Adhesion Forces between Hybrid Colloidal Particles and


Brazil, and Max Planck Insitut for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. ReceiVed NoVember 15, 2005. In Final Form: February 6, ...
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Adhesion Forces between Hybrid Colloidal Particles and Concanavalin A Lizandra B. R. Castro,† Michael Kappl,‡ and Denise F. S. Petri*,† Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, AV. Prof. Lineu Prestes 748, 05508-900 Sa˜ o Paulo, Brazil, and Max Planck Insitut for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ReceiVed NoVember 15, 2005. In Final Form: February 6, 2006 Hybrid particles of poly(methyl methacrylate) and carboxymethylcellulose, PMMA/CMC, were attached to atomic force microscopy cantilevers and probed against concanavalin A (ConA) films formed either on Si wafers or on CMC substrate. Regardless of the substrate, the approach curves showed different inclinations, indicating that the probe first touches a soft surface and then a hard substrate. The distance corresponding to the soft layer was estimated as 20 ( 10 nm and was attributed to the CMC layers attached to the hybrid particles surfaces. Probing PMMA/CMC particles against ConA adsorbed onto Si wafers yielded retract curves with a sawlike pattern. The average range of adhesion forces (maximum pull-off distance) and mean adhesion force were estimated as 100 ( 40 nm and -11 ( 7 nN, respectively, evidencing multiple adhesions between CMC sugar residues and ConA. However, upon probing against ConA adsorbed onto CMC substrates, the mean pull-off distance and mean adhesion force were reduced to 37 ( 18 nm and -3 ( 1 nN, respectively, indicating that the ConA molecules immobilized onto CMC films are less available to interact with the hybrid particle than the ConA molecules adsorbed onto Si wafers. Another set of experiments, where PMMA/CMC particle probed against ConA-covered Si wafers in the presence of mannose, showed that the addition of mannose led to a considerable decrease in the mean adhesion force from -11 ( 7 to -3 ( 1 nN. Two hypotheses have been considered to explain the effect caused by mannose addition. The first suggested the desorption of ConA from the substrate so that the hybrid particle would probe bare Si wafer (weak adhesion). The second proposed the adsorption of mannose onto the ConA layer so that mannose layer would probe against another mannose layer, leading to low adhesion forces. In situ ellipsometry and capillary electrophoresis have been applied to check the hypotheses.

Introduction Carbohydrates have been known as a class of molecules for more than 100 years. Their importance, however, for cellular recognition and intercellular adhesion has been recognized only during the last two decades.1 In fact, most microorganisms and viruses and many proteins have either carbohydrate or carbohydrate binding sites at their surface. Lectins are proteins that specifically interact with carbohydrates. They play a prominent role in cell adhesion and recognition of pathogens by specific surface carbohydrates by the immune system and are useful probes in studying the carbohydrates of cell surfaces. Apart from their physiological importance, carbohydrates and lectins are expected to become important tools for recognition of bioanalytes2 or drug targeting.3 In biochemical processes, specific intermolecular recognition might involve electrostatic interaction, van der Waals forces, hydrogen bonding and hydrophobic interactions between geometrical complementary surfaces. Atomic force microscopy (AFM) has been used to measure such interaction forces with piconewton (pN) resolution. When a binding event takes place, the AFM detects the additional force required to break the molecular adhesion. Colloidal particles attached to tipless AFM cantilevers can work as probes, enabling one to measure forces between the spheres and the samples of interest.4,5 * To whom correspondence should be addressed. E-mail: [email protected] † Universidad de Sa ˜ o Paulo. ‡ Max Planck Institut for Polymer Research. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Chinnayelka, S.; McShane, J. M. J. Fluorescence 2004, 14, 585-595. (3) Bies, C.; Lehr, C.-M.; Woodley, J. F. AdV. Drug DeliVery ReV. 2004, 56, 425-435. (4) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239-241. (5) Butt, H.-J. Biophys. J. 1991, 60, 1438-1444.

