Rapid and Sensitive Polarization Measurement for Characterizing

Article pubs.acs.org/JPCC

Rapid and Sensitive Polarization Measurement for Characterizing Protein Adsorption at the Solid−Liquid Interface Shaun A. Hall, Paul A. Covert, Benjamin R. Blinn, Saba Shakeri, and Dennis K. Hore* Department of Chemistry, University of Victoria, Victoria, British Columbia, V8W 3V6, Canada ABSTRACT: We have constructed an ellipsometer capable of rapid polarization measurements based on two photoelastic modulators. It is capable of simultaneously measuring all elements of the Stokes vector on a 10-ms time scale, allowing for a complete polarization analysis including any depolarization that may occur at rough and inhomogeneous surfaces. We overcome the traditional challenges associated with electro-optic modulators through a novel scheme for the calibration of the retardation amplitude and a means for precisely controlling the temperature of the optics. This instrument is therefore especially suited for studying the real-time adsorption of proteins from solution onto solid surfaces. We provide a demonstration of bovine serum albumin adsorption from saline solution onto a hydrophilic silica surface.

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INTRODUCTION The adsorption of proteins onto surfaces1−6 is a fundamental aspect of biofouling,7 implant compatibility,8,9 and hemocompatibility.10−12 From a technological perspective, the design and optimization of biosensors relies on controlling enzyme− substrate interactions that enable immobilization yet leave the protein in a functional state.13−15 Perhaps of even more widespread importance, the separation of proteins by chromatographic methods is based on subtle differences in the molecules’ affinity for surfaces of varying hydrophobicity.16−21 It is also known that the structure of proteins is heavily affected by the nature of the surface it is adsorbing to and the solution conditions.22−25 Various methods have been adopted for studying protein− surface interactions, including X-ray based techniques, vibrational and electronic spectroscopy, atomic force microscopy, surface plasmon resonance, dual polarization waveguide interferometry, and quartz crystal microbalance measurements.26−29 Methods that are able to operate at the solid− liquid interface are particularly valued, as the substrate surface environment and the adsorbed proteins may be considerably different if the sample has been dried or subsequently placed in vacuum. Among these, surface plasmon resonance (SPR) has proven to be an extremely sensitive probe, as it can detect changes in refractive index on the order of one part per 10− 100 000, and is therefore employed in a wide variety of adsorption-based biosensors.21,30 While SPR is typically performed on metal surfaces for maximum sensitivity, it is possible to use polymer overlayers31,32 so long as the polymer films are more than 50 nm thick (avoiding exposed metal surfaces) and less than 100 nm (ensuring significant sensitivity).30 Optical methods are attractive as they can probe a wider variety of substrate−solution interfaces so long as at least one side of the interface is sufficiently transparent to the wavelengths of interest. Such measurements are aided by a © 2013 American Chemical Society

quantitative analysis of the change in light polarization upon interaction with the sample. Ellipsometry prepares light in a known polarization state and then analyzes the polarization after reflecting from a sample surface.33−35 When the energy of the probe light is close to a molecular resonance, the optical anisotropy may be further related to structural organization in the adsorbed layers. There have been many constructed and proposed designs of polarimeters and ellipsometers. The most basic designs employ polarizers before and/or after the sample. More complete characterization additionally requires the use of a retarding element. This can be a static retarder for a fixed wavelength such as quartz or calcite, a tunable range of wavelengths such as a Soleil-Babinet compensator or Pockels cell, or a broadband device such as a Fresnel rhomb. Alternatively, a photoelastic modulator (PEM) may be used.36−40 These typically consist of an optically isotropic material (such as fused silica) fused onto a piezoelectric material (such as quartz). When an ac field is applied to the piezoelectric transducer, a time-varying deformation of the fused silica results in a time-varying phase modulation of a transmitted beam. As the modulation frequency is usually in the tens of kilohertz range, polarization information is extracted using lock-in amplifiers or by digitizing the detector waveform and subsequent Fourier analysis. Although there are several specific merits to the individual instrument designs, a useful generalization is that PEM-based devices offer high speed and precision, yet compromise accuracy. Instruments based on static retarders or nulling schemes are known for their high accuracy, but are slower and are often not able to detect as small a change in polarization. The difficulty in obtaining accurate results with PEMs largely stems from challenges in Received: November 23, 2012 Revised: December 30, 2012 Published: January 7, 2013 1796

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calibrating their retardation amplitude and drifts in retardation with respect to temperature. Here we describe an instrument that is capable of characterizing the complete polarization state of a light beam, including the fraction of the light that has been depolarized upon interaction with the sample. This is especially useful for rough surfaces such as those consisting of adsorbed proteins as they tend to scatter some of the incident light. We take advantage of the millisecond time-resolution and high precision afforded by PEMs to study rapid yet small changes in polarization accompanying protein adsorption. This is particularly important for the adsorption of globular proteins onto hydrophilic surfaces as the amount adsorbed and accompanying structural changes are much less pronounced than on hydrophobic surfaces,41 resulting in smaller changes in the probe beam polarization. When working off-resonance, the changes in beam polarization are even smaller. However, longer wavelength probes are of interest as they are able to penetrate a wider variety of substrates. At the same time, we are able to overcome the typical challenges associated with accuracy in PEM-based measurements through a novel calibration scheme and fine control of the temperature. A demonstration is provided for the case of bovine serum albumin adsorption at the silica−solution interface.

