Results

We begin by discussing certain important optoelectronic properties of the ITO-coated sensor chip. In Fig. 4, we present the effective refractive index, the current, and the ITO and platinum electrode potentials versus applied voltage in the presence of deionized water (of pH 5.5-6 and conductivity 1.30 ± 0.05 |S at 25 °C) and HEPES buffer [10 mM N-(2-hydroxyethyl)

Fig. 4. The asymptotic current, the electric potential of the ITO and Pt electrodes, and the effective refractive index of the ITO-coated sensor chip as a function of applied voltage for a water and b HEPES buffer [10 mM N-(2-hydroxyethyl) piperazine-N'-(ethanesulfonic acid) in 100 mM NaCl, adjusted to pH 7.4 by addition of 6 N NaOH] solvents. The increases in current at 1.5 V and 1.0 V, respectively, indicate the onset of water electrolysis. NtE Effective refractive index of the transverse electric mode. Reproduced with permission from Brusatori et al. (2003)

Fig. 4. The asymptotic current, the electric potential of the ITO and Pt electrodes, and the effective refractive index of the ITO-coated sensor chip as a function of applied voltage for a water and b HEPES buffer [10 mM N-(2-hydroxyethyl) piperazine-N'-(ethanesulfonic acid) in 100 mM NaCl, adjusted to pH 7.4 by addition of 6 N NaOH] solvents. The increases in current at 1.5 V and 1.0 V, respectively, indicate the onset of water electrolysis. NtE Effective refractive index of the transverse electric mode. Reproduced with permission from Brusatori et al. (2003)

piperazine-N'-(ethanesulfonic acid) in 100 mM NaCl, adjusted to pH 7.4 by addition of 6 N NaOH]. We observe the effective refractive index to increase with applied potential up to AV = VITO - VPt > 1.4 V. Upon further voltage increase, NTE decreases in the water system and plateaus in the HEPES system. These changes suggest an alteration in optical density of the ITO-coated waveguiding film due to accumulation of charged species at the interface and/or within the film, and possibly also to mild oxidative reactions. A voltage-induced orientation of water in the porous ITO

film is also a possible explanation, as suggested elsewhere (Stankowski and Ramsden 2002). The somewhat sharp increase in current above an applied voltage of 1.5 V (water) and 1.0 V (HEPES) is due to the onset ofwater electrolysis. However, we observe no H2 or O2 gas formation and attribute this absence to the low overall current density.* Increasing the applied voltage increases VITo and decreases VPt; the latter becomes negative relative to a standard hydrogen electrode (SHE) when AV exceeds 1.7 V and 1.5 V for water and HEPES, respectively. We find prolonged cathodic polarization (i. e., A V < 0) to result in increased opacity of the ITO film. This is probably due to electrochemical reduction and renders the sensor chip unusable in an OWLS experiment.

We wish to use the measured effective refractive index to calculate the adsorbed protein density, but doing so brings forth two important questions: (1) Must the ITO layer be considered explicitly, and if so, must the imaginary portion of its complex refractive index be considered? (2) Do the model Eqs. 12-19 hold under an applied voltage? To answer these questions, we measure the refractive index of a (non-adsorbing) 5.0 mg/ml glucose solution in water, with and without an applied potential, using three different waveguide models: (1) a three-layer model in which the STO and ITO films are considered to be a single layer (i. e., both reflection coefficients are determined via Eq. 12), (2) a four-layer model in which nF is real (i. e., the F' layer is a dielectric), and (3) a four-layer model in which nF' is complex (i.e., the F' layer is a conductor). The glucose solution has a known refractive index of 1.33173 ± 1 x 10-5 at 25 °C. In Table 1, we show refractive index values measured using OWLS within each of the three waveguide models for cases of OCP and an applied voltage of A V = 5 V. We observe all values to be within 3x 10-5 of one another. Since the difference (nF - nC) appearing in Eq. 18 is typically around 0.5, uncertainty of this magnitude has no appreciable effect on a measured adsorbed density. Thus, we conclude that an applied potential difference has only a negligible effect on the instrument's detection and that one may consider the STO and ITO films as a single effective film.

