Fabrication of a 3-dimensional nanostructured binary colloidal crystal within a confined channel.

The reproducible fabrication of nanostructured 3Dimensional (3D) binary colloidal crystal (bCC) in a defined geometric space through a simple one step process is detailed. This method allows for the potential fabrication of a bCC in a sealed µchip within a defined area or channel by capillary forces, unlike other bCC formation methods such as dip-drawing, where the substrate must be submerged in a suspension to form a bCC, or bCC monolayers, which are fabricated at the water air interface. Through simple variation in volume fraction ratio (VF(S/L)) of nano-(denoted small, S) and macro-sized (denoted large, L) polystyrene (PS) spheres and diameter size ratio (D(S/L)), the manipulation of bCC structures was also achieved. Variation of nano-sized PS sphere number within the interstitial voids formed between neighbouring macro-sized spheres enabled the reproducible fabrication of LS2 and LS6 structures, which contain 1 and 3 nano-spheres respectively in each interstitial void. It must be noted that while VF(S/L) allows for control of the final LSn structure, thickness of bCC formation in this manner is independent of VFS/L.

Evaluation of a silver-based electrocatalyst for the determination of hydrogen peroxide formed via enzymatic oxidation.

Silver paste electrodes modified with lyotropic phases formed from dodecyl benzenesulphonic acid and KCl were used as the reductant in the determination of the hydrogen peroxide released from the enzymatic reaction of glucose oxidase with glucose and oxygen. The response of the modified electrode to hydrogen peroxide reduction (-0.1 V vs. Ag/AgCl) was shown to suffer from interference resulting from co-localization of enzyme and substrate at the electrode surface. This interference was eradicated by the introduction of a perm-selective membrane in the form of cellulose acetate. This further facilitated immobilization of the enzyme while allowing diffusion of the generated peroxide to the electrode. The resulting configuration was shown to be capable of the analytical determination of glucose.

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor.

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.

Amperometric microsensor for direct probing of ascorbic acid in human gastric juice.

This article reports on a novel microsensor for amperometric measurement of ascorbic acid (AA) under acidic conditions (pH 2) based on a carbon fiber microelectrode (CFME) modified with nickel oxide and ruthenium hexacyanoferrate (NiO-RuHCF). This sensing layer was deposited electrochemically in a two-step procedure involving an initial galvanostatic NiO deposition followed by a potentiodynamic RuHCF deposition from solutions containing the precursor salts. Several important parameters were examined to characterize and optimize the NiO-RuHCF sensing layer with respect to its current response to AA by using cyclic voltammetry, and scanning electron microscopy-energy dispersive X-ray spectroscopy methods. With the NiO-RuHCF coated CFME, the AA oxidation potential under acidic conditions was shifted to a less positive value for about 0.2 V (E(p) of ca. 0.23 V vs. Ag/AgCl) as compared to a bare CFME, which greatly improves the electrochemical selectivity. Using the hydrodynamic amperometry mode, the current vs. AA concentration in 0.01 M HCl, at a selected operating potential of 0.30 V, was found to be linear over a wide range of 10-1610 µM (n=22, r=0.999) with a calculated limit of detection of 1.0 µM. The measurement repeatability was satisfactory with a relative standard deviation (r.s.d.) ranging from 4% to 5% (n=6), depending on the AA concentration, and with a sensor-to-sensor reproducibility (r.s.d.) of 6.9% at 100 µM AA. The long-term reproducibility, using the same microsensor for 112 consecutive measurements of 20 µM AA over 11 h of periodic probing sets over 4 days, was 16.1% r.s.d., thus showing very good stability at low AA levels and suitability for use over a prolonged period of time. Moreover, using the proposed microsensor, additionally coated with a protective cellulose acetate membrane, the calibration plot obtained in the extremely complex matrix of real undiluted gastric juice was linear from 10 to 520 µM (n=14, r=0.998). These results demonstrated the unique featuring of the proposed NiO-RuHCF microsensor under acidic conditions with enhanced sensitivity and stability and proved its promising potentiality for direct amperometric probing of AA at physiological levels in real gastric juice environments.

Advanced printing and deposition methodologies for the fabrication of biosensors and biodevices.

Advanced printing and deposition methodologies are revolutionising the way biological molecules are deposited and leading to changes in the mass production of biosensors and biodevices. This revolution is being delivered principally through adaptations of printing technologies to device fabrication, increasing throughputs, decreasing feature sizes and driving production costs downwards. This review looks at several of the most relevant deposition and patterning methodologies that are emerging, either for their high production yield, their ability to reach micro- and nano-dimensions, or both. We look at inkjet, screen, microcontact, gravure and flexographic printing as well as lithographies such as scanning probe, photo- and e-beam lithographies and laser printing. We also take a look at the emerging technique of plasma modification and assess the usefulness of these for the deposition of biomolecules and other materials associated with biodevice fabrication.