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Figure 1. Schematic of the optics (black), electronics (green), and temperature control (blue). A 632.8-nm HeNe laser (beam path shown in red) is modulated by a mechanical chopper at 1 kHz. Steering mirrors (SM1 and SM2) are used to align the beam through all the subsequent optics. The light passes through a polarizer (P1), quarter-wave plate (QWP), second polarizer (P2), and Soleil-Babinet compensator (BSC) before approaching the sample interface through a prism. Reflected light passes through a 60 kHz photoelastic modulator (PEM1), 50 kHz photoelastic modulator (PEM2), analyzer (P3), and is collected on a high-speed Si photodiode. The signal waveform is demodulated at the fundamental and harmonic components of the input frequencies by four lock-in amplifiers.

BACKGROUND The polarization of any light source may be completely described by the 4-element Stokes vector ⎤ ⎡ ⎡ s0 ⎤ ⎢ Itotal ⎥ ⎢s ⎥ ⎢ I − I ⎥ 0 90 1 s=⎢ ⎥=⎢ ⎥ ⎢ s2 ⎥ ⎢ I+45 − I −45 ⎥ ⎢s ⎥ ⎢ ⎥ ⎣ 3⎦ ⎣ Ircp − Ilcp ⎦

(1)

where s0 represents the total intensity of the light; s1 is the excess of horizontally polarized over vertically polarized light; s2 the excess of light polarized at +45° over that at −45°; s3 is the excess of right circular polarization of the two circularly polarized states. Measurement of the complete Stokes vector also yields the degree of polarization since its elements are related by s0 2 ≥ s12 + s2 2 + s32

modulation (nominally 1 kHz) is applied by a mechanical chopper (Thorlabs MC1000A) fitted with a 30-slot blade. The light then passes through a calcite Glan Taylor polarizer (P2, Thorlabs GT10) with polarization ratio better than 200,000:1. Finally the light passes through a Soleil-Babinet compensator (BSC) that has been set to λ/4 retardation for our working wavelength. This comprises the polarization state generator (PSG) arm of the instrument. Details of our custom sample compartment have been published previously.42 Briefly, it consists of a Teflon cell housed in an aluminum block that is plumbed with a circulating water supply connected to an external heater/chiller (VWR 1167P) to achieve constant sample temperature. The front face of the Teflon cell is in contact with a fluoropolymer O-ring (Marco Rubber, Seabrook NH) pressed against a fused silica prism (Del Mar Phototonics, San Diego CA). The incident and exit faces of the prism are cut at 70° so the beam approaches the air-fused silica interface and leaves the fused silica-air interface at normal incidence. The polarization state analyzer (PSA), that is our Stokes polarimeter, consists of two fused silica photoelastic modulators (PEM1 and PEM2, Hinds Instruments, Hillsboro OR), antireflection coated for the 400−700 nm wavelength range. The crystal in the first modulator is cut to vibrate at nominally 60 kHz; the second at 50 kHz. The light then passes through an analyzer (P3), another Glan Taylor polarizer. Since we need to demodulate the signal at high frequencies, we use a reversebiased amplified Si photodiode (Thorlabs DET10A) with

(2)

where the equality holds in the case of perfectly polarized light. Normalized stokes vectors sj/s0 are reported when one wishes to describe the polarization irrespective of the intensity. In other words, the normalized Stokes vector (s0 = 1) describes the azimuth and ellipticty of the polarization ellipse irrespective of its size. By measuring the polarization of light that has been transmitted or reflected at the solid−liquid interface, one is able to determine how the polarization has changed from the incident light. This may be traced back to the optical constants of the interface and, ultimately, to the molecules present in this region. The instrument described here is based on photoelastic modulators, and so is capable of monitoring structural changes that occur on the millisecond time scale.

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INSTRUMENT DESIGN AND CALIBRATION Overview. A schematic of our instrument appears in Figure 1. On the incident arm, we use a 632.8-nm HeNe laser (Thorlabs HRP170) as a light source. A low frequency 1797

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1 mm2 active area and 1 ns rise time. The azimuths of all optical elements are controlled with stepper motors (Standa, Lithuania). If we set the azimuth of the first PEM to 0°, the second PEM to 45°, and the analyzer to 22.5° then it has been demonstrated38 that the light reaching the detector has a time-dependence according to I(t ) = I0 + I1 cos 2ω1t + I2 cos 2ω2 + I3 cos ω1t