Using OWLS and employing a four-layer waveguide model - in which the STO and ITO layers are treated as a single layer, as justified above -we measure the adsorption kinetics of human serum albumin and horse heart cytochrome c, in flowing water or HEPES solutions at a surface shear rate of 1.5/s, onto ITO as a function of applied potential (Brusatori et al. 2003; Brusatori and Van Tassel 2003). The raw data of a typical experiment are shown in Fig. 5. Following a baseline measured under flowing solvent (deionized water in this case), application of a potential difference (1.0 V)

* A current of 1 |A results in the production of approx. 0.5 x 10-7 M/s of H2 in our flow cell. The time needed to achieve the solubility limit of approx. 10-3 M is thus approximately 2x104 s; this is much greater than the space time of the flow cell, which is about 50 s.

Table 1. The refractive index, nC, of a 5.0 mg/ml glucose solution - for an open circuit potential (OCP) and an applied voltage of 5V - as determined using (1) a three-layer waveguide model where the silicon titanium oxide (STO) and indium tin oxide (ITO) layers are treated as a single layer, (2) a four-layer waveguide model where the ITO film is treated as a separate dielectric layer (i.e., of real refractive index), and (3) a four-layer waveguide model where the ITO film is treated as a separate conductinglayer (i. e., of complex refractive index). The closeness of the values demonstrates both the validity of treating the STO and ITO films as a single optical layer and the accuracy of detection in the presence of an applied voltage

Table 1. The refractive index, nC, of a 5.0 mg/ml glucose solution - for an open circuit potential (OCP) and an applied voltage of 5V - as determined using (1) a three-layer waveguide model where the silicon titanium oxide (STO) and indium tin oxide (ITO) layers are treated as a single layer, (2) a four-layer waveguide model where the ITO film is treated as a separate dielectric layer (i.e., of real refractive index), and (3) a four-layer waveguide model where the ITO film is treated as a separate conductinglayer (i. e., of complex refractive index). The closeness of the values demonstrates both the validity of treating the STO and ITO films as a single optical layer and the accuracy of detection in the presence of an applied voltage

OCP

Three-layer waveguide model with composite

nc =

1.33177 ± 1X10

STO-ITO layer

OCP

Four-layer waveguide model with dielectric ITO layer

nc =

1.33178 ± 1X10

OCP

Four-layer waveguide model with conducting ITO

nc =

1.33177 ± 1X10

layer

Ay =

5 V

Three-layer waveguide model with composite

nc =

1.33175 ±2X10

STO-ITO layer

Ay =

5 V

Four-layer waveguide model with dielectric ITO layer

nc =

1.33175 ±2X10

Ay =

5 V

Four-layer waveguide model with conducting ITO

nc =

1.33175 ±2X10

layer layer

Cytochrome C in H2O

Cytochrome C in H2O

« o Current ^

r~

^V* J

°o oo o

2000 4000 6000 8000 10000

Time(s)

2000 4000 6000 8000 10000

Time(s)

Fig. 5. The effective refractive index (the raw output signal during an OWLS measurement) and the current during cytochrome c adsorption from water onto ITO as a function of time. At point i, a voltage difference of 1.0 V is applied. At point ii, the protein solution is introduced. At point iii, the protein solution is replaced by a buffer solution. At point iv, the system is returned to an open-circuit potential (OCP), and at point v, the 1.0 V voltage difference is reapplied. At point vi, the protein solution is reintroduced. Reproduced with permission from Brusatori et al. (2003)

between the adsorbing substrate and a platinum counterelectrode (i) yields an increase in the measured effective refractive index, the fundamental output signal of OWLS (see Eq. 10). This increase is not fully understood, but is likely to be due to penetration of small ions into the ITO and/or the underlying silicon titanium oxide films (even in deionized water, carbonate and other ionic impurities are present), or to mild oxidation of the ITO. The current increases initially but then decreases, as would occur for a resistor and capacitor in series (see Fig. 3). Upon the addition of cytochrome c in water (ii), the signal greatly increases due to optical changes at the interface brought about by adsorption. A return to the deionized water (iii) causes only a small signal reduction, indicating that only a small quantity of protein desorbs. When the electric field is removed (iv), a decreased signal results; however, this is not due to further desorption as a re-application of the field (v) returns the signal to its previous level. Finally, a re-introduction of the protein (vi) yields an additional signal increase. This additional adsorption is likely to be due to an increased amount of area on the surface open for adsorption. The cause of this increased available area is probably aggregation among the adsorbed molecules.

