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Langmuir 2007, 23, 1948-1952
Adsorption Kinetics of Bovine Serum Albumin on Fused Silica: Population Heterogeneities Revealed by Single-Molecule Fluorescence Microscopy K. C. Kwok, K. M. Yeung, and N. H. Cheung* Department of Physics, Hong Kong Baptist UniVersity, Kowloon Tong, Hong Kong, China ReceiVed June 20, 2006. In Final Form: NoVember 10, 2006 The adsorption of bovine serum albumin (BSA) on fused silica at neutral pH was investigated at the single-molecule level by total internal reflection fluorescence microscopy. Dye-labeled BSA molecules that adsorbed on the quartz surface lit up as discrete, fluorescent dots which eventually disappeared upon desorption. Movies of these events offered unprecedented details for kinetics modeling. The results suggested that 99.3% of the BSA was not sticky, and even if adsorbed, it would desorb in minutes. In contrast, the remaining 0.7% was not only sticky, but would anchor in due course. Such population heterogeneity, otherwise masked in ensemble measurements, sheds new light on our understanding of protein adsorption. The methodology is also generally applicable to the studies of macromolecules at interfaces.
Introduction Adsorption of protein to surfaces occurs frequently. Blood clotting, organ transplants, biofouling, and embryogenesis are illustrative examples.1 Current proteomics also hinges on the control of protein adsorption.2 While ensemble adsorption measurements have greatly advanced the field,3 they may be complemented by the latest single-molecule approach in three ways.4,5 First, instead of measuring the averaged behaviors of a huge number of protein molecules, population heterogeneities can be easily revealed. Second, the very initial stage of sparse coverage may evade ensemble detection, yet it is best investigated at the level of single molecules when adsorbate-adsorbate interaction is negligible.6 Third, bulk methods measure net coverage, but single-molecule detection readily distinguishes desorption from adsorption. Among the various single-molecule detection schemes, total internal reflection fluorescence microscopy (TIRFM) is particularly suited for monitoring adsorption because of its wide field of view.7 Novel adsorption studies have since been demonstrated.8,9 Here, we report an investigation of the adsorption of single molecules of bovine serum albumin (BSA) on fused silica at neutral pH using TIRFM. BSA was selected for four reasons. First, the protein itself has long been studied.10 Second, its adsorption is exploited extensively to prevent nonspecific binding in proteomics applications.2 Third, it is widely adopted * To whom correspondence should be addressed. Phone: +852 34117034. Fax: +852 3411-5813. E-mail:
[email protected]. (1) For a recent review of protein adsorption, see: Malmsten, M., Ed. Biopolymers at Interfaces, 2nd ed.; Marcel Dekker: New York, 2003. See also: Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110-115. (2) For a recent review of immobilization strategies for proteomics, see: Yeo, D.; Panicker, R.; Tan, L.; Yao, S. Comb. Chem. High Throughput Screening 2004, 7, 213-221. (3) Ramsden, J. J. Q. ReV. Biophys. 1993, 27, 41-105. (4) Ha, T. Methods 2001, 78, 78-86. (5) Moerner, W. E.; Fromm, D. P. ReV. Sci. Instrum. 2003, 74, 3597-3619. (6) Tie, Y.; Claudio, C.; Van Tassel, P. R. J. Colloid Interface Sci. 2003, 268, 1-11. (7) Axelrod, D.; Hellen, E. H.; Fulbright, R. M. In Topics in fluorescence spectroscopy, Volume 3: Biochemical applications; Lakowicz, J. R., Ed.; Plenum Press: New York, 1992. (8) Yeung, E. S. Annu. ReV. Phys. Chem. 2004, 55, 91-126. (9) Ludes, M. D.; Wirth, M. J. Anal. Chem. 2002, 74, 386-393. (10) For a review of the family of serum albumins, including BSA, see: Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153-203.
