Real-Time Dynamic Adsorption Processes of Cytochrome <i>c</i> on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

<div><p>An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome <i>c</i> (cyt <i>c</i>) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt <i>c</i> adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt <i>c</i> molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt <i>c</i> using EC-HS-AFM. The root mean square roughness (<i>R</i>_RMS) from the AFM images and the number of the electrochemically active cyt <i>c</i> molecules adsorbed onto the electrode (<i>Γ</i>) simultaneously increased in positive cooperativity. Cyt <i>c</i> molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.</p></div

Real-time cyt <i>c</i> desorption processes from the MUA-modified gold electrode.

<p>(A) Continuous AFM images of desorbing cyt <i>c</i> molecules. Frame rate, 2 frames/s; image area, 150 × 150 nm². (B) Time evolution of <i>R</i>_RMS values at the higher ionic strengths. (C) Schematic of desorbing cyt <i>c</i> molecules from the MUA-modified electrode. </p

AFM images show (A) the MUA-modified gold surface and (B) cyt <i>c</i> adsorbed on the MUA SAM at 450 sec.

<p>Continuous AFM images of adsorbing cyt <i>c</i> molecules with real-time labels. Frame rate, 2 frames/s; image area, 150 × 150 nm². The cross sections of the images in (A) and (B), along the short white line, are shown in the lower right. </p

CVs of cyt <i>c</i> adsorbed on the electrode.

<p>(A) CVs of cyt <i>c</i> molecules adsorbed on the MUA electrode in a 10 mM phosphate buffer solution (pH 7.0) from 333 to 522 s (from −0.35 V to 0.1 V, each segment is 4.5 s). (B) Background-subtracted CVs from the voltammogram from 313 to 324 s and 333 to 522 s (from inside to outside, each segment is 4.5 s). The voltammograms were collected at a scan rate of 100 mVs⁻¹. Ag wire was used as a reference electrode. </p

Time-course analysis of <i>R</i>_RMS and <i>Γ</i> values.

<p>(A) Time evolution of <i>R</i>_RMS values from the HS-AFM images (open circle) and the level of electrochemically active cyt <i>c</i> (<i>Γ</i>) from the cyclic voltammograms (open square). (B) Schematic of the adsorbing cyt <i>c</i> molecules on the MUA-modified electrode at each time point.</p

Titration of the apo-form of DH_PDH with PQQ.

The purified apo-form of DHPDH (50 nM) was pre-incubated with various concentrations of PQQ in 50 mM MES buffer, pH 6.5, containing 1 mM CaCl2. After 1 min, the enzyme activity was determined according to the procedure described in the Experimental Procedures.</p

Cyclic voltammogram of <i>Cc</i>PDH immobilized on a plastic-formed carbon electrode modified with 27 nm carbon nanoparticles.

The voltammogram was obtained in 100 mM HEPES buffer, pH 7.0, at a scan rate of 20 mV/s.</p

Enzyme concentration dependence of the amount of adsorbed <i>Cc</i>PDH.

Closed circle, highly crystalline cellulose from Cladophora; open circle, PASC. The adsorption of CcPDH was measured after incubation for 120 min with 1 mg/mL of cellulose at 30°C as described in the Experimental Procedures.</p

UV-visible absorption spectra of the apo- and holo-forms of DH_PDH.

<p>Dotted line, apo-form of DH_PDH; black solid line, holo-form of DH_PDH; blue solid line, reduced form by addition of 1 mM l-fucose. All spectra were recorded in 50 mM HEPES buffer, pH 7.0 at room temperature.</p

Specificity constant values of <i>Cc</i>PDH for various monosaccharides.

<p>Specificity constant values of <i>Cc</i>PDH for various monosaccharides.</p