamine on the controlled degrees of exerted forces. Cysteamine molecules covalently bridged between magnetic particles and Ag nanoparticles (NPs). The magnetic particles were used as force transducers, and the unidirectional magnetic field gradients were applied to the magnetic particles with Nd-Fe-B magnets. The resultant forces were exerted to the bridged cysteamine molecules. The degree of the forces per magnetic particle was regulated from 0 to 100 pN by controlling distance between a sample surface and a magnet surface. The changes in the equilibrium states of cysteamine isomerization, depending on the degrees of pulling forces, were studied from their SERS spectra.

Experimental Section Materials. Silver nitrate (99.9999% trace metal basis, Aldrich), sodium citrate tribasic dehydrate (Sigma-Aldrich), (2-(N-morpholino)ethanesulfonic acid monohydrate (MES, Fluka), N-ethylN0 -(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDAC, Fluka), N-hydroxysuccinimide (NHS, Aldrich), cysteamine hydrochloride (Sigma), and carboxyl-functionalized superparamagnetic particle (diameter 1 μm, MagSense Life Sciences, Inc.) were used as received. All the aqueous solutions were prepared with distilled and deionized water purified by a Milli-Q system (∼18.2 MΩ cm). Preparation of SERS Substrates. Ag NPs were prepared by the reduction of silver nitrate with sodium citrate.23 A silver nitrate aqueous solution (1.06 mM, 200 mL) was stirred and purged with nitrogen gas for 15 min. The solution was boiled, and 10 mL of a citrate solution (34 mM) was added. The solution was refluxed for 90 min to complete the reduction of Ag ions. The Ag sol solution was centrifuged for 30 min (3700g), and the supernatant solution was used as the Ag NP dispersed solution. As a SERS substrate, Matsunami adhesive silane (MAS, amino-functionalized)-coated glass slide (Matsunami Glass Ind., Ltd., Japan) was cut (20  26 mm) and ultrasonicated in ethanol and water for each 15 min. The Ag NP solution (200 μL) was dropped on the cleaned glass slide and kept for overnight to immobilize Ag NPs. Then, the slide was washed by immersing it in water and vacuum-dehydrated at 25
C to agglomerate Ag NPs on the slide. Preparation of Cysteamine Bound Magnetic Particles. To form the covalent bonds of cysteamine molecules to the carboxylfunctionalized magnetic particles, the carboxyl groups of the magnetic particles were chemically modified to succinimidyl ester. The magnetic particle stock sol (0.5 mL) was washed three times in 3 mL of MES buffer (pH 5.8, 10 mM). An EDAC solution (200 mM, 0.25 mL, MES buffer) and a NHS solution (200 mM, 0.25 mL, MES buffer) were added to the sol solution and mixed for 15 min at room temperature. The particles were washed three times in 3 mL of MES buffer. A cysteamine hydrochloride solution (20 mM, 1 mL, MES buffer) was added and mixed for 2 h. The particles were washed 15 times in 3 mL of MES buffer and resuspended in 2 mL of MES buffer. The cysteamine bound magnetic particle solution (0.2 mL) was dropped on the SERS substrate and kept at least for 2 h. The substrate was washed by immersing it in water just before SERS measurements. In the (22) Goto, T.; Shigeki, T.; Watarai, H. Anal. Sci. 2007, 23, 891–893. (23) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 88, 3391–3395.

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supernatant solutions, no SERS bands attributable to cysteamine were observed, and therefore the contribution of Raman scattering from the free cysteamine molecules was not considered.

