Different carbon-based materials have been compared for the development of NADH sensors: glassy carbon electrodes (GCE), multiwalled carbon nanotubes (MWCNT), and carbon black (CB). The GCE and MWCNT has been subjected to oxidative pretreatment to study the influence of oxidative groups for NADH oxidation. The materials had been characterized by FT-IR to identify the surface composition. The response of bare (GC) and GC/modified electrodes toward potassium ferricyanide have been employed to obtain information about the electroactive area and electron transfer rate. Studies of NAD+/NADH redox behavior showed that MWCNT and GCE exhibit high degree of passivation while CB shows no fouling effects. Catalytic effect of surface-oxygenated groups was also proved for GCE and MWCNT, and both, O-GCE and O-MWCNT, exhibited a lower oxidation overpotential compared to the respective untreated materials. Chronoamperometric quantification showed a linear dependence between 2–18
The electrochemical behavior of NADH/NAD+ redox couple has been widely studied because of the importance of this compound as cofactor in several biochemical processes. Oxidation of NADH occurs at high anodic overpotentials employing the most common electrode materials (i.e., glassy carbon and graphite) and implies the transfer of 2 electrons and one proton and the formation of NAD+ molecule, which is reduced at high cathodic overpotentials for regenerating NADH molecule [
A significant decrease in the overpotential for NADH oxidation has been achieved using carbon-based nanomaterials. Different studies have demonstrated that oxygen-rich groups and quinone-like structures formed on the surface of carbon-based materials can promote the oxidation rate of NADH, working as “redox mediators” in electron transfer [
Depending on the electrode material, it has been demonstrated that NAD+ produced from NADH oxidation can be strongly adsorbed on the electrode surface, causing fouling and loss of electrochemical response. This behavior limits the use of sensors for practical applications and suggest using them as disposable. The adsorption of NADH onto surface electrodes has been well studied by Elving at the end of the 1970s and the beginning of the 1980s [
The majority of studies of NADH oxidation are focused on the oxidation processes, while NAD+ adsorption and reduction are not widely studied and the decrease in electrochemical response over reused electrodes is not commonly reported.
In this work, a careful comparison study of the electrochemical behavior of NAD+/NADH redox couple has been carried out for sensors made of different materials: the classical glassy carbon and the recently widely used nanomaterial CB and MWCNTs. The aim was to properly correlate the electrochemical activity of NADH with the presence of oxygenated groups over the surface of the sensors and to understand the electrode surface modification during the sensing process. The study demonstrated that for complex redox reaction different considerations are needed to select the best material for the development of the appropriate sensors.
Carbon-based materials used in this research were glassy carbon electrodes (GCE, CH Instruments Inc.), multiwalled carbon nanotubes (MWCNT), and carboxylate-modified MWCNT (O-MWCNT), supplied by Nano-Lab (EE.UU); carbon black (CB type N220 from Cabot Corporation, Ravenna, Italy).
All electrochemical measurements were carried out with a Metrohm-Autolab PGSTAT101 potentiostat-galvanostat. Typical three electrode configuration composed of GCE and modified GCE, Ag/AgCl, and Pt wire as working, reference, and counter electrodes, respectively, was used.
The GCE was polished with alumina powder of different size (1, 0.3, and 0.05
CB and O-MWCNT were suspended in Milli-Q water and sonicated for 2 hours until a stable 1 mg·mL−1 dispersion was obtained. The same protocol was carried out for MWCNT using ethanol as solvent. The preparation of GCE/CB, GCE/MWCNT, and GCE/O-MWCNT was carried out by one drop casting of 10
Samples were characterized using qualitative infrared spectroscopy analysis in a Nicolet 6700 spectrometer, equipped with an MCT/A detector, and operated in a wavenumber ranging from 800 cm−1 to 4000 cm−1. For MWCNT, O-MWCNT, and CB, pellets were prepared with a sample/KBr weight ratio of 1/3000. Samples and pellets were heated, before the analysis, at 150°C in order to remove the water. For GCE and O-GCE, an ATR module was used.
The effective working area of the electrodes was determined from the slopes obtained for the graphs of
The heterogeneous rate constants (
Cyclic voltammetry (CV) at different potential ranges and scan rates were performed. Chronoamperometric (CA) experiments were carried out by fixing oxidation potentials according to the cyclic voltammetric data.
Plots of scan rate and the square root of scan rate were compared versus peak current in order to verify the type of electrochemical control of the NADH oxidation over the carbon-based materials. After each scan rate, every electrode was carefully washed with Milli-Q water in order to remove NADH oxidation products. Finally, chronoamperometric curves were obtained for NADH concentrations in the range of 2–18
FT-IR spectra obtained for the different carbon-based materials studied are reported in Figure
Infrared spectra for GC (a), O-GC (b), MWCNT (c), CB (d), and O-MWCNT (e).
MWCNTs and O-MWCNTs (Figure
CB spectra (Figure
Figure
Anodic and cathodic peak currents versus square root of scan rate potential in potassium ferricyanide 1 mmol·L−1, scan rates: 5, 10, 20, 40, 60, 80, 100, and 150 mV·s−1. GCE (□), B O-GCE (○), GCE/CB (∆), GCE/MWCNT (∇), and GCE/O-MWCNT (◊).
Electroactive area and heterogeneous transfer constant obtained from ferro/ferricyanide redox couple.
GCE | 16.03 | 1.9 |
O-GCE | 29.23 | 4.5 |
GCE/CB | 23.99 | 3.6 |
GCE/MWCNT | 24.02 | 4.6 |
GCE/O-MWCNT | 36.74 | 10.1 |
The smallest electrochemical area was obtained for GCE that is consistent with the lower value for
Figure
Cyclic voltammetry for different carbonaceous materials. Scan rate: 5 mV·s−1, NADH concentration: 1 mmol·L−1. C NADH: 1 mM;
From cyclic voltammetry, it is possible to obtain an expression for heterogeneous transfer constant for the NADH oxidation process, using the correlation between the peak current (
In order to evaluate the type of electrochemical control for NADH oxidation on the electrodes surface, graphs of scan rate and the square root of scan rate versus
Anodic peak currents versus potential scan rate (a) and square root of potential scan rate (b). NADH concentration: 1 mmol·L−1; scan rates: 5, 10, 20, 40, 60, 80, 100, 150, and 200 mV·s−1. GCE (□), B O-GCE (○), GCE/CB (∆), GCE/MWCNT (∇), and GCE/O-MWCNT (◊).
The surface covering parameters obtained for GCE/MWCNT and GCE/O-MWCNT were 4.17 × 10−7 and 3.43 × 10−7
Control of NADH oxidation for the different carbon-based materials tested resulted in different main control adsorption for the MWCNTs sensors, diffusion for O-GC and CB-GCE, and mixed control for O-GC. This is a key parameter in developing sensors since not only the chemical species reacting at the electrodes are significant but also the reaction product that can migrate to the bulk solution or remain adsorbed at the electrode surface. The latter case leads both to electrode passivation (fouling), which implies a decrease in the electroactive area, and in the decrease of electrocatalytic current for NADH oxidation. Evaluation of NAD+ adsorption after NADH oxidation was carried out running 5 consecutive CVs (Figure
Cyclic voltammetries obtained for NADH over different carbonaceous materials. 5 cycles,
Cyclic voltammetries obtained for NADH over (a) GCE/MWCNT and (b) GCE/O-MWCNT at an initial potential before to the reduction potential peak. Scan rate: 100 mV·s−1, NADH concentration: 1 mmol·L−1.
According to these voltammograms, the following mechanism can be proposed for NADH/NAD+ over GCE/MWCNT and GCE/O-MWCNT:
In the first anodic scan,
Reduction process at the first cathodic scan
Oxidation from the second scan onwards
The reported mechanism is very similar to that described by Moiroux and Elving [
Recent works for NADH oxidation studies onto chemically oxidized single-walled carbon nanotubes have shown experimental data in which two peaks are obtained for the oxidation process, but no discussion was reported [
Finally, chronoamperometric experiments were carried out to check linearity of the current response in function of NADH concentration. Different fixed potentials were employed according to Ep,a obtained by voltagrams reported in Figure
Calibration curve for NADH concentrations in the range of 2–18
Slope value, determination coefficient (
Parameters extracted from the calibration curve.
Material | Slope × 10−4 |
LOD |
LOQ | |
---|---|---|---|---|
GCE | 9.20 | 0.9957 | 6.2 | 31 |
O-GCE | 3.05 | 0.9660 | 5.4 | 27 |
GCE/CB | 11.1 | 0.9993 | 3.2 | 16 |
GCE/MWCNT | 2.22 | 0.9960 | 9.6 | 47.5 |
GCE/O-MWCNT | 6.73 | 0.9963 | 4.9 | 24.5 |
Satisfactory determination coefficients were observed for all sensors but there are significant differences in the slopes and limits of detection.
Highest slope and lowest detection limits were obtained for GCE/CB, indicating the good behavior of this material for NADH oxidation. On the other hand, the lowest slope was obtained for GCE/MWCNT with a high limit of detection compared with the other materials. This can be attributed to the passivation process after NADH oxidation which produces a decrease in the current response. Also, O-GCE exhibits a poor amperometric response and loss of linearity for the last points. This sensor did not exhibit the peaks due to dimer formation; however, the voltammogram (Figure
The electrochemical response of NADH of different sensors made of carbon-based materials has been evaluated. The effect of surface oxygen-rich functionalities and the type of structural proprieties of the material were related with catalytic effect for the NADH oxidation. According to the oxidation peaks obtained for NADH, oxygen-rich materials confers a catalytic effect for the oxidation of the molecule.
As expected, and reported in the literature, MWCNT and O-MWCNT exhibited the lowest potential for the oxidation and high catalytic activity. CB which has a graphite-based structure and nanometric size exhibited also good catalytic activity.
A study of the passivation of the sensors gave high passivation for GCE/MWCNT and GCE/O-MWCNT, probably due to the morphology of the material. Both materials showed the appearance of a cathodic peak that can be assigned to the formation of a (NAD)2 dimer due to a strong NAD+ adsorption over the surface of the electrode. Chronoamperometric measurement confirmed that passivation strongly influences the analytical behavior of the sensors indicating as the best sensor the CB-GCE despite the higher oxidation potential and lower heterogeneous rate constant with respect to O-MWCNTs. This study clearly demonstrates that a careful study is needed for the selection of the appropriate material to develop chemical sensors for organic compounds. This selection must not be only directed to assess the highest response or the lower applied potential useful for the redox reaction. Complex phenomena occurring at the electrode surface, as those reported in this paper for NADH oxidation, can result in the wrong choice of material and voltammetric technique used. For example, in the case reported, O-MWCNTs appear to be the best carbon-based material among those tested, but only if a very low applied potential is required for the analysis (i.e., analysis directed in a medium with potentially oxidizable interfering compounds) and it should be used as disposable sensor with a rapid voltammetric technique (i.e., differential pulse voltammetry). If the potential is low enough and it will be necessary to run calibration measurement cycles or to carry an analysis for few seconds or minutes (as for enzymatic analysis), the CB-GCE option would be definitively more applicable.
The authors declare that they have no conflicts of interest.
Lucas Blandón-Naranjo and Jorge Hoyos-Arbeláez would like to thank to COLCIENCIAS for the doctoral scholarship. Mario V. Vázquez would like to thank to CODI-University of Antioquia for the support of the Project 2015-7523. Flavio Della Pelle and Dario Compagnone acknowledge the financial contribution of the Italian Ministry of Foreign Affairs for the Project “materiali nanostrutturati per sistemi (bio) chimici sensibili ai pesticidi.”