The morphology and evolution of bipyramidal gold nanoparticles.

We examine the growth and evolution with time of bipyramidal gold nanoparticles grown by a seed-mediated process. The nanoparticles are characterized both by their physical dimensions determined by transmission electron microscopy and by the wavelength position of their localized surface plasmon resonance. Each growth's physical dimensions correspond to particular initial conditions, and we observe two distinct modes of temporal evolution during growth. The effects of varying silver nitrate concentration and growth time are also explored. We observe a linear relationship between the tip radius of curvature and the wavelength of the longitudinal localized surface plasmon resonance (LSPR) peak. Critical parameters for synthesizing bipyramidal nanoparticles with sharp tips and correct length to width ratio are determined.

Fluorogenic assay for beta-glucuronidase using microchip-based capillary electrophoresis.

Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with beta-glucuronidase of the conjugated glucuronide, fluorescein mono-beta-D-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess beta-glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yielded the Km value. The results were compared with those from experiments done by conventional CE. The Km value for beta-glucuronidase with FMG is being reported for the first time as 18 microM. The inhibition of beta-glucuronidase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant beta-glucuronidase and FMG concentrations. A dose-response plot was acquired and the IC50 value for SL was determined to be 3 microM.

Small volume bead assay for ovalbumin with electrochemical detection.

A bead based sandwich enzyme immunoassay coupled to electrochemical detection for ovalbumin has been developed. The enzyme label alkaline phosphatase was used to convert the substrate 4-aminophenyl phosphate to electroactive product 4-aminophenol. The detection was done in a microdrop by continuously monitoring the enzyme turnover with a rotating disk electrode. This reduces dilution of the enzyme product, a key to achieving low detection limits. The assay developed has a detection limit of 0.1 ng ml-1. Assay sensitivity in complex matrices such as food and serum was compared.

Spatially addressed deposition and imaging of biochemically active bead microstructures by scanning electrochemical microscopy.

A new procedure is described to deposit paramagnetic beads on surfaces to form microscopic agglomerates. By using surface-modified beads, microscopic structures with defined biochemical activity are formed. The shape and size of agglomerates were characterized by scanning electron microscopy (SEM), and the biochemical activity was mapped with scanning electrochemical microscopy (SECM). This approach is demonstrated using beads modified with anti-mouse antibodies (Ab). After allowing them to react with a conjugate of mouse IgG and alkaline phosphatase (ALP), the beads were deposited as agglomerates of well-defined size and shape. The biochemical activity was recorded in the generation-collection SECM mode by oxidizing 4-aminophenol formed in the ALP-catalyzed hydrolysis of 4-aminophenyl phosphate at the surface of the beads. The signal height correlated with both the amount of beads present in one agglomerate and the proportion of Ab binding sites saturated with the ALP mouse IgG conjugate. The feedback mode of the SECM was used to image streptavidin-coated beads after reaction with biotinylated glucose oxidase.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 9. Incorporation of planar waveguide technology.

Incorporation of planar waveguide technology into a spectroelectrochemical sensor is described. In this sensor design, a potassium ion-exchanged BK7 glass waveguide was over-coated with a thin film of indium tin oxide (ITO) that served as an optically transparent electrode. A chemically selective film was spin-coated on top of the ITO film. The sensor supported five optical modes at 442 nm and three at 633 nm. Investigations on the impact of the ITO film on the optical properties of the waveguide and on the spectroelectrochemical performance of the sensor are reported. Sensing was based on the change in attenuation of light propagated through the waveguide resulting from an optically absorbing analyte. By applying either a triangular or square wave excitation potential waveform, electromodulation of the optical signal has been demonstrated with Fe(CN)6(3-/4-) as a model electroactive couple that partitions into a PDMDAAC-SiO2 film [where PDMDAAC = poly(dimethyldiallylammonium chloride)] and absorbs at 442 nm.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 5. Simulation of sensor response for different excitation potential waveforms.

