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Electrospray-mass spectrometry (ES?MS) was employed to confirm the formation of the [Cu.sup.2+] complex with [alpha]-syn or with an N-terminal peptide, [alpha]-synuclein which play an important role in neuropathology in ParkinsonEs disease (PD). The results provide insight into partial role of oxidative stress for the loss of dopaminergic cells in PD brain and the multifaceted role of the [alpha]-syn-[Cu.sup.2+] complex in oxidative stress associated with PD symptoms.

The complexation of Cu(II) by amyloid-[beta] (A[beta]) is examined through the tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. With the help of tyrosine fluorescence spectroscopy has provided mechanistic insights into the oxidation state of copper ions bound to A[beta], the binding heterogeneity and the impact of solution conditions on complex formation.

The redox reactions of copper complexes and the binding stoichiometry between Cu(II) and the full-length [beta]-amyloid A[beta](1-42) was determined. A stoichiometric ratio of 1:1 was observed which remains the same in the presence of more than 10--fold excess of Cu(II) and the A[beta]-Cu(II) complex could be involved in the consumption of antioxidants causing their depletion in neurons and damaging the neuronal defense system.

Electrochemical surface plasmon resonance (EC-SPR) and electrochemical quartz crystal microbalance (EQCM) are employed to study the redox-induced orientation change of 11-ferrocenylundecanethiol self-assembled monolayers (FcC11SH) (SAM) in two electrolyte solutions. It is seen that the tandem use of EC-SPR and EQCM is an attractive approach for delineating the various redox-triggered surface processes of FcF11SH SAMs.

Two six-port injector valves and one selector valve commonly used in flow injection analysis are combined with a surface plasmon resonance (SPR) instrument wherein solutions introduced from the two inlets counter-flow inside the flow cell. The system is versatile as the same or different solutions can be rapidly and repeatedly introduced to the two fluidic channels in series or in parallel. Unlike most commercial SPR instruments employing a single injector valve, solutions separately injected from the two injector valves can be readily exchanged (<1 s) between the two channels. This new method, referred to as the alternate injection mode, not only saves analysis time but also facilitates efficient and facile surface reactions for ligand immobilization and prevents immobilized species from desorbing. These advantages are demonstrated with the measurements of binding of acetazolamide (222.2 Da) to histidine-tagged human carbonic anhydrase II (his-tagged HCA). Amine-containing residues of his-tagged HCA molecules tethered at Ni-nitrilotriacetic acid (NTA) sensors were rapidly cross-linked to the underlying carboxymethylated dextran. The higher ligand densities and more stable surfaces are essential for SPR detection of small molecule binding. In a different application, microglobulin solutions of increasing concentrations were introduced for continuous binding to the preimmobilized antibody. The kinetic and affinity measurements can be conducted without performing repeated dissociation and surface regeneration reactions. [ABSTRACT FROM AUTHOR]

The combination of high-performance liquid chromatography (HPLC) with surface plasmon resonance (SPR) for continuous separation and label-free detection of protein samples is described. The detection was realized by electrostatic adsorption of proteins bearing positive and negative charges onto chemically modified SPR sensors in two separate SPR channels. One SPR channel is coated with carboxymethylated dextran which facilitates the detection of positively charged proteins, whereas the other, devoted to the monitoring of negatively charged proteins, is covered with ethylenediamine molecules attached onto a dextran surface. Renewal of the sensor surface in the channels can be accomplished by introducing regeneration solutions through two six-port valves. The coupled technique (HPLC-SPR) was assessed for its analytical figures of merit and applied to the quantification of lysozyme in human milk samples. Unlike the SPR detection of bulk solution refractive index changes during chromatographic peak elutions, the highest sensitivity of SPR is retained in this work since the measurement is performed at the SPR sensor surface where the evanescent field is the strongest. The renewable SPR detection of continuous separations is reproducible and versatile and does not require the separated proteins to contain chromophores or to be prelabeled with a tag (e.g., a redoxactive or fluorescent molecule). Such generality makes SPR complementary to other types of chromatographic detectors. [ABSTRACT FROM AUTHOR]

Scanning electrochemical microscopy (SECM) has been employed in the imaging of DNA microarrays fabricated on gold substrates using methylene blue (MB) as a redox-active intercalator and ferrocyanide as the SECM mediator in solution. MB intercalated between base pairs of immobilized ds-DNA is electrochemically reduced via electron transfer from the underlying gold substrate, and the product is reoxidized in solution by SECM tip-generated ferricyanide. The resulting feedback current allows a heterogeneous electron-transfer rate constant for the MB-intercalated DNA to be deduced. Moreover, DNA microarray spots can be imaged at a detection level of 14 fmol/spot for ds-DNA consisting of 15 base pairs. Microarrays prepared using 20 M DNA solutions are easily visualized, and the feasibility of detecting base pair mismatches is also demonstrated. [ABSTRACT FROM AUTHOR]

