Waldemar Gorski

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.

Facilitation of NADH Electro-oxidation at Treated Carbon Nanotubes

The relationship between the state of the surface of carbon nanotubes (CNTs) and their electrochemical activity was investigated using the enzyme cofactor dihydronicotinamide adenine dinucleotide (NADH) as a redox probe. The boiling of CNTs in water, while nondestructive, activated them toward the oxidation of NADH, as indicated by a shift in the anodic peak potential of NADH (E(NADH)) from 0.4 V to 0.0 V. The shift in E(NADH) was due to the redox mediation of NADH oxidation by traces of quinone species that were formed on the surface of treated CNTs. The harsher treatment that was comprised of microwaving CNTs in concentrated nitric acid had a similar effect on the E(NADH), and, additionally, it increased the anodic peak current of NADH. The latter correlated with the formation of defects on the surface of acid-microwaved CNTs, as indicated by their Raman spectra. The increase in current was discussed, considering the role of surface mediators on the buckled graphene sheets of acid-microwaved CNTs. The other carbon allotropes, including the edge-plane pyrolytic graphite, graphite powder, and glassy carbon, did not display a comparable activation toward the oxidation of NADH.

Ruthenium catalyst for amperometric determination of insulin at physiological pH

A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and −0.85V vs. SSCE at 100 V s−1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH 7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 μM (five points) had slopes of 72 ± 2 pA μM−1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t90%) was 40 ms or less. The electrode response to 0.2 μM insulin was stable for 3 days. The modified electrode was used for amperometric detection of exocytosis from individual pancreatic s-cells.

Electrocatalyst for non-enzymatic oxidation of glucose in neutral saline solutions

An electroactive coating was deposited on the surface of carbon microelectrodes from solutions containing Pb(NO3)2, Na2IrCl6 and KNO3 by scanning their potentials from 0.0 to 1.15 V. The coating, believed to be IrO2 entrapped inside a PbO2 matrix, catalyzes oxidation of glucose, and other carbohydrates (e.g. mannose), in a phosphate buffered saline solution at pH 7.4. The microelectrodes coated with iridium and lead were used as amperometric detectors at 0.75 V (versus a sodium saturated calomel electrode) for flow injection analysis of glucose samples. The linear dynamic range extended from 0.1 mM up to at least 20 mM, and the detection limit was 50 μM for glucose. The stability and selectivity of the oxide-modified electrodes were improved by application of Nafion® coating. The response of the Nafion®-coated electrode to 15 mM glucose was stable for four days.

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 β-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 β-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.

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.

Spectral analysis of synthesized nanocrystalline aggregates of Er3+:Y2O3

The absorption and fluorescence spectra of nanocrystalline aggregates of Y2O3 doped with Er3+ are reported between 8K and room temperature. The nanocrystalline particles were synthesized from a homogenous solution of the metal ions and urea at elevated temperatures in order to control the precipitation of the mixed hydroxides by a slow uniform reaction throughout the solution. The morphology of the calcinated materials revealed uniformly spherical aggregates 200nm or less depending on the ratio of the metal ions in the initial solution. Spectra obtained from these particles were analyzed in detail for the crystal-field splitting of the LJ2S+1 multiplet manifolds of Er3+(4f11) including the ground-state manifold I15∕24, and excited manifolds I9∕24, F9∕24, S3∕24, H11∕22, F7∕24, F5∕24, and F3∕24. Fluorescence lifetimes and results from an analysis of the intensities of manifold-to-manifold transitions are also reported. The sharp-line absorption and emission spectra are comparable to spectra reported earlier...

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.

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.

Preparation and spectroscopic characterization of Nd3+:Y2O3 nanocrystals suspended in polymethyl methacrylate

We describe a method to fabricate polymethyl methacrylate (PMMA), a polymeric host, in which nanocrystals of Nd3+:Y2O3 are suspended. The spectroscopic properties of this material are analyzed using the standard Judd–Ofelt technique. The phenomenological Judd–Ofelt intensity parameters are used to calculate the radiative decay rates and the branching ratios of the F43/2→I4J (J=9/2, 11/2, 13/2, and 15/2) intermanifold transitions. The room temperature fluorescence lifetime has been measured for the most intense F43/2→I411/2 emission transition. Emission cross sections for the intense intermanifold transitions and peak emission cross sections for the intense inter-Stark transitions are also reported. Assignments to individual Stark levels of the I4J manifolds have been made and compared with the calculated splittings reported earlier. Finally, the spectroscopic properties of the Nd3+:Y2O3 nanocrystals suspended in PMMA are compared with those of Nd3+ doped in various host materials. Detailed optical analysi...