Temperature Stabilization. Although photoelastic modulator-based polarimeters offer the possibility for rapid and precise polarization measurements, their accuracy is often hindered by the extent to which the modulation amplitude can be calibrated and controlled. In the following section, we outline our procedure for this amplitude calibration. However, in order to achieve a stable calibration and maintain these operating conditions during the measurement, it is necessary to have fine control and stability of the modulator temperature. We have enclosed the entire instrument in a plexiglass box on top of our optical table, and have employed a cascading proportional-integral-derivative (PID) routine to control the temperature inside the box. A recirculating heater/chiller was connected to 5 m of copper tubing, wound into a helical radiator with a length of 50 cm and diameter of 20 cm. A small computer fan was used to flow air over this coil inside the box, and a plastic shield deflected this air toward the photoelastic modulators. A software PID routine written in python was used to read the heater/chiller’s external RTD sensor (inside the box) and control the internal temperature of the water bath (outside the box) through a serial connection. A nested fixedparameter hardware PID on the bath controller was used to maintain the internal temperature. With the appropriate software PID settings, we were able to stabilize the temperature to within ±0.02 °C over an arbitrary amount of time, following a 30-min equilibration period. Calibration of Modulator Retardation Amplitudes. Photoelastic modulators induce a time varying retardation δ(t) on a transmitted probe beam according to the amplitude A of their modulation in δ(t) = A cos (ωt). Now that the temperature has been stabilized, it is important to calibrate the applied electric field so that A = 2.4048 rad for each of the two PEMs. This value corresponds to a zero-crossing of the zero-order Bessel function J0(A), greatly simplifying the extraction of the Stokes vector elements from the Fourier components of the detector waveform. A common procedure is to determine A for each PEM individually from the ratio of the fundamental to the third harmonic response, or the second over the fourth harmonic response.43 While this works reasonably well for the 50 kHz PEM, such a calibration scheme is problematic for the 60 kHz modulator. This is a result of the high frequencies required for the demodulations of the higher harmonics, 180 kHz for 3ω and 240 kHz for 4ω. Such measurements allow for the use of a only a small terminal resistor on the detector output, placing the signal in a range close to the noise level. At the same time, it is desirable to calibrate the amplitudes of both PEMs while they are installed together, so the system is ready for subsequent experiments in the same configuration. We have achieved this by first setting the azimuths of all optics to their final experimental configuration with PEM1 at 45°, PEM2 at 0°, and the analyzer P3 at 22.5°. The intensity waveform at the detector may be written as

(3)

where ω1/2π ≈ 60 kHz and ω2/2π ≈ 50 kHz. The Stokes vector elements sj of the light transmitted or reflected by the sample are encoded in the intensities sj = kjIj, where kj is a constant to be determined in the calibration. This expression illustrates that all four elements of the Stokes vector are present at unique frequency components of the signal. The output of the photodiode is therefore demodulated using four lock-in amplifiers (Signal Recovery 7265). The s1 element is extracted from the amplitude of the second harmonic of PEM1, s2 from the second harmonic of PEM2, and s3 from the fundamental of PEM1. s0 is measured in the DC component, obtained by demodulating at the chopper frequency. Since the highest frequency of interest is 120 kHz, we do not need all of the available detector bandwidth and are able to increase our signalto-noise with a 10 kΩ resistor. Calibration of Optic Azimuths. An initial reference polarization state is required prior to calibrating the azimuth of any other optics in our system. This is obtained by setting the angle of incidence to Brewster’s angle for a thick piece of glass mounted in the sample position. All of the optical elements are mounted on computer controller rotation stages with a resolution of 1 arcsecond. A polarizer (P2 in Figure 1) is rotated to set p-polarization by extinction of the reflection at Brewster’s angle. We avoid any attenuation due to crossing the laser’s output polarization by generating circularly polarized light prior to P2. This is achieved with an initial polarizer (P1 in Figure 1) and quarter-wave retarder (QWP). We now remove the glass sample and put back the instrument into a straightthrough transmission geometry, with the polarization state generator and polarimeter in line with each other. The analyzer (P3) is then set by extinction upon crossing with P2. The BSC and PEMs are now installed one at a time; theirs azimuth are calibrated by monitoring I0 to achieve extinction between P2 and P3 (crossed). In the case of the PEMs, it is typical to carry out this procedure with the detector signal demodulated at 2ω. However, as is illustrated in Figure 2, the DC signal provides a much more sensitive measure of the extinction.

I(t ) = 2 2 I0 − 2I3 sin δ1(t ) + (I1 − I2) cos δ1(t ) + (I1 + I2) cos δ2(t ) + 2I1 sin δ1(t ) sin δ2(t ) + 2I3 cos δ1(t ) sin δ2(t ) − I1 cos δ1(t ) cos δ2(t ) Figure 2. Photoelastic modulator signal vs azimuth between crossed polarizers, showing variation in the DC component I0 (solid line), and demodulating at the second- (dotted line) and fourth harmonic (very low intensity dashed line) of the PEM frequency.

(4)

where I1, I2, and I3 are the Fourier components from eq 3, δ1 is the time-varying retardation of PEM1, and δ2 is the retardation of PEM2. This expression may be expanded using 1798

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Stokes parameters. Such data was collected over a range of factory settings for the amplitude of each PEM (as displayed along the horizontal and vertical axes of Figure 3b) and then fit to a sine function of adjustable amplitude, frequency, and phase. The resulting amplitude is plotted in Figure 3b. Values of A1 and A2 that result in the minimum dc variation represent the values of the retardation amplitude closest to 2.4048 rad for each PEM. A fit of the dc signal data to a two-dimensional Gaussian function (Figure 3c) using a BFGS routine44,45 allowed for a more accurate determination of the desired amplitude settings. Materials. The prism was made of fused silica (Del Mar Photonics, San Diego CA) with the incident and exit faces cut at 70°. Prior to use, it was cleaned by dipping in a solution of 0.1 v/v% nitric acid in sulfuric acid, followed by copious rinsing in 18 MΩ·cm deionized water (Nanopure, Barnstead Thermo). The three prism surfaces of interest (incident, exident, samplecontact faces) were lightly polished using a 3 μm polycrystalline diamond suspension (Buehler Metadi supreme), followed by 0.05 μm silica suspension (Buehler Masterprep) and again rinsed with deionized water. Phosphated-buffered saline (PBS, 1X Dulbecco’s formula without magnesium, MPBio) was produced by dissolving one tablet containing 20 mg KCl, 20 mg KH2PO4, 800 mg NaCl and 115 mg Na2HPO4 in 200 mL of 18 MΩ·cm deionized water. Bovine serum albumin (MW 66430 g/mol, lyophilized powder ≥96% Sigma) stock solutions were prepared by dissolving in PBS solution.