The simulation of the optical response in spectroelectrochemical sensing has been investigated. The sensor consists of a sensing film coated on an optically transparent electrode (OTE). The mode of detection is attenuated total reflection. Only species that partition into the sensing film, undergo electrochemistry at the potentials applied to the OTE, and have changes in their absorbance at the wavelength of light propagated within the glass substrate of the OTE can be sensed. A fundamental question arises regarding the excitation potential waveforms employed to initiate the electrochemical changes observed. Historically, selection has been based solely upon the effectiveness of the waveform to quickly electrolyze any analyte observable by the optical detection method employed. In this report, additional requirements by which the waveform should be selected for use in a remote sensing configuration are discussed. The effectiveness of explicit finite difference simulation as a tool for investigating the applicability of three different excitation potential waveforms (square, triangle, sinusoid) is demonstrated. The simulated response is compared to experimental results obtained from a prototype sensing platform consisting of an indium tin oxide OTE coated with a cation-selective, sol-gel-derived Nafion composite film designed for the detection of a model analyte, tris(2,2'-bipyridyl)ruthenium(II) chloride. Using a diffusion coefficient determined from experimental data (5.8 x 10(-11) cm2 s for 5 x 10(-6) M Ru(bipy)3(2+)), the simulator program was able to accurately predict the magnitude of the absorbance change for each potential waveform (0.497 for square, 0.403 for triangular, and 0.421 for sinusoid), but underestimated the number of cycles required to approach steady state. The simulator program predicted 2 (square), 3 (triangle), and 5 cycles (sinusoid), while 5 (square), 15 (triangle), and 10 (sinusoid) cycles were observed experimentally.

Capillary electrochemical enzyme immunoassay (CEEI) for phenobarbital in serum.

A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 microl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product (p-aminophenol) from the catalytic conversion of the substrate (p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 microg l(-1) (2.8 pmoles or 0.65 ng) and a range of 30-3000 microg l(-1). The assay could be used to determine the phenobarbital serum concentration in a 4 microl clinical serum sample without pretreatment.

Feasibility studies of simultaneous multianalyte amperometric immunoassay based on spatial resolution.

A multianalyte immunoassay concept based on the geometric separation of different analyte-specific antibodies has been demonstrated. The assay and amperometric detection are done in a cell with two working electrodes controlled at the same potential, and the amperometric signal at each electrode is monitored. The distance between any two adjacent electrodes in this prototype is 2.5 mm, and during the course of amperometric measurement, the product formed at one electrode does not reach the other working electrode within 20 min after the addition of enzyme substrate. Thus, the method relies on the spatial resolution between the different antibodies being such that measurements are taken before cross-interference due to diffusion can occur. Identical enzyme labels (alkaline phosphatase, ALP) and substrates (p-aminophenyl phosphate, PAPP) are used for all analytes. Alkaline phosphatase-conjugated rat anti-mouse IgG was immobilized by passive adsorption. Our studies showed that this concept is feasible and can be applied to the simultaneous measurement of multiple analytes.

Spectroscopic and electrochemical evaluation of a perfluorosulfonated ionomer and its gel as preconcentrating media for [ReI(DMPE)3]+, where DMPE = 1,2-bis(dimethylphosphino)ethane.