Application of Magnetic Field Gradients on Magnetic Particles. For the exertion of unidirectional (z-direction) pulling forces on the magnetic particles bound to Ag NPs through cysteamine molecules, magnetic field gradients were applied to a sample slide with a magnet set, which was arranged with two Nd-Fe-B magnets (0.55 T, 40  80  10 mm) and two iron pole pieces (5  3  2 mm) (Supporting Information, Figure S1). Two iron pole pieces were set on the magnets to intensify the magnetic flux (1.5 T) and its gradients (1050 T/m). A gap between the pole pieces was 2 mm. The magnet set can be moved separately under the sample slide. The magnetic flux density at each position was measured with a gauss meter (HGM-3000P, ADS Corp., Japan). The probe of the gauss meter was a hole-effect probe FS-7S (Toyojiki Industry Co., Ltd.), and its detection area was 70  70 μm. The magnetic flux density is related to the magnetic force (Fm) by the following equations: Fm ¼ MðBÞrB

MðBÞ ¼ BV

χp ðBÞ -χm μ0

ð1Þ

ð2Þ

where M(B) is the relative magnetization of a superparamagnetic particle in comparison with the solvent, rB is the magnetic field gradient at the given position, V is the volume of a superparamagnetic particle, μ0 is the permeability of free space, and χp and χm are the volume magnetic susceptibilities of a magnetic particle and solvent (χp = 333 at 0.6 T and χglycerol = -9.74  10-6).

Determination of Magnetic Force on a Single Magnetic Particle. A magnetic force (Fm) at the z-direction exerted on a magnetic particle was determined independently using the Stokes drag equation. The magnetic particles were washed in water several times and vacuum-dried at 30
C. The magnetic particles were dispersed in glycerol, and the dispersed solution was introduced in a hollow square capillary tube (0.4  0.4 mm in the internal dimension, VitroCom, Inc.). The magnet set was rotated 90
for the z-axis to be horizontal. The capillary was set horizontally between the pole pieces along the rotated z-axis. The average magnetophoretic velocities of magnetic particles, which moved toward the pole pieces, for 40 μm at different distances from the pole pieces were measured with a long working distance objective lens (9, NA 0.28) equipped with a zoom CCD camera. The magnetic forces at the given distance were calculated using Stokes drag equation9,24-26 Fm ¼ 6πrηv

ð3Þ

where r is the radius of a magnetic particle, η the viscosity of glycerol (934 mPa s at 25
C), and v the average magnetophoretic velocity at the given position. Spectroscopic Measurements. SERS spectra were measured with a microscope Raman system (Photon Design Corp., Japan), which included an objective lens (18, NA 0.42), an argon ion gas laser (514.5 nm, Stabilite 2017, Spectra-Physics Laser Inc.), a spectrograph (spectral slit width 7.0 cm-1, HR-320, Jobin Yvon Ltd.), and a liquid nitrogen cooled CCD detector (LN/CCD1100-PB/UVAR/1, Roper Scientific). The spectrograph was calibrated with the Raman frequencies of toluene. The incident angle of the laser was 74
, and the laser power at the sample was 2 mW. The spectral measurement area through the objective lens (200  200 μm) was regulated by a spatial filter. (24) Assi, F.; Jenks, R. J. Appl. Phys. 2002, 92, 5584–5586. (25) Watarai, H.; Namba, M. Anal. Sci. 2001, 17, 1233–1236. (26) Suwa, M.; Watarai, H. Anal. Chem. 2002, 74, 5027–5032.

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The microscopic plasmon absorption spectra of immobilized Ag NP slides were measured with a fiber-optic spectrophotometer (USB2000, Ocean Optics). A xenon arc lamp light source was collimated and focused on the slides through an objective lens (18, NA 0.42), and the transmitted light was collected through the other objective lens (9, NA 0.28). The collected light was focused on the entrance of a fiber and delivered to the spectrophotometer. The exposure time on the CCD detector was 2 s at each absorption measurement. The plasmon absorption spectra of Ag NPs dispersed in an aqueous solution were measured with a UV/vis/NIR spectrophotometer (path length 1 mm, V-570, JASCO Corp., Japan).

