Langmuir 2004, 20, 1827-1832
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Lysozyme Adsorption Studies at the Silica/Water Interface Using Dual Polarization Interferometry Jian R. Lu,*,† Marcus J. Swann,‡ Louise L. Peel,‡ and Neville J. Freeman*,‡ Biological Physics Group, Department of Physics, UMIST, PO Box 88, Manchester M60 1QD, United Kingdom, and Farfield Sensors Ltd., Salford University Business Park, Leslie Hough Way, Salford, Greater Manchester M6 6AJ, United Kingdom Received October 28, 2003. In Final Form: December 15, 2003 Lysozyme adsorption at the silica/water interface has been studied using a new analytical technique called dual polarization interferometry. This laboratory-based technique allows the build up or removal of molecular layers adsorbing or reacting on a lightly doped silicon dioxide (silica) surface to be measured in terms of thickness and refractive index changes with time. Lysozyme adsorption was studied at a range of concentrations from 0.03 to 4.0 g dm-3 and at both pH 4 and pH 7. Adsorbed layers ranging from 14 to 43 ( 1 Å in thickness and 0.21 to 2.36 ( 0.05 mg m-2 in mass coverage were observed at pH 4 with increasing lysozyme concentration, indicating a strong deformation of the monolayer over the low concentration range and the formation of an almost complete sideways-on bilayer toward the high concentration of 4 g dm-3. At pH 7, the thickness of adsorbed layers varied from 16 to 54 ( 1 Å with significantly higher surface coverage (0.74 to 3.29 ( 0.05 mg m-2), again indicating structural deformation during the initial monolayer formation, followed by a gradual transition to bilayer adsorption over the high concentration end. The pH recycling performed at a fixed lysozyme concentration of 1.0 g dm-3 indicated a broadly reversible adsorption regardless of whether the pH was cycled from pH 7 to pH 4 and back again or vice versa. These observations are in good agreement with earlier studies undertaken using neutron reflection although the fine details of molecular orientations in the layers differ subtly.
Introduction The study of interfacial behavior at wet interfaces where one of the bulk phases is aqueous solution is of importance to researchers involved in a wide range of scientific endeavors from tribology to the life sciences.1-5 Although various analytical tools can be deployed, their inability in resolving interfacial structures is often characterized by two most common features: lack of sensitivity and ambiguity in data interpretation. A range of analytical methods has been employed to obtain a better understanding of the behavior of molecules at surfaces and interfaces and subsequent structural changes. These include spectroscopic techniques such as infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectrometry, and circular dichroism (CD), which can, for example, provide indirect evidence of putative conformational changes in adsorbed proteins when adsorption is studied via particulate dispersions.5 Techniques such as radioactive and fluorescence labeling have been deployed to provide information on the amounts of material adsorbed at the interface, but they are not able to elucidate structural data. In addition, labels may, in the case of large flexible molecules such as proteins, perturb the native conformation.1,2 Optical techniques such as ellipsometry are highly sensitive with regard to the amount of material adsorbed at an interface and can also provide * To whom all correspondence should be made. † UMIST. ‡ Farfield Sensors Ltd. (1) Rapola, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (2) Baszkin, A.; Boissonnade, M. M. J. Biomed. Mater. Sci. 1993, 27, 145. (3) Malmsten, C. J. Colloid Interface Sci. 1994, 166, 333. (4) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657. (5) Proteins at Interfaces II: Fundamentals and Interfaces; Horbett, T. A., Brash, T. A., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995.