sin δ1(t ) = 2 ∑ J2j − 1(A1) sin[(2j − 1)(ω1t )] (5a)

j=1

sin δ2(t ) = 2 ∑ J2j − 1(A 2 ) sin[(2j − 1)(ω2t )] (5b)

j=1

cos δ1(t ) = J0 (A1) + 2 ∑ J2j (A1) sin(2jω1t ) j=1

(5c)

cos δ2(t ) = J0 (A 2) + 2 ∑ J2j (A 2) sin(2jω2t ) j=1

(5d)

where A1 is the retardation amplitude of PEM1 and A2 is the amplitude of PEM2. The key to our amplitude calibration is to consider the J0(A) contribution to the signal. In the event that J0(A) = 0 (when A = 2.4048 rad), this has no effect upon the DC component. However if it is not set to this value, a sinusoidal variation in the DC signal is observed when the input polarization state is varied. Prior to calibration, this variation in may be seen in Figure 3a upon the application of varying input

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RAPID STOKES VECTOR MEASUREMENT Obtaining the Stokes Vectors. Following demodulation of the detector waveform, the four acquired signals Ij(j = 0−3) are proportional to the four corresponding elements sj of the Stokes vector. The constants of proportionality ki are obtained in an additional calibration step as instrument parameters such as impedance matching between different lock-in amplifiers may then be included in the ki values.36 The procedure is based on that in ref 38. with the exception that, rather than a choosing a few arbitrary values of the retardation, we use a Soleil-Babinet compensator (BSC) calibrated to λ/4 retardation. The first step is to normalize all intensities according to Ii = Ii/I0. We then form a column vector of normalized I02 (ones) with length equal to the number of data points n. ⎡ (I 2 ) ⎤ ⎢ 0 1 ⎥ ⎡1⎤ ⎢(I 2) ⎥ ⎢ ⎥ ⎢ 0 2 ⎥ ⎢1⎥ a = ⎢(I 2) ⎥ = ⎢1⎥ ⎢ 0 3⎥ ⎢ ⎥ ⎢ ⋮ ⎥ ⎢⋮⎥ ⎢ ⎥ ⎣1⎦ ⎢⎣(I0 2)n ⎥⎦

(6)

We are working under the assumption that when the PSG is directly inline with the PSA with no sample present, there are no depolarizing elements and therefore Figure 3. Simultaneous calibration of the retardation amplitude of both photoelastic modulators. (a) Variation in the DC signal as a function of the BSC azimuth for fixed modulator retardations. A fit to a sine function returned the amplitude of the variation. (b) Plot of the amplitude variation as a function of the modulator amplitudes. Darker red points indicate smaller amplitudes. The position of the crosshairs indicates the values of the modulator retardations for which the smallest DC variation was obtained. (c)Fit of the data to obtain the final calibrated modulator retardations.

s12 + s2 2 + s32

(7)

I0 2 = k12I12 + k 2 2I2 2 + k 32I32

(8)

s0 = becomes

having set k0 = 1. We place the measured intensities into the matrix 1799

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The Journal of Physical Chemistry C ⎡ (I 2 ) ⎢ 1 1 ⎢(I 2) ⎢ 1 2 B = ⎢ (I 2 ) ⎢ 1 3 ⎢ ⋮ ⎢ ⎢⎣(I12)n

(I2 2)1 (I32)1 ⎤ ⎥ (I2 2)2 (I32)2 ⎥ ⎥ (I2 2)3 (I32)3 ⎥ ⎥ ⋮ ⋮ ⎥ ⎥ (I2 2)n (I32)n ⎥⎦

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(9)

Finally we define a vector containing the squares of the desired coefficients ⎡k 2⎤ ⎢ 1 ⎥ k = ⎢k 22 ⎥ ⎢ ⎥ ⎢k 2 ⎥ ⎣ 3 ⎦

(10)

The relationship in eq 8 is then expressed as a = Bk and so the desired coefficients are obtained from k = B−1a

(11)

Results of the fit are shown in Figure 4. With the kj values determined in this procedure, we are now able to transform the measured Ij into the sj elements of the Stokes vector.

