No abstract

The electrochemistry of horse heart cytochrome c was studied by cyclic voltammetry at a glassy carbon electrode modified with single-wall carbon nanotubes (SWNTs). A pair of well-defined redox waves was obtained in cytochrome c aqueous solution at an activated SWNT film-modified electrode. The optimal conditions for activating the SWNT film-modified electrode has been determined. The electrode reaction of cytochrome c is a diffusion-controlled process. The peak current increases linearly with the concentration of cytochrome c in the range from 3.0 x 10(-5)-7.0 x 10(-4) M. The detection limit is 1.0 x 10(-5) M. The activated SWNT film was characterized by scanning electron microscopy. Furthermore, interaction of cytochrome c with adenine was characterized by electrochemical and spectral methods.

The binding stoichiometry between Cu(II) and the full-length beta-amyloid Abeta(1-42) and the oxidation state of copper in the resultant complex were determined by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and cyclic voltammetry. The same approach was extended to the copper complexes of Abeta(1-16) and Abeta(1-28). A stoichiometric ratio of 1:1 was directly observed, and the oxidation state of copper was deduced to be 2+ for all of the complexes, and residues tyrosine-10 and methionine-35 are not oxidized in the Abeta(1-42)-Cu(II) complex. The stoichiometric ratio remains the same in the presence of more than a 10-fold excess of Cu(II). Redox potentials of the sole tyrosine residue and the Cu(II) center were determined to be ca. 0.75 and 0.08 V vs Ag/AgCl [or 0.95 and 0.28 V vs normal hydrogen electrode (NHE)], respectively. More importantly, for the first time, the Abeta-Cu(I) complex has been generated electrochemically and was found to catalyze the reduction of oxygen to produce hydrogen peroxide. The voltammetric behaviors of the three Abeta segments suggest that diffusion of oxygen to the metal center can be affected by the length and hydrophobicity of the Abeta peptide. The determination and assignment of the redox potentials clarify some misconceptions in the redox reactions involving Abeta and provide new insight into the possible roles of redox metal ions in the Alzheimer's disease (AD) pathogenesis. In cellular environments, the reduction potential of the Abeta-Cu(II) complex is sufficiently high to react with antioxidants (e.g., ascorbic acid) and cellular redox buffers (e.g., glutathione), and the Abeta-Cu(I) complex produced could subsequently reduce oxygen to form hydrogen peroxide via a catalytic cycle. Using voltammetry, the Abeta-Cu(II) complex formed in solution was found to be readily reduced by ascorbic acid. Hydrogen peroxide produced, in addition to its role in damaging DNA, protein, and lipid molecules, can also be involved in the further consumption of antioxidants, causing their depletion in neurons and eventually damaging the neuronal defense system. Another possibility is that Abeta-Cu(II) could react with species involved in the cascade of electron transfer events of mitochondria and might potentially sidetrack the electron transfer processes in the respiratory chain, leading to mitochondrial dysfunction.

Redox-induced orientation changes (or monolayer thickness changes) of self-assembled monolayers (SAMs) of 11-ferrocenylundecanethiol (FcC 11 SH) were quantified by the electrochemical surface plasmon resonance (EC-SPR). EC-SPR enables one to determine the collective effect of the monolayer thickness and refractive index changes resulted from the oxidation of ferrocene (Fc) to ferrocenium. To measure the monolayer volume variation associated with the molecular orientation change, an electrochemical quartz crystal microbalance (EQCM) was used to determine the total number of water molecules accompanying with the ion-pairing between the ferrocenium cation and the counteranion in the solution. With the maximum void space within the SAM for water incorporation known, the Lorentz-Lorenz equation was used to correlate the SPR dip shift to the maximum monolayer thickness variation. In the presence of 0.1 M HClO 4 and 0.1 M HNO 3 , the monolayer thickness changes were deduced to be 0.09 and 0.08 nm, respectively. Thus, upon electrochemical oxidation of the FcC 11 SH SAM, the swinging of the alkyl chain farther away from the electrode (Ye et al., Langmuir, 1997, 13, 3157) or the rotation or flipping of the Fc cyclopentyldiene ring around the bond between the Fc group and the alkyl chain (Viana et al., J. Electroanal. Chem. 2001, 500, 290) can both lead to the observed film thickness changes, with the former probably being the more important process.

The voltammetric behavior of norepinephrine (NE) was studied at a glassy carbon (GC) electrode modified with single wall carbon nanotubes (SWNTs). In pH 5.72 B‐R buffer solution, the SWNT‐modified electrode shows high electrocatalytic activity toward NE oxidation. One well‐defined reversible redox couple is obtained at scan rates lower than 0.15 V s−1. The peak current increases linearly with the concentration of NE in the range of 1.0×10−5 ‐ 1.1×10−3 mol dm−3. The detection limit is 6.0×10−6 mol dm−3 and the diffusion coefficient (D) of NE is 8.53×10−6 cm2 s−1. The SWNT was characterized with scanning electron microscope (SEM). Furthermore, the SWNT‐modified electrode has favorable electrocatalytic activity with dopamine, epinephrine, and ascorbic acid.

No abstract

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 (Fc) 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 (ELISA), and complements ELISA 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.

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17–25 nucleotides) and extremely low concentrations (typically < pM) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (RSD < 5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease) and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10-HL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.