Recently, AFM has been used to investigate the interactions between ConA and solid surfaces. Gad et al.6 modified ConA with dithiothreitol to make thiol groups available for the reaction with gold coated AFM tips. The reaction between gold and thiol groups ensures the covalent attachment of ConA to the tip. Yeast cells with mannan polymer were used as substrates. The adhesion forces measured between the ConA modified tip and the substrate were estimated in the range of 75-200 pN. Chen and Moy7 used Avitin-Biotin coupling of ConA to AFM probes to measure the interaction of ConA with the sugar residues at the surface of fibroblast cells. The Dufrene group8,9 measured the interaction between a ConA functionalized AFM tip and a yeast cell surface as well as the forces between an AFM-tip functionalized with hexamylose molecules and lectins on the yeast cell surface. Lekka et al.10 determined the force between ConA and mannose-type glycans present on the surface of human prostate carcinoma cells as being 117 pN. Carbohydrate-modified polymeric particles7,11-13 can be advantageous substrates for lectins because they offer large surface areas and their physical characteristics (surface charge, size, and particle number density) can be easily tailored. Hybrid particles (6) Gad, M.; Itoh, A.; Ikai, A. Cell Biol. Int. 1996, 21, 697-702. (7) Chen, A.; Moy, V. T. Biophys. J. 2000, 78, 2814-2820. (8) Touhami, A.; Hoffmann, B.; Vasella, A.; Denis, F. A.; Dufreˆne, Y. F. Microbiology 2003, 149, 2873-2878. (9) Touhami, A.; Hoffmann, B.; Vasella, A.; Denis, F. A.; Dufreˆne, Y. F. Langmuir 2003, 19, 1745-1751. (10) Lekka, M.; Laidler, P.; Dulinska, J.; Labedz, M.; Pyka, G. Eur. Biophys. J. 2004, 33, 644-650. (11) De Souza Delgado, A.; Le´onard, M.; Dellacherie, E. Langmuir 2001, 17, 4386-4391. (12) Castro, L. B. R.; Soares, K. V.; Naves, A. F.; Carmona-Ribeiro, A. M.; Petri, D. F. S. Ind. Eng. Chem. Res. 2004, 43, 7774-7779. (13) Castro, L. B. R.; Petri, D. F. S. J. Nanosci. Nanotechnol. 2005, 5, 20632069.