information on structure. However, due to the nature of the assumptions that must be made in order to derive structural data, the degree of certainty is generally low.3 It should be noted that recent developments in quartz microbalance (QCM),6 infrared-visible sum frequency generation (SFG),7 and IRRAS spectroscopy8 offer a great deal of potential for unraveling different aspects of structural details of interfacially adsorbed protein molecules. In the past decade neutron reflection (NR) has been developed which allows interfacial molecular behavior to be studied in terms of both the amount of material adsorbed and the structural conformation.4,9 NR has been used successfully to study surfactant, polymer, and protein behavior at air/liquid and solid/liquid interfaces. Although NR has revealed an extensive range of molecular structures of adsorbed protein layers, it is not possible to obtain real time data on the temporal behavior of adsorbed species as, at high resolution, data points typically take several minutes to collect. This particular feature can, however, be complemented by the new analytical technique to be introduced in this study: dual polarization interferometry (DPI). The adsorption of lysozyme at the silica/water interface has been well characterized by a number of analytical techniques including NR. For comparison, the lysozyme system has been employed in this work to demonstrate the interfacial structural characteristics from DPI measurement. Protein adsorption is an important interfacial molecular event that underpins many technological processes. For example, protein adsorption is used to a good effect in a range of biomimic systems including (6) Reimhult, E.; Hook, F.; Kasemo, B. Langmuir 2003, 19, 1681. (7) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150. (8) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9395. (9) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995.
10.1021/la0360299 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/22/2004
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Figure 1. Schematic representation of the dual slab waveguide interferometer. The sensor chip comprises five layers of deposited silicon oxynitride. A window is opened in the final layer to expose the sensing waveguide.
biosensors and in the stabilization of emulsions. The control of protein adsorption is also critical in areas such as the biocompatibility of medical devices and the fouling of filtration membranes in bioseparation/bioremediation.1,2,5 The study of interfacial behavior of proteins is particularly challenging in terms of the determination of in situ structural conformations. Proteins are capable of complex changes in tertiary and quaternary structures as a consequence of interfacial adsorption, including conformational changes and unfolding. Typical forces involved in a seemingly straightforward interfacial adsorption include electrostatic, dispersion, van der Waals, and hydrogen bonding. The interplay between these forces together with the possible structural deformation and unfolding of protein molecules has made it extremely difficult to rationalize the general characteristics of protein adsorption at interfaces.5,9,10 When attractive forces are present, e.g., hydrophobic attraction between a protein and the surface, proteins may undergo irreversible unfolding processes (denaturation) during interfacial adsorption. Even where only forces such as electrostatic interactions are involved, irreversible adsorption can result if the number of surface contacts with the protein is numerous. The precise fate of the adsorbed protein depends on numerous factors including the nature of the surface it comes into contact with, its concentration, the isoelectric point of the molecule, the pH and the ionic strength of the medium, and the size and stability of protein molecule itself. The deployment of novel analytical techniques such as DPI will strengthen our ability in unraveling the in situ structural conformations, leading to improved understanding of the complex molecular processes involved in protein adsorption. Theory Section Dual Polarization Interferometry. The dual polarization interferometer consists of slab waveguides in which the dual orthogonal modes of the structure were used for studying lysozyme adsorption at the silica/water interface. The sensing principle has the capability to provide structural information on layer growth at a resolution equal to or higher than currently available methods. Figure 1 shows a schematic representation of the interferometer comprised of a four-layer dielectric stack of silicon oxynitride on a silicon wafer surface. The second and fourth layers of the stack are of higher refractive index than their adjacent layers thus forming the reference and sensing optical waveguiding paths, respectively, in a dual (10) Haynes, C. A.; Norde, W. Colloid Surf., B 1994, 2, 517.
Lu et al.