as a reference adsorbate.11 Fourth, its pure forms are available commercially. Quartz was chosen for its surface consistency, while silica is a common chromatographic adsorbent.12 In what follows, we will show that single molecules of BSA adsorbed onto quartz in unexpected ways and that population heterogeneities are critically important. Experimental Section Sample solutions of Cy3B-labeled BSA (dye:protein ) 1.6:1) in citrate phosphate buffer of pH 6.6 was prepared in the standard way, at a protein concentration of about 500 pM.13 The concentration was chosen to produce well-separated adsorbates. About 20 µL of the solution was transferred to a sample cell placed on the translation stage of an inverted microscope for TIRFM at 532 nm excitation, as shown in Figure 1. The thickness of the liquid layer inside the cell was 30 ( 5 µm, while the penetration depth of the evanescent wave was about 150 nm. The oval laser spot size (full width at half-maximum) was 110 µm × 40 µm, giving laser irradiance (traveling wave) of 24 W cm-2. Because adsorption sensitively depended on the surface cleanliness of the adsorbent, the quartz slides were cleaned most thoroughly.14 Further instrumentation details are given in the figure caption. The filtered emissions were imaged onto an intensified CCD camera which started recording once the sample solution was introduced, at 24 frames per second for about 17 s. The laser beam was then blocked for 1 min, and recording was resumed for another 25 frames with the laser unblocked. These 1 min cycles were repeated till the 10th minute. The viewed area was (11) Tarasevich, Y. I.; Monakhova, L. I. Colloid J. 2002, 64, 482-487. (12) Iler, R. K. The Chemistry of Silica; Wiley-Interscience: New York, 1979. (13) BSA (Sigma, catalog no. A7638, powder, g99% purity) was used as received. It was dissolved in water at 0.4 mg/mL and labeled with Cy3B mono NHS ester (Amersham Bioscience, catalog no. PA63101, g99% purity) per the vendor’s instruction. Labeled BSA was stored in 500 µL aliquots at -20 °C until use, when it was thawed and diluted to about 500 pM in citrate phosphate buffer (CPB) at pH 6.6. Because of the low concentration, the sample solution was immediately transferred to the sample cell for detection to avoid excessive loss of BSA to container walls. The CPB solution contained 27 mM citric acid and 146 mM dibasic sodium phosphate. All reagents were the highest biochemical grade (Fluka BioChemika Ultra), and water was dispensed from a water purifier (Millipore, model Milli-Q). (14) The slides were sonicated twice in acetone for 15 min at room temperature and then rinsed with doubly distilled water before going through two cycles of sonication in ethanol (15 min, room temperature) and KOH (60 min, 60 °C) and a doubly distilled water rinse. They were finally sonicated in 0.1 M acetic acid for 15 min at 60 °C and rinsed with ultrapure water. Water droplets on cleaned slides gave contact angles of less than 2°. See ref 4.
10.1021/la061779e CCC: $37.00 © 2007 American Chemical Society Published on Web 01/05/2007
Adsorption Kinetics of BSA on Fused Silica
Figure 1. The TIRFM setup was built around an inverted microscope. The sample cell on the traveling stage was coupled via immersion oil (O) to a Suprasil isosceles Brewster prism (P) fixed relative to the microscope stand with the apex side down. The cell could freely translate without moving the prism. A p-polarized 532 nm laser beam from a diode-pumped solid-state laser was incident through the prism on the quartz-water interface at 69° and was totally reflected internally. The fluorescence signal was collected by a 60× NA 1.4 objective (M) oil-coupled to the cover slip, band-filtered (F), and imaged onto an intensified CCD camera mounted at one of the microscope exit ports. The camera format was 1300 × 1030. At the operating gain of 60K, about 20 photons per pixel were required to produce a signal-to-dark noise ratio of 3. The spatial resolution of the entire system was checked to be diffraction limited. For enhanced sensitivity, pixels were 2 × 2 binned, and each binned pixel corresponded to an object size of 350 nm × 350 nm. The inset shows the top and section views of the sample cell. It was made up of a 1 in. × 3 in. × 1 mm fused quartz slide (Q) and a 22 mm × 40 mm cover glass (C) sealed with high-vacuum grease (G). Both the slide and the cover slip were carefully cleaned and dried in nitrogen before fabrication. Two columns of 2 mm diameter holes, three each, were predrilled in the quartz slide, and the sealant was patterned to form three separate flow channels. The rims of the inlet ports were greased to prevent wetting. To fill the channels, 20 µL of blank buffer was pipetted into each inlet port. The channels would then be filled automatically by capillary action. Once the channel was filled, liquid could be made to flow by dipping