CE-LIF method for the separation of anthracyclines: application to protein binding analysis in plasma using ultrafiltration.

Anthracyclines are chemotherapeutic drugs that are widely used in the treatment of cancers such as lung and ovarian cancers. The simultaneous determination of the anthracyclines, daunorubicin, doxorubicin and epirubicin, was achieved using CE coupled to LIF, with an excitation and emission wavelength of 488 and 560 nm, respectively. Using a borate buffer (105 mM, pH 9.0) and 30% MeOH, a stable and reproducible separation of the three anthracyclines was obtained. The method developed was shown to be capable of monitoring the therapeutic concentrations (50-50 000 ng/mL) of anthracyclines. LODs of 10 ng/mL, calculated at an S/N = 3, were achieved. Using the CE method developed, the in vitro protein binding to plasma was measured by ultrafiltration, and from this investigation the estimated protein binding was determined to be in the range of 77-94%.

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 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 40min to just under 4min, 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 the 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.

The application of conducting polymer nanoparticle electrodes to the sensing of ascorbic acid.

An ascorbic acid sensor was fabricated via the drop-casting of dodecylbenzene sulphonic acid (DBSA)-doped polyaniline nanoparticles onto a screen-printed carbon-paste electrode. The modified electrode was characterised with respect to the numbers of drop cast layers, optimum potential and operating pH. The sensor was found to be optimal at neutral pH and at 0V vs. Ag/AgCl. Under these conditions, the sensor showed good selectivity and sensitivity in that it did not respond to a range of common interferents such as dopamine, acetaminophen, uric acid and citric acid, but was capable of the detection of ascorbic acid at a sensitivity of 0.76 microA mM(-1) or 10.75 microA mM(-1) cm(-2) across a range from 0.5 to 8mM (r2=0.996, n=6), and a limit of detection of 8.3 microM (S/N=3). The sensor was compared to a range of other conducting polymer-based ascorbate sensors and found to be comparable or superior in terms of analytical performance.

An aqueous ammonia sensor based on an inkjet-printed polyaniline nanoparticle-modified electrode.

A sensor for the amperometric detection of aqueous ammonia was fabricated using the inkjet printing of dodecylbenzene sulfonate (DBSA)-doped polyaniline nanoparticles (nanoPANI) onto a screen-printed carbon paste electrode. The combination of the environmentally inert, aqueous nanoparticle dispersion with the inkjet printing technique allowed the rapid fabrication of sensors based on polyaniline that was not easily achievable in the past due to the lack of processability of bulk forms of the conducting polymer. The resulting modified electrode was characterised with respect to its operating pH and number of print layers and was found to perform optimally at near neutral pH with four nanoPANI inkjet-printed layers. The sensor was tested in a flow injection system for its response to aqueous ammonia using amperometric detection at -0.3 V vs. Ag/AgCl pseudo-reference and was found to have reproducibility to injections of ammonia of below 5% RSD and good sensitivity with an experimental detection limit of 20 microM and a theoretical detection limit of 3.17 microM (0.54 ppm). The sensor was also tested for its day-to-day stability and its response towards a range of interferents common to refrigerant waste waters. This system allows the rapid production of an ultra-low-cost, solid state, polyaniline-based aqueous ammonia sensor.

Inkjet printable polyaniline nanoformulations.

Aqueous polyaniline (PANI) nanodispersions doped with dodecylbenzenesulfonic acid (DBSA) were synthesized and successfully inkjet-printed using a piezoelectric desktop printer. This paper examines the optimization and characterization of the nanoparticulate formulation for optimal film electrochemistry and stability. PANI nanoparticle synthesis was optimized in terms of the ratio of monomer (aniline) to oxidant (ammonium persulphate, APS) and dopant (DBSA). Particle size, UV-vis spectroscopy, electrochemical, and conductivity analyses were performed on all materials. Optimal synthesis conditions were found to be at a molar ratio of 1.0:0.5:1.2 aniline/APS/DBSA. This resulting nanodispersion showed a uniform particle size distribution of approximately 82 nm, and UV-vis analysis indicated a high doping level. These synthetic conditions resulted in the highest conductivity, and the electrochemistry of the resulting films was well-defined and stable. Surface tension analysis and rheological studies demonstrated that the aqueous nanodispersions were suitable for inkjet printing. Successful inkjet printing of these polyaniline nanoparticulate formulations is demonstrated.