In Figs. 6 and 7, we show the adsorbed density versus time for albumin and cytochrome c in both water and HEPES solvents. In all cases, we observe the total amount adsorbed to increase with increasing applied potential. For water systems, adsorption curves reach a virtual plateau during OCP (AVoCp = -0.02 ± 0.01 V) but fail to do so in the presence of an applied voltage. The amount adsorbed during 1 h is greatly increased (by up to a factor of 4) by an applied potential difference. We observe a similar, but less pronounced voltage dependence in the HEPES systems (where AyOCP = +0.02 ± 0.01 V). Variability is approximately 20% for runs using different ITo-coated sensor chips (most likely due to subtle variations in surface microstructure) and approximately 8% for runs on the same sensor chip.

The principal advantage of OWLS over other detection methods is the precision of the output signal. We exploit this advantage in Fig. 8 by plotting the discrete time derivative of the adsorbed density, dr/dt, as a function of surface density, r. These derivative data are themselves sufficiently precise so as to allow for a clear delineation of transport- and adsorption-limited regimes. Adsorption is initially limited by the rate of transport to the surface; this occurs by a combination of diffusion and electrophoretic migration in the presence of a flow field. We observe the adsorption rate to continuously increase during this regime, as would occur initially for convective diffusion in laminar flow (see Theoretical Prediction). If the surface were a perfect sink, a plateau would eventually be reached upon achievement of a steady-state concentration profile above the surface. In the example shown here, surface availability begins to reduce the rate of adsorption before such a steady, transport-limited rate is achieved. Theoretical treatments of the surface-limited regime (Eq. 11) predict an initial linear decrease in adsorption rate with increasing surface coverage, followed by a nonlinear asymptotic dependence; our observations are consistent with a) Albumin in H?0 1.4 i-

0 1000 2000 3000 4000

0 1000 2000 3000 4000

b) Cytochrome c In HiO

1.4

• IS V o 1 0 V O OCP

0 2 ■

00

—1-

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Fig. 6. The density of adsorbed protein versus time for a albumin and b cytochrome c in water onto ITO at OCP and under various applied potentials (1.0, 1.5, and 2.0 V). Since monolayer densities are approximately 0.46 and 0.33 |g/cm2 for albumin and cytochrome c, respectively, multilayer adsorption is occurring under an applied potential these predictions. Extrapolation of data in the linear portion of the surface-limited regime to zero density yields an estimate of the apparent adsorption rate constant, ka< (Calonder et al. 2001; Calonder and Van Tassel 2001). Rate curves such as these also allow for an estimate of the free protein concentration at the surface, and thus of the perfect sink approximation employed in the analysis shown in "Theoretical Prediction". For a given adsorbed density, this is just the ratio of the adsorption rate to the corresponding value of the extrapolated linear region.

Fig. 7. The density of adsorbed protein versus time for a albumin and b cytochrome c in HEPES buffer onto ITO at OCP and under various applied potentials (1.0 and 1.5 V)

In Fig. 8, we show the adsorption rate as a function of the adsorbed amount (and, in the insets, as a function of time) for albumin and cy-tochrome c in water. In the insets, we also show predictions based upon purely convective diffusion (Eq. 2). For albumin, we observe the adsorption rate to increase with applied voltage over the entire range of surface coverage. As shown by the inset, the initial rate is increased significantly by the presence of an applied potential, indicating a transport mechanism to which both convective diffusion and electrophoretic migration contribute. It should be noted that the observed behavior is far from the limit of