Sequential surface plasmon resonance imaging (SPRI) and imaging ellipsometry (IE) measurements are realized with a single set of optical components mounted on a goniometer. The sample situated on top of a triangular prism is positioned in the center of the goniometer. The resultant setup (SPRI/IE) can be used to examine the same surface region above (IE) and below (SPRI) the sample. Thickness values of silver stripes sputtered onto a gold substrate were determined by SPRI and IE, and the results were compared to establish the validity of the method. The SPRI/IE setup was also used to monitor the thickness of phospholipid films of different layer numbers. SPRI measurements were found to be more accurate for ultrathin films, whereas the IE results are more reliable for films whose thicknesses approach or exceed the distance encompassed by the evanescent wave of the surface plasmon. Thus, utilizing these two techniques sequentially facilitates the continuous monitoring of film thickness variation over a wide thickness range with high fidelity and provides a viable approach to image the surface regions with large topographic fluctuations. [ABSTRACT FROM AUTHOR]

The combination of scanning electrochemical microscopy (SECM) with surface plasmon resonance (SPR) is described. By oxidizing ferrocenylalkanethiol self-assembled monolayer (SAM) with SECM-generated Ce4+, the coupled technique, SECM-SPR, is shown to be viable for determining local variations in thin film thickness. Factors (tip/substrate distance, tip potential scan rate, and solution composition change) affecting the SECM-SPR response and operation are also discussed. The approach was further extended to the determination of conformational changes of cytochrome e molecules attached electrostatically onto a negatively charged SAM during its reduction by the tip-generated methyl viologen monocation. The high sensitivity of the SPR equipped with a bicell detector facilitates the measurement of infinitesimal film thickness changes accompanying redox reactions, while the SECM provides a means to obviate the necessity of applying a potential to the SPR substrate, which tends to cause unwanted interferences and complications. The approach also affords an avenue for determining film thickness variations that are not subject to certain effects, such as the surface charge, the heterogeneity of the substrate, and the distance between the redox center of the immobilized molecule and the underlying substrate electrode. [ABSTRACT FROM AUTHOR]

A direct-injection high-efficiency nebulizer (DIHEN) is used to couple a thin-layer electrochemical flow cell online with an ICP-mass spectrometer to perform anodic stripping voltammetry (ASV) at a thin mercury film followed by subsequent ICPMS measurements for the stripped metal analytes. The resultant hyphenated technique (ASV-DIHEN-ICPMS) is capable of analyzing select heavy metals present at ultratrace levels (down to lowppt to sub-ppt levels) that are lower than the detection limits obtained by conventional ICPMS. In addition to its good analytical performance, the technique offers other attractive features such as the ability to eliminate detrimental matrix effects that can compromise ICPMS analyses and the possibility of probing electrode reactions involving trace amounts metal species with ICPMS. For conducting ASV on-line with ICPMS, the DIHEN was found to be more advantageous than the microconcentric nebulizer in terms of minimizing memory effects and potential artifacts caused by the erosion of the Hg film into the flowing solution stream. Compared to a direct injection nebulizer (DIN), the DIHEN was easier to operate. Moreover, its simpler design and the lack of back pressure from the DIHEN capillary made it more compatible with coupling to the thin-layer electrochemical cell than a DIN system. [ABSTRACT FROM AUTHOR]

p53, a tumor suppressor protein and a transcription factor, is capable of inhibiting the growth of tumor cells by eliciting either cell-cycle arrest or apoptosis through a cascade of events. p53 binds sites within the promoters of several genes that conform to a sequence commonly defined as the consensus site. In more than 50% of cancer cases, the p53 gene has been found to be mutated and the p53 protein loses its ability to bind the consensus DNA. In this work, double-stranded (ds-) oligonucleotides (ODNs) containing the consensus site are immobilized onto gold electrodes to capture wild-type p53. The cysteine residues on the exterior of the p53 molecule were derivatized for the attachment of gold nanoparticle/ streptavidin conjugates capped with multiple ferrocene (Pc) groups. Well-defined voltammetric peaks of high signal intensity were obtained, and p53 concentration as low as 2.2 pM was measured. The peak heights were found to be dependent on the surface density of the consensus ds-ODN, the sequence of the immobilized ODNs, and the p53 concentration. With base pair(s) in the full consensus binding sequence altered, the level of p53 binding was found to decrease sharply, and no p53 binding occurred at electrodes covered with nonconsensus ds-ODNs The amenability of this method to the analyses of p53 from normal and cancer cell lysates was also demonstrated. Owing to the p53 mutation in the cancer cells, the concentration of the wild-type p53 was found to decrease significantly (by about 50-182 times). The sensitivity and amenability for real sample analysis of the method compared well with enzyme-linked immunosorbant assay (EUSA), and complements EUSA in that wild-type p53, instead of total p53 (wild-type and mutant p53) concentration, is measured. The method described herein is simple and selective and does not require the use of p53 antibodies. [ABSTRACT FROM AUTHOR]

Focuses on amplified voltammetric detection of DNA hybridization via oxidation of ferrocene caps on gold nanoparticle/streptavidin conjugates. Account of materials used in this detection process; Overview of the preparation of 6-ferrocenylhexanethiol-capped gold nanoparticle/streptavidin conjugates; Overview of the sequence of the hepatitis B virus pre-S gene; Highlights of probe immobilization and hybridization procedures; Schematic representation of the amplified electrochemical detection of DNA hybridization via oxidation of the ferrocene caps on the gold nanoparticle/streptavidin conjugates.