Electrochemical preparation of distinct polyaniline nanostructures by surface charge control of polystyrene nanoparticle templates.

A family of nanostructured polyaniline (PANI) materials including polystyrene (PS)/PANI core/shell particles, PANI hollow spheres, PANI/PS nanocomposite and nanoporous PANI, were conveniently prepared by surface charge control of PS nanoparticle templates which resulted in different polymer growth mechanisms when PANI was electropolymerized around the templates.

Double-codified gold nanolabels for enhanced immunoanalysis.

A novel double-codified nanolabel (DC-AuNP) based on gold nanoparticle (AuNP) modified with anti-human IgG peroxidase (HRP)-conjugated antibody is reported. It represents a simple assay that allows enhanced spectrophotometric and electrochemical detection of antigen human IgG as a model protein. The method takes advantage of two properties of the DC-AuNP label: first, the HRP label activity toward the OPD chromogen that can be related to the analyte concentration and measured spectrophotometrically; second, the intrinsic electrochemical properties of the gold nanoparticle labels that being proportional to the protein concentration can be directly quantified by stripping voltammetry. Beside these two main direct determinations of human IgG, a secondary indirect detection was also applicable to this system, exploiting the high molar absorptivity of gold colloids, by which, the color intensity of their solution was proportional to the concentration of the antigen used in the assay. Paramagnetic beads were used as supporting material to immobilize the sandwich-type immunocomplexes resulting in incubation and washing times shorter than those typically needed in classical ELISA tests by means of a rapid magnetic separation of the unbound components. A built-in magnet graphite-epoxy-composite electrode allowed a sensibly enhanced adsorption and electrochemical quantification of the specifically captured AuNPs. The used DC-AuNP label showed an excellent specificity/selectivity, as a matter of fact using a different antigen (goat IgG) a minimal nonspecific electrochemical or spectrophotometric signal was measured. The detection limits for this novel double-codified nanoparticle-based assay were 52 and 260 pg of human IgG/mL for the spectrophotometric (HRP-based) and electrochemical (AuNP-based) detections, respectively, much lower than those typically achieved by ELISA tests. The developed label and method is versatile, offers enhanced performances, and can be easily extended to other protein detection schemes as well as in DNA analysis.

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.

Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography-tandem mass spectrometry.

A method has been developed to analyse for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in salmon. Salmon samples were extracted with acetonitrile:McIIIvain pH 3 buffer (90:10 v/v), sample extracts were purified on a Bakerbond strong cation exchange solid phase extraction cartridge. Aliquots of the extracts were analysed by LC-MS/MS. The method was validated in salmon, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCalpha) was 0.17, 0.15, 0.35 and 0.17 microg kg(-1), respectively, for MG, LMG, CV and LCV and for the detection capability (CCbeta) values of 0.30, 0.35, 0.80 and 0.32 microg kg(-1), respectively, were obtained. Fortifying salmon samples (n=6) in three separate assays, show the accuracy to be between 77 and 113% for MG, LMG, LCV and CV. The precision of the method, expressed as RSD values for the within-laboratory reproducibility, for MG, LMG and LCV at the three levels of fortification (1, 1.5 and 2.0 microg kg(-1)), was less than 13%. For CV a more variable precision was obtained, with RSD values ranging between 20 and 25%.

Nanocomposite and nanoporous polyaniline conducting polymers exhibit enhanced catalysis of nitrite reduction.

Nanostructured polyaniline (PANI) conducting polymer films were prepared on electrochemically pretreated glassy carbon electrodes, which were previously modified with multilayers of polystyrene (PS) nanoparticles with a diameter of 100 nm. PANI was electropolymerised and grown through the interstitial spaces between the PS nanoparticles, which formed a nanocomposite film of PANI and PS nanoparticles on the electrode surface. Furthermore, a nanoporous PANI film was fabricated through the removal of the PS nanoparticles by dissolution in toluene. As a result of their nanostructure, both of the PANI films (before and after removal of the PS nanoparticles) exhibited enhanced electrocatalytic behaviour towards the reduction of nitrite relative to bulk-PANI films; however, partial collapse or shrinkage may have occurred with the removal of the nanoparticles and could have resulted in a less enhanced response. Under optimised conditions, the nanocomposite-film-modified electrode exhibited a fast response time of 5 s and a linear range from 5.0 x 10(-7) to 1.4 x 10(-3) M for the detection of nitrite; the detection limit was 2.4 x 10(-7) M at a signal-to-noise ratio of 3.

