Maogen Zhang

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.

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...

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...

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.

Optical characterization of gold and Er3+:Y2O3 nanoparticles for biosensor applications

Highly luminescent nanoparticles, such as, trivalent erbium-doped yttrium-oxide, Er3+:Y2O3, are expected to have a wide range of applications, including imaging, range finding, flash lidar, and other remote-sensing possibilities as well as medical applications. These particles are synthesized by the precipitation from a homogeneous solution of the metal ions and urea at elevated temperatures. The morphology of the calcinated materials, revealed through SEM, shows uniformly spherical aggregates 200 nm or less depending on the ratio of the metal ions in the initial solution. Room temperature optical absorption and emission spectra show that the trivalent erbium ions in Er3+:Y2O3 nanocrystals possess sharp absorption lines and strong emission in near infrared region that are characteristic of Er 3+:Y2O3 grown as large single crystals. Low temperature (8 K) spectra obtained from these particles were analyzed in detail for the crystal-field splitting of the 2S+1LJ multiplet manifolds of Er3+(4f11) including the ground-state manifold 4I15/2, and excited manifolds 4I9/2, 4F9/2, 4S3/2, 2H11/2, 4F7/2, 4F5/2, and 4F3/2. Fluorescence lifetimes and results from an analysis of the intensities of manifold-to-manifold transitions are also reported. Similarity of the nanocrystalline and large single crystal Er3+:Y2O3, we propose that the simple, inexpensive method described in this study will lead to further investigation of these nanocrystals for their optical properties, especially in the near infrared region of the spectrum.