Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method

Cytochrome c was immobilized on a mixed-thiol (mercaptoundecanoic acid/mercaptoundecanol) modified gold electrode (MUA:MU/cyt c electrode). Characterization of the cyt c electrode showed a quasi-reversible, electrochemical redox behavior with a formal potential of −13±5 mV (versus Ag/AgCl) for the surface adsorbed protein and 3±5 mV for covalently immobilized cyt c. The heterogeneous electron transfer rate constants were determined to be about 70 and 40 s⁻¹ for both states of the protein, respectively. They were found to be significantly higher than those of pure MUA-modified cyt c electrodes (MUA/cyt c electrodes). The interaction of superoxide radicals (O₂⁻) with the (MUA:MU)/cyt c electrode was characterized and used for an amperometric O₂⁻ detection. The influence of H₂O₂ and uric acid on the sensor signal was investigated. The sensitivity of the (MUA:MU)/cyt c electrode to O₂⁻ was significantly improved compared with that of the MUA/cyt c electrode. Based on a kinetic model for the superoxide detection system, a new calibration method was established. This simple and fast method used the spontaneous dismutation of KO₂ and was compared with the enzymatic superoxide generation system using xanthine oxidase.     

Quantum dots on electrodes—new tools for bioelectroanalysis

The review covers recent developments in which quantum dots (QDs) are combined with electrodes for detection of analytes. Special focus will be on the generation of photocurrents and the possibility of spatially resolved, light-directed analysis. Different modes for combining biochemical reactions with QDs will be discussed. Other applications involve the use of QDs as labels in binding analysis. Different methods have been developed for read-out. In addition to photocurrent analysis, voltammetric detection of metals and electrochemiluminescence (ECL) can be used. In the latter, light is the sensor signal. ECL-based systems combine the advantage of very sensitive analytical detection with rather simple instrumentation.

First Observation of Hyperfine Structure in K(2)

The hyperfine structure of the (v'-v") = (27-0) band of the b(3)Pi(u0(+)) <-- X(1)Sigma(+)(g) transition has been observed by laser excitation spectroscopy in a highly collimated molecular beam. Hyperfine parameters for magnetic dipole and electric quadrupole interaction are derived from least-squares fitting of the hyperfine pattern and deperturbation between A(1)Sigma(+)(u) and b(3)Pi(u0(+)). Copyright 2000 Academic Press.     

Line shape analysis of two-colour photoassociation spectra on the example of the Cs ground state

Two-photon photoassociation spectra in a Λ-type excitation scheme are analysed under the systematically varied experimental conditions of frequency detunings and laser intensities. Line shape fits are presented as well as the investigation of intensity and detuning dependent line shifts. From both we determine the attained spectroscopic precision, that is corrected for a systematic line shift due to the thermal distribution of atoms in the trap. An energy correction for this effect is given. Information about the feasibility of generating translationally cold molecules in a well defined rotational and vibrational level by the photoassociation process is derived from the analysis. Received 21 May 2002 Published online 15 October 2002 RID="a" ID="a"Current address e-mail: christian.lisdat@ptb.de RID="b" ID="b"e-mail: pierre.pillet@lac.u-psud.fr     

Fast protein detection using absorption properties of gold nanoparticles

In this study we describe the use of gold nanoparticles as a fast detection system for the sensitive analysis of proteins. The immunological method allows for protein analysis at the nanogram level, as required for clinical diagnosis. Initially a test protein is used for the development of the assay. The system is subsequently adopted for alpha-fetoprotein, which is a relevant tumor marker. This work demonstrates that antibody functionalized gold nanoparticles can be used for the detection of proteins by forming gold nanoparticle aggregates. The influence of the size of the gold nanoparticles on the sensitivity of the assay is investigated in the range from 20-60 nm particles; the larger particles show here the highest relative changes. The formation of antigen-gold nanoparticle aggregates is detected by an increase in hydrodynamic diameter by dynamic light scattering (DLS). UV/Vis spectroscopy also allows assay monitoring by quantifying the red shift of the plasmon resonance wavelength. Alpha-fetoprotein can be analysed in the concentration range of 0.1-0.4 μg ml(-1). The influence of pH, ionic strength and ratio of sample to Au-NP solution is studied. With this method, the protein AFP can be rapidly detected as demanded for clinical diagnosis.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

Bienzyme Sensor Based on an Oxygen Reducing Bilirubin Oxidase Electrode

Based on an oxygen reducing electrode combining bilirubin oxidase and multiwalled carbon nanotubes modified gold (BOD‐MWCNT‐Au electrode) a bienzyme sensor is developed. Therefore the BOD‐MWCNT‐Au electrode is covalently coupled to enzymes catalysing oxygen‐consuming reactions (glucose oxidase and ascorbate oxidase) to result in a membrane‐free bienzyme electrode. The electrochemical characterisation of these bienzyme sensors reveals an enzyme substrate sensitivity down to 250 μM glucose and 100 μM ascorbate. In addition, the assembled sensor systems allow amperometric measurements in a potential range where the influence of interfering substances reacting directly at the transducing electrode is minimised. The results indicate that the BOD electrode provides a suitable platform for sensing analytes of medical and environmental interest for which oxidases of high activity are available.     

Characterization of antioxidants using a fluidic chip in aqueous/organic media

Application of antioxidants in the cosmetic industry demands control of the efficiency of ROS-scavenging within the cream matrix. Our goal was to construct a system for the simultaneous detection of superoxide and hydrogen peroxide and their possible scavengers. DMSO is a good solvent for many cosmetic products, and thus the system should work in mixed aqueous-organic media. The fluidic chip developed consists of an ROS-generation chamber, a mixing section and a compartment for the biosensor chip. This electrode chip had two sensors: one sensor for each species. Cytochrome c was used as the sensing protein. Both the superoxide and the hydrogen peroxide sensors demonstrated sufficient sensitivity in DMSO-buffer mixtures within the concentration range 0.4 nM-1.2 nM (superoxide) and 50 microM-1000 microM (hydrogen peroxide). The influence of the flow conditions on the generation of ROS was investigated and the optimal parameters for the antioxidant detection were evaluated. The efficiency of ROS-scavenging was tested with typical antioxidants of enzymatic and non-enzymatic origin, as well as complex cosmetic creams.     

An electrochemical method for quantification of the radical scavenging activity of SOD

An electrochemical technique for the determination of enzymatic superoxide scavenging activity is described. In amperometric mode of sensor operation ¶the oxidation current was altered by additions of superoxide dismutase (SOD). The 50% inhibition value (I50) corresponded to 0.165 U of the standard photometric test of McCord and Fridovich. Influence by inert proteins was much less than in the standard method: The SOD/BSA ratio was 4 × 10⁵. Potentially interfering substances, e.g. ascorbic acid and glutathione, were tested. Plasma samples of rats which were treated with modified SOD during cerebral ischaemia, were assayed with the new method and the standard photometric test. A good correlation was found for the analysis in these physiological samples (r = 0.97).