Fig. 8. The rate of adsorption versus adsorbed density (and in the inset, versus time) for a albumin and b cytochrome c in water onto ITO at OCP and under various applied voltages (1.0, 1.5, and 2.0 V). Also shown are predictions (solid lines) for pure convective diffusion given by Eq. 2. Both convective diffusion and electromigration influence transport of the negatively charged albumin, but only convective diffusion influences the transport of the positively charged cytochrome c. If electromigration were the dominant transport mechanism, an initial nonzero adsorption rate would result, as predicted by Eq. 10. r Surface density, dr/ di time derivative of the adsorbed density, t time

Fig. 8. The rate of adsorption versus adsorbed density (and in the inset, versus time) for a albumin and b cytochrome c in water onto ITO at OCP and under various applied voltages (1.0, 1.5, and 2.0 V). Also shown are predictions (solid lines) for pure convective diffusion given by Eq. 2. Both convective diffusion and electromigration influence transport of the negatively charged albumin, but only convective diffusion influences the transport of the positively charged cytochrome c. If electromigration were the dominant transport mechanism, an initial nonzero adsorption rate would result, as predicted by Eq. 10. r Surface density, dr/ di time derivative of the adsorbed density, t time pure electrophoretic migration characterized by an initially nonzero, time-independent adsorption rate, as predicted by Eq. 10. For cytochrome c, we observe the transport-limited rates to be essentially independent of ap-

Table 2. The apparent adsorption rate constant kai determined by extrapolating the linear region of a time derivative of the adsorbed density (dT/ di) versus surface density (r) curve (see Fig. 8), and the first-order density expansion coefficient to the cavity function C1 (Eq. 11), for human serum albumin and horse heart cytochrome c (Cyt c) in water and HEPES [10 mM N-(2-hydroxyethyl) piperazine-N'-(ethanesulfonic acid) in 100 mM NaCl, adjusted to pH 7.4 by addition of 6 N NaOH] buffer onto ITO at an OCP and under an applied voltage (1.0,1.5, or 2.0 V)

Table 2. The apparent adsorption rate constant kai determined by extrapolating the linear region of a time derivative of the adsorbed density (dT/ di) versus surface density (r) curve (see Fig. 8), and the first-order density expansion coefficient to the cavity function C1 (Eq. 11), for human serum albumin and horse heart cytochrome c (Cyt c) in water and HEPES [10 mM N-(2-hydroxyethyl) piperazine-N'-(ethanesulfonic acid) in 100 mM NaCl, adjusted to pH 7.4 by addition of 6 N NaOH] buffer onto ITO at an OCP and under an applied voltage (1.0,1.5, or 2.0 V)

Protein

Applied potential (V)

H2O solvent ka' C1 (10-5 cm/s) (cm2/|g)

HEPES solvent ka' C1 (10-5 cm/s) (cm2/|g)

Albumin

OCP

1.5 ±0.1

-3.6 ±0.1

0.4 ± 0.01

-9.2 ± 0.6

1.0

3.4 ±0.1

-2.8 ± 0.1

1.1 ± 0.1

-8.2 ± 1.0

1.5

5.2 ± 0.2

-3.2 ± 0.3

1.5 ± 0.1

-7.1 ± 0.7

2.0

9.1 ± 0.2

-2.1 ± 0.1

-

-

Cyt c

OCP

9.9 ± 0.3

-4.5 ± 0.4

3.8 ± 0.2

-8.1 ± 0.7

1.0

9.5 ± 0.2

-4.7 ± 0.1

4.5 ± 0.3

-6.7 ± 0.8

1.5

9.0 ± 0.3

-4.5 ± 0.3

4.3 ± 0.6

-6.7 ± 2.0

2.0

8.3 ± 0.3

-4.3 ± 0.4

-

-

plied potential and the surface-limited rates to be enhanced only at higher coverage (i. e., in the asymptotic region). In Table 2, we show the apparent adsorption rate constant, ka<, versus voltage obtained by extrapolating data in the linear regions. For albumin in both water and HEPES solvents, we find ka to increase significantly with voltage. For cytochrome c, the effect is solvent dependent: we find a decrease in ka> with voltage for water but no appreciable change for HEPES. We also observe the linear coefficient to the density expansion of the cavity function (as introduced in Eq. 11), obtained from the slope of the linear region, to decrease (in magnitude) with voltage for albumin in both solvents but to remain roughly unchanged for cytochrome c.

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