slab waveguide structure. On illumination of the end face of the interferometer, the upper and lower modes are excited approximately equally and propagate through the structure. At the output, the two modes are allowed to diffract into the far-field where they form the well-known pattern of Young’s interference fringes on an array photodiode.11,12 The interference pattern represents the relative phase position of upper and lower modes at the output face of the device. Should this change, the spatial intensity distribution changes, thus providing a transduction method for determining changes in the condition of the surface immediately adjacent to the sensing waveguide. Operating Principles. The phase changes of interest, ∆φ, involve changes to the effective refractive index, Ns, of the mode in the upper (sensing) waveguide. The effective index of the lower (reference) mode, Nr, is unaffected by changes occurring at the surface since the evanescent field of this mode decays rapidly in the region between the two guiding layers. The phase difference is given by
∆φ ) k0L∆Ns
(1)
where k0 is the free space wavenumber, L is the interaction length, and ∆Ns is the effective index change in the upper waveguide mode. Direct measurement of ∆φ is possible by continuously monitoring the relative phase position of the fringe pattern by performing a Fourier transformation relating intensity to position. Thus we can easily convert the experimental data to changes in effective refractive index since the path length is fixed. It is assumed that the lower mode’s effective index remains unchanged and therefore that the measured change relates solely to that experienced by the upper waveguide mode. The absolute effective index of a waveguide mode is found by solving the equations of electromagnetism for a system of uniform multiple dielectric layers in which the fields in the semi-infinite bounding layers are exponentially decaying solutions. The parameters required are the refractive index and thickness of each layer (except the bounding layers where index only is specified) for each of the two allowed states of polarization, transverse magnetic (TM) and transverse electric (TE). Provided the input information is complete, an effective index value for each mode is obtained which is representative of the distribution of optical power among the layers. This is the starting point for any analysis of the measured phase changes. If a new layer is introduced to (or removed from) the system, it will alter the effective index of the mode. For each of the two polarization states, the new effective index can satisfy a continuous range of thickness and refractive index values for the new layer. However, there will only be one unique combination that satisfies the effective index of the TE and TM modes simultaneously. The methodology to practically utilize this approach has been fully described elsewhere.12 We show in Figure 2 an embodied DPI system used in this study. Experimental Section Lysozyme from chicken egg white was purchased from Sigma. It has a molecular weight of 14600. Its isoelectric point is around pH 11. It is ellipsoidal in shape with its long axis of 45 Å and short axis of 30 Å. Ultrahigh quality (UHQ) H2O was processed from an Elgastat ultrapure water system. Phosphate buffer was (11) Cross, G. H.; Ren, Y.; Freeman, N. J. J. Appl. Phys. 1999, 86, 6483. (12) Cross, G. H.; Freeman, N. J.; Swann, M. J. 2001; Sensor Assembly, WO-A-01/36946.
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quite consistent13-17 with typical values of 1.465, it is possible to determine the mass of material deposited on the sensor surface using eq 4
FL ) Fp(nL - ns)/(np - ns) mL ) FLτL
(4)
where FL is the adsorbed layer density, Fp is the protein density, nL is the adsorbed layer refractive index, np is the protein refractive index, ns is the solution (bulk) refractive index, mL is the mass loading per unit area, and τL is the adsorbed layer thickness. From the mass loading it is straightforward to calculate the area per molecule according to eq 5 Figure 2. Physical embodiment of dual polarization interferometry.
A ) Mw/(NamL)