Results and Discussion Figure S1 (Supporting Information) shows a schematic illustration of the cysteamine structures (gauche and trans conformations) covalently bridging between the magnetic particle and the Ag NPs and a microscopic setup of the magnetic force-SERS spectroscopic measurements. The magnets and the pole pieces generate magnetic field gradients at a gap between two pole pieces, which exert pulling forces on the superparamagnetic particles. The center line between the pole pieces is defined as a vertical line (z-axis) with respect to the horizontal sample surface. To study the effects of pulling forces on the cysteamine molecules, the microscopic SERS spectra on the sample slide were measured at various distances along the center line of the pole pieces. The measured SERS spectra only included the vibrational information from the cysteamine molecules, which formed covalent bonds with both Ag NPs and magnetic particles. The Raman scattering from the cysteamine molecules not bonding with Ag NPs was very weak and negligible. The packing of magnetic particles (1 μm in diameter, sphere shape) on the Ag NP substrate was thought as a closed-packing monolayer. But it was difficult to examine the real packing state of magnetic particles from the microscopic observation. The AFM image (1  1 μm) of the Ag NP substrate (Supporting Information, Figure S2) corresponds to the space occupied by a single magnetic particle. The Ag NP size was observed as 62 ( 21 nm measured with AFM.27 The topographic images showed that the surface roughness (maximum heights depending Ag NP size and shape) was between 30 and 90 nm. Because of the uncertainty of the real packing state of magnetic particle and the surface roughness of SERS substrates, the measured SERS spectra represent the ensemble averages over some distributions of bridged cysteamine molecular density and angle on the substrate. Stokes Drag. The degree of pulling forces at the given distance was determined from the microscopic observations of the average magnetophoretic velocities of magnetic particles in glycerol using the Stokes drag equation (3). The microscopic images of magnetic particle trajectories toward the gap of pole pieces were recorded, and the average velocities for 40 μm (N = 100) were measured at each position. Figure 1 shows the magnetic force determined from the observed velocities (9, the left axis), the force calibration fitting curve (solid line), and the magnetic field gradients measured with the gauss meter (dotted line, right axis). The spread in the observed forces mainly results from the variation in the iron oxide content and in the size of magnetic particles. The manufacture (MagSense Life Sciences, Inc.) reported that the saturated magnetization of this particle was 50 emu/g, and the variant coefficient of each particle was less than 20%. The observed magnetic force showed the linear dependence on the magnetic field gradient above 0.6 T, at which the magnetization of magnetic (27) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012–1024.

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Figure 1. Pulling forces applied to the 1 μm superparamagnetic

particles suspended in glycerol (η = 934 mPa s at 25
C). The forces (9) were determined from the observed magnetophoretic velocities (N = 100). The distance shows a given point from the magnet set surface. The dotted line shows the measured dB/dz (right axis) with a gauss meter, and the solid line shows the force calibration fitting curve.

particle was saturated at 74 emu/g. The measured saturated magnetization was 50% larger than the reported value. The values of rB in the z-direction were much more intense (1050 T/m) than in the x- and y-directions (120 T/m) at the center line of the gap, and thus the z-directional pulling force on the bridged cysteamine molecules was predominant. Plasmon Absorption Spectra of Ag NPs. The absorption spectrum of dispersed Ag NPs in an aqueous solution (path length 1 mm) and the microscopic absorption spectra of SERS substrates, on which Ag NPs were not agglomerated or agglomerated, were measured to confirm the plasmon resonance states. Figure 2 shows the absorption spectra of three samples (9, dispersed in a solution; 2, not agglomerated; and O, agglomerated on the slide). The plasmon spectra reflect the shape, the size, and the architecture of the aggregates of Ag NPs. The spectrum of Ag NPs dispersed in the aqueous solution showed the maximum wavelength, λmax at 400-405 nm, depending on a batch. The maximum wavelength of Ag NPs immobilized on the glass slide was 395 nm corresponding to the monomers or transversal plasmons.28 The immobilized Ag NPs were monolayer coverage on the glass slide. A broad band around 600 nm was assigned to the longitudinal plasmons in dimers.28 Since the strong plasmon induced by dimers and oligomer was observed at the excitation wavelength (514.5 nm) in the agglomerated Ag NP slide, it was used as a substrate for the SERS spectra measurements of covalently bridged cysteamine molecules. SERS Spectra of Covalently Bridged Cysteamine Molecules. SERS spectra of covalently bridged cysteamine molecules were measured three times at different distances from the pole pieces of the magnet set. The exposure time of laser was 50 s for each measurement. Figure 3 shows the typical SERS spectra of covalently bridged cysteamine without a magnetic force (12 mm, 0 pN, a)) and with a magnetic force (0 mm, 100 pN, b)). The notation of “0 mm” is the position at which the sample slide contacts the magnet set. But the SERS measurement point on the sample slide was located at the center of the pole piece gap and was never touched to the magnet set. Spectrum c shows the cysteamine monolayer as a reference, which was prepared by the (28) Guerrini, L.; Garcia-Ramos, J. V.; Domingo, C.; Sanchez-Cortes, S. Anal. Chem. 2009, 81, 1418–1425.