The interaction of [ReI(DMPE)3]+, where DMPE = 1,2-bis(dimethylphosphino)ethane, a nonradioactive analogue of a heart imaging agent, with Nafion gel, which is Nafion plasticized with tri-n-butyl phosphate, has been evaluated spectroscopically and electrochemically. Thin-layer spectroelectrochemistry on the rhenium compound yields a linear Nernst plot with an n value of 0.99 and E degree' of 0.049 V vs Ag/AgCl. The electrochemistry is consistent with a reversible one-electron transfer between the mono- and dicationic forms of the complex. The UV-visible spectrum of electrogenerated [ReII(DMPE)3]2+ is identical to that obtained by air oxidation of [ReI(DMPE)3]+. Thin, free-standing films of Nafion gel and Nafion that were sufficiently clear to record visible spectra were cast. Spectroscopic measurement of the partitioning of [ReI-(DMPE)3]+ from aqueous solution into these films shows a more rapid uptake of the complex by the Nafion gel. Preconcentration factors into Nafion gel and Nafion were 350 and 50, respectively, after 4 h of soaking. Cyclic voltammetry of 1.0 x 10(-4)-1.0 x 10(-7) M (ReI(DMPE)3]+ in 0.15 M supporting electrolyte aqueous solution at bare gold and spectroscopic graphite electrodes suggests that the complex adsorbs to these electrodes. By comparison, the well-defined cyclic voltammograms at Nafion gel-modified electrodes exhibit diffusion-controlled behavior. The formal reduction potential at Nafion gel-modified electrodes is shifted positively compared to bare electrodes. A current enhancement of approximately 4 was observed at Nafion gel-modified spectroscopic graphite over a bare electrode. A calibration plot of peak current for differential pulse voltammetry vs concentration at Nafion gel-modified spectroscopic graphite was linear in the 10(-7)-10(-5) M concentration range, with a detectable signal down into the 10(-9) M range.

The analysis of fountain pen inks by capillary electrophoresis with ultraviolet/visible absorbance and laser-induced fluorescence detection.

Information on the identity of inks adds to the circumstantial evidence in legal cases involving fraudulent documents. In combination with optical methods, multiple thin-layer chromatography (TLC) is currently the analytical tool used by forensic chemists to separate, compare and distinguish inks based on their dye composition. In our studies, capillary electrophoresis (CE) was used for the analysis of water-soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The investigations included the development of an electrophoretic separation method, the optimization of an extraction procedure for inks from paper as well as the evaluation of ultraviolet/visible (UV/VIS) absorbance and laser-induced fluorescence (LIF) detection for the analysis of inks by CE. Good results for the separation of 17 blue and black inks of different manufacturers and countries of origin were obtained with 100 mM borate buffer, pH 8.0, containing 20% methanol. The electropherograms of the inks and their extracts from paper showed patterns that were in most cases distinctly different from each other. Ultraviolet/visible scans can be used to compare spectra of the separated main and trace components of inks. Fluorescence detection at different excitation and emission wavelengths was more sensitive, but added to the complexity of the electropherograms due to the excitation of coextracted fluorescing paper components. The resolution power of CE combined with the information content provided by the detection modes investigated prove CE to be a powerful tool for the identification of water-soluble inks used on paper documents.

Separation of aromatic acids, DOPA, and methyl-DOPA by capillary electrophoresis with dendrimers as buffer additives.

Polyamidoamine starburst dendrimers with terminal carboxylate groups are used as a pseudo-stationary phase in electrokinetic chromatography for separating the selected aromatic amino acids phenylalanine, phenylglycine, homophenylalanine, and tyrosine and the catecholamines 3-(3,4-dihydroxyphenyl)alanine and 3-(3,4-dihydroxyphenyl)-2-methylalanine at different pH levels. A significant difference in analyte selectivity is observed between dendrimers of different generations and at different pH levels. At pH 7.0, the utility of the dendrimers for separating these analytes is limited by relatively low selectivity and a noisy baseline. However, good separation and selectivity are obtained with low generation dendrimers (G0.5 and G1.5) at low pH. Strong association of the solute with dendrimers is observed for high generations (G2.5, G3.5, and G5.5).

Dual-analyte spectroscopic sensing in sol-gel derived polyelectrolyte-silica composite thin films.

Ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid, monosodium salt hydrate), an iron indicator, and HTPS (8-hydroxyl-1,3,6-pyrenetrisulfonic acid, trisodium salt), a pH indicator, were immobilized in sol-gel derived PDMDAAC-SiO(2) (where PDMDAAC stands for poly(dimethyldiallylammonium chloride), composite thin films via ion-exchange. The two indicators were immobilized in two adjacent sections of the same PDMDAAC-SiO(2) film which was supported on a glass optical substrate. The spectroscopic response of the film to both Fe(2+) and H(+) in solutions was investigated by attenuated total reflection (ATR) spectrometry at two well-separated wavelengths, 562 nm for Fe(2+) and 460 nm for H(+). The Ferrozine/HPTS immobilized PDMDAAC-SiO(2) films had the following characteristics: linear range, 2.5x10(-6)-5.0x10(-5) M for Fe(2+), pH 4.1-6.8 for H(+); sensitivity, 2.2x10(4) DeltaA/M for Fe(2+), 0.583 DeltaA/pH for H(+).

Electrochemical behavior of [ReI(DMPE)3]+, where DMPE = 1,2-bis(dimethylphosphino)ethane, at perfluorosulfonated ionomer-modified electrodes.

The perfluorosulfonated ionomer Nafion shows potential utility as a polymer film to enhance the electrochemical detection of [ReI(DMPE)3]+, where DMPE = 1,2-bis-(dimethylphosphino)ethane. [ReI(DMPE)3]+, a nonradioactive radiopharmaceutical analog for heart imaging, partitions strongly into Nafion films on glassy carbon. Well-defined, chemically reversible cyclic voltammograms are obtained for the [ReI(DMPE)3]+/[ReII(DMPE)3]2+ couple with Eo' shifted positively by 60 mV relative to its value on bare glassy carbon. [ReI(DMPE)3]+ partitions into Nafion more strongly than the oxidized form, [ReII-(DMPE)3]2+. The detection limit for [ReI(DMPE)3]+ by cyclic voltammetry was improved by 2-3 orders of magnitude by the Nafion film. Differential pulse voltammetry for oxidation of [ReI(DMPE)3]+ at the Nafion-modified electrode has a detection limit of 2.5 x 10(-9) M compared to 1.0 x 10(-7) M at the bare electrode. A preconcentration factor of 1 x 10(6) for partitioning of [ReI(DMPE)3]+ from 0.05 M NaCl into Nafion on a glassy carbon electrode was measured.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 2. Demonstration of selectivity in the presence of direct interferences.

Three modes of selectivity based on charge-selective partitioning, electrolysis potential, and spectral absorption wavelength were demonstrated simultaneously in a new type of spectroelectrochemical sensor. Operation and performance of the three modes of selectivity for detection of analytes in the presence of direct interferences were investigated using binary mixture systems. These binary mixtures consisted of Fe(CN)(6)(3-) and Ru(bpy)(3)(2+) and of Fe(CN)(6)(4-) and Ru(CN)(6)(4)(-) in aqueous solutions. Results on the Fe(CN)(6)(3-)/Ru(bpy)(3)(2+) binary mixture showed that an anion-exchange coating consisting of PDMDAAC-SiO(2) [where PDMDAAC is poly(dimethyldiallylammonium chloride)] and a cation-exchange coating consisting of Nafion-SiO(2) can trap and preconcentrate analytes with charge selection. At the same time, such coatings exclude interferences carrying the same type of charge as that of the exchange sites in the sensor coating. Using the Fe(CN)(6)(4-)/Ru(CN)(6)(4-) binary mixture, the Fe(CN)(6)(4-) component can be selectively detected by restricting the modulation potential cycled to a range specific to the redox-active Fe(CN)(6)(4-) component and simultaneously monitoring the optical response at the overlapping wavelength of 420 nm. It was also shown that, when the wavelength for optical monitoring was chosen as 500 nm, which is specific to the Ru(CN)(6)(4-) component, interference from the Fe(CN)(6)(4-) component for spectroelectrochemical detection of Ru(CN)(6)(4-) was significantly suppressed, even though the cyclic modulation potential encompassed the redox range for the Fe(CN)(6)(4-) component.

