Electrochemical Control of Protein Monolayers at Indium Tin Oxide Surfaces for the Reagentless Optical Biosensing of Nitric Oxide

Abstract

Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.

Lack of nano size effect on electrochemistry of dopamine at a gold nanoparticle modified indium tin oxide electrode

“…In our work, AuNPs were synthesized by the wet chemistry method, and their size was varied by adjusting the amount of citrate in the reaction mixture [19][20][21][22]. AuNPs were attached to ITO substrates via well established silane surface chemistry with APTES [24,25]. Figure 1 shows SEM image of 11 nm AuNPs deposited on ITO substrate.…”

“…After the reaction, the slices were washed ultrasonically in dry toluene (5 min, twice) to remove physically adsorbed APTES from the surface, followed by anhydrous acetone and water (5 min, twice). The APTES modified ITO slices were dried under a stream of nitrogen [24,25]. They were stored in a sealed box till use.…”

Effect of Different Pretreatments on Indium-Tin Oxide Electrodes

“…6,7 However, the effect of pretreatment methods on indium-tin oxide (ITO) electrodes has been rarely investigated, although many pretreatment methods have been employed to clean ITO electrodes. [10][11][12][13][14][15][16][17][18][19][20] Recently, the authors studied the effect of thermal treatment on the electrocatalytic activities and surface roughness of ITO electrodes: the electrocatalytic activities and surface roughness of ITO electrodes increase with increasing the incubation temperature. 10 The electrocatalytic activities of ITO electrodes are much lower than those of common metal and carbon electrodes.…”

“…[11][12][13][14][15][16][17][18][19][20] However, the electrocatalytic activities, surface roughness, hydrophilicity, and surface composition of ITO electrodes obtained after these pretreatments have never been intensively investigated and compared. In this study, the authors have studied the effect of pretreatment methods on the electrochemical properties, surface roughness, contact angle, and surface composition of ITO electrodes.…”

Nitric Oxide Biosensors Based on the Immobilization of Hemoglobin on Mesoporous Titania Electrodes

“…These methods involve heme proteins encapsulated in sol -gels [2], a peroxidase amperometric electrode [3], a fiber-optic biosensor based on Guanylate cyclase [4], myoglobin adsorbed on carbon nanotubes [5], Hb-DNA films [6], a fiber-optic chemiluminescent sensor [7], Hb immobilized on tin oxide electrodes [8], myoglobin clay electrodes [9] and a protein monolayer at an optical protein -indium tin oxide surface [10]. Real time in vivo NO measurements in biomedical samples have also taken place [11,12].…”