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of poly(methyl methacrylate), PMMA, and carboxymethylcellulose, CMC, are convenient model systems because (i) CMC chains are tightly bound to the PMMA particle core, avoiding any kind of CMC desorption, (ii) the particles are uniform, (iii) the mean particle size can be easily controlled, and (iv) the synthesis is low cost.12 Such characteristics open the possibilities to apply PMMA/CMC hybrid particles to the development of biomedical assays. In this work, the interaction forces between hybrid particles of PMMA/CMC attached to the AFM cantilever and concanavalin A (ConA) films were studied by means of AFM. On one hand, this system is not as close to physiological conditions than cell surfaces studied in the mentioned studies. On the other hand, the components of the system are well defined by design and should largely exclude unspecific binding events. Experimental Section Hybrid PMMA/CMC Particles Preparation and Characterization.12 The synthesis of PMMA in the presence of CMC, a cellulose derivative, was carried out by emulsion polymerization using a cationic surfactant, cetyltrimethylammonium bromide (CTAB). First, the complex formation between CTAB and CMC was studied by surface tension measurements.14 The polymerization condition chosen was that corresponding to CMC chains fully saturated with CTAB and to the onset of pure surfactant micelles formation, namely at 0.25 mmol‚L-1 of CTAB and 1.0 g/L of CMC. The medium was purged with N2 during 30 min, while the temperature was brought to 82 ( 2 °C. Afterward the initiator, K2S2O8, at the concentration of 0.214 g/L was added. Two minutes later, MMA at the concentration of 66 g/L was thrown in the system without any particular procedure. The polymerization was carried out under reflux and mechanical stirring (500 rpm). After 3 h the system was cooled to room temperature and dialyzed (dialysis membrane 14 000 MW, Viskase Corporation, USA) against water with 4 changes daily during one week or until the conductivity of dialysis water reached 5 µS/cm. In this process, no buffer was used. The dialyzed dispersions presented pH in the range of 4.5 to 4.8. This novel procedure12 brings the advantage of synthesizing and stabilizing particles with D-glucopyranoside units of CMC on the particle surface in a one-step method using very small amounts of surfactant, a friendly condition for the environment. The hybrid particles presented a mean diameter of 350 ( 50 nm and a mean zeta potential of -50 ( 5 mV, evidencing the presence of CMC on the particle surfaces. A detailed report about the synthesis and characterization of PMMA/CMC hybrid particles can be found elsewhere.12 Adsorption of ConA onto Flat Substrates. Si wafers were rinsed in a standard manner15,16 prior to the ConA immobilization. Si wafers were also modified by reacting with aminopropyltriethoxy silane (APS, Acros, USA).15 The resulting amino-terminated substrates became cationic at pH < 6.0. Under these conditions CMC chains attached to the substrates due to electrostatic attraction, as described in details elsewhere.16 Adsorption experiments of CMC onto aminoterminated surfaces were performed from CMC solution in NaCl 0.001 mol L-1 at a fixed concentration of 1.00 g L-1 and pH 3.5, for 3 h. After that period, the substrates were removed from the CMC solution, washed 10 times in pure water, and dried under a stream of N2. The adsorption of ConA onto films of CMC or silicon wafers was performed with ConA solutions prepared in the range of 0.001-0.5 g/L at pH 4.5 in the presence of 0.01 mol/L MnCl2 and 0.01 mol/L CaCl2. At pH 4.5, ConA molecules are found predominantly as dimers.17 (14) Naves, A. F.; Petri, D. F. S. Colloids Surf. A: Physicochem. Eng. Aspects 2005, 254, 207-217. (15) Petri, D. F. S.; Wenz, G.; Schunk, P.; Shimmel, T. Langmuir 1999, 15, 4520-4523. (16) Fujimoto, J.; Petri, D. F. S. Langmuir 2001, 17, 56-60. (17) Gupta, D.; Dam, T. K.; Oscarson, S.; Brewer, C. F. J. Biol. Chem. 1997, 272, 6388-6392.

