Gold Plating and Biofunctionalization of Ferromagnetic Magnetic

Magnetic Tweezers: Application for Local Studies of Soft ... and iron particles for applications as magnetic tweezers to characterize the elasticity o...
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Langmuir 2001, 17, 2129-2136

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Gold Plating and Biofunctionalization of Ferromagnetic Magnetic Tweezers: Application for Local Studies of Soft Surface-Grafted Polymer Films C. Abdelghani-Jacquin,*,† M. Dichtl,† L. Jakobsmeier,‡ W. Hiller,‡ and E. Sackmann† Physik Department, Institut fu¨ r Biophysik E22, and Lehrstuhl fu¨ r Anorganische und Analytische Chemie, Technische Universita¨ t Mu¨ nchen, D-85747 Garching, Germany Received July 1, 2000. In Final Form: November 24, 2000 We present a method for the gold plating and biofunctionalization of micrometer-sized, ferromagnetic cobalt and iron particles for applications as magnetic tweezers to characterize the elasticity of surfacegrafted polymer films and to measure viscoelastic moduli at cell surfaces locally on micrometer length scales. Gold plating of ferromagnetic particles is achieved by reduction of tetrachlorogold(III) on the metallic surface in acidic solutionsa spontaneous reactionswithout any electrical current. Agglomeration of the ferromagnetic particles during the plating reaction is avoided by dispersing the beads in an aqueous dextrane solution. The resulting gold layer is a versatile platform for further biofunctionalization using a wide variety of standard coupling protocols based on thiol chemistry. Several methods, such as electron microscopy, elemental analysis, X-ray powder diffraction, and X-ray photoelectron spectroscopy, have been used for quantitative and qualitative characterization of the coatings. The magnetic tweezers are used for local quantitative characterization of the elasticity of soft surface-grafted films of hyaluronic acid. A method for the calibration of the magnetization of each bead chosen for the measurement is introduced which is based on the simultaneous analysis of the Brownian motion.

Introduction Magnetic bead microrheometry has been proven as a powerful new technique to measure local viscoelastic moduli of networks of biopolymers such as the actin-based cytoskeleton1 or of the cell envelope and, moreover, to measure intracellular transport forces.2 Such applications require forces imposed on the beads of up to 10 nN, while the bead diameter should be in the submicron range. With commercially available biofunctionalized and superparamagnetic microspheres such as Dynabeads, these conditions cannot be met and it is thus highly desirable to use transducers composed of high permeability ferromagnetic materials such as cobalt and iron. However, these ferromagnetic materials are toxic and cannot be directly applied for studies of cells. In the first part of this paper, a method for producing gold-covered magnetic particles is described. Because gold is one of the most noble metals, such passivated beads do not elicit appreciable cell damages. Moreover, gold can be readily biofunctionalized by deposition of ultrathin bio-organic films through thiol chemistry.3 The gold coating has been achieved by the electroless plating technique, which exhibits several advantages. Electroless plating by chemical reduction yields metal deposits of a uniform thickness. This technique does not require electrodes (no electrical current is required) and can thus be applied to passivate colloidal beads in aqueous suspensions, which can be stabilized by the addition of polymers (such as dextran). Moreover, this technique * Corresponding author. Fax: (++49) 89 2891 2469. E-mail: [email protected]. † Physik Department, Institut fu ¨ r Biophysik E22. ‡ Lehrstuhl fur Anorganische und Analytische chemie. (1) Ziemann, F.; Ra¨dler, J.; Sackmann, E. Biophys. J. 1994, 66, 2210. (2) Bausch, A. R.; Ziemann, F.; Boulbitch, A. A.; Jacobson, K.; Sackmann, E. Biophys. J. 1998, 75, 2038. (3) Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723.

