Hormone glucagon: electrooxidation and determination at carbon nanotubes.

The oxidation of glucagon, which is one of the key hormones in glucose homeostasis, was studied at electrodes modified with carbon nanotubes (CNT) that were dispersed in a polysaccharide adhesive chitosan (CHIT). Such electrodes displayed improved resistance to fouling, which allowed for the investigation of both the electrolysis/mass spectrometry and electroanalysis of glucagon. The off-line electrospray ionization and tandem mass spectrometric analyses showed that the -4 Da mass change to glucagon upon electrolysis at CNT was due to the electrooxidation of its tryptophan (W25) and dityrosine (Y10, Y13) residues. The methionine residue of glucagon did not contribute to its oxidation. The amperometric determination of glucagon yielded the limit of detection equal to ∼20 nM (E = 0.800 V, pH 7.40, S/N = 3), sensitivity of 0.46 A M(-1) cm(-2), linear dynamic range up to 2.0 µM (R(2) = 0.998), response time <5 s, and good signal stability. Free tryptophan and tyrosine yielded comparable analytical figures of merit. The direct amperometric determination of unlabeled glucagon at CHIT-CNT electrodes is the first example of a rapid alternative to the complex analytical assays of this peptide.

Morphology of Gold Nanoparticles and Electrocatalysis of Glucose Oxidation.

The shape-dependent activity of gold nanoparticles (AuNP) was studied by testing them as electrocatalysts for the notoriously slow non-enzymatic oxidation of glucose in neutral solutions. The AuNP of spherical and irregular (including polyhedral) morphologies were synthesized and attached to glassy carbon electrodes with chitosan. Voltammetric and mass spectrometric studies showed that the irregular AuNP were more catalytically active toward the oxidation of glucose to gluconic acid. No obvious differences between both morphologies were found based on their X-ray diffraction patterns and HRTEM images suggesting that the crystallographic orientation alone did not account for their catalytic properties. While both morphologies contain the (111) crystallographic planes that are catalytic toward glucose oxidation, the better activity of irregular AuNP was ascribed to a higher surface density of incipient gold oxide acting as a fast redox mediator for glucose oxidation. Supporting this, the AuNP of both morphologies oxidized glucose after their anodic activation, although not to the same extent. The amperometric (0.30 V) determination of glucose at electrodes made of irregular AuNP yielded a wide linear calibration plot (0.20-110 mM; R2, 0.998), sensitivity of 66 µA M-1 cm-2, limit of detection of 100 µM (S/N, 3), and a response time below 5 s. The advantage of low-cost irregular AuNP over macro gold is that they are catalytic toward glucose oxidation without any need for prior activation.

Electrochemical coupled-enzyme assays at carbon nanotubes.

The recently developed internally calibrated electrochemical continuous enzyme assay (ICECEA) has proved to work well for single-enzyme systems. In the present work, its relevance to more challenging coupled-enzyme measurements was investigated by using a model enzyme pair comprising aspartate transaminase (AST) and malic dehydrogenase. The ICECEA was performed at an electrode modified with carbon nanotubes (CNTs), which were dispersed in a polysaccharide chitosan that acted as an adhesive. The 7 min assay required a 100 µL sample and relied on an AST-free calibration. It had a limit of detection equal to 5.0 pM AST (0.10 U L(-1)) with no need for the incubation period. Its linear range extended up to 3500 pM (70 U L(-1)). Perhaps the most promising was the fact that the assay and its calibration could be performed in the same solution even though the composition of the assay solution for the coupled-enzyme assays is typically more complex than that for the single-enzyme assays. This and the fast electrode kinetics of the signal transducing reaction of nicotinamide adenine dinucleotide at CNTs accounted for the low limit of detection. The unique shape of the ICECEA amperogram allowed for the selective determination of AST in the complex matrix of serum samples containing redox active potentially interfering species. Given these advantages, the prospects for the ICECEA in the development of other coupled-enzyme assays were also discussed.

On the direct electron transfer, sensing, and enzyme activity in the glucose oxidase/carbon nanotubes system.