Figure 5. BSA adsorption at the PBS solution−silica interface, showing plots of (a) s1, (b) s2, (c) s3, (d) the degree of polarization, and (e) temperature over a 10-min period. Initial data represents the beam polarization at the PBS−silica interface; BSA solution was injected at t = 1 min, indicated by the vertical dashed line. The horizontal dashed lines shows the value of the output beam polarization as predicted from the solution refractive index change alone.

linearly polarized incident probe beam. The exit beam polarization was measured as indicated by the data from 0 < t < 1 min in Figure 5. The normalized values of s1, s2, and s3 as shown in panels a−c are what is expected for a PBS solution with a refractive index of 1.333046 and fused silica with an index of 1.4570.47 The degree of polarization (Figure 5d) is very close to unity, as expected for a Fresnel-type interface. Small deviations are observed when we move from the transmission geometry used in the calibration of the Stokes vector element to the reflection geometry of the experiment. The temperature inside the box, measured at a point equidistant from the photoelastic modulators and the sample cell, is shown in Figure 5e. At the 1 min mark, the lid of the box was opened, 0.5 mL of a 24.94 mg/mL solution of BSA in PBS was injected over a period of ≈0.5 s, and the box lid was closed. During this procedure, the box lid was ajar for ≈4 s, and we can see that there was a negligible change in the temperature of the system. As a result of the stirring in the sample cell, the change in refractive index of the solution was rapid. On the basis of the final bulk concentration of BSA, we calculate the refractive index of the solution after mixing to be 1.3334.48−50 We have considered a change of 0.190 from the PBS solution refractive

Figure 4. Calibration of the instrument response in the absence of depolarization. Assigning k0 = 1 and simultaneously fitting the data from all of the input polarization states resulted in k1 = 2.4864, k2 = 2.5926 and k3 = 1.8005. Linearity of the plots and proximity of the slopes to unity is shown for (a) s1, (b) s2, and (c) s3. Experimental data are plotted as points; the solid line has unit slope. The degree of polarization (d) is shown for consistency, as this calibration is based on a DOP of unity.).

Protein Adsorption onto a Silica Surface. As a demonstration, we have studied the adsorption of bovine serum albumin (BSA) in phosphate-buffered saline (PBS) solution onto a fused silica surface. The substrate was a 70° dove prism. For a 70°-incidence geometry, the beams therefore approached the air−prism interface and exited the prism−air interface at normal incidence. This assisted in the critical alignment of the beam as back reflections could be monitored, and simplified the analysis as no change in polarization occurred upon entering or leaving the prism. Prior to protein addition, we placed 5 mL of PBS in the sample cell and set the polarizer and λ/4 compensator to 45° azimuth to create a 1800

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on two photoelastic modulators. As a result of the high modulation frequency, changes in the sample response may be monitored on the millisecond time scale. We have demonstrated the off-resonance monitoring of serum albumin adsorption onto a hydrophilic silica surface. This illustrates the potential of such a technique for sensitive, rapid, and quantitative characterization of adsorption at the solid−liquid interface.

index for every gram of protein per milliliter added, as determined in a previous surface plasmon resonance study.51 The expected Stokes vector elements for BSA dissolved in PBS are indicated by the horizontal dashed lines in Figure 5. One notices that there is not much change in s1 ≈ 0 as the beam is incident above the critical angle. Variation in s1 about its mean value is within ±0.0001, roughly the accuracy in our experiment. A simultaneous inspection of Figure 5d does not provide any indication that the degree of polarization is reduced over the course of the experiment. The largest change upon protein addition to the solution was observed in s2. Figure 5b indicates that s2 immediately reached its BSA-solution value (a change of ≈0.001, indicated by the dashed line), and continued to drop by an additional ≈0.001. The signal then reached a level value around 3 min after sample injection. The slight increase in the signal toward long time may be slightly correlated with a weak trend in the temperature (Figure 5e) for this region (t > 6 min), but is only the order of 0.0001, within the limit of our accuracy. Further evidence of protein adsorption is found in the s3 data, as the change commensurate with the refractive index of the solution is calculated to be only 0.0001, yet a change of ≈0.0008 was observed. The time scale of the change in s3 was similar to what was observed for s2, with a stable signal obtained around 3 min following injection. An ellipsometer based on second-harmonic generation52,53 has been used to study BSA coadsorpting with a dye onto a fused silica substrate.54 At protein concentrations comparable to those in our study, the authors found that a rapid change in signal occurred immediately following BSA introduction, and was stable after ≈5 min, in agreement with the time scale of the s2 and s3 response we have observed. The change in conformation of BSA on silica has been found to be small and reversible.55−57 Transmission electron microscopy studies of BSA adsorption onto a variety of planar solid surfaces have concluded that the adsorbed amount increases as the wettability of the surface decreases, and that the protein forms islands on hydrophobic surfaces, but more uniformly coats hydrophilic surfaces.58,59 A compact, uniform BSA coating on silica particles was also found in a study employing partial proteolysis followed by identification of cleavage sites by MALDI mass spectroscopy.60 As a result of this uniformity and our degree of polarization near unity, we can use the equilibrium values of the Stokes vector elements in a three-phase model61 to estimate the amount of BSA adsorbed on the silica surface. We use fused silica as the entrance phase, BSA as the layer in which multiple internal reflections occur, and the BSA/PBS solution as the adjacent third phase. The refractive index of dry BSA films has been reported as 1.6, and hydrated BSA as 1.57.62,63 A study that considered a reduced occupied fraction has arrived at a lower effective refractive index of 1.50.64 We have used our polarization data together with BSA layer refractive indices in the range 1.5−1.6 and determined that the layer thickness is in the range 4−10 nm. This is in good agreement with values in the range 5−8 nm obtained with dual polarization interferometry and neutron reflectivity.65 Considering a dry density66 of 1.37 g/cm3, this results in an upper limit of 5−11 ng/mm2 of BSA at the silica− solution interface.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support of this science with a Discovery Grant. Instrumentation was purchased with assistance from an NSERC RTI Grant. We thank Prof. Joel Harris (University of Utah) for advice regarding the temperature control, and Prof. David Harrington (UVic Chemistry) for the use of his lock-in amplifiers. S.A.H. is grateful to NSERC for an Alexander Graham Bell graduate scholarship.