yields a coating of high uniformity and is independent of the geometry of the particles.4 Therefore, particles can be plated evenly and completely, even if they exhibit sharp edges and deep recesses.5 Immersion metal plating is sometimes referred to as electroless plating, but the mechanism is chemical displacement.6 The metal substrate acts as a reducing agent, resulting in the deposition of gold ions from the solution on the surface. Plating occurs only when the substrate metal has a lower oxidation potential than the metal ion in solution and continues only as long as the substrate metal is exposed. The gold-coated particles were analyzed first qualitatively by powder X-ray diffraction (XRD). The method is based on the fact that every crystalline powder produces a characteristic diffraction pattern. Identification is usually accomplished by systematic comparison of an unknown pattern with a catalog of standard data.7 Quantitative characterization of the coatings has been achieved by elemental analysis and X-ray photoemission spectroscopy (XPS). Finally electron microscopy (EM) has been applied to evaluate the smoothness of the coatings. To demonstrate the functionality of the magnetic beads designed by our method, we studied the local elastic properties of thin surface-grafted films of hyaluronic acid (HA) in a magnetic tweezers microrheology. This type of macromolecule was chosen because it is a major component of the extracellular matrix and plays a key role for the regulation of cell-cell and cell-tissue interaction8 for (4) Bhatgadde, L. G.; Joseph, S.; Kulkarni, S. C. Met. Finish. 1996, 94, 45. (5) Keuler, J. N.; Lorenzen, L.; Sanderson, R. D.; Prozesky, V.; Przybylowicz, W. J. Thin Solid Films 1999, 347, 91. (6) Baudran, D. Plat. Surf. Finish. 1997, 84, 55. (7) Whistom, C. In X-ray Methods; Prichard, F. E., Ed.; John Wiley & Sons: London, 1987; p 66. (8) Toole, B. P. In Cell Biology of Extracellular Matrix; Hay, E. D., Ed.; Plenum Press: New York, 1991; p 305.

10.1021/la000930w CCC: $20.00 © 2001 American Chemical Society Published on Web 02/27/2001

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which the elasticity is a key physical parameter. Moreover, because of its high degree of charging (0.7 negative charges/ monomer at pH 7.5), it swells strongly in water in a pH and ionic strength dependent manner and thus readily forms soft polymer cushions on solid surfaces (often called a surface-grafted hydrogel) which are well suited for the immobilization of proteins and cells under nondenaturing conditions. The detailed knowledge of the structure and the elastic and hydrodynamic properties of such surfacegrafted hydrogels is of great importance for applications in biotechnology.9 We describe methods for the surface anchoring of HA and the coupling of the magnetic tweezers to the HA film surface. The thermal and magnetic force induced motion of the beads was followed by highresolution ultramicroscopy using a rapid scanning confocal microscope and methods of particle tracking developed previously.10 Materials and Methods Materials. Cobalt powder (mean diameter 1.6 µm; 99.8% metals basis) and iron powder (diameter < 10 µm; 99.998% metals basis) were purchased from Alfa (Karlsruhe, Germany). The powder was filtered to generate a more narrow distribution of bead sizes ranging from 0.1 to 1.2 µm with a mean diameter of 0.6 µm. HAuCl4‚3H2O was a product from Aldrich (Steinheim, Germany). The dextran (T500) of average monomer number 500 was from Pharmacia Biotech (Uppsala, Sweden). (4-Aminobutyl)dimethylmonomethoxysilane (ABDMS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer (HEPES), N-hydroxysuccinimide (NHS), and 2-aminoethanethiol (cysteamine, 98%) were purchased from Fluka (Fluka, Germany). Bacterial hyaluronic acid from streptococcus zooepidemicus was a product from Aldrich (Aldrich Chemie GmbH, Steinheim, Germany). N-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Pierce (Pierce, Germany). Preparation of Solutions. A total of 0.5 g of dextran (T500) was added to 10 mL of Millipore water. This solution was agitated by using a “Vortex Mixer” (Bender & Hobein Ag) and stored for 1 day at room temperature. An Au(III) ion standard bath was prepared by dissolving 0.05 g of HAuCl4‚4H2O in 50 mL of Millipore water. The pH was then adjusted to pH 1 using a 1 M HCl solution. Gold Coating. A total of 0.1 g of metallic powder was dispersed in 10 mL of the dextran solution to obtain a stable suspension. The gold bath was then added at a 1:1 volume ratio under vigorous stirring. The mixture was stirred and ultrasonicated for 30 min and then washed twice with 10 mL of Millipore water and once with ethanol. The particle suspension in ethanol was stored at room temperature. To concentrate the gold-coated magnetic particles, the resulting suspension was then placed in a magnet separator (Dynal MPC magnetic particle concentrators; Hamburg, Germany). XRD. The XRD experiments were performed with a Philips diffractometer PW 1730 using Cu KR1 and Cu KR2 radiation. The powder was deposited on a glass slide placed in the middle of an aluminum frame by spreading of a suspension of the powder in ethanol on the slide and evaporation of the ethanol. Intensity data were collected at room temperature (298 K) in a stepwise mode from 5 to 70° (2θ). TEM. To check the smoothness of the gold coating, some particles were analyzed by TEM. For this purpose the powder was dispersed in ethanol. A drop of this suspension was deposited on a grid, and the ethanol was allowed to evaporate. The particles were observed by a Philips CM 100 electron microscope. Elemental Analysis. The total percentage of gold and iron or cobalt of the coated particles was obtained by the analytical laboratory of the Technische Universita¨t of Muenchen. The reported values have a probability of 95%. (9) Sackmann, E.; Tanaka, M. Trends Biotechnol. TIBTECH, 1999, 18, 58. (10) Dichtl, M. A.; Sackmann, E. New J. Phys. 1999, 1, 18.1.