a wig made of lens paper into the outlet port while liquid was added at the inlet port. Experience showed that the flow speed was proportional to the net water head at the inlet and would stop when liquid at both ports leveled regardless of the wig. The entire assembly was simple and yet practical and allowed for recycling of used slides. then photobleached for more than a minute for background counting. The cell was finally translated to three nearby regions while 25 frames of each were taken. The entire movie was then stored on disk for off-line processing. Image processing was performed with a MATLAB program. Frames of the raw movie were first averaged to produce clearer images.15 A typical image is shown in Figure 4 (inset). Each dot normally occupied 2 × 2 pixels, with the brightest pixel about 90 counts above the background. The dark background for nonirradiated areas was 24 ( 1.4 counts. The bright dots were sharply in focus, and no lateral diffusion was seen. They were photobleached in a stepwise fashion, indicating that they were single dye molecules. The position (x-y pixel coordinate) of each dot in a central area of 53 µm × 27 µm was recorded, together with its appearance and disappearance time.16 Variation of laser irradiance over this central area was no more than (7%. Because of possible loss of BSA (15) Starting with the onset of the fluorescence background, the first 20 frames were averaged to give image 1. The next 50 frames were skipped, and the next 20 frames were averaged to give image 2. This was repeated to exhaust the first 400 frames. After the first minute pause, the leading 4 frames were skipped, and the next 20 frames were averaged to give the first min image. Subsequent nth minute images were obtained similarly. (16) A dot was considered an adsorbate if its brightness was 50 counts above the average background.
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Figure 2. Solutions of various [BSA] values and DPRs at pH 6.6 were flowed into the sample cell for adsorption coverage measurements as functions of time: triangles, [BSA] ) 61 nM and DPR ) 1.64; circles, [BSA] ) 67 nM and DPR ) 1.62; times signs, [BSA] ) 100 nM and DPR ) 2.74. Coverage was based on the fluorescence intensity of the central irradiated patch, normalized by laser irradiance, [BSA], and DPR. Two BSA samples (circles and times signs) were from the same vendor’s lot, while the third (triangles) was from another lot. during sample preparation, the background counts subsequent to photobleaching were used to determine the true concentration of labeled BSA. It was found to range from 400 to 660 pM from run to run. The sample flow rate was estimated by analyzing the first 17 s of the movie. The uniform fluorescence background rose upon sample introduction and reached equilibrium after about 4 s. In another 16 s or less, fluid flow was stopped, and adsorption subsequently occurred in a static cell. Since adsorption events were on the minute scale (see later), adsorption kinetics could be deconvoluted from fluid flow. Control experiments were performed with blank buffer and with solutions of free Cy3B dye. No bright dots were observed in either case. In separate experiments, the photobleaching rate was measured to be 11.7 s (1/e). To study the complications arising from dyelabeling, protein impurities, and adsorbent cleanliness, experiments were also performed using BSA of different lot numbers and dyeto-protein ratios, as well as different slide cleaning protocols. Higher concentrations of BSA in phosphate-buffered saline were used in some of these studies.
Results and Discussion We will first address the possible complications arising from the protein samples as well as the silica adsorbents. The adsorption of single BSA molecules on silica will be reported at the end. Protein Labeling and Impurities. The effect of dye-labeling on protein adsorption was a concern.17 That was investigated by flowing into the sample cell three different stocks of BSA each labeled with different dye-to-protein ratios (DPRs). For this series of experiments, ensemble averages were measured when coverage was determined from the fluorescence intensity of the central illuminated patch instead of counting discrete dots. Accordingly, higher BSA concentrations of 60-100 nM in citrate phosphate buffer were used for better signal-to-noise statistics. The results are plotted in Figure 2, and the DPRs are also listed. The fluorescence intensity was normalized by laser irradiance, BSA concentration, and DPR. As can be seen, the three samples exhibited very similar adsorption kinetics. Although one could not extrapolate to zero DPR, one could at least conclude that variations in DPR, from 1.6 to 2.7, had a negligible effect on adsorption. Meanwhile, of the three BSA samples shown in Figure (17) Gajraj, A.; Ofoli, R. Y. Langmuir 2000, 16, 8085-8094.