used to control the solution pH, and the ratio of Na2HPO4, NaH2PO4, and H3PO4 was varied to obtain the pH required. The thickness and refractive index of the upper waveguide of the sensor chip were calibrated by measuring the phase transitions of both TE and TM polarizations when the surface was subjected to a known refractive index change. A bulk refractive index calibration for each buffer solution was performed by measuring the phase changes of the two polarizations when a flow of UHQ H2O over the chip surface was exchanged for the buffer. For the measurements at different lysozyme concentrations, an unmodified dual channel sensor chip was loaded into the instrument and phosphate buffer solution (PBS), 2 mM, pH 7, was flowed over the chip surface at 100 µL/min. With the temperature stabilized at 20 ( 0.002 °C, a solution of ethanol in water (80% v/v) was injected for 2 min. This injection was repeated and followed by an injection of UHQ H2O for 2 min for the calibrations described above. The flow rate was reduced to 50 µL/min, and once the temperature had stabilized, lysozyme solutions (0.03, 1.0 and 4.0 mg/mL in PBS, pH 7.0) were injected in sequence for 7 min each. The flow was returned to buffer solution for about 5 min between each injection. The adsorption was reversed by washing the chip surface with a detergent (Decon) (5% v/v) for 2 min. Without removal of the chip from the instrument or change of the fringe image settings, the experiment was repeated at pH 4. The pH cycling at fixed lysozyme concentration was done using the same chip, fringe positions, and temperature settings as in the previous experiments. PBS (2 mM, pH 4) was flowed over the chip surface at 100 µL/min. Decon (5% v/v) was injected for 2 min to wash the chip surface, and then ethanol in water (80% v/v) followed by UHQ H2O was injected for 2 min each for the calibrations described above. The flow rate was reduced to 30 µL/min, and once the temperature had stabilized, 1.0 mg/mL lysozyme in PBS (pH 4) was injected for 11 min and then incubated for 20 min. Without returning the flow to the running buffer, 1.0 mg/mL lysozyme in PBS (pH 7) was injected at 30 µL/min for 11 min and then incubated for 20 min. This step was repeated using the first lysozyme solution (pH 4) before the flow was returned to the running buffer (pH 4) at 30 µL/min. The adsorption was reversed by washing the chip surface with a detergent (Decon) (5% v/v) for 2 min. The pH cycling procedure was repeated using PBS (2 mM, pH 7) as the running buffer and switching the pH of the lysozyme solution from 7 to 4 and then back to 7.
where A is the area per molecule, Mw is the protein molecular weight, and Na is Avogadro’s number. By use of the measured values for the refractive index of the bulk solution, the volume fraction of the layer occupied by protein (φp) can also be calculated
Results and Discussion By using the interferometric technique described above, it is possible to calibrate the refractive index values for bulk solutions such as the PBS running buffers and subsequently obtain thickness and refractive index data for layers deposited upon the surface of the sensing waveguide. Given the refractive indices of proteins are
φp ) nL2 - ns2/np2 - ns2
(5)
(7)
After calculation of the parameters described above, it is possible to draw inferences regarding not only the gross structures of the deposited protein layers but also the likely orientation of the protein molecules within the layers. While the primary purpose of the work reported here was to make a comparison of the DPI technique with neutron reflection data, some caution is required when comparing the results with those obtained from neutron reflection experiments. First, the surface of the sensor, while predominantly composing silicon dioxide, had been doped with silicon nitride during the manufacturing process. Second, the surface deposition had been undertaken using chemical vapor deposition and the surface roughness could be different from the features on the silicon blocks prepared for neutron studies. This together with the differing surface chemical composition may contribute to surface adsorption and the reproducibility of surface coverage, as will be described in the following. Repeated DPI measurements at a lysozyme concentration of 1.00 g dm-3 at pH 7 demonstrated a variation of 15% from measurement to measurement as compared with a percentage variation of some 10% reported for repeated neutron reflection measurements at a lysozyme concentration of 0.03 g dm-3.18,19 Finally, the measurements in the DPI experiments were taken in a flowing cell in order to prevent the attendant fluidic system from becoming obstructed. This was unlike the neutron reflection studies where the samples were held in stagnant conditions. It should be noted that during the second DPI experiment when the flow rates were reduced for the examination of the effects of pH cycling, the measured film characteristics more closely correlated with those observed in neutron (13) Arwin, H. Thin Solid Films 2000, 377-378, 48. (14) Wen, J.; Arakawa, T. Anal. Biochem. 2000, 280, 327. (15) Benesch, J.; Askendal A.; Tengvall P. Colloids Surf., B 2000, 18, 71-81. (16) Davis, T. M.; Wilson, W. D. Anal. Biochem. 2000, 284, 348. (17) Light Scattering from Polymer Solutions; Huglin, M. B., Ed.; Academic Press: NewYork, 1972. (18) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. J. Colloid Interface Sci. 1998, 203, 419. (19) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438.