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

Imaging of immobilized antibody layers with scanning electrochemical microscopy.

Visualization of immobilized antibodies can be achieved with scanning electrochemical microscopy (SECM) by saturation of the antigen binding sites with an alkaline phosphatase-antigen conjugate, which catalyzes hydrolysis of the redox-inactive 4-aminophenyl phosphate to the redox-active 4-aminophenol (PAP). PAP was detected in the collection mode at an amperometric SECM tip. The tip current reflects the density of active binding sites in the immobilized antibody layer. The application of this approach for immunosensing research has been demonstrated with the optimization of a covalent immobilization procedure of antibodies on glass. The special advantages and present limitations of the procedures are discussed.

Electrochemical dehydrogenase-based homogeneous assays in whole blood.

An electrochemical method has been developed for determining NADH in whole blood for dehydrogenase-based assays by flow-injection analysis. NADH generated by dehydrogenase is oxidized by an electron-transfer coupling reagent, 2,6-dichloroindophenol (DCIP). The reduced form of DCIP (DCIPH2) is measured amperometrically by flow-injection analysis. Endogenous interferents were inhibited by p-hydroxymercuribenzoate. Electrode fouling by proteins was not observed under assay conditions. The Emit theophylline enzyme immunoassay and the hexokinase glucose assay were used as models. For the glucose assay, the intraassay CVs were 15% at 0.31 g/L and 3.5% at 1.82 g/L. Recoveries of glucose from whole blood (compared with that for aqueous standards) were 109%, 97.9%, and 101% at 0.050, 2.00, and 5.00 g/L glucose, respectively, and 104%, 101%, and 102% for theophylline at concentrations of 5.0 (low), 16.4 (medium), and 30.2 (high) mg/L, respectively, with corresponding precisions of 12%, 9.5%, and 8.8%. Both assays correlated well with results by reference methods. These studies demonstrate that this method can measure NADH in whole blood without prior separation and that it is potentially applicable to other dehydrogenase-based assays in whole blood.

Simultaneous immunoassay using electrochemical detection of metal ion labels.

The concept of a simultaneous dual analyte immunoassay based on two different metal ion labels is demonstrated. The model system consists of two proteins, human serum albumin (HSA) and immunoglobulin G (IgG). Bismuth and indium ions have been coupled to these proteins through the bifunctional chelating agent diethylenetriamine-pentaacetic acid (DTPA). A maximum molar labeling ratio of 6:1 and 10:1 was obtained for HSA and IgG, respectively. Following a competitive equilibrium between unlabeled and labeled protein for a limited amount of specific antibody immobilized on polystyrene, the bound metal ion labels were released by acidification and detected by differential pulse anodic stripping voltammetry (ASV). Limits of detection for HSA and IgG are 1.8 and 0.6 microgram/mL, respectively. Application of the dual immunoassay to human serum samples gave results that were comparable to those obtained by nephelometry.

gamma-irradiation-induced grafting of poly (styrenesulfonate) to poly(tetrafluoroethylene) shielded microelectrodes.

A new method for the fabrication and polymer modification of microelectrodes is described. These electrodes are constructed by heat sealing the electroactive material in dual shrink/melt poly(tetrafluoroethylene) (PTFE) under vacuum. The PTFE shield may be activated to provide a support upon which polymers of interest may be grafted. gamma-Irradiation was used to graft polymerize styrene to the surface. The poly-(styrene) was subsequently sulfonated with chlorosulfonic acid to form poly(styrenesulfonate). Scanning electron microscopy and Raman microspectroscopy provide evidence that the poly-(styrenesulfonate) film has been formed and extends over the electrode material. Voltammetry indicates that hexaammine-ruthenium(III) cation is preconcentrated and stabilized via an association with the polymer film.