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Figure 2. Plasmon absorption spectra of Ag NPs: (9) dispersed Ag NPs in an aqueous solution (path length 1 mm); (2) Ag NPs immobilized on a glass slide; and (O) Ag NPs immobilized and agglomerated on a glass slide. The immobilized Ag NPs spectra were taken with a microscopic spectrophotometer.

adsorption of a cysteamine solution (20 mM) on the SERS substrate for 2 h. Table 1 shows the assignments of major vibration bands in the observed SERS spectra.29-31 Both spectra of (a) and (b) show very weak bands around 547 cm-1, attributed to the ν(S-S) between cysteamine molecules on magnetic particles. Most of the disulfide bonds faced to the Ag NPs were dissociated, and the thiol terminal groups formed the covalent bonds with Ag NPs, showing the intense ν(C-S) bands. The appearance of bands at 917 and 1390 cm-1 indicates the amide bonds of chemically modified cysteamine molecules. The SC-CR bond of an alkanethiol chain, including cysteamine, can take two conformations: gauche and trans.30,31 The two conformations are distinguishable from the Raman frequencies of C-S and C-C stretching vibrations. In Figure 3, the bands around 630 cm-1 are attributable to ν(C-S)Gauche and the bands around 720 cm-1 to ν(C-S)Trans. The bands of 947 and 1023 cm-1 correspond to ν(C-C)Trans. Long alkanethiol molecules, as the concentration and the reaction time increase, tend to form a self-assemble monolayer (SAM) on a substrate, and their SERS spectra show only the trans bands.30 The cysteamine molecule, however, is a short alkanethiol (C = 2) and does not form a complete SAM due to weak lateral van der Waals interactions among the carbon chains. The SERS spectrum (c) of cysteamine monolayer on the SERS substrate shows the gauche band together with the larger trans band. By comparing (c) with (a) and (b), the trans conformation of the cysteamine monolayer (c) was thermodynamically much more stable than those of the covalently bridged cysteamine (a) and (b). Since the adsorption reaction time was the same for the three conditions, the surface concentrations of covalently bridged cysteamine molecules on the Ag NPs in( a) and (b) were expected to be much lower than that of the condition (c). With the increase in the pulling forces, the relative intensity of the trans conformation was increased as shown in Figure 3a,b, indicating that the trans conformation was more stable than the gauche conformation under the forced condition. The intensity (29) Socrates, G. Infrared and Raman Characteristic Group Frequencies, 3rd ed.; John Wiley & Sons, Ltd.: Chichester, 2001. (30) Wrzosek, B.; Bukowska, J.; Kudelski, A. J. Raman Spectrosc. 2005, 36, 1040–1046. (31) Kudelski, A.; Hill, W. Langmuir 1999, 15, 3162–3168.