Abdelghani-Jacquin et al. XPS. For the quantitative analysis of the chemical composition of the Fe/Au and Co/Au samples, XPS was used. The XPS analyses were performed on the ellipsoidal samples over areas of typically 8 × 6 mm (analysis area: 1-2 mm diameter) and, therefore, represent laterally averaged (over the surface) chemical compositions. The XPS spectra were recorded with a Leybold-Heraeus 10 spectrometer with a hemispherical electron energy analyzer. Nonmonochromatic Al KR (1486.6 eV) radiation was used to excite photoemission, and photoelectrons were detected at an angle of 90° with respect to the plane of the surface. First wide-scan spectra (0-1000 eV binding energy) were taken, while highresolution scans were recorded of the relevant core-level and valence-level photoemission peaks of all of the main coating and impurity elements. Binding energies were determined to an accuracy of (0.2 eV at a pass energy of 18 eV, based on the Au 4f7/2 standard set at 84.0 eV. The base pressure of this equipment was typically 2 × 10-8 mbar during the analysis. Laser Scanning Confocal Microscopy and Magnetic Tweezers Setup. The magnetic tweezers experiments were performed by ultramicroscopy with a laser scanning confocal microscope. This microscopic technique enables high spatial resolution compared to standard microscopy and exhibits a very high temporal resolution, which makes it possible to study the bead dynamics down to milliseconds. Analysis of the samples was performed with a NORAN Odyssey XL (Middleton, WI) confocal microscope using a long-distance objective with a magnification of 32×. The Magnetic Tweezers Setup was used in the “High Force” mode.2 A square pulse current with a frequency f ) 200 mHz and an amplitude of I ) 2.5 A was used to displace the magnetic beads. The whole experiment was performed under room temperature.

Results and Discussion Basic Steps in Metal Deposition. The metal deposition from an aqueous solution follows the reaction

(Mz+(H2O)n)sol + Ze- f Mlattice + nH2O where Mz+(H2O)n represents a hydrated metal ion in the bulk solution and M is a metal atom in the bulk of the metal phase. Gold Plating of Cobalt Powder. The beads were prepared in an aqueous solution by reduction of HAuCl4 and oxidation of cobalt according to the half-reactions6