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Figure 3. Adsorption isotherms of BSA on nitric acid-washed slides (solid triangles) and KOH-washed slides (solid circles). The dotted curves are just visual aids. The sample solutions were 5 nM Cy3Blabeled BSA mixed with appropriate concentrations of blank BSA in phosphate-buffered saline at pH 7.5. Coverage was based on the fluorescence intensity of the central irradiated patch divided by the typical intensity of single BSA molecules. The third isotherm (open triangles) was adapted from ref 22, while the last one (open circles) was adapted from ref 23.
2, two (times signs and circles) were from one vendor’s lot while the third (triangles) was from another.18 The similarity of the three curves indicates that the data trends were unlikely to be caused by impurities. Adsorbent Surface Cleaning. As previously mentioned, for the cleaning of quartz slides, we adopted a very stringent potassium hydroxide (KOH) protocol required for single-molecule studies because even a few fluorescent dirt particles within the area of interest would be unacceptable.14 More conventional cleaning, such as an acid wash,19 would produce different wetting behavior. For example, slides cleaned with the KOH protocol gave contact angles of less than 2°, while those cleaned in nitric acid gave contact angles of 12°.20 A pristine, fully hydroxylated silica surface should give zero contact angle.21 Adsorption behaviors were significantly different as well. This is shown in Figure 3, where the equilibrated coverages over KOH-washed slides (solid circles) and nitric acid-washed slides (solid triangles) are plotted for a few [BSA] values. Sample solutions were 5 nM Cy3B-labeled BSA mixed with micromolar concentration of blank BSA in phosphate-buffered saline at pH 7.5. Coverage was based on the fluorescence brightness of the irradiated patch. Because the typical fluorescence intensity of a single BSA was known, the actual coverage, mg m-2, could be determined from the patch brightness with the ratio of the blank-to-labeled BSA taken into account. So converted, our isotherms could be compared against those reported in the literature where experimental conditions were similar to ours. Two isotherms, adapted from two such published works (open triangles, ref 22; open circles, ref 23), are also plotted in Figure 3 for easy comparison. Evidently, our nitric acid-washed slides gave a jamming limit that was consistent with the published results. KOH-washed slides were notably (18) Two from Sigma lot no. 100k7420 and the third from Sigma lot no. 014k7670. (19) Forciniti, D.; Hamilton, W. A. J. Colloid Interface Sci. 2005, 285, 458468. (20) We used a procedure of two 15 min sonication cycles in acetone at room temperature, then a rinse with doubly distilled water, then sonication in 20% nitric acid for 60-90 min at 60 °C, and finally a rinse with ultrapure water. (21) Lamb, R. N.; Furlong, D. N. J. Chem. Soc., Faraday Trans. 1982, 78, 61-73. (22) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87-93. (23) Norde, W.; Anusiem, A. C. I. Colloids Surf. 1992, 66, 73-80.
Kwok et al.
Figure 4. Coverage, in number of bright dots N over the central area of 53 µm × 27 µm and normalized to 500 pM BSA, plotted against time. Solid circles are the averages of five experimental data sets, together with the standard deviations as error bars. The solid square at t ) 600 s is the coverage unaffected by photobleaching. The dotted curve and open triangles are theoretical fits when photobleaching was, respectively, ignored and included. The inset shows a typical image, averaged over 20 frames with the background subtracted, at t ) 300 s. The number indicates pixels that were 350 nm apart at the object plane.