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Figure 3. SERS spectra of covalently bridged cysteamine: (a) shows 12 mm distance (0 pN) and (b) shows 0 mm distance (100 pN). (c) SERS spectrum of simply adsorbed cysteamine (20 mM, 2 h) on Ag NPs for a reference. The notation of “0 mm” is the position at which the sample slide contacts the magnet set. But the SERS measurement point on the sample slide was located at the center of the gap of iron pole pieces and was never touched to the magnet set.

ratio of the trans to the gauche C-S stretching modes (T/G) was about 0.6 under 0 pN, and it changed to 1.08 ( 0.09 under 100 pN. Figure 4 shows the dependence of the T/G intensity ratio on distance between the sample slide and the edge line of the pole pieces for four different sample slides. For the range from 12 to 1 mm, the T/G ratios were almost constant, showing about 0.6. The inset of Figure 4 shows the range below 2 mm. The T/G ratios were dramatically increased, when the force was increased above 60 pN (below 0.3 mm). Also, the Raman frequencies of ν(C-S)Gauche were not dependent on the exerted forces, but those of ν(C-S)Trans were shifted from 713 cm-1 (0 pN) to 719.2 ( 0.9 cm-1 (100 pN). Figure 5 shows the dependence of ν(C-S)Trans frequency on the distance. The ν(C-S)Trans frequencies were constant from 12 to 1 mm and dramatically shifted to higher frequency above 60 pN (below 0.3 mm). The plus and minus ranges of Figures 4 and 5 mean that the magnet set was first approaching to the sample slide until the contact position (0 mm), and then it was retracted from the slide. The both changes of the T/G ratios and the Raman frequencies of ν(C-S)Trans did not completely revert back to the original states, even after the retraction to -5 mm. This was probably because the strong magnetic field gradients induced the some changes of the intermagnetic particle architecture and their relative positions, although the definite changes of the magnetic particle positions were not observed with the microscopic images. The rotational isomerization of SC-CN from the gauche to the trans form induced by the pulling forces might correspond to the extension of distance between the magnetic particle surface and the Ag NP surface at a molecular scale. First, Figure 3 shows the diminishment of bands (813, 917, and 1390 cm-1) associated with the amide bonds by exerting the forces. The Raman scattering enhancement of adsorbed molecules on Ag NPs is strongly dependent on the distance of a molecule from the Ag NP surface. Theoretically, the electric field enhancement by a metal nanoparticle declines with (a/r)3, where a is the radius of a metal sphere and r is the distance from the center of a metal sphere.32 The (32) Birke, R. L.; Lombardi, J. R. In Spectroelectrochemistry Theory and Practice; Gale, R. J., Ed.; Plenum Press: New York, 1988; p 263.

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Goto and Watarai Table 1. Raman Frequencies (cm-1) and the Assignments of Observed Bands of Cysteamine under the Different Conditionsa

0 pN (12 mm) (a)

100 pN (0 mm) (b)

no magnetic particle (c)

547 547 631 632 634 713 720 726 813 813 818 917 947 946 944 1023 1019 1018 1390 a The assignments of vibration modes were referred from the literature (refs 29-31).

Figure 4. Correlation of trans/gauche ν(C-S) intensity ratio with distance between the pole piece surface and the sample slide. The magnet set approached the sample slide from 12 to 0 mm and then was retracted (a minus region of the distance). The inset shows the region shorter than 2 mm.

