Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dN^FcXTP s were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A ( dA^Fc ) and FcPa-labelled C ( dC^FcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dA^Fc and dC^FcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.     

The use of cationic surfactants to control the structure of zinc oxide films prepared by chemical vapour deposition

This paper describes a powerful and versatile new method for controlling the structure of zinc oxide thin films prepared by aerosol assisted chemical vapour deposition, based on the use of a common surfactant. The technique combines the benefits of solution and vapour based methods and leads to high quality morphologically-defined and orientated thin films.     

Rapid determination of total hardness in water using fluorescent molecular aptamer beacon

A double-labelled synthetic oligonucleotide is used as a fluorescent molecular aptamer beacon for the reagentless determination of total hardness in tap and bottled waters. Modified thrombin binding aptamer (5′-NH-C3-GGTTGGTGTGGTTGG-C3-SH-3′) carrying 6-carboxyfluorescein (FAM) and 7-amino-4-methyl-coumarin labels at 5′ and 3′, respectively, was used for the simultaneous combined measurement of Mg²⁺ and Ca²⁺ cations. Interference from the K⁺ cation is eliminated via selective tuning of the assay conditions, increasing the temperature beyond the melting point of the potassium-stabilised quadruplex facilitating its liberation from the quadruplex, whilst maintaining the integrity of the magnesium/calcium-stabilised structure. No interference from other cations found in tap or bottled water was observed. The detection limit of the aptamer beacon is 0.04 mmol L⁻¹, with a dynamic linear range of 0-0.5 μM and is very reproducible, with an R.S.D. = 8%, n = 3. The fluorescent molecular beacon is applied to the determination of total hardness in tap and bottled waters and its’ performance compared to that of the standard method of complexiometric titration and atomic absorption spectroscopy, with an excellent correlation observed. Further work is focused on the immobilization of the aptamer for the development of a re-usable fluorescent/electrochemical aptasensor, for the determination of water hardness.     

The role of auditory feedback in sustaining vocal vibrato

Vocal vibrato and tremor are characterized by oscillations in voice fundamental frequency (F0). These oscillations may be sustained by a control loop within the auditory system. One component of the control loop is the pitch-shift reflex (PSR). The PSR is a closed loop negative feedback reflex that is triggered in response to discrepancies between intended and perceived pitch with a latency of approximately 100 ms. Consecutive compensatory reflexive responses lead to oscillations in pitch every approximately 200 ms, resulting in approximately 5-Hz modulation of F0. Pitch-shift reflexes were elicited experimentally in six subjects while they sustained /u/ vowels at a comfortable pitch and loudness. Auditory feedback was sinusoidally modulated at discrete integer frequencies (1 to 10 Hz) with +/- 25 cents amplitude. Modulated auditory feedback induced oscillations in voice F0 output of all subjects at rates consistent with vocal vibrato and tremor. Transfer functions revealed peak gains at 4 to 7 Hz in all subjects, with an average peak gain at 5 Hz. These gains occurred in the modulation frequency region where the voice output and auditory feedback signals were in phase. A control loop in the auditory system may sustain vocal vibrato and tremorlike oscillations in voice F0.     

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.     

Controlled Zn‐Mediated Grafting of Thin Layers of Bipodal Diazonium Salt on Gold and Carbon Substrates

A controlled, rapid, and potentiostat‐free method has been developed for grafting the diazonium salt (3,5‐bis(4‐diazophenoxy)benzoic acid tetrafluoroborate (DCOOH)) on gold and carbon substrates, based on a Zn‐mediated chemical dediazonation. The highly stable thin layer organic platforms obtained were characterized by cyclic voltammetry, AFM, impedance, XP, and Raman spectroscopies. A dediazonation mechanism based on radical formation is proposed. Finally, DCOOH was proved as a linker to an aminated electroactive probe.     

Multilayered catalytic biosensor self-assembled on cyclodextrin-modified surfaces

In this paper we describe the deposition of enzyme layers on cyclodextrin-modified surfaces through self-assembly with adamantane-appended alkaline phosphatase using cyclodextrin-capped gold nanoparticles as supramolecular linkers. The system was studied by surface plasmon resonance and electrochemical methods (cyclic voltammetry, impedance spectroscopy). Up to three enzyme layers were formed on the cyclodextrin coated electrodes and the modified surface was used for the electrochemical detection of heavy metals (Cd²⁺, Ag⁺) based on the inhibition of enzymatic activity by these metal cations.     

Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection

Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. Among the 1,000 known cystic fibrosis-related mutations, DF508 is the most common, with a frequency varying between 50% and 70% according to geographical areas and population typology. In this work, we report the use of methylene blue as an electrochemical reporting agent in the discrimination of synthetic PCR analogue of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt). At optimum experimental condition, a discrimination factor between mutant and wild type of approximately 1.5-fold was found. The proposed assay was quantitative and linear in the range of 10–100 nM, exhibiting a limit of detection of 2.64 nM. Electrochemical studies at variable ionic strength conditions allowed further elucidation of the mechanism of the methylene blue (MB)–DNA interaction. To the best of our knowledge, this is the first report of detection of hybridisation solely via guanine-specific MB–DNA interaction simultaneously in MB solution, independent of electrostatic interaction as demonstrated in the ionic strength study. The introduction of formamide in the hybridization buffer, to improve discrimination, was also investigated. Finally, mutant wild type discrimination was demonstrated, at 10 nM concentration, with the use of a multi-sensor setup.