less sticky. The difference in the initial slope of the various curves was probably due to a slight difference in the adsorbent surface treatments. This illustrates the challenge that one faces when one tries to reproduce adsorption measurements quantitatively. Single-Molecule Measurements. For adsorption studies at the level of single BSA molecules, KOH-washed slides and low subnanomolar BSA concentrations were used. Coverage was determined by counting the number of discrete bright dots N over the central area. N is plotted against time in Figure 4 (solid circles). Shown are the averages of five data sets. The standard deviations are shown as error bars, which are about 1.8N1/2. Given the very sensitive dependence of protein adsorption on the pH, ionic strength, and substrate cleanliness, the relatively small error demonstrates good reproducibility. The data point at t ) 600 s (solid square) is the coverage over the three neighboring areas that had not been exposed to laser light. The effect of photobleaching is apparent. Adsorption events were analyzed by subtracting the image taken at time t - ∆t from the image taken at time t. In the difference image, the bright dots (positive brightness) corresponded to adsorbates that landed during ∆t without desorbing while the missing dots (negative brightness) indicated desorption or photobleaching. Figure 5 (solid circles) shows the number of “positive” dots, ∆N+, as a function of time t, for ∆t ) 1 min. Because each image required only 1 s of exposure to laser light, ∆N+ was not affected by photobleaching. Desorption could be analyzed analogously by plotting the number of “negative” dots. However, a more revealing approach was adopted. To start off, dots that landed without desorbing during the first minute, i.e., ∆N+(60 s) in Figure 5, were selected. Among this cohort, the percentage of dots that remained, Pre, as a function of time is plotted in Figure 6 (solid circles). As mentioned earlier, dots would blink off upon desorption or photobleaching. If they blinked off while irradiated, photobleaching presumably had occurred, and those dots were removed from the sample population. Accordingly, the decay shown in Figure 6 was due to desorption only. Similar plots of Pre for the second, sixth, and tenth minute cohorts are also shown (triangles,
Adsorption Kinetics of BSA on Fused Silica
Langmuir, Vol. 23, No. 4, 2007 1951
Figure 5. Coverage increase per minute, ∆N+, as a function of time. The averages of the five data sets are shown as solid circles, together with the standard deviations as error bars. The darker dotted curve is a theoretical fit, which could be decomposed into normal (lighter horizontal line) and sticky (lighter decaying curve) components.
Figure 6. Percentage of dots that remained adsorbed, Pre, as a function of time for the first (circles), second (triangles), sixth (tilted squares), and tenth (squares) minute cohorts. For each cohort, the averages of three data sets are plotted. The error bars are the standard deviations. For clarity, error bars for the second and sixth minute cohorts are not shown. Each data set had more than 60 dots initially to guarantee reliable statistics.
tilted squares, and squares, respectively). For graphical clarity, error bars for the second and sixth minute cohorts are not drawn. They were comparable to those of the other two cohorts. Adsorption Kinetics Modeling. As apparent from the data, a simple rate equation
N˙ ) kaC - kdN
(1)
with constant adsorption and desorption rate coefficients ka and kd and protein concentration C would predict neither a timevarying ∆N+ nor a cohort-dependent Pre (Figures 5 and 6). A more realistic model was to assume two types of BSA: a normal type that could be described by a rate equation analogous to eq 1
N˙ n ) kna Cn - kndNn
(2)
Figure 7. Adsorption isotherm for coverage plotted against the concentration of Cy3B-labeled BSA. Coverage was based on the fluorescence brightness over the central 53 µm × 27 µm at room temperature and after 10 min of incubation time. Each data point is the average of five measurements over five different locations on the slide. The standard deviation gives the y error bar. [BSA] was determined from the background fluorescence either initially before extensive adsorption or after photobleaching at 240 W cm-2 for over 20 s. The displayed [BSA] is the average, while the x error bar is the deviation. For the lowest [BSA], the background fluorescence was measured after photobleaching at either 240 W cm-2 for over 20 s or 24 W cm-2 for over 1 min. The best linear fit through the origin is also shown, together with the value of the correlation coefficient R2. At [BSA] ) 0.6 nM, the number of bright dots over the central 53 µm × 27 µm was also counted at five locations. The average was 133, to be compared with 111 dots at [BSA] ) 0.5 nM shown in Figure 4.
