The Study of Solid/Liquid Interfaces with X-ray Standing Waves.

The principles and applications of the x-ray standing wave technique are described. Emphasis is placed on its utility for the study of structure, composition, and distribution of interfacial species especially at solid/liquid interfaces.

Ultrafast voltammetry of adsorbed redox active dendrimers with nanometric resolution: an electrochemical microtome.

Megavolt-per-second cyclic voltammetry is used to control the expansion with time of a diffusion layer created within the redox outer shell of a fourth generation dendrimer molecule adsorbed onto an electrode; one quarter of the dendrimer is shown in the picture. This allows the measurement of the degree of communication between the ruthenium(II/III) bis(terpyridyl) ("LRuL") redox centers borne at the extremity of the dendrimer tethers, as well as the characterization of the shape of the adsorbed dendrimer molecule.

Analytical strategies for amperometric biosensors based on chemically modified electrodes.

Various strategies based on the use of chemically modified electrodes for the development of amperometric biosensors are described. Particular emphasis is placed on materials capable of catalyzing the oxidation of NADH and coupling these with enzymatic activities for biosensor construction. In terms of electrocatalysts, the discussion will centre on electrodeposited films of 3,4-dihydroxy benzaldehyde (3,4-DHB) and related analogs as well as on electrodeposited films of transition metal complexes of 1,10-phenanthroline-5,6-dione (phen-dione). Electrodeposited films of these materials have been coupled to the enzymatic activity of aldehyde dehydrogenase and