Amperometric Detection of Aqueous Silver Ions by Inhibition of Glucose Oxidase Immobilized on Nitrogen-Doped Carbon Nanotube Electrodes.

An amperometric glucose biosensor based on immobilization of glucose oxidase on nitrogen-doped carbon nanotubes (N-CNTs) was successfully developed for the determination of silver ions. Upon exposure to glucose, a steady-state enzymatic turnover rate was detected through amperometric oxidation of the H2O2 byproduct, directly related to the concentration of glucose in solution. Inhibition of the steady-state enzymatic glucose oxidase reaction by heavy metals ions such as Ag(+), produced a quantitative decrease in the steady-state rate, subsequently creating an ultrasensitive metal ion biosensor through enzymatic inhibition. The Ag(+) biosensor displayed a sensitivity of 2.00 × 10(8) ± 0.06 M(-1), a limit of detection (σ = 3) of 0.19 ± 0.04 ppb, a linear range of 20-200 nM, and sample recovery at 101 ± 2%, all acquired at a low-operating potential of 0.05 V (vs Hg/Hg2SO4). Interestingly, the biosensor does not display a loss in sensitivity with continued use due to the % inhibition based detection scheme: loss of enzyme (from continued use) does not influence the % inhibition, only the overall current associated with the activity loss. The heavy metals Cu(2+) and Co(2+) were also detected using the enzyme biosensor but found to be much less inhibitory, with sensitivities of 1.45 × 10(6) ± 0.05 M(-1) and 2.69 × 10(3) ± 0.07 M(-1), respectively. The mode of GOx inhibition was examined for both Ag(+) and Cu(2+) using Dixon and Cornish-Bowden plots, where a strong correlation was observed between the inhibition constants and the biosensor sensitivity.

H2O2 Detection at Carbon Nanotubes and Nitrogen-Doped Carbon Nanotubes: Oxidation, Reduction, or Disproportionation?

The electrochemical behavior of hydrogen peroxide (H2O2) at carbon nanotubes (CNTs) and nitrogen-doped carbon nanotubes (N-CNTs) was investigated over a wide potential window. At CNTs, H2O2 will be oxidized or reduced at large overpotentials, with a large potential region between these two processes where electrochemical activity is negligible. At N-CNTs, the overpotential for both H2O2 oxidation and reduction is significantly reduced; however, the reduction current from H2O2, especially at low overpotentials, is attributed to increased oxygen reduction rather than the direct reduction of H2O2, due to a fast chemical disproportionation of H2O2 at the N-CNT surface. Additionally, N-CNTs do not display separation between observable oxidation and reduction currents from H2O2. Overall, the analytical sensitivity of N-CNTs to H2O2, either by oxidation or reduction, is considerably higher than CNTs, and obtained at significantly lower overpotentials. N-CNTs display an anodic sensitivity and limit of detection of 830 mA M(-1) cm(-2) and 0.5 µM at 0.05 V, and a cathodic sensitivity and limit of detection of 270 mA M(-1) cm(-2) and 10 µM at -0.25 V (V vs Hg/Hg2SO4). N-CNTs are also a superior platform for the creation of bioelectrodes from the spontaneous adsorption of enzyme, compared to CNTs. Glucose oxidase (GOx) was allowed to adsorb onto N-CNTs, producing a bioelectrode with a sensitivity and limit of detection to glucose of 80 mA M(-1) cm(-2) and 7 µM after only 30 s of adsorption time from a 81.3 µM GOx solution.

Facile fabrication of carbon ultramicro- to nanoelectrode arrays with tunable voltammetric response.

The voltammetric response for nano- to micrometer-sized electrode arrays are represented by two major regimes: a sigmoidal shaped i-v response for arrays acting as individual electrodes in parallel and peak-shaped i-v response for arrays acting as an ensemble in concert. Here, we present a facile and versatile technique to fabricate ultramicro- to nanoelectrode arrays using atomic layer deposition of insulating Al2O3 on conductive carbon films masked by 1.54, 11, or 90-µm-diameter polystyrene spheres (PSS). The ratio between the interelectrode distance and the electrode radii of the electrode arrays is a predictable function of the PSS radius used in fabrication, resulting in electrode arrays with a tunable voltammetric response. Arrays are characterized utilizing cyclic voltammetry and electrochemical impedance spectroscopy, which provides the critical scan rate, νcrit, the scan rate at which the radial diffusion layers of the individual electrodes overlap and appear as a single linear diffusion layer. Thus, below ν(crit), the electrode has a peak-shaped i-v response associated with semi-infinite linear diffusion, whereas above this critical scan rate, the i-v response is sigmoidal as a result of hemispherical radial diffusion. Results indicate that the critical scan rates are 6.6, 1.0, and 0.01 V/s for the 1.54, 11, and 90 µm PSS prepared electrode arrays, respectively.

Electrochemical behavior of flavin adenine dinucleotide adsorbed onto carbon nanotube and nitrogen-doped carbon nanotube electrodes.

Flavin adenine dinucleotide (FAD) is a cofactor for many enzymes, but also an informative redox active surface probe for electrode materials such as carbon nanotubes (CNTs) and nitrogen-doped CNTs (N-CNTs). FAD spontaneously adsorbs onto the surface of CNTs and N-CNTs, displaying Langmuir adsorption characteristics. The Langmuir adsorption model provides a means of calculating the electroactive surface area (ESA), the equilibrium constant for the adsorption and desorption processes (K), and the Gibbs free energy of adsorption (ΔG°). Traditional ESA measurements based on the diffusional flux of a redox active molecule to the electrode surface underestimate the ESA of porous materials because pores are not penetrated. Techniques such as gas adsortion (BET) overestimate the ESA because it includes both electroactive and inactive areas. The ESA determined by extrapolation of the Langmuir adsorption model with the electroactive surface probe FAD will penetrate pores and only include electroactive areas. The redox activity of adsorbed FAD also displays a strong dependency on pH, which provides a means of determining the pKa of the surface confined species. The pKa of FAD decreases as the nitrogen content in the CNTs increases, suggesting a decreased hydrophobicity of the N-CNT surface. FAD desorption at N-CNTs slowly transforms the main FAD surface redox reaction with E1/2 at -0.84 V into two new, reversible, surface confined redox reactions with E1/2 at -0.65 and -0.76 V (vs Hg/Hg2SO4), respectively (1.0 M sodium phosphate buffer pH = 6.75). This is the first time these redox reactions have been observed. The new surface confined redox reactions were not observed during FAD desorption from nondoped CNTs.

Electrochemical oxidation of dihydronicotinamide adenine dinucleotide at nitrogen-doped carbon nanotube electrodes.

Nitrogen-doped carbon nanotubes (N-CNTs) substantially lower the overpotential necessary for dihydronicotinamide adenine dinucleotide (NADH) oxidation compared to nondoped CNTs or traditional carbon electrodes such as glassy carbon (GC). We observe a 370 mV shift in the peak potential (Ep) from GC to CNTs and another 170 mV shift from CNTs to 7.4 atom % N-CNTs in a sodium phosphate buffer solution (pH 7.0) with 2.0 mM NADH (scan rate 10 mV/s). The sensitivity of 7.4 atom % N-CNTs to NADH was measured at 0.30 ± 0.04 A M(-1) cm(-2), with a limit of detection at 1.1 ± 0.3 µM and a linear range of 70 ± 10 µM poised at a low potential of -0.32 V (vs Hg/Hg2SO4). NADH fouling, known to occur to the electrode surface during NADH oxidation, was investigated by measuring both the change in Ep and the resulting loss of electrode sensitivity. NADH degradation, known to occur in phosphate buffer, was characterized by absorbance at 340 nm and correlated with the loss of NADH electroactivity. N-CNTs are further demonstrated to be an effective platform for dehydrogenase-based biosensing by allowing glucose dehydrogenase to spontaneously adsorb onto the N-CNT surface and measuring the resulting electrode's sensitivity to glucose. The glucose biosensor had a sensitivity of 0.032 ± 0.003 A M(-1) cm(-2), a limit of detection at 6 ± 1 µM, and a linear range of 440 ± 50 µM.

Influence of surface adsorption on the interfacial electron transfer of flavin adenine dinucleotide and glucose oxidase at carbon nanotube and nitrogen-doped carbon nanotube electrodes.

The adsorption of flavin adenine dinucleotide (FAD) and glucose oxidase (GOx) onto carbon nanotube (CNT) and nitrogen-doped CNT (N-CNT) electrodes was investigated and found to obey Langmuir adsorption isotherm characteristics. The amount adsorbed and adsorption maximum are dependent on exposure time, the concentration of adsorbate, and the ionic strength of the solution. The formal potentials measured for FAD and GOx are identical, indicating that the observed electroactivity is from FAD, the redox reaction center of GOx. When glucose is added to GOx adsorbed onto CNT/N-CNT electrodes, direct electron transfer (DET) from enzyme-active FAD is not observed. However, efficient mediated electron transfer (MET) occurs if an appropriate electron mediator is placed in solution, or the natural electron mediator oxygen is used, indicating that GOx is adsorbed and active on CNT/N-CNT electrodes. The observed surface-confined redox reaction at both CNT and N-CNT electrodes is from FAD that either specifically adsorbs from solution or adsorbs from the holoprotein subsequently inactivating the enzyme. The splitting of cathodic and anodic peak potentials as a function of scan rate provides a way to measure the heterogeneous electron-transfer rate constant (k(s)) using Laviron's method. However, the measured k(s) was found to be under ohmic control, not under the kinetic control of an electron-transfer reaction, suggesting that k(s) for FAD on CNTs is faster than the measured value of 7.6 s(-1).

Amperometric detection of L-lactate using nitrogen-doped carbon nanotubes modified with lactate oxidase.

Nitrogen-doped carbon nanotubes (N-CNTs) provide a simple, robust, and unique platform for biosensing. Their catalytic activity toward the oxygen reduction reaction (ORR) and subsequent hydrogen peroxide (H(2)O(2)) disproportionation creates a sensitive electrochemical response to enzymatically generated H(2)O(2) on the N-CNT surface, eliminating the need for additional peroxidases or electron-transfer mediators. Glassy carbon electrodes were modified with 7.4 atom % N-CNTs, lactate oxidase (LOx), and a tetrabutylammonium bromide (TBABr)-modified Nafion binder. The resulting amperometric l-lactate biosensors displayed a sensitivity of 0.040 ± 0.002 A M(-1) cm(-2), a low operating potential of -0.23 V (vs Hg/Hg(2)SO(4)), a repeatability of 1.6% relative standard deviation (RSD) for 200 µM samples of lactate, a fabrication reproducibility of 5.0% (RSD), a limit of detection of 4.1 ± 1.6 µM, and a linear range of 14-325 µM. Additionally, over a 90 day period, the repeatability for 200 µM samples of lactate remained below 3.4% (RSD). Direct electron transfer was observed between the LOx redox-active center and the N-CNTs with the electroactive surface coverage determined to be 0.27 nmol cm(-2).

Toxicological effects of military fog oil obscurant on Daphnia magna and Ceriodaphnia dubia in field and laboratory exposures.

Our purpose was to determine if the acute and sub-lethal effects of fog oil, an obscurant used for military training, could be observed in realistic field exposures. To this end, we exposed Daphnia magna to oil fogs under actual release conditions at a U.S. Army training site. Guided by field investigations, acute toxicity experiments were conducted in the laboratory with the more sensitive species Ceriodaphnia dubia to test the hypothesis that dissolution of fog oil constituents into water is minimal and actual contact by organisms with the water surface is required to cause toxicity. We conducted further experiments to test the hypothesis that vaporization of fog oil alters its chemical composition and toxicity to freshwater invertebrates. In the field, daphnid mortality was minimal more than 5 m from the point of fog generation, but sub-lethal effects were more extensive. Both field and laboratory experiments suggested that physical contact with oils on the water surface was the most important factor driving toxicity. To our knowledge, this is the first attempt to evaluate toxicological endpoints with freshwater invertebrates in field exposures with fog oil.