Separation of oxidatively damaged DNA Nucleobases and Nucleosides on Packed and Monolith C18 Columns by HPLC-UV-EC

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and in each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40 min. to just under 4 min., was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV-vis and EC detector, which is especially useful for analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2’-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal

Inverse-opal conducting polymer monoliths in microfluidic channels

Inverse opal monolithic flow-through structures of polyaniline (PANI) were achieved in microfluidic channels for lab-on-a-chip (LOC) applications. In order to achieve the uniformly porous monolith, polystyrene (PS) colloidal crystal (CC) templates were fabricated in channel. An inverse opal PANI structure was achieved on-chip, through a two-step process involving the electrochemical growth of PANI and subsequent removal of the template. The effect of electropolymerisation on these structures is discussed. It was found that growth time is critical in achieving an ordered structure with well-defined flow-through pores. This is significant in order to fabricate optimal porous PANI structures that maximise surface area of the monolith and also provide well-defined flow profiles through the micro-channel

Nickel(II)-catalysed oxidative guanine and DNA damage beyond 8-oxoguanine

Oxidative DNA damage is one of the most important and most studied mechanisms of disease. It has been associated with a range of terminal diseases such as cancer, heart disease, hepatitis, and HIV, as well as with a variety of everyday ailments. There are various mechanisms by which this type of DNA damage can be initiated, through radiation and chemical oxidation, among others; however, these mechanisms have yet to be fully elucidated. A HPLC-UV-EC study of the oxidation of DNA mediated by nickel(II) obtained results that show an erratic, almost oscillatory formation of 8-oxoguanine (8-oxoG) from free guanine and from guanine in DNA. Sporadic 8-oxoG concentrations were also observed when 8-oxoG alone was subjected to these conditions. A HPLC-MS/MS study showed the formation of oxidised-guanidinohydantoin (oxGH) from free guanine at pH 11, and the formation of guanidinohydantoin (GH) from DNA at pH 5.5

Creatinine biosensors: principles and designs

Creatinine biosensors, based on both potentiometric and amperometric devices, have been created. However, there are significant problems still to be addressed, including the balance between sensitivity and selectivity, interference rejection and sensor stability. In addition, many devices still rely on a dual-sensor approach for creatine and creatinine subtractive measurements. However, creatinine biosensors appear close to attaining the performance goals necessary for their widespread application. This article looks at the operating principle and design of both potentiometric and amperometric creatinine biosensors, and shows how the design of these devices affects their performance

Elucidation of the mode of action of a conductive polymer-based electrochemical immunosensor.

An amperometric biosensor has been developed, incorporating the electroactive polymer, polyaniline (PANI), which undergoes redox cycling, and can couple electrons directly from the enzyme active site, to the electrode surface. Construction of this sensor was achieved by electropolymerisation of polyvinylsulphonate-doped aniline onto the surface of a screen-printed carbon-paste electrode. Biomolecules could then be doped onto the surface of the polymer by electrostatic interactions with the polymer backbone. A key component in a biosensor is the recognition molecule and its immobilisation. This study investigates this process of protein immobilisation using amperometric and colorimetric techniques. Immobilisation of protein (enzyme or antibody) onto the transducer is achieved by electrostatic interactions. By applying bovine serum albumin (BSA) electrostatically at the electrode, efficient blocking of the electrode surface from the bulk solution was achieved above approximately 0.75 mg/ml. When horseradish peroxidase was immobilised on the electrode surface at various concentrations, optimal amperometric responses were achieved at approximately the same protein concentration. Determination of the number of molecules of protein immobilised on the surface of the electrode at this concentration was done using a colorimetric enzyme assay. It was found that under optimal immobilisation conditions, a protein monolayer was formed at the electrode surface. In the case of enzymes such as horseradish peroxidase (HRP), this provides simultaneous blocking of the electrode surface from bulk solution interactions as well as yielding optimal electron transfer properties

