Transparent Gold as a Platform for Adsorbed Protein Spectroelectrochemistry: Investigation of Cytochrome c and Azurin

Abstract

The majority of protein spectroelectrochemical methods utilize a diffusing, chemical mediator to exchange electrons between the electrode and the protein. In such methods, electrochemical potential control is limited by mediator choice and its ability to interact with the protein of interest. We report an approach for unmediated, protein spectroelectrochemistry that overcomes this limitation by adsorbing protein directly to thiol self-assembled monolayer (SAM) modified, thin (10 nm), semitransparent gold. The viability of the method is demonstrated with two diverse and important redox proteins: cytochrome c and azurin. Fast, reversible electrochemical signals comparable to those previously reported for these proteins on ordinary disk gold electrodes were observed. Although the quantity of protein in a submonolayer adsorbed at an electrode is expected to be insufficient for detection of UV-vis absorption bands based on bulk extinction coefficients, excellent spectra were detected for each of the proteins in the adsorbed state. Furthermore, AFM imaging confirmed that only a single layer of protein was adsorbed to the electrode. We hypothesize that interaction of the relatively broad gold surface plasmon with the proteins' electronic transitions results in surface signal enhancement of the molecular transitions of between 8 and 112 times, allowing detection of the proteins at much lower than expected concentrations. Since many other proteins are known to interact with gold SAMs and the technical requirements for implementation of these experiments are simple, this approach is expected to be very generally applicable to exploring mechanisms of redox proteins and enzymes as well as development of sensors and other redox protein based applications.

Highly Stable Perylenediimide‐Based Self‐Assembled Monolayers Studied with Spectroelectrochemistry

“…[1] The ease of SAM preparation, their stability,a nd the possibility to introduce different functional groups makes it easy to obtain surfaces that exhibit tailoredp roperties. [2][3][4] Our interest in using these modified surfaces concerns the investigationa nd identification of electroactive speciesa td ifferent redox states or products arising from redoxr eactions by using time-resolved absorption and emission spectroelectrochemistry.C ontrary to molecules studied in solution, this approach is still uncommon for these materials, [5][6][7][8][9][10] because the nanometric scale of the electroactive layer remains amajor limiting factor to characterizing SAMs with the usual absorption and fluorescence spectroscopic methods. Thereby,such characterizationsr equire the development of efficient time-resolved spectroelectrochemicalm easurements that are capable of accuratelym onitoring the evolution of the spectroscopics ignature as afunction of an electrical perturbation, such as apotential step or linear scan, and of probing very low-intensitys ignals at high signal-to-noiseratios.…”

Spectroelectrochemistry on electroactive self-assembled monolayers: Cyclic voltammetry coupled to spectrophotometry

“…To the best of our knowledge, less than a dozen publications report experimental results involving an electrochemical/spectroscopic coupling dedicated to monolayers, essentially in potentiostatic conditions (i.e. fluorescence [8], PM-IRRAS [9], SERS [7], UV-visible absorption [10,11], fluorescence microscopy [12] …). This work clearly show that spectroelectrochemical coupling provides excellent tools to establish detailed structure-property relationships.…”

Electron Transfer at Gold Nanostar Assemblies: A Study of Shape Stability and Surface Density Influence

“…Nowadays, molecular nanoelectronics represent and important research field in which organic molecules and inorganic nanomaterials are combined to form electronic components [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. For this purpose, self-assembled monolayers (SAMs) of bifunctional organic thiols have been extensively used for the chemical binding of metallic nanomaterials to various substrates and studies of electron transport [1][2][3][4][5][6][7].…”

“…For this purpose, self-assembled monolayers (SAMs) of bifunctional organic thiols have been extensively used for the chemical binding of metallic nanomaterials to various substrates and studies of electron transport [1][2][3][4][5][6][7]. The bifunctional organic thiols are good molecular candidates to be used for surface modifications and as linkers because of their easy preparation, high stability and ordered structures [8][9][10][11][12][13][14][15][16][17].…”