diminishment of amide bands implies that the pulling forces tend to move away the bridged cysteamine molecules from the Ag NP surface. In this situation, the amide bond groups also leave from the Ag NP surface, and their SERS bands are diminished. Second, the shift of ν(C-S)Trans to the higher frequency corresponds to the extension of the bridging distance. It has been reported that the Raman frequencies ν(C-S)Trans of HOOC(CH2)mSH adsorbed on Ag metal tend to be shifted to the higher frequency as the number of methylene group, m, is increased (m5: 701 cm-1, m7: 710 cm-1, m10: 721 cm-1, and m11: 712 cm-1).33 Third, the bridging distance (SC-CN) of the trans conformation is longer than that of the gauche by 0.84 A˚ (Supporting Information, Figure S3). From the above evidence, we concluded that the exerted forces induced the extension of interparticle distance between the magnetic particle and the Ag NP, which resulted in the isomerization shift from the gauche to the trans conformation with the longer molecular length. The exerted magnetic forces shifted the equilibrium of the rotational isomerization of SC-CN toward the trans conformation. If the Raman cross sections of ν(C-S)Trans and ν(C-S)Gauche are assumed to be equal and all the magnetic energies are used as the work done, then the variations in the Gibbs free energy change ΔG(F) = -RT ln([trans/gauche]) of the isomerization equilibria induced by the force can be estimated from the measured T/G intensity ratios. Figure 6 shows the dependence of the observed ΔG(F) on the exerted magnetic forces. Two kinds of linear dependence of ΔG(F) on the applied force were observed in Figure 6 with the critical point 80 pN. The slope value is -4.2 J mol-1 pN-1 for the lower force region and -49 J mol-1 pN-1 for the higher force region. The almost pulling work (33) Maeda, Y.; Yamamoto, H.; Kitano, H. J. Phys. Chem. 1995, 99, 4837–4841.

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assignments ν(S-S) ν(C-S) gauche ν(C-S) trans (N-H) wagging secondary amide ν(CCON) ν(C-C) trans ν(C-C) trans ν(C-N) secondary amide

Figure 5. Correlation of ν(C-S)Trans with distance between the pole piece surface and the sample slide. The magnet set approached to the sample slide from 12 to 0 mm and then was retracted (a minus region of the distance). The inset shows the region shorter than 2 mm.

Figure 6. Dependence of Gibbs free energy change of two SC-CN conformations on the exerted magnetic forces. The ΔG values were determined from the trans/gauche intensity ratios. The dotted lines show linear fitting curves for the low and high magnetic force regions.

exerted with below 80 pN might be used to overpass the van der Waals interactions between the magnetic particle and the Ag NP surface,34 and the work exerted with above 80 pN was used for the isomerization of bridged cysteamine molecules. This agrees with the spectroscopic observations, in which the amide peaks were diminished above 80 pN. The reaction rate of the rotational isomerization of an alkane chain is very fast, and the activation Gibbs free energy ΔG‡ is comparable to thermal energy RT at room temperature. Although the isomerization kinetic of (34) Iiguni, I.; Watarai, H. Bull. Chem. Soc. Jpn. 2006, 79, 47–52.

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increment Δx was expected to be much longer than the fluctuation of the equilibrium bond length induced by the molecular vibrations. The intrinsic fluctuation in the bond length based on a harmonic oscillating model (δx) can be calculated by the following equation: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h2 4 δx ¼ Æx2 æ -Æxæ2 ¼ 16π2 kμ

Figure 7. Schematic free energy diagram of the SC-CN isomerization reaction with (dotted line) and without (solid line) a pulling force.

cysteamine molecules adsorbed on a metal surface is not reported, Zheng et al. reported that the rotational isomerization time constant of 1-fluoro-2-isocyanatoethane in CCl4 at room temperature was 43 ( 10 ps with 2D IR echo spectroscopy.35 So, the isomerization reaction was expected to be almost completed under the present experimental conditions. The measurement time scale from one position to the other was about 5 min. Figure 7 illustrates the expected energy diagram of the transgauche isomerization reaction, which was shifted by the pulling force. The broken potential curve shows G(F), and the solid one shows G(0). The number of covalently bridged molecules per magnetic particle (n) can be estimated from the observed dependence of ΔG(F) on F. For the trans-gauche isomerization reaction, the Gibbs free energy difference ΔG(F) under the force F can be described as the following equation: ΔGðFÞ ¼ ΔGð0Þ -FΔx=n