Immunoelectrochemical methods immunoelectrochemical of hormone anaylsis

Over the last decade, there has been considerable interest in the development of immunoelectrochemical assays, mainly due to the advantages offered by the combination of the selectivity of immunoassays with the great sensitivity and simplicity of modern electroanalytical techniques. However, immunoelectrochemical assays have not yet been exploited commercially, as transitions from the laboratory bench to large-scale manufacturing has proved difficult. The system described is an amperometric peroxide biosensor prepared by electrochemical deposition of horseradish peroxidase on an electroactive polymer, polyaniline. Polyaniline brings about mediatorless redox coupling between the electrode and biomolecular components attached to the surface. This assay will be the stepping-stone to developing an immunoelectrochemical method for the analysis of human chorionic gonadotropin (hCG), and other female hormones. The assay initially utilises glassy carbon electrodes for preliminary data, before being replaced with a commercially available electrode Euroflash TM, produced by Inverness Medical Limited TM. This electrode was utilised with a view to producing an electrochemical assay that complies with manufacturing requirements

Reagentless glucose biosensor based on the direct electrochemistry of glucose oxidase on carbon nanotube-modified electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)-modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about -455 mV (vs. Ag/AgCl) and shows a surface-controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD

Production of polystyrene spheres for use as a templating material for polyaniline monolith structures.

Polystyrene (PS) spheres are potentially useful as a reproducible, sacrificial templating material for monolith columns once they can be utilised to create a uniform microstructured packing which enables a higher monolith batch to batch reproducibility. To achieve PS spheres which can meet these requirements, their synthesis was optimised. Parameters investigated included variation of reactant concentrations, along with optimisation of reaction conditions temperature, agitation speed and nitrogen flow during aeration. Temperature and agitati on played vital roles in the size and homogeneity of the synthesised PS spheres. Temperature affected the equilibrium concentration of monomer in the aqueous phase. When reaction temperature was increased, sphere size reduced and as reaction temperature decreased sphere size increased. A similar trend was seen when agitation speed was varied. At higher agitation speed average PS sphere size decreased as the rate of polymerisation increased. At lower agitation speed the average PS sphere size increased as the rate of polymerisation decreased. Ensuring fluctuations in both temperature and agitation were kept to a minimum was key to maintaining reproducibility. Any fluctuation above ~10% in either temperature or agitation speed affected standard deviation irreversibly. The facile dissolution of the PS spheres was also investigated. If the spheres produced could not be dissolved, their use as a sacrificial templating material would not be possible. By decreasing the original concentration of cross-linker, dissolution increased dramaticall

Polyaniline-silver hybrid materials for ink-jet printing

The synthesis of PANI/Ag hybrid materials has been carried out via a rapid chemical oxidative, in-situ polymerisation method, using silver nitrate and ammonium peroxydisulfate as oxidant precursors. These species react together to produce Ag2+, an oxidising intermediate which has greater oxidising power than either persulphate or Ag+ alone. The reaction thus proceeds at a much accelerated rate than that of pure PANI, approximately 6 times faster. Various characterisation techniques were used to characterise this new hybrid material, PANI-Ag. UV-vis absorption spectroscopy was used to follow the formation of polymer over time and monitor the rate at which the reaction progresses for both PANI and PANI-silver. It is evident from the absorption spectra that the polymer forms via the fully-oxidised pernigraniline state initially, before reducing back into the more stable emeraldine salt form. This process is significantly accelerated using both APS and AgNO3, as opposed to when using APS or AgNO3 alone. These new composites could potentially find use as functional materials for the printed electronics industry where new processable, hybrid materials as required for applications in sensing, memory, logic and photovoltaics

Electroactive monolith μchips based on nanostructured polyaniline

The extensive application of monolithic columns for HPLC is severely hindered by a lack of column-to-column reproducibility. EMμ(Electroactive Monolithic μChip) is a new concept that solves the significant reproducibility problems, as well as allowing miniaturization and improving overall efficiency through electrochemically controlled dynamic separations. This novel μchip has a micro-structured monolith fabricated from intelligent, electroactive polymer. By application of a specific potential, conducting polymers such as polyaniline (PANI) can be reproducibly grown and readily fine-tuned in terms of porosity, hydrophobicity an d ionic capacity. This unique chip provides for an Electroac tive Monolithic μchip capable of multi-dimensional chromatographic separations. The monolith microstructuring (provided by templating) wi ll provide reproducibility and improve efficiency by decre asing the A-term of the Van Deemter equation. Furthermore, the use of these intelligen t materials will enable gradient control and redox reaction s to be exploited during separations of large biomolecules