OWLS Experiments

A schematic of our OWLS system (BIOS-1, MicroVacuum, Budapest, Hungary), as modified to allow for detection in the presence of an applied potential difference, appears in Fig. 2. The adsorbing surface is an indium tin oxide (ITO, or In2-2xSnxO3-x with x = 0.50 ± 0.02)-coated optical waveguide 2400 sensor chip (MicroVacuum). The ITO layer has a thickness of approximately 10 nm and complex refractive index 1.80-0.03i and rests on a silicon titanium oxide layer (STO, or Si1-xTixO2 with x = 0.25 ± 0,05) of thickness ca. 200 nm and refractive index 1.77 ± 0.03. The water contact angle of the ITO coating is 48.1 ± 0.1 this value does not change within the range of applied potential investigated here. The sensor chip serves as the base of a temperature-controlled flow cell of volume 70 pl A flat platinum counterelectrode is situated at the top of the flow cell 1.0 mm from the ITO surface. The sensor chip/flow cell assembly rests on the head of a precision goniometer.

An electric circuit schematic appears in Fig. 3. A potential difference between the ITO working electrode and the platinum counter is applied via an external power supply. The current is measured across a 100-kW resistor via a voltmeter, and the magnitude of the potential difference is determined using a second voltmeter. The potential of the ITO surface relative to a reference electrode (situated in the inlet solution) is measured using an electrometer.

Fig. 2. A schematic of our modified optical waveguide lightmode spectroscopy (OWLS) system for the continuous measurement of macromolecular adsorption under an applied potential. The adsorbing substrate is an approximately 10-nm indium tin oxide (ITO) layer on a silicon titanium oxide (STO) waveguiding film of approximately 200 nm, itself supported on a glass substrate (not shown). A potential difference (A V) is applied between the ITO layer and a flat platinum (Pt) counterelectrode, situated 1 mm above at the top of the flow cell. A polarized HeNe laser beam directed toward a grating coupler at the STO/ITO interface (not shown) from below excites a guided mode at a resonant angle, from which adsorbed layer mass and thickness maybe determined

Fig. 2. A schematic of our modified optical waveguide lightmode spectroscopy (OWLS) system for the continuous measurement of macromolecular adsorption under an applied potential. The adsorbing substrate is an approximately 10-nm indium tin oxide (ITO) layer on a silicon titanium oxide (STO) waveguiding film of approximately 200 nm, itself supported on a glass substrate (not shown). A potential difference (A V) is applied between the ITO layer and a flat platinum (Pt) counterelectrode, situated 1 mm above at the top of the flow cell. A polarized HeNe laser beam directed toward a grating coupler at the STO/ITO interface (not shown) from below excites a guided mode at a resonant angle, from which adsorbed layer mass and thickness maybe determined

Fig. 3. A schematic of the electric circuit of our OWLS system. The ITO- and Pt-solution interfaces maybe considered as resistors (R™ andfiptt, respectively) and capacitors (C1™ and Cptt, respectively) in parallel. The ITO film and the solution within the flow cell act as resistors (Rito and Rsol, respectively). A potential difference is applied through a power supply (V) and the current is measured via the voltage drop across a 100-kQ resistor. A second voltmeter is used to measure the potential difference between the ITO and Pt electrodes. The potentials of the ITO and Pt electrodes versus a reference electrode (e.g., Ag/AgCl) are measured using an electrometer (EM)

Fig. 3. A schematic of the electric circuit of our OWLS system. The ITO- and Pt-solution interfaces maybe considered as resistors (R™ andfiptt, respectively) and capacitors (C1™ and Cptt, respectively) in parallel. The ITO film and the solution within the flow cell act as resistors (Rito and Rsol, respectively). A potential difference is applied through a power supply (V) and the current is measured via the voltage drop across a 100-kQ resistor. A second voltmeter is used to measure the potential difference between the ITO and Pt electrodes. The potentials of the ITO and Pt electrodes versus a reference electrode (e.g., Ag/AgCl) are measured using an electrometer (EM)

Prior to each experiment, the flow cell, tubing, and sensor chip are cleaned by exposure to a 2% Hellmanex (Hellma, Mülheim, Germany) solution in ultrapure water, followed by an intensive rinse with ultrapure water. An experiment begins with the introduction of a pure buffer to the flow cell via a peristaltic pump. An angular scan is performed, resulting in a mode spectrum from which NTE and NTM are obtained at approximately 20-s intervals. During an open-circuit potential (OCP) experiment, a flowing protein solution then replaces the flowing buffer solution once stable values of NTE and NTM are achieved. In other experiments, a voltage difference AV is applied (AV = VITO - VPt), resulting in further changes to Nte and NTM. Once steady values are recovered, a protein solution is introduced.

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