Castro et al. Ellipsometry.18 The mean thickness (d) of each layer was calculated from the ellipsometric angles ∆ and Ψ, using a multilayer model composed by the substrate, the unknown layer, and the surrounding medium with the fundamental ellipsometric equation and iterative calculations with Jones matrixes. Details about the CMC films are described elsewhere.16 The index of refraction of ConA, nConA, was considered as 1.50, which is an usual value for proteins, and of the bulk solution, n0 was measured with an Abbe´ refractometer at 24 ( 1 °C. The ellipsometer DRE-X02C Ellipsometer (Ratzeburg, Germany), equipped with a He-Ne laser (632.8 nm), operated with the angle of incidence set to 70°. The immobilization of ConA onto silicon wafers or CMC films was monitored in situ at 24 ( 1 °C. The substrates were immersed into a special cell16 containing ConA solution. After approximately 3 h of adsorption, the mean thickness values of ConA layers from solution 0.1 g/L onto Si wafers and CMC films amounted to 2.0 ( 0.2 nm and 4.5 ( 0.5, respectively. Atomic Force Microscopy. Multimode Nanoscope IIIa AFM with Picoforce add-on from Veeco/Digital Instruments operating in the force calibration mode was used for the adhesion force measurements. PMMA/CMC hybrid particles were glued (UHU plus) onto the apex of the tipless V-shaped silicon nitride cantilevers (Veeco NP-OW) with the help of a micromanipulator (LN, Ratingen, Germany) and a Leica DMIRB microscope. The spring constants of the cantilevers (all taken from the same wafer) were determined using the thermal noise method19 as 0.29 ( 0.03 N/m. AFM cantilevers with the attached PMMA/CMC hybrid particle were mounted in a special fluid cell (Veeco/Digital Instruments) that allows measurements of the interaction forces in liquids. The top part of the liquid cell consists of the cantilever holder made from glass, the sidewalls are formed by an elastic O-ring, and the bottom is given by the sample surface. The cell was filled with about 50 µL of MnCl2 0.01 mol/L and CaCl2 0.01 mol/L solution in the absence or in the presence of mannose at 0.005 mol/L. Cantilever deflections versus sample position curves were acquired using the AFM software of the manufacturers at a scan rate of 1 Hz. The reverse delay time, defined as the time after the loading force has reached the set value until the probe begins to retract, was set at 1 s. Force measurements were performed at four different locations over every substrate. About 250 force curves were obtained at each site. Recorded deflection versus piezo position data were converted into force versus distance data using software developed by the Max Planck Institute for Polymer Research. The adhesion force is the value measured at the point of maximum deflection during the colloid probe retraction from the surface. Replicates were obtained for each system. The mean values of pull-off distance and adhesion force were determined from a set of at least 100 force versus distance curves taken at different positions on the samples. Topographic images were obtained in the fluid cell using a PicoSPM-LE Molecular Imaging system with cantilevers operating in the Magnetic AC mode (MAC Mode), slightly below their resonance frequency of approximately 40 kHz. MAC Mode is a gentle nondestructive AFM imaging technique. It uses a magnetic field to drive a magnetically coated cantilever, yielding precise control over oscillation amplitude and, thus, excellent force regulation. Only the tip is driven. This greatly increases the signal-to-noise ratio, providing tremendous improvement in fluid imaging. In this work, MAClevers type II, which are silicon cantilevers with a thin magnetic coating on the backside, were used. They are manufactured from highly doped, single-crystal silicon and contain one cantilever per chip. The tip has the shape of a pyramid with a polygon as the base. The tip radius is typically 10 nm, and its height is approximately 10 µm. All topographic images represent unfiltered original data and are displayed in a linear gray scale. At least two samples of the same material were analyzed at different areas of the surface. (18) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and polarized light; NorthHolland Publication: Amsterdam, 1987. (19) Hutter, J. L.; Bechhoefer, J. ReV. Sci. Instrum. 1993, 64, 1868-1873.

Adhesion Colloidal Particles and ConcanaValin A

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Figure 1. Topographic images of ConA adsorbed onto (a) Si wafers (z range ) 28 Å) and (b) CMC films (z range ) 100 Å) with the corresponding cross sections. Table 1. Mean Pull-Off Distances and Mean Adhesion Forces Determined from Retract Curves Obtained for PMMA/CMC Hybrid Particles Probing Surfaces under Different Conditions substrate

substrate roughness (nm)

ConA adsorbed onto Si wafer

8(1

ConA adsorbed onto Si wafer

8(1

ConA adsorbed onto CMC film Si wafer

80 ( 9 0.20 ( 0.05

Results and Discussion Probing PMMA/CMC Hybrid Particles and ConA Layers in the Absence of Mannose. Typical topographic images of ConA-covered Si wafers and ConA-covered CMC films obtained in the fluid cell with cantilevers operating in the MAC mode are shown in Figure 1, panels a and b, respectively. Upon adsorbing ConA onto Si wafers and CMC films, the surface mean roughness increased to 8 ( 1 and 80 ( 9 nm, as shown in Table 1. Actually, CMC films are rougher substrates than Si wafers, because long CMC chains probably adsorb onto the APS layer forming loops, tails, and trains.20 Figure 2a shows a typical curve of deflection as a function of separation distance obtained for PMMA/CMC hybrid particle probing onto ConA-covered Si wafers. Figure 2b presents the corresponding force distance curve. After touching the surface, the approach curves show different inclinations, indicating that the probe first touches a soft surface and then a hard substrate. The distance corresponding to the soft layer was estimated as 20 ( 10 nm and might be attributed to the highly hydrated CMC layer. This finding supports the high colloidal stability observed for such hybrid particles.12 The colloidal stability of stock (20) Pancera, S. M.; Salvadori, M. C.; Petri, D. F. S. Acta Microsc. 2003, 12Suppl. A, 103.