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Lu et al. Table 1
a. Lysozyme Adsorption at pH 4 Using Neutron Reflection Techniques18 concn, g dm-3
layer thickness, Å
mass loading, mg m-2
area per molecule, Å2
protein vol fraction
0.03 1.00 4.00
30 ( 3 37 ( 3 40 ( 3a 30 ( 5b
0.95 ( 0.3 2.10 ( 0.3 3.20 ( 0.3a 0.70 ( 0.3b
2552 1154 758a 3463b
0.22 ( 0.05 0.40 ( 0.05 0.54 ( 0.05a 0.16 ( 0.05b
b. Lysozyme Adsorption at pH 4 Using the DPI Technique
a
concn, g dm-3
layer thickness, Å ((1)
RI ((0.001)
mass loading, mg m-2 ((0.05)
area per molecule, Å2
protein vol fraction ((0.001)
0.03 1.00 4.00
14 21 43
1.362 1.401 1.435
0.21 0.75 2.36
11540 ( 500 3250 ( 150 1025 ( 50
0.139 0.343 0.525
Inner layer (closest to substrate) of two-layer model. b Outer layer (furthest from substrate) of two-layer model. Table 2 a. Lysozyme Adsorption at pH 7 Using Neutron Reflection Techniques18 concn, g dm-3
layer thickness, Å
mass loading, mg m-2 ((0.3)
area per molecule, Å2
protein vol fraction ((0.05)
0.03 1.00
30 ( 3 30 ( 3a 30 ( 5c 40 ( 3a 30 ( 3b 30 ( 5c
1.70 2.30a 1.20c 3.10a 1.20b 0.40c
1426 1054a 2020c 782a 2020b 6060c
0.39 0.55a 0.28c 0.55a 0.28b 0.09c
4.00
b. Lysozyme Adsorption at pH 7 Using DPI Technique concn, g dm-3
layer thickness, Å ((1)
RI ((0.001)
mass loading, mg m-2 ((0.05)
area per molecule, Å2
protein vol fraction ((0.001)
0.03 1.00 4.00
16 34 54
1.420 1.447 1.446
0.74 2.10 3.29
3280 ( 150 1152 ( 50 737 ( 35
0.446 0.590 0.585
a Inner layer (closest to substrate) of multilayer model. b Middle layer of multilayer model. c Outer layer (furthest from substrate) of multilayer model.
studies. In general, the results obtained using DPI techniques demonstrate reasonable agreement with those reported using neutron reflection techniques. Although the overall amounts of protein adsorbed to the surface of the sensor in the experiments reported here were significantly lower than those reported using neutron techniques, the general trend (increase in adsorbed material with free lysozyme concentration) was similar (see Tables 1 and 2). A comparison of Tables 1 and 2 shows that significantly more lysozyme was adsorbed at pH 7 at all three concentrations investigated than was the case at pH 4, which is again in broad agreement with neutron reflection work. In analogous studies, approximately twice as much adsorbed lysozyme was observed at the silica surface at pH 7 than was the case at pH 4. This has also been found to be the case in the current study, although the mass adsorbed was significantly less, suggesting that the sensor surface was less attractive to lysozyme than the silica surfaces used in neutron reflection studies. In addition to the roughness effect, difference in surface chemical composition may also play a role in the form of electrostatic contributions. Lysozyme with an isoelectric point of pH 11 will be positively charged across the pH range studied and the sensor surface containing a proportion of silicon nitride might be expected to be less negatively charged over the pH range studied than a pure silica surface. Lysozyme Adsorption at pH 4. At 0.03 g dm-3, a layer 14 Å thick was observed with a mass coverage of 0.21 mg m-2. Using eq 5, the area per molecule was calculated to be 11543 Å2. Given the dimensions of
lysozyme, if the molecules adsorbed with short axis normal to the sensor surface without distorting (sideways-on), a layer thickness of 30 Å would be expected. The observed area per molecule is large when compared with the volume fraction of 0.14 calculated from eq 7. This is consistent with some distortion and spreading of the lysozyme molecules on adsorption (a nondistorted layer with a volume fraction of protein of 0.14 would be expected to have a measured area per molecule of around 9700 Å2). The strong deformation indicates the effect of surface chemistry of the sensor oxide surface, as this was not observed from the native oxide used in neutron reflection. When the free concentration of protein was increased to 1.00 g dm-3, the layer thickness increased to 21 Å, which is closer to the short axis of lysozyme of 30 Å. The area per molecule calculated was 3249 Å2. The expected area per molecule for an undistorted short axis normal adsorbed lysozyme layer of approximately 3900 Å2 was estimated for the measured protein volume fraction of 0.343. The results suggest that there is still significant distortion of the lysozyme molecules at this concentration. At 4.0 g dm-3, the layer thickness increases to 43 Å, which implies either the formation of a sideways-on bilayer or a headways-on monolayer with the long axis of the molecule almost perpendicular to the surface. The hypothesis of a bilayer adsorption is supported by the measured area per molecule which has now dropped to 1026 Å2, which is below 1350 Å2 expected for a short axis normal monolayer. The average volume fraction is calculated to be 0.525. It is envisaged to be difficult for the lysozyme molecules to transform into headways-on con-