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REFERENCES

(1) Poksinski, M.; Arwin, H. Total internal reflection ellipsometry: ultrahigh sensitivity for protein adsorption on metal surfaces. Opt. Lett. 2007, 32, 1308−1310. (2) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. A Comparative Study of Protein Adsorption on Titanium Oxide Surfaces Using in situ Ellipsometry, Optical Wavefuide Lightmode Spectroscopy, and Quartz Crystal Microbalance/Dissipation. Colloid Surf. B 2002, 24, 155−170. (3) Seitz, R.; Brings, R.; Geiger, R. Protein Adsorption on Solid− Liquid Interfaces Monitored by Laser Ellipsometry. Appl. Surf. Sci. 2005, 252, 154−157. (4) Gill, K. J. S.; Isaacson, K.; Dew, S.; Kwok, D. Y. Reversible Protein Detection Method Based on Self-Assembled Monolayers Using Ellipsometry. Int. Conf. MEMS, NANO, Smart Syst. 2004, 640− 643. (5) Elwig, H. Protein Absorption and Ellipsometry in Biomaterial Research. Biomaterials 1998, 19, 397−406. (6) Pokinski, M.; Arwin, H. Protein Absorption and Ellipsometry in Biomaterial Research. Thin Solid Films 2004, 455−456, 716−721. (7) Fusetani, N. Biofouling and Antifouling. Nat. Prod. Rep. 2004, 21, 94−104. (8) Kasemo, B. Biological Surface Science. Curr. Opin. Solid State Mater. Sci. 1998, 3, 451−459. (9) Pavritha, D.; Doble, M. Biofilm Formation, Bacterial Adhesion and Host Response on Polymeric ImplantsIssues and Prevention. Biomed. Mater. 2008, 3, 034003−034003 + 13. (10) Logothetidis, S. Haemocompatibility of Carbon-based Thin Films. Diam. Relat. Mater. 2007, 16, 1847−1857. (11) Lousinian, S.; Logothetidis, S. In-situ and Real-time Protein Adsorption Study by Spectroscopic Ellipsometry. Thin Solid Films 2008, 516, 8002−8008. (12) Xu, F.; Nacker, J. C.; Crone, W. C.; Masters, K. S. The Haemocompatibility of Polyurethanehyaluronic Acid Copolymers. Biomaterials 2008, 29, 150−160. (13) Miyata, T.; Uragami, T.; Nakamae, K. Biomolecule-sensitive Hydrogels. Adv. Drug Delivery Rev. 2002, 54, 79−98. (14) Adhikari, B.; Majumdar, S. Polymers in Sensor Applications. Prog. Polym. Sci. 2004, 29, 699−766.

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CONCLUSIONS We have demonstrated how a combination of calibration procedures and temperature stabilization may be used to operate a high-accuracy and high-precision ellipsometer based 1801