(AuCl4)- + 3e- f Au + 4ClCo f Co2+ + 2e-

E° ) 0.994 V NHE

E° ) -0.277 V NHE

The H3O+ ions are reduced according to 2H3O+ + 2e- f H2 + 2H2O. Gold Plating of Iron Powder. The same procedure as that for cobalt particles was used. The iron was oxidized according to the reaction

Fe f Fe2 + 2e-

E° ) -0.440 V NHE

which was also accompanied by H2 production. Powder XRD. The XRD patterns of cobalt powder before (Figure 1a) and after the reaction (Figure 1b) clearly exhibit the presence of gold on the coated particles. The deposition of gold on the iron particles is also clearly demonstrated by powder XRD (compare parts a and b of Figure 2). Characterization of Particles by TEM. Figure 3a shows TEMs for an iron particle before the deposition of gold. The surface of the iron particle is rather smooth. After the deposition of gold (Figure 3b), we can observe characteristic rounded mounds of gold11 of about 1-2 nm height. In fact, gold deposits exhibit the same surface

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Figure 1. Powder XRD patterns of (a) pure cobalt and (b) cobalt after gold reaction.

Figure 2. Powder XRD patterns of (a) pure iron and (b) iron after gold reaction.

morphology on flat surfaces, and this is attributed to the nucleation and growth process of the film formation. This provides at least some indications on the morphology of gold films on the surface of the particles. Elemental Analysis and XPS. To gain insight into the variation of the amount of deposited gold as a function of the used gold salt concentration, we applied two techniques: elemental analysis and XPS. The elemental analysis shows that the percentage of gold deposited varies only slightly with the concentration (11) Liu, Z. H.; Brown, N. M. D. Thin Solid Films 1997, 300, 84.

Figure 3. TEMs of (a) an iron particle and (b) an iron particle after the deposition of gold using 10% atomic percentage of gold salt in solution. This was obtained by using 10 mL of a gold solution of 19.9 mmol/L.

of the gold salt in the bulk solution (Table 1). Two gold concentrations have been tested for each metal. For covering the iron powders, we used respectively 10 mL of gold salt solutions of 2.7 and 5.5 mmol/L to obtain atomic percentages of gold of 1.5% and 3%. With the cobalt powders, we used again 10 mL of gold salt solutions of 2.7 and 5.9 mmol/L and so we obtained atomic percentages of gold of 1.6% and 3.4%. The reaction is not complete; the chemical yield is about 75% for the deposition of gold on an iron particle and 86% on cobalt. The smaller chemical

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Table 1. Elemental Analyses on Iron and Cobalt Powders Coated with Gold atomic percentage of atomic percentage of gold in solution (%)a gold in particles (%)b iron powders coated with gold cobalt powders coated with gold

1.5 3.0 1.6 3.4

1.0 2.5 1.4 2.9

a Calculated from initial conditions. b Results from elemental analysis. “atomic percentage of gold” ) [number of Au moles]/ [number of Au moles + number of Fe or Co moles]. The percentage of gold, cobalt, and iron are given with respectively (0.3%, (0.6%, and (0.25%.

yield in the case of iron could be due to the oxidation of iron according to the following reaction:

(AuCl4)- + 3e- f Au + 4ClFe2+ f Fe3+ + e-

E° ) 0.994 V NHE

E° ) 0.771 V NHE

The XPS spectra of cobalt and iron beads after deposition of gold are given in parts a and b of Figure 4, respectively. With this technique one can measure the percentage of gold in a surface layer of thickness of about 2-5 nm. We identified each peak belonging to iron, cobalt, and gold. Because the samples were exposed to air during the measurements and the used sample substrate was silicon, we could also detect carbon, oxygen, and silicon peaks. Figure 4c shows the results of the XPS analysis for iron and cobalt. The atomic percentage of gold of the surface increased with the percentage of gold in the salt solution, and it saturated at a total coverage, which is considerably smaller than 100%. Despite of the fact that the electron microscopy pictures show that the particles were totally covered by gold (no smooth surface of pure iron remains, but only rounded mounds of gold all around the particle exist). We know that the X-rays used during XPS measurements can penetrate the metal for about 2-5 nm. This result could indicate that the gold layer is thinner than this limit at some places on the particles. The elemental analysis and XPS results are consistent. The reaction is not complete, but by increasing the concentration of gold in the salt solution, we can increase the coverage of the particle to 100%, as shown in Figure 3b. Preparation of Hyaluronic Acid (HA) Cushions on Glass Substrates. Bacterial HA from streptococcus zooepidemicus exhibits an average molecular weight of 106 Da and is mostly dissociated at neutral pH, yielding f ) 0.7 charges/monomer unit.12 The polymer was anchored on glass cover slides (24 × 24 × 0.1 mm3) through aminosilane monolayers. The monolayers of ABDMSwere deposited from the gas phase as described previously13 (as illustrated in Figure 5). To anchor HA at the silanized surface, the polymer was dissolved in 10 mM HEPES buffer at a concentration of 1 mg/mL and pH 7.0. To functionalize the carboxyl groups of HA with active ester, 0.05 M NHS and 0.2 M EDC were added. After an incubation of 5 min, the silanized glass slides were immersed into the activated HA solution and incubated for 12 h at room temperature. Then the slides were washed in Millipore water. The flasks were shaken on an incubator table for at least 2 days, while the water was changed daily. This procedure ensured that only covalently bound HA remains on the glass surface. (12) Rinoudo, M.; Milas, M.; Jouon, N.; Borsali, R. Polymer 1993, 34, 3710. (13) Albersdorfer, A.; Sackmann, E. Eur. Phys. J. B 1999, 10, 663.

Coupling of Gold-Plated Magnetic Beads to an HA Cushion. In the following experiments, we used iron beads. We filtered the particles so we could work with small beads (diameters of between 0.1 and 1.2 µm). The gold-plated magnetic beads were coupled to the activated HA layers anchored on the glass slides again through amide bonds. For this purpose the beads were functionalized with cysteamine by a technique developed previously in our laboratory.14 The gold-plated beads were washed twice in 1 mL of ethanol. They were then immersed in a 1 mM solution of cysteamine of 98% purity and incubated for 12 h at room temperature. The particles were then rinsed twice in 1 mL of ethanol and 1 mL of Millipore water. The HA-covered glass slides are then immersed in a 10 mM HEPES solution containing again 0.05 M NHS and 0.2 M EDC and adjusted to pH 7.0. After 5 min, the functionalized magnetic beads were added (1 vol %) and the system was shaken for 12 h at room temperature. The substrates were again washed in Millipore water as described above. The distribution of the gold beads on the HA surface was then characterized by confocal microscopy (cf. Figure 6 for a typical example). Evaluation of the Local Viscoelastic Properties of the HA Layer. The functionalized magnetic tweezers were applied to characterize the local viscoelastic properties of the soft polymer film with micrometer lateral resolution. By application of confocal ultramicroscopy, it is possible to measure the time dependence of the displacement of a magnetic bead induced by application of a magnetic force pulse with submicrometer resolution. For such small deflections the applied magnetic force fmag(t) and the bead displacement of the beads studied dmag(t) can be related by the linear viscoelastic law

dmag(t) ) G(t) fmag(t)

(1)

where G(t) is the viscoelastic response modulus which characterizes the viscoelastic properties of the system studied. In Figure 7 a time sequence of the tangential displacement x(t) of a bead coupled to the HA layer is shown. The displacement was generated by application of a sequence of magnetic force pulses with a frequency f ) 200 mHz onto that bead. The response curves exhibit very pronounced noise because of Brownian motion of the bead in the lateral and normal directions. Consequently, eq 1 connecting displacement dmag(t) and magnetic force fmag(t) must be generalized in the following way:

x(t) ) dmag(t) + dtherm(t) ) G(t) (fmag(t) + ftherm(t)) ) G(t) f(t) (2) where dmag(t) and dtherm(t) denote the displacements caused by the magnetic (fmag(t)) and thermal (ftherm(t)) forces, respectively. By analyzing the thermal fluctuations and the enforced bead deflection, one can characterize the elasticity of the polymer film in terms of a force constant. This information allows the measurement of the unknown magnetic moment of the bead. However, first the question is addressed as to whether the response of the bead to the external field pulses is purely elastic or viscoelastic. In the case of pure elastic response, the beads should follow the square pulse signal instantaneously. This cannot be checked directly by analysis of the bead displacement curves x(t) because of the strong thermal fluctuations of the beads. The thermal (14) Brink, G.; Schmitt, L.; Tampe, R.; Sackmann, E. Biochim. Biophys. Acta 1994, 1196, 227.

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Figure 4. (a) XPS spectra of Co-Au particles. (b) XPS spectra of Fe-Au particles. (c) XPS quantitative analysis of Co-Au and Fe-Au particles. The atomic percentage of gold at the surface of the beads is represented as a function of the percentage of gold salt used for the reaction.

fluctuations can, however, be averaged out by calculation of the mean-square displacement (MSD) 〈(x(t) - x(0))2〉 of

the beads, provided the correlation times of thermal processes are much smaller than the correlation times of

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Figure 7. Time sequence of magnetic bead displacement coupled to the HA layer (frame rate: 25 frames/s). The first 15 s no magnetic force was applied so that here the bead only exhibits thermal driven motion. After that the magnetic force was applied. The resulting signal consists of the thermal driven part and the imposed square pulse force signal.

Figure 5. Chemical structure of HA composed of repeating N-acetyl-D-glycosamine and D-glucoronic acid disaccharide and mechanism of anchoring on aminosilane monolayers. Figure 8. MSD curves calculated from the displacement data of several magnetic beads to which a square pulse magnetic force was applied. The strict triangular shape of the MSD for all beads demonstrates the fully elastic response of the HA film to the force pulse. In addition, it is also shown that the force pulse frequency of f ) 200 mHz is fully reproduced. Because of the numerical aspects of the evaluation procedure, only the first period of the MSD curve is shown here.

essential for the analysis of the bead motion in the presence of a magnetic force. We now evaluate the local, lateral stiffness of the HA film by analyzing only the thermally driven random walk of the bound beads within the plane of the layer by application of the model of Brownian motion in a harmonic potential

V(x) ) V0 +

Figure 6. Confocal scanning microscopy picture of iron beads (plain arrows) and colloidal gold beads (diameter 30 nm, dotted arrow) coupled onto the glass substrates covered with HA. The image was taken using the reflection channel of the microscope so that only the high reflective beads are visualized as bright spots, whereas the HA layer with much lower reflectivity appears as a homogeneous dark area.

the used force pulses (here 5 s). Under this condition the MSD signal of a square pulse displacement should exhibit a triangular shape. As shown in Figure 8, for four different beads this condition is very well-fulfilled, demonstrating the purely elastic nature of the response of the bead coupled to the HA layer. This qualitative result is

1 k(x - x0)2 2

(3)

where k ) d2V/dx2, the local spring constant, characterizes the local stiffness of the HA film with respect to tangential deformations and is a measure for the shear elastic modulus. To analyze the thermal motion dtherm(t) in eq 2, the time sequences (cf. Figure 7) were analyzed in the following way. The sequence x(t) was averaged over times t with an averaging time T ) 5 s corresponding to the force pulse frequency of f ) 200 mHz. The resulting average signal x˜ (t) ) 〈x(t)〉t)5 s was subtracted from the total motion dtherm ) x(t) - x˜ (t). An example of the resulting time sequence of the random walk is shown in Figure 9a (bottom trace). All random walk sequences resulting from this evaluation procedure were checked by calculating the probability distribution function P(dtherm) of the thermally driven

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The saturation values of the MSD obtained by the above procedure are shown in Figure 10 for several beads. The spring constant k varies between 1.6 × 10-5 and 2.8 × 10-5 N/m. After measurement of the force constant k, the force exerted by the magnets on the bead considered can be determined from the measured maximum displacement dmag(max) according to

fmag ) kdmag(max) For the bead of the measurement in Figure 9a, one obtains fmag = 0.8 pN. Concluding Remarks