medium MnCl2 0.01 mol/L + CaCl2 0.01 mol/L MnCl2 0.01 mol/L + CaCl2 0.01 mol/L + mannose 0.005 mol/L MnCl2 0.01 mol/L + CaCl2 0.01 mol/L MnCl2 0.01 mol/L + CaCl2 0.01 mol/L

mean pull-off distance (nm)

mean adhesion force (nN)

100 ( 40

-11 ( 7

22 ( 10

-3 ( 1

37 ( 18

-3 ( 1

6(4

-3 ( 1

dispersions (6.9 × 1012 particles/mL) of PMMA/CMC in the presence of NaCl 2.0 mol L-1 was observed during a period of at least 4 days. The stability was attributed to the presence of CMC hydrated layers surrounding the particles, which are strongly bound to the particle surfaces. In the retract curves, strong adhesion with a sawlike pattern is observed. The average range of adhesion forces (maximum pull-off distance) and mean adhesion force were estimated as 100 ( 40 nm and -11 ( 7 nN, respectively. In comparison, Gad et al.6 measured the adhesion forces between ConA modified tip and mannan polymer as 75-200 pN, evidencing multiple adhesions between CMC sugar residues and ConA. In a recent study, the adsorption constant (Kads) of ConA onto the CMC film was determined as 2.1 ( 0.2 × 106 L mol-1, which is similar to Kads of 5.6 ( 1.7 × 106 L mol-1 found for ConA adsorbing onto a mannose surface.21 These findings show that the affinity of ConA for glucose residues is similar to that for mannose residues. Lekka and co-workers10 determined by scanning force microscopy the adhesion force between ConA and carboxypeptidase Y as 940 ( 39 pN, evidencing that carboxylic groups on the substrate favor the adhesion. Figure 2c shows schematically a PMMA/CMC hybrid particle probing Si wafers. (21) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140-6148.

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Figure 3. (a) Force distance curve obtained for PMMA/CMC hybrid particles probing onto ConA-covered CMC films. Approach and retract curves are represented by blue and black lines, respectively. The red lines are guides for the eyes. (b) Schematic representation of PMMA/CMC hybrid particles probing onto ConA-covered CMC films. The sizes of scheme elements are not to scale.

Figure 2. (a) Deflection as a function of piezo position and (b) the corresponding force distance curve obtained for PMMA/CMC hybrid particles probing onto ConA-covered Si wafers. Approach and retract curves are represented by blue and black lines, respectively. The red lines are guides for the eyes. (c) Schematic representation of PMMA/ CMC hybrid particles probing onto ConA-covered Si wafers. The sizes of scheme elements are not to scale.

Force distance curves obtained from deflection curves (deflection curves not shown any more from now on, but is available on request) for PMMA/CMC hybrid particle probing onto ConAcovered CMC films (Figure 3b) presented features similar to those observed in Figure 2b. The system is schematically depicted in Figure 3b. The approach curves also show different inclinations (red lines) indicating that the probe first touches a soft layer 18 ( 11 nm thick and then a hard surface. However, the mean pull-off distance and mean adhesion force were estimated as 37 ( 18 nm and -3 ( 1 nN, indicating that the ConA molecules immobilized onto CMC films are less available to interact with the hybrid particle than the ConA molecules adsorbed onto Si wafers. The adsorption of ConA onto CMC films is due to specific binding, where both the number of sugar residues together with the respective propinquity confer to the glycosylated clusters