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(15) Jin, W.; Brennan, J. Properties and Applications of Proteins Encapsulated Within Sol-gel Derived Materials. Anal. Chim. Acta 2002, 461, 1−36. (16) Lu, J. R.; Zhao, X.; Yaseen, M. Protein Adsorption Studied by Neutron Reflection. Curr. Opin. Colloid Interface Sci. 2007, 12, 9−16. (17) Chena, H.; Yuana, L.; Song, W.; Wub, Z.; Lia, D. Biocompatible Polymer Materials: Role of Protein−surface Interactions. Prog. Polym. Sci. 2008, 33, 1059−1087. (18) Zhang, L.; Sun, Y. Molecular Simulation of Adsorption and Its Implications to Protein Chromatography: A Review. Biochem. Eng. J. 2010, 48, 408−415. (19) Krishnan, S.; Weinman, C. J.; Ober, C. K. Advances in Polymers for Anti-biofouling Surfaces. J. Mater. Chem. 2008, 18, 3405−3413. (20) Elbert, D. L.; Hubbell, J. A. Surface Treatments of Polymers for Biocompatibility. Annu. Rev. Mater. Sci. 1996, 26, 365−394. (21) Green, R. J.; Frazier, R. A.; Shakesheff, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. Surface Plasmon Resonance Analysis of Dynamic Biological Interactions with Biomaterials. Biomaterials 2000, 21, 1823−1835. (22) Lu, J. R.; Perumal, S.; Hopkinson, I.; Webster, J. R. P.; Penfold, J.; Hwang, W.; Zhang, S. Interfacial Nano-structuring of Designed Peptides Regulated by Solution pH. J. Am. Chem. Soc. 2004, 126, 8940−8947. (23) Churchill, H.; Teng, H.; Hazen, R. M. Correlation of pHdependent Surface Interaction Forces to Amino Acid Adsoprtion: Implications for the Origin of Life. Am. Mineral. 2004, 89, 1048−1055. (24) Read, M. J.; Mayes, A. M.; Burkett, S. L. Effects of Temperate and pH on the Helicity of a Peptide Adsorbed to Colloidal Silica. Colloid Surf. B: Biointerfaces 2004, 37, 113−127. (25) Soderquist, M. E.; Walton, A. G. Structural Changes in Proteins Adsorbed on Polymer Surfaces. J. Colloid Interface Sci. 1980, 75, 386− 396. (26) Wudy, F.; Multerer, M.; Stock, C.; Schmeer, G.; Gores, H. J. Rapid Impedance Scanning QCM for Electrochemical Applications Based on Miniaturized Hardware and Highperformance Curve Fitting. Electrochim. Acta 2008, 53, 6568−6574. (27) Liu, S.; Kim, J. Application of Kevin-Voigt Model in Quantifying Whey Protein Adsorption on Polyethersulfone Using QCM-D. J. Lab. Auto. 2009, Aug, 213. (28) Berglin, M.; Pinori, E.; Sellborn, A.; Andersson, M.; Hulander, M.; Elwing, H. Fibrinogen Adsorption and Conformational Change on Model Polymers: Novel Aspects of Mutual Molecular Rearrangement. Langmuir 2009, 25, 5602. (29) Gormally, M.; McKibben, R.; Johal, M.; Selassie, C. Controlling Tyrosinase Activity on Charged Polyelectrolyte Surfaces: A QCM-D Analysis. Langmuir 2009, 25, 10014. (30) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Surface Plasmon Resonance for Real Time in situ Analysis of Protein Adsorption to Polymer Surfaces. Biomaterials 1997, 18, 405− 413. (31) Tokareva, I.; Minko, S.; Fendler, J. H.; Hutter, E. Nanosensors Based on Responsive Polymer Brushes and Gold Nanoparticle Enhanced Transmission Surface Plasmon Resonance Spectroscopy. J. Am. Chem. Soc. 2004, 126, 15950−15951. (32) Dong, J.; Peng, Y.; Gao, N.; Bai, J.; Ning, B.; Liu, M.; Gao, Z. A Novel Polymerization of Ultrathin Sensitive Imprinted Film on Surface Plasmon Resonance Sensor. Analyst 2012, 137, 4571−4576. (33) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland Publishing Company: Amsterdam, 1977. (34) Tompkins, H. G., Irene, E. A., Eds. Handbook of Ellipsometry; William Andrew: Norwich, NY, 2005. (35) Schubert, M. Another Century of Ellipsometry. Ann. Phys 2006, 15, 480−497. (36) Guan, W.; Jones, G. A.; Liu, Y. W.; Shen, T. H. The measurement of the Stokes parameters: A Generalized Methodology Using a Dual PhotoelasticModulator System. J. Appl. Phys. 2008, 103, 043104.