Figure 9. (a) Extraction of thermal fluctuations dtherm(t). Displayed are the original data set x(t) (dots), the average signal x˜ (t) ) 〈x(t)〉t)5 s (dotted line) and the resulting thermal fluctuations dtherm(t) (straight line). Data correspond to that of Figure 7. (b) Probability distribution P(dtherm) for extracted thermal fluctuations. The data are clearly Gaussian distributed (dotted line), which fits the model of the motion of a Brownian particle within a harmonic potential.

Figure 10. MSD curves calculated for the thermal driven fluctuations of several magnetic beads. It is obvious that the curves for all beads saturate at times t ) 250 ms. The level of saturation varies between 150 and 270 µm and is used to obtain the local spring constant k of the HA layer.

displacements dtherm(t). All distributions exhibited Gaussian shapes, demonstrating that the lateral bead motion is random (Figure 9b). The maximum value of the MSD 〈(x(t) - x(0))2〉sat for Brownian motion within a harmonic potential is given by15

〈(x(t) - x(0))2〉sat ) lim〈(x(t) - s(0))2〉 ) kBT/k tf∞

(15) Doi M.; Edwards S. F. The Theory of Polymer Dynamics; Clarendon Press: Oxford, U.K., 1986.

The main purpose of the present work was to show that magnetic beads can be biofunctionalized in a universal way by electroless chemical plating with gold films and by subsequent coupling of biofunctional molecules through thiol chemistry. The biomolecules can be directly coupled to the gold surface or to polymer cushions, e.g., composed of polysaccharides. Such hydrogels are routinely used in surface plasmon resonance biosensors or surface acoustic wave devices and chemical preparation procedures are thus well developed.16,17 The gold-plated biofunctionalized beads offer several advantages as magnetic tweezers for measurements of local viscoelastic properties of cell membranes or cytoplasms through microrheometry. Owing to the higher magnetic moments of pure iron compared to iron oxide, the bead size for such applications can be reduced, while the gold coats prevent cell damages normally encountered with pure iron or cobalt beads. One disadvantage of the present technique of fabrication of magnetic beads, namely, the relatively broad distribution of bead sizes and the unknown magnetization, can be overcome by measuring the force constants of the beads coupled to soft materials through parallel analysis of the Brownian motion and the force-induced deflections. As an example, the technique was applied to characterize the elasticity of thin surface-grafted films of HA. A true elastic modulus of the film could not be determined yet. This requires the knowledge of the decay length of the strain field in the lateral direction to apply the application of a recently developed method of analysis.18,19 Until now, our attempts to measure this decay length only showed that it is smaller than 1 µm (Dichtl, M., unpublished results). Separate recent measurements with larger beads yielded similar results and also showed that the HA filaments are fixed strongly to the glass surface (Vonna, L., unpublished work). List of Abbreviations Electroless: name given by A. Brenner and G. Riddel (1946) for the coating with metal without any electrical current; electrodeless became electroless Chemicals: Au (gold), Co (cobalt), Fe (iron) Mz+: hydrated metal ion M: metal atom (16) Wegener, J.; Janshoff, A.; Galla, H. J. Eur. Biophys. J. 1998, 28, 26. (17) Malqvist, M. Nature 1993, 361, 186. (18) Bausch, A. R.; Mo¨ller, W.; Sackmann, E. Biophys. J. 1999, 76, 573. (19) Boulbitch, A. A. Phys. Rev. E 1999, 59, 3402.

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E°(Mz+/M): standard electrode potentials for the couple Mz+/M at 25 °C given using as a reference the normal hydrogen electrode (NHE)

Acknowledgment. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 266) and the

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Fonds der Chemischen Industrie. C.A.-J. gratefully acknowledges financial support through a Curie doctoral fellowship by the EU, Grant BMH4-CT98-5141 in the program Biomedicine and Health. LA000930W