to account for the asymptotic ∆N+ of about 25 counts shown in Figure 5 and a second type that made up the rest which decayed and soon ceased to adsorb. Termination of adsorption of this second type could be due to solute or site depletion. In separate experiments, the leveled ∆N+ was found to increase again when a new solution of the same [BSA] was flowed into the cell, suggesting that it was solute depletion. An adsorption isotherm was also measured for the low [BSA] range. This is shown in Figure 7. The apparent linearity also argues against site depletion. We therefore concluded that the decay in ∆N+ (Figure 5) was due to solute depletion and that this second type of BSA must be scarce. Accordingly, different cohorts would comprise different mixes of normal and scarce proteins. That would produce cohort-dependent Pre. Figure 5 indicates that the first minute adsorbates were mostly the scarce type. Decay of this cohort (Figure 6, circles) was slower than exponential and leveled at about 50%. A plausible explanation was the postadsorption structural change that prevented further desorption.24-26 Adsorption and postadsorption transition of the scarce BSA could be described by
N˙ s ) ksaCs - ksdNs - ktNs
(3)
N˙ t ) ktNs
(4)
and
The notations are self-explanatory. Cs in eq 3 is subject to mass conservation:
Cs(t) ) Cs(0) -
N s + Nt Aτ
(5)
where A is the central area of 53 µm × 27 µm and τ is the cell
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Kwok et al.
Table 1. Values of Kinetic Parameters That Best Fit the Experimental Data normal concentration, C (m-3) adsorption sticking coefficient, s
scarce
3.0 × 1017
(2.0 ( 0.7) × 1015
+2.1 5.0-1.1 × 10-5
+0.26 0.17-0.05
+0.006 +0.01 activation energy, ∆Ga (eV) 0.25-0.009 0.04-0.02 desorption +6 rate coefficient, kd (s-1) (1.7 ( 0.4) × 10-2 6-5 × 10-3 +0.007 a +0.04 a activation energy, ∆Gd (eV) 0.68-0.004 0.70-0.02 post-adsorption transition +17 rate coefficient, kt (s-1) × 10-3 8-5 +0.02 activation energy, ∆Gr (eV) 0.70-0.03 a a
A frequency factor of 1010 s-1 was assumed (see ref 27).
thickness of 30 µm. The measured coverage was then given by the sum of all adsorbates:
N ) N n + Ns + Nt
(6)
The coefficient ka that appears in eqs 2 and 3 is related to the diffusion constant D, diffusion distance δ, sticking coefficient s, and area A by
ka ) A(D/δ)s
(7)
D for BSA in water is 3.26 × 10-11 m2 s-1 27 and was assumed to be the same for both types of protein. For low concentration, δ is approximated by [BSA]-1/3.28 Six unknowns were therefore left: sticking and desorption rate coefficients for normal and scarce BSA and the initial concentration Cs(0) and postadsorption transition rate coefficient kt of the scarce type. By solving the rate equations numerically and best-fitting the empirical data shown in Figures 4-6, the six unknowns could be easily determined.29 They are summarized in Table 1. Because the six parameters could be optimized sequentially,29 their associated tolerances could be evaluated from empirical error bars. They are listed in Table 1 as well. As can be seen, while some parameters were resolved to no better than factors of 3, others were determined to within tens of percent. The fitted curves are shown alongside (24) For a review of the thermodynamics of the postadsorption structural change, see: Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517-566. (25) For kinetics modeling of the postadsorption change, see: Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 1190-1199. See also: Van Tassel, P. R.; Guemouri, L.; Ramsden, J. J.; Tarjus, G.; Viot, P.; Talbot, J. J. Colloid Interface Sci. 1998, 207, 317-323. (26) Servagent-Noinville, S.; Revault, M.; Quiquampoix, H.; Baron, M. H. J. Colloid Interface Sci. 2000, 221, 273-283. (27) Kurrat, R.; Prenosil, J. E.; Ramsden, J. J. J. Colloid Interface Sci. 1997, 185, 1-8. (28) In a static cell, a BSA molecule diffused the full height of the sample cell in 27 s. On the minute time scale, [BSA] would be reasonably homogeneous spatially. Once a BSA molecule was adsorbed, the distance for the next nearest protein molecule to diffuse to the substrate was therefore [BSA]-1/3. (29) The values of the six parameters were fitted sequentially. From Figure 3, ∆N+ from normal BSA could be estimated, and its contribution toward the final coverage N (Figure 1) could thus be determined. The remaining N came from the sticky kind. Because all the sticky BSA was used up, its total population Cs(0) could thus be approximated. For the normal BSA, kd could be estimated from the decay of the 10th minute cohort shown in Figure 6 while ka could then be determined from the constant ∆N+ of Figure 5. For the scarce BSA, its contribution toward ∆N+ at the initial and late times helped determine ka and kd, respectively. Finally, kr was fitted using the decay curve of the first minute cohort shown in Figure 6. All parameter values were then optimized after a few rounds of iteration. (30) The photobleaching time constant of 11.7 s was measured in a separate experiment. There was no adjustable parameter in the simulation of bleaching effects. (31) The value of the sticking coefficient was estimated on the basis of the results given in ref 27. We assumed that the TiSiO2 used there as a sorbent for BSA was sufficiently similar to SiO2 used in the present study. (32) Giacomelli, C. E.; Avena, M. J.; De Pauli, C. P. J. Colloid Interface Sci. 1997, 188, 387-395. (33) Fernandez, A.; Ramsden, J. J. J. Biol. Phys. Chem. 2001, 1, 81-84.