their improved overall binding affinity.22 On the other hand, the adsorption of ConA onto Si wafers is driven by nonspecific binding.13 As a control experiment, force distance curves were also obtained for PMMA/CMC hybrid particles probing onto Si wafers (Figure 4). Similarly to the other approach curves, the probe first forms a soft contact with a layer thickness of approximately 20 nm and then gets into hard contact. The mean pull-off distance and mean adhesion force of 6 ( 4 nm and -3 ( 1 nN, respectively, are indicative of nonspecific interaction between CMC and Si wafers. The adsorption of CMC chains from solution onto Si wafers does not take place, as evidenced by ellipsometric measurements. Probing PMMA/CMC Hybrid Particles and ConA Layers in the Presence of Mannose. To study the effect of mannose on the adhesion force between PMMA/CMC hybrid particle and ConA layers, ConA-covered Si wafers were chosen as substrates. This system was chosen because it presented the highest mean adhesion force, so that changes due to the presence of mannose could be more easily monitored. Force distance curves obtained for PMMA/CMC hybrid particle probing onto ConA-covered Si wafers in the presence of mannose solution showed a distinct behavior, as shown in Figure 5. The mean pull-off distance and mean adhesion force were estimated as 22 ( 10 nm and -3 ( 1 nN, respectively. Table 1 comprises the mean pull-off distances and mean adhesion forces determined from retract curves obtained for PMMA/CMC hybrid particle probing surfaces under different conditions. The presence of mannose led to a considerable (22) Lee, R. T., Lee, Y. C., Eds.; Neoglycoconjugates: Preparation and Application; Academic Press: San Diego, CA, 1994; p 23.

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Figure 4. (a) Force distance curve obtained for PMMA/CMC hybrid particles probing onto Si wafers. Approach and retract curves are represented by blue and black lines, respectively. The red lines are guides for the eyes. (b) Schematic representation of PMMA/CMC hybrid particles probing onto Si wafers. The sizes of scheme elements are not to scale.

Figure 6. (a) Schematic representation of ellipsometric set up. (b) ∆ and Ψ values measured as a function of time for the adsorption of ConA (0.001 g/L) onto Si wafers in the presence of 0.01 mol/L MnCl2 and 0.01 mol/L CaCl2 up to 4 h. The mannose was added to the cell so that the final concentration was 0.005 mol/L (I). A second mannose addition took place in (II), increasing the mannose concentration in the solution to 0.014 mol/L. (c) ∆ and Ψ values measured as a function of time using ConA covered Si wafers in the presence of mannose at the concentration of 0.014 mol/L.

Figure 5. (a) Force distance curve obtained for PMMA/CMC hybrid particles probing onto ConA-covered Si wafers in the presence of mannose. Approach and retract curves are represented by blue and black lines, respectively. The red lines are guides for the eyes. (b) Schematic representation of PMMA/CMC hybrid particles probing onto ConA-covered CMC films. The sizes of scheme elements are not to scale.

decrease in the mean adhesion force between PMMA/CMC hybrid particle and ConA-covered Si wafers from -11 ( 7 to -3 ( 1 nN. Two hypotheses may be put forward to explain this behavior. The first hypothesis considers the desorption of ConA from the substrate, so that free complexes of ConA and mannose would be formed in the solution, whereas the hybrid particle would probe the bare Si wafer. As shown in Figure 4, the mean adhesion force between bare Si wafer and hybrid particle amounted to -3 ( 1 nN, which is similar to that observed for ConA-covered Si wafers in the presence of mannose. The second hypothesis regards the adsorption of mannose onto the ConA layer, so that the mannose layer would probe against CMC at the surface of the hybrid particle, leading to low adhesion forces. The first hypothesis can be checked off by means of ellipsometric measurements. Changes in the ellipsometric angles ∆ and Ψ larger than 0.1° (the accuracy due to the optics and step motors) evidence changes (adsorption of desorption) on the reflecting surface, as schematically depicted in Figure 6a. Figure 6b shows a decrease in ∆ and an increase in Ψ as a function of