(37) Mackey, J. R.; Salari, E.; Tin, P. Modulation, Alignment, and Calibration Methods for Phasemodulated Optical Polarimeters. Opt. Eng. 2003, 42, 1460−1466. (38) Liu, Y. W.; Jones, G. A.; Peng, Y.; Shen, T. H. Generalized Theory and Application of Stokes Parameter Measurements Made with a Single Photoelastic Modulator. J. Appl. Phys. 2006, 100, 063537. (39) Kuldkepp, M.; Hawkes, N. C.; Rachlew, E.; Schunke, B. Accurate Polarization Measurements with a Dual Photoelastic Modulator. Appl. Opt. 2005, 44, 5899−5904. (40) Cross, L. J. K.; Hore, D. K. Dual-modulator Broadband Infrared Mueller Matrix Ellipsometry. Appl. Opt. 2012, 51, 5100−5110. (41) Hlady, V.; Buijs, J. Protein Adsorption on Solid Surfaces. Curr. Opin. Biotechnol. 1996, 7, 72−77. (42) Jena, K. C.; Covert, P. A.; Hore, D. K. The Effect of Salt on the Water Structure at a Charged Solid Surface: Differentiating Secondand Third-order Nonlinear Contributions. J. Phys. Chem. Lett. 2011, 2, 1056−1061. (43) Wang, M. W.; Chao, Y. F.; Leou, K. C.; Tsai, F. H.; Lin, T. L.; Chen, S. S.; Liu, Y. W. Calibrations of Phase Modulation Amplitude of Photoelastic Modulator. Jap. J. Appl. Phys. 2004, 43, 827−832. (44) Zhu, C.; Byrd, R.; Nocedal, J. L-BFGS-B: Algorithm 778: LBFGS-B, FORTRAN Routines for Large Scale Bound Constrained Optimization. ACM Trans. Math. Software 1997, 23, 550−560. (45) Byrd, R.; Lu, P.; Nocedal, J. A Limited Memory Algorithm for Bound Constrained Optimization. SIAM J. Sci. Stat. Comp. 1995, 16, 1190−1208. (46) Zhernovaya, O.; Sydoruk, O.; Tuchin, V.; Douplik, A. The Refractive Index of Human Hemoglobin in the Visible Range. Phys. Med. Biol. 2011, 56, 4013−4021. (47) Malitson, I. H. Interspecimen Comparison of the Refractive Index of Fused Silica. J. Opt. Soc. Am. 1965, 55, 1205−1209. (48) Akimoto, T.; Sasaki, S.; Ikebukuro, K.; Karube, I. Refractive Index and Thickness Sensitivity in Surface Plasmon Resonance Spectroscopy. Appl. Opt. 1999, 38, 4058−4064. (49) Jin, Y. L.; Chen, J. Y.; Xu, L.; Wang, P. N. Refractive Index Measurement for Biomaterial Samples by Total Internal Reflection. Phys. Med. Biol. 2006, 51, N371−N379. (50) Lin, Y.-C.; Tsai, W.-H.; Tsao, Y.-C.; Tai, J.-K. An Enhanced Optical Multimode Fiber Sensor Based on Surface Plasmon ResonanceWith Cascaded Structure. IEEE Photonics Techol. Lett 2008, 20, 1287−1289. (51) Tumolo, T.; Angnes, L.; Baptista, M. S. Determination of the Refractive Index Increment (dn/dc) of Molecule and Macromolecule Solutions by Surface Plasmon Resonance. Anal. Biochem. 2004, 333, 273−279. (52) Begue, N. J.; Moad, A. J.; Simpson, G. J. Nonlinear Optical Stokes Ellipsometry. 1. Theoretical Framework. J. Phys. Chem. C 2009, 113, 10158−10165. (53) Begue, N. J.; Everly, R. M.; Hall, V. J.; Haupert, L.; Simpson, G. J. Nonlinear Optical Stokes Ellipsometry. 2. Experimental Demonstration. J. Phys. Chem. C 2009, 113, 10166−10175. (54) Polizzi, M. A.; Plocinik, R. M.; Simpson, G. J. Ellipsometric Approach for the Real-time Detection of Label-free Protein Adsorption by Second Harmonic Generation. J. Am. Chem. Soc. 2004, 126, 5001−5007. (55) Giacomelli, C. E.; Norde, W. The Adsorption−Desorption Cycle. Reversibility of the BSA− Silica System. J. Colloid Interface Sci. 2001, 233, 234−240. (56) Gee, M. L.; Lensun, L.; Smith, T. A.; Scholes, C. A. Timeresolved EvanescentWave-induced Fluorescence Anisotropy for the Determination of Molecular Conformational Changes of Proteins at an Interface. Eur. Biophys. J. 2004, 33, 130−139. (57) Rezwan, K.; Meier, L. P.; Gauckler, L. J. Lysozyme and Bovine Serum Albumin Adsorption on Uncoated Silica and AlOOH-coated Silica Particles: the Influence of Positively and Negatively Charged Oxide Surface Coatings. Biomaterals 2005, 26, 4351−4357. (58) Uyen, H. M. W.; Schakenraad, J. M.; Sjollema, J.; Noordmans, J.; Jongebloed, W. L.; Stokroos, I.; Busscher, H. J. Amount and Surface Structure of Albumin Adsorbed to Solid Substrata with Different 1802

dx.doi.org/10.1021/jp311573q | J. Phys. Chem. C 2013, 117, 1796−1803

The Journal of Physical Chemistry C

Article

Wettabilities in a Parallel Plate Flow Cell. J. Biomed. Res. 1990, 24, 1599−1614. (59) Busscher, H. J.; van der Mei, H. C.; Schakenraad, J. M. Analogies in the Two-dimensional Spatial Arrangement of Adsorbed Proteins and Adhering Bacteria: Bovine Serum Albumin and Streptococcus Sanguis 12. J. Biomater. Sci., Polym. Ed. 1991, 3, 85−94. (60) Larsericsdotter, H.; Oscarsson, S.; Buijs, J. Structure, Stability, and Orientation of BSA Adsorbed to Silica. J. Colloid Interface Sci. 2005, 289, 26−35. (61) Hecht, E.; Zajac, A. Optics; Addision-Wesley: Reading, MA, 1979. (62) Putzeys, M. P.; Brosteaux, J. L’Indice de Refraction des Proteides. Bull. Soc. Chim. Biol. 1936, 18, 1681−1763. (63) Armstrong, S. H.; Budka, M. J. E.; Morrison, K. C.; Hasson, M. Preparation and Properties of Serum and Plasma Proteins. XII. The Refractive Properties of the Proteins of Human Plasma and Certain Purified Fractions. J. Am. Chem. Soc. 1947, 69, 1747−1753. (64) Fisk, A. A. The Thickness of Hemoglobin and Bovine Serum Albumin Molecules as Unimolecular Layers Adsorbed onto Films of Barium Stearate. Proc. Natl. Acad. Sci. U.S.A. 1950, 36, 518−523. (65) Cowsill, B. J.; Coffey, P. D.; Yaseen, M.; Waigh, T. A.; Freeman, N. J.; Lu, J. R. Measurement of the Thickness of Ultra-thin Adsorbed Globular Protein Layers with Dual-polarisation Interferometry: A Comparison with Neutron Reflectivity. Soft Matter 2011, 7, 7223− 7230. (66) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundström, I. Molecular Packing of HSA, IgG, and Fibrinogen Adsorbed on Silicon by AFM Imaging. Thin Solid Films 1998, 324, 257−273.

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