ð4Þ

where Δx is the increase of molecular length required for the isomerization from the gauche to the trans through the activation barrier. In this experiment, the number of n was assumed as a constant value for all the measured regions, since the exerted force was much weaker than 1 nN per bond, which is required to rupture a single covalent bond.36,37 The molecular models of gauche and trans isomers show that the distance between S and N atoms is longer in the trans form by 0.84 A˚ (Supporting Information, Figure S3). If the average molecular length increment Δx was assumed as 0.84 A˚, the value of n was determined as 1.0 molecule per magnetic particle from the slope value (Δx2/n) of Figure 6. A typical value for surface concentration of alkanethiol self-assemble monolayer (SAM) was reported as 4.7  106 molecules/μm2.38 The surface concentration of bridged cysteamine molecules were calculated as 1.3 molecules/μm2, which was less than that of SAM state by 6 orders. The excitation laser spot on the sample slide was an elliptic shape (100 and 40 μm for the long and short axes). If the magnetic particles formed a closepacked monolayer on the substrate, one Raman spectrum corresponded to the Raman scattering of 1.0  104 molecules (∼104 magnetic particles in the laser spot area). The molecular length (35) Zheng, J.; Kwak, K.; Xie, J.; Fayer, M. D. Science 2006, 313, 1951–1955. (36) Grandbois, M.; Beyer, M.; Rief, M.; Hauke, C. S.; Gaub, H. E. Science 1999, 283, 1727–1730. (37) Afrin, R.; Arakawa, H.; Osada, T.; Ikai, A. Cell Biochem. Biophys. 2003, 39, 101–117. (38) Tsen, T.; Sun, L. Anal. Chim. Acta 1995, 307, 333–340.

Langmuir 2010, 26(7), 4848–4853

ð5Þ

where h is the Planck constant, k the force constant, and μ the reduced mass. For the ν(C-S) band at 720 cm-1, δx is calculated as 0.052 A˚, indicating much smaller than the molecular length increment, Δx = 0.84 A˚. The present experimental results showed that the trans conformation of covalently bridged cysteamine molecules was stabilized by the external pulling force. At 94 pN, ΔG(F) was reduced to 0 kJ/mol, indicating that the original free energy difference of ΔG(0) = 1.5 kJ/ mol was completely compensated by the pulling force. Further studies concerning the effects of pulling force on the equilibrium and the kinetics of analogous molecules with cysteamine are promising for the elucidation of molecular structural effects on the Gibbs free energy diagram of trans-gauche isomerization.

Conclusion In the present work, the effects of unidirectional pulling forces on covalently bridged cysteamine between magnetic particles and Ag NPs were studied with SERS spectroscopy. With an increase in the exerted force from 0 to 100 pN per magnetic particle, the ν(C-S)Trans/ν(C-S)Gauche intensity ratio, corresponding to the isomerization equilibrium constant, was increased from 0.6 to 1.08, the Raman frequency of ν(C-S)Trans was shifted from 716 to 719 cm-1, and the Raman bands associated with the amide groups were diminished. From these observations, it was concluded that the pulling forces induced the extension of distance between the magnetic particle surface and the Ag NP surface and caused the shift of SC-CN isomerization equilibrium to the more stable trans conformation. The combination of a magnetic pulling force and SERS spectroscopy was demonstrated to be a highly useful method to study the force-induced effects on chemical kinetics and equilibria, and this method will be applicable to various molecular events, especially for the protein folding dynamics. Acknowledgment. This research was financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (JSPS) and Grant-in-Aid for Scientific Research (A) (No. 21245022) and (S) (No. 16105002), and “Special Coordination Funds for Promoting Science and Technology: Yuragi Project” of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Supporting Information Available: Schematic illustration of the cysteamine structures (gauche and trans conformations) bridging between the magnetic particle and Ag NPs, a microscopic force--SERS measurement setup, an AFM image of the agglomerated Ag NP substrate, and the bridged molecular models of two conformations. This material is available free of charge via the Internet at http:// pubs.acs.org.

DOI: 10.1021/la903637t

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