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time, evidencing the adsorption of ConA (0.001 g/L) onto Si wafers in the presence of MnCl2 0.01 mol/L and CaCl2 0.01 mol/L up to 4 h. Then mannose was added to the cell so that the final concentration was 0.005 mol/L (I). Just after the addition, the increase in ∆ and Ψ values reveal oscillations in the bulk concentration, but then values of ∆ and Ψ come to the similar values to those observed before adding mannose. A second mannose addition (II) took place after approximately 2 h, so that the final mannose concentration in the solution was 0.014 mol/ L. Again just after the addition, ∆ and Ψ values increased, but then after ca. 15 min, ∆ and Ψ came back to the original values. This behavior indicated that ConA molecules do not desorb from Si wafers in the presence of mannose. One could doubt about the role played by the free ConA molecules in the solution. Therefore, another experimental set was tested, where first ConA adsorbed onto Si wafer, yielding a lectin layer of 1.5 ( 0.1 nm, and then it was inserted into another ellipsometric cell containing 0.01 mol/L MnCl2, 0.01 mol/L CaCl2, and 0.014 mol/L mannose. Figure 6c shows ∆ and Ψ values measured as a function of time. The small increase in ∆ and decrease in Ψ values after 3 h did not evidenced any desorption. The thickness of the ConA layer amounted to 1.4 ( 0.1 nm. Furthermore, an aliquot of the bulk solution was taken from the cell and analyzed by means of UV spectrophotometry at 280 nm, where ConA peptides would absorb. The absorbance was null, confirming that desorption of ConA in the presence of mannose did not take place. These results are very interesting because they show that ConA layers are very stable, contradicting the studies of Anzai’s group23 on the stability of ConA/glycogen multilayer films onto quartz slides in the presence of sugar by means of spectrophotometry. They observed that exposing the films to 0.020 mol/L of mannose and 0.020 mol/L of glucose the film desorbed 80% and 50%, respectively. The second hypothesis, regarding the adsorption of mannose onto the ConA layer is hard to be checked off by simple analytical methods. An attempt to detect the decrease in the concentration of a bulk solution of mannose after 1 h in contact with ConAcovered Si wafer was capillary electrophoresis. First of all, a calibration curve was obtained for mannose in the concentration

range of 0.0005-0.005 mol/L. Then a ConA-covered Si wafer was added to 100 µL of mannose, 0.005 mol/L. After 1 h of contact, the mannose solution was separated from the ConAcovered Si wafer and analyzed by capillary electrophoresis. Unfortunately, it was not possible to detect any variation in the mannose concentration from the initial one, because it is much smaller than the detection limit. Although the ellipsometric measurements and capillary electrophoresis did not yield any evidence about the adsorption of mannose onto the ConA layer because the variations are negligible, this seems to be a plausible explanation for the low adhesion force. Hydrogen bonding between mannose and ConA has been well characterized by X-ray crystallographic studies,24 where the distances between the hydroxyl groups and ConA residues have been well reported. Therefore, mannose molecules bind specifically to binding sites on the ConA surface. The low adhesion force observed probably stems from the nonspecific interactions between ConA-mannose complexes and CMC on the hybrid particles.

(23) Sato, K.; Imoto, Y.; Sugama, J.; Seki, S.; Inoue, H.; Odagiri, T.; Anzai, J. Anal. Science 2004, 20, 1247-1248. Sato, K.; Imoto, Y.; Sugama, J.; Seki, S.; Inoue, H.; Odagiri, T.; Hoshi, T.; Anzai, J. Langmuir 2005, 21, 797-799.

LA053080Z

Conclusions Regardless of the substrate, the approach curves indicate that the PMMA/CMC hybrid particle probe is composed of a hard core and a soft layer approximately 20 nm thick, giving support to explain the outstanding colloidal stability of such particles. The strongest adhesion and the longest pull-off distance were observed for PMMA/CMC probing ConA adsorbed onto an Si wafer. In this situation, only the CMC sugar residues are available to interact with the ConA molecules. However, upon enriching the system with sugar residues, either by adding mannose to the solution or by adsorbing ConA onto CMC film, less ConA molecules are available to bind to the PMMA/CMC probe, decreasing considerably the mean adhesion forces and pull-off distances values. Therefore, probing the ConA covered Si wafer with PMMA/CMC hybrid particles can work as a qualitative method for sugar detection. Acknowledgment. The authors thank FAPESP (2003/100153), CNPq, and DAAD for financial support. The authors also thank Dr. Marina F. M. Tavares and Fernando G. Tonin from IQUSP, Brazil, for capillary electrophoresis experiments.

(24) Naismith, J. H.; Field, R. A. J. Biol. Chem. 1996, 271, 972-976.