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Table 3. Expected DPI Responses and Calculated Parameters (using assumption of single layer model) from the Multilayer Model for Lysozyme Adsorption at pH 7 As Proposed from Neutron Reflection Data concn, g dm-3
layer thickness, Å
0.03 1.00 4.00
30 54 75
RI
mass loading, mg m-2
area per molecule, Å2
protein vol fraction
1.386 1.395 1.393
0.852 1.796 2.414
2845 1350 1004
0.390 0.457 0.442
formation under the circumstance because the interfacial adsorption has already formed a strongly deformed sideways-on inner layer at this pH as described under the low lysozyme concentration regime. It is therefore logical to speculate that the overall layer thickness may correspond to the inner deformed sublayer of some 15 Å and an outer sublayer of some 30 Å, corresponding to a sideways-on bilayer adsorption. This outcome is in contrast with our earlier speculation of formation of headways-on monolayer from neutron reflection under the same solution conditions, indicating the distinct advantage from DPI in revealing structural details at the low surface coverage. Lysozyme Adsorption at pH 7. At a concentration of 0.03 g dm-3 it can be seen that three times as much protein was adsorbed than was the case at pH 4 although there was little difference in the layer thickness measured. Despite the higher levels of adsorption, the area per molecule of 3280 Å2 suggests that the coverage was significantly lower than that expected for a monolayer. Again, the measured thickness at 16 Å suggests that there was significant deformation of the adsorbed lysozyme molecules which was not the case with the pure silica surface. At 1.0 g dm-3 there are some significant changes occurring compared to the situation at pH 4. The thickness measured at 35 Å is over 50% greater than is the case at the same concentration at pH 4. The area per molecule at 1152 Å2 is midway between the value expected for a saturated sideways-on monolayer and the saturated headways-on. This together with the thickness progression tends to suggest the formation of tilted monolayer. This result shows that the adsorbed protein molecules retain their globular framework rather well under this condition. At the highest concentration of 4.0 g dm-3, a total protein thickness of some 55 Å is observed. The thickness and surface loading measured is consistent with the oblique layer observed at 1.0 g dm-3 having a second layer of protein adsorbed to it with its short axis normal to the sensor surface. The layers obtained in the above experiments are in broad terms thinner and denser than those found in analogous neutron studies. Although this might be expected as a consequence of the subtle differences in the two surfaces employed, the proposed multilayer models were used to generate the expected phase responses, were they to occur on respective DPI measurements. These responses were then resolved in the same way as the experimental data, and the results are shown in Table 3. These data suggest that the layer structure observed in the neutron studies could not account for the responses observed during the current experimentation and supports the hypothesis that differences in the fine structure of the protein layers are due to genuine differences in the surface morphology and energetics between the two experiments. pH Cycling. The pH was cycled between pH 4 and pH 7 at 1.0 g dm-3 and the adsorption behavior appeared to be partially reversible. This is in good agreement with both neutron reflection and ellipsometery measurements and other related studies.20-22 For example, at pH 7 and
Table 4. pH Cycling Profile Low-High-Low with Lysozyme Concentration Fixed at 1.0 g dm-3 layer mass loading, thickness, RI mg m-2 pH Å ((1) ((0.001) ((0.05) 4 7 4
25 49 37
1.401 1.443 1.431
0.89 2.91 2.01
area per molecule, Å2
protein vol fraction ((0.001)
2733 ( 100 832 ( 50 1208 ( 60
0.343 0.568 0.520
Table 5. pH Cycling Profile High-Low-High with Lysozyme Concentration Fixed at 1.0 g dm-3 layer mass loading, area per protein vol fraction thickness, RI mg m-2 molecule, ((0.001) pH Å ((1) ((0.001) (( 0.05) Å2 7 4 7
43 30 50
1.443 1.411 1.444
2.56 1.25 3.00
947 ( 65 1933 ( 80 808 ( 50
0.568 0.396 0.574
1.0 g dm-3, surface excess values at the silica/water interface have been reported in the range 2.5-3.5 mg m-2 and the current DPI measurements as shown in Tables 4 and 5 appear to match these reported values well. Two pH cycles were undertaken, one cycling from pH 4 to pH 7 and back (Table 2) and one running the inverse profile (pH 7 to pH 4 and back again), with the results listed in Tables 4 and 5. These experiments were carried out at slower flow rates and were allowed somewhat longer to equilibrate. The thickness of the lysozyme layers and surface mass loadings can be seen to be slightly greater as a result. Regardless of the history of pH experienced by the sensor surface, the adsorption process appears to be similar to that obtained at the corresponding single pH values, thinner and more diffuse layers being obtained at the lower pH values. The layer recovery in cycling back to pH 4 from pH 7 appears to show some latency although it is anticipated that if the experiment had been allowed to progress for a significantly longer time period a complete recovery would have been observed. While this is in good agreement with neutron reflection results as has already been discussed, this is a surprising result in terms of electrostatic forces19 as the lysozyme molecules would be expected to be more positively charged at low pH and therefore adhere more strongly to the negatively charged silica surface. It has been proposed that the increased electrostatic repulsion between adsorbed molecules on the surface might be responsible for this surprising trend. There is a general trend in surface excess values observed during pH cycling experiments. The third and final pH condition surface excess values are somewhat greater than those observed under the first (initial) identical pH conditions. The agreement between surface excess values under the initial and final (identical) pH conditions during the same cycling experiments was, in the case of neutron reflection studies, rather better, again indicating the effects of differing surface properties. Conclusions Lysozyme adsorption has been extensively studied by techniques such as neutron reflection and ellipsometry. The results obtained from the DPI technique are in good general agreement with the published data on lysozyme adsorption. This is demonstrated by the observation that (20) Claesson, P. M.; Blomberg, E.; Froberg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (21) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (22) Wahlgren, M.; Arnebrant, T.; Lundstrom, L. J. Colloid Interface Sci. 1995, 175, 506.
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more (approximately twice as much in general) lysozyme is adsorbed to the silica surface at pH 7 than at pH 4. In addition, the adsorption process appears to be largely reversible as the surface excess of lysozyme can be cycled by appropriate adjustment of the pH. There are some significant deviations between the neutron reflection and DPI data sets. The absolute levels of adsorbed protein appear to be significantly less in the DPI study, and the detailed surface structures also appear to be different. Given that there are subtle differences between the pure silica surface deployed in the published neutron reflection work and the lightly doped silica surfaces used in the present study, these differences are not surprising.
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DPI offers the potential of providing information to complement data obtained from other analytical methods such as neutron reflection in the laboratory environment. DPI can complement neutron reflection in providing structural information on the subsecond time scale enabling a greater understanding of the molecular mechanistic processes to be revealed during adsorption process. Work is currently in progress to utilize the real time capabilities of the method to obtain temporal information on the adsorption of lysozyme at the silica/water interface with well-controlled surface chemistry and topology on the sensor surfaces. LA0360299