the data points (Figures 4-6). Their match with empirical data was satisfactory in all cases. As evident from Figure 4, the measured coverage N (circles) was affected by photobleaching while the model (dotted curve) did not take that into account. Incorporation of photobleaching in the model resulted in excellent agreement with empirical data (Figure 4, open triangles).30 During the experiment, photobleaching commenced once the labeled protein was adsorbed, much like starting a timer carried by each dye label. The excellent fit once again demonstrated the correct timing of the adsorption events as predicted by the model. From the sticking coefficients, the activation energies could be estimated. If a frequency factor of 1010 s-1 for molecular vibrations was assumed,27 the activation energies for desorption and postadsorption transition could also be determined. These energies, together with the uncertainties, are listed in Table 1 as well. Our results are unexpected for two reasons. First, the majority of the BSA molecules are not as sticky as we thought. The sticking coefficient of 5 × 10-5 is much less than that of 4 × 10-4 to 3 × 10-2 reported in ensemble measurements performed under conditions comparable to ours.31 The adsorption lifetime of 1 min is also much shorter than the hours reported.27 Second, while most BSA molecules hardly adsorb, a small minority are thousands of times more sticky and will remain permanently adsorbed. This is contrary to the common belief that all BSA molecules are alike. It should be pointed out that a sticking coefficient of 10-210-4 and an adsorption lifetime of hours might be observed if the adsorption behaviors of the two kinds of BSA were “averaged”, as in ensemble measurements. This is especially true for flow cell configurations when the scarce BSA was continuously replenished. Another complication with ensemble measurements is the possible interference of adsorbate-adsorbate interactions.6 A third factor is the adsorbent cleaning protocol. All these factors could explain the higher sticking coefficients reported in the literature. The structural difference of the two kinds of BSA is presently unknown. The depletion of the scarce type indicated that it was not interchangeable with the normal type, at least on the hour time scale. That ruled out landing orientation as their intrinsic difference.27 Diverse conformations or aggregate forms were possibilities.32 It is well-known that pH affects both the BSA structure and its adsorption kinetics.10,26,32 For example, at the isoelectric point, the protein would be softer because of charge neutrality. That would allow more extensive postadsorption structural change. The resultant higher entropy gain would favor adsorption.33 We have studied pH effects, and our preliminary results were consistent with that hypothesis. Yet, more interestingly, our results showed that pH affected the normal-to-scarce mix rather than the kinetic rates. To elucidate the underlying mechanism, more systematic study is currently under way.
Conclusions In summary, the adsorption of single BSA molecules on fused silica at neutral pH was investigated. This yielded unprecedented details for unique modeling of the adsorption kinetics. Our results indicated that two types of BSA were present: a minority that was sticky and a dominant majority that was not. This heterogeneity will have wider implications in our understanding of protein adsorption. For example, the effects of pH and adsorbate clustering may require reinterpretation. Experiments along those lines are ongoing. Acknowledgment. This work was supported by the Faculty Research Grant of Hong Kong Baptist University. LA061779E