Potential of CE for the determination of inorganic and acidic anions in cyanoacrylate adhesives.

In this work, a CZE method with indirect UV detection was developed for the simultaneous determination of the inorganic and acidic anions, chloride, sulfate, nitrate, fluoride, formate, phosphate, diethylphosphate, methyl sulfonate, cyanoacetate, and methacrylate present in cyanoacrylate adhesives. Chromate was employed as the probe ion, and the EOF was reversed by incorporating CTAB into BGE. Detection limits of 0.7-4.6 microg/mL were obtained for all the anions studied. The CE method developed is a significant improvement on traditionally used chromatographic methods such as ion chromatography, as it resulted in shorter analysis times with enhanced separation efficiencies. This method was successfully employed for the analysis of inorganic and acidic anions in cyanoacrylate adhesive samples.

Production and characterization of a polyclonal antibody for Os(II) and Ru(II) polypyridyl complexes.

The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)(2)dcbpy] hapten is described. Bpy is 2,2'-bipyridine and dcbpy is 2,2'-bipyridine-4,4'-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 x 10(8) and 5.0 x 10(8)M(-1) for [Os(bpy)(2)dcbpy] and [Ru(bpy)(2)dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H(2)O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D(2)O/H(2)O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)(2)dcbpy] and 80% of [Ru(bpy)(2)dcbpy] from excited state deactivation by the aqueous solvent.

Enhancement of a conducting polymer-based biosensor using carbon nanotube-doped polyaniline.

A biosensor with improved performance was developed through the immobilization of horseradish peroxidase (HRP) onto electropolymerized polyaniline (PANI) films doped with carbon nanotubes (CNTs). The effects of electropolymerization cycle and CNT concentration on the response of the biosensor toward H2O2 were investigated. It was found that the application of CNTs in the biosensor system could increase the amount and stability of the immobilized enzyme, and greatly enhanced the biosensor response. Compared with the biosensor without CNTs, the proposed biosensor exhibited enhanced stability and approximately eight-fold sensitivity. A linear range from 0.2 to 19 microM for the detection of H2O2 was observed for the proposed biosensor, with a detection limit of 68 nM at a signal-to-noise ratio of 3 and a response time of less than 5s.

Optimisation and characterisation of biosensors based on polyaniline.

With lower limits of detection and increased stability constantly being demanded of biosensor devices, characterisation of the constituent layers that make up the sensor has become unavoidable, since this is inextricably linked with its performance. This work describe the optimisation and characterisation of two aspects of sensor performance: a conductive polymer layer (polyaniline) and the immobilised protein layer. The influence of the thickness of polyaniline films deposited electrochemically onto screen-printed electrode surfaces is described in this work in terms of its influence on a variety of amperometric sensor performance characteristics: time to reach steady state, charging current, catalytic current, background current and signal/background ratios. The influence of polymer film thickness on the conductivity and morphology of finished films is also presented. An electrostatic method of protein immobilisation is used in this work and scanning electron microscopy in conjunction with gold-labelled antibodies and back-scattered electron detection has enabled the direct visualisation of individual groups of proteins on the sensor surface. Such information can provide an insight into the performance of sensors under influence of increasing protein concentrations.

Analyzing the mechanisms of selectivity in biomimetic self-assemblies via IR and NMR spectroscopy of prepolymerization solutions and molecular dynamics simulations.

Molecularly imprinted polymers (MIPs) for 2,4-dichlorophenoxyacetic acid were synthesized via a noncovalent approach with 4-vinylpyridine as functional monomer and ethylene glycol dimethacrylate as cross-linker in a methanol/water mixture. Templated polymers synthesized in this self-assembly approach rely on complex formation between the target analyte and functional monomers in porogenic solution prior to radical polymerization. Consequently, the achievable selectivity is governed by the nature and stability of these complexes. The nature of noncovalent interactions responsible for complex formation during imprinting of the template 2,4-dichlorophenoxyacetic acid (2,4-D) with the functional monomer 4-vinylpyridine has been investigated. Fourier transform infrared and 1H NMR spectroscopies provide the fundamental analytical basis for rationalizing the mechanisms of recognition during the imprinting process probing the governing interactions for selective binding site formation at a molecular level. Molecular modeling studies in explicit solvent (chloroform and water) corroborate the importance of hydrogen bonding in aprotic solvents and of hydrophobic interactions in protic media in agreement with the experimental spectroscopic investigations of prepolymerization solutions. Furthermore, chromatographic studies of the synthesized MIPs provided insight on the importance of size, shape, and functionality during selective 2,4-D rebinding processes confirming the results obtained during the prepolymerization studies.