The signal transduction and enzyme activity were investigated in biosensors based on the glucose oxidase (GOx) and carbon nanotubes (CNT) embedded in a bioadhesive film of chitosan (CHIT). The voltammetric studies showed that, regardless of CHIT matrix, the GOx adsorbed on CNT yielding a pair of surface-confined current peaks at -0.48 V. The anodic peak did not increase in the presence of glucose in an O2-free solution indicating the lack of direct electron transfer (DET) between the enzymatically active GOx and CNT. The voltammetric peaks were due to the redox of enzyme cofactor flavin adenine dinucleotide (FAD), which was not the part of active enzyme. The presented data suggest that DET may not be happening for any type of GOx/CNT-based sensor. The biosensor was sensitive to glucose in air-equilibrated solutions indicating the O2-mediated enzymatic oxidation of glucose. The signal transduction relied on the net drop in a biosensor current that was caused by a decrease in a 4-e(-) O2 reduction current and an increase in a 2-e(-) H2O2 reduction current. The enzyme assays showed that CNT nearly doubled the retention of GOx in a biosensor while decreasing the average enzymatic activity of retained enzyme by a factor of 4-5. Such inhibition should be considered when using a protein-assisted solubilization of CNT in water for biomedical applications. The proposed analytical protocols can be also applied to study the effects of nanoparticles on proteins in assessing the health risks associated with the use of nanomaterials.

Signal amplification in enzyme-based amperometric biosensors.

A unique mode of current amplification was investigated in reagentless biosensors based on the clinically significant enzymes including alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, glycerol 3-phosphate dehydrogenase, and glucose oxidase. The biosensors were designed by sandwiching the enzyme-polymer film between an electrode and Nafion film. In particular, each enzyme and its cofactor were covalently attached to the chains of polysaccharide chitosan and mixed with carbon nanotubes on the electrode surface. The coating of such biosensors with Nafion resulted in the current increase by up to 1000%, depending on the enzyme. The results were analyzed considering the interplay between the enzyme activity-pH profiles and the Nafion-induced pH increase in the underlying chitosan film. The data were collected by using the rapid (<5 min) amperometric enzyme assays and pH-sensitive iridium oxide films. The increase in the biosensor current was attributed to the pH-driven increase in the enzyme activity inside the two-film interface. Such signal amplification should also be feasible in other biosensors based on the polyelectrolytes and sandwiched enzymes providing that a proper match is made between the enzyme activity-pH profiles and the pH of buffer solutions.

Rapid electrochemical enzyme assay with enzyme-free calibration.

The internally calibrated electrochemical continuous enzyme assay (ICECEA, patent pending) was developed for the fast determination of enzyme activity unit (U). The assay depends on the integration of enzyme-free preassay calibration with the actual enzyme assay in one continuous experiment. Such integration resulted in a uniquely shaped amperometric trace that allowed for the selective picomolar determination of redox enzymes. The ICECEA worked because the preassay calibration did not interfere with the enzyme assay allowing both measurements to be performed in succession in the same solution and at the same electrode. The method displayed a good accuracy (relative error, <3%) and precision (relative standard deviation (RSD), <3%) when tested with different working electrodes (carbon nanotubes/chitosan, glassy carbon, platinum) and enzymes (alcohol dehydrogenase, ADH; lactate dehydrogenase, LDH; xanthine oxidase, XOx; glucose oxidase, GOx). The limit of detection for the ADH, LDH, XOx, and GOx was equal to 0.18, 0.14, 0.0031, and 0.11 U L(-1) (or 4.2, 0.72, 89, and 6.0 pM), respectively. The simplicity, reliability, and short analysis time make the ICECEA competitive with the optical enzyme assays currently in use.

Electrochemistry and current control in surface films based on silica-azure redox nanoparticles, carbon nanotubes, enzymes, and polyelectrolytes.

The redox active nanoparticles were developed by covalently attaching redox dye Azure C (AZU) to commercial silica nanoparticles (SN) via the silylated amine and glutaric dialdehyde links. The SN-AZU nanoparticles were studied as redox mediators for the oxidation of reduced ß-nicotinamide adenine dinucleotide (NADH) in two polymeric films. The first film (F1) was composed of SN-AZU, carbon nanotubes, and cationic polyelectrolyte chitosan. The second film (F2) contained also added enzyme glucose dehydrogenase and its cofactor ß-nicotinamide adenine dinucleotide (NAD(+)). The films F1 and F2 were cast on the glassy carbon electrodes, covered with an anionic polyelectrolyte Nafion, and their electrochemical properties were probed with NADH and glucose, respectively, using voltammetry, amperometry, and potentiometry. The Nafion overcoat reduced the sensitivity of F1/Nafion film electrodes to NADH by >98%. In contrast, depending on the concentration of Nafion, the sensitivity of the F2/Nafion film electrodes (reagentless biosensors) to glucose increased by up to 340%. The amplification of glucose signal was ascribed to the Donnan exclusion and ensuing Nafion-gated ionic fluxes, which enhanced enzyme activity in films F2. The proposed model predicts that such signal amplification should be also feasible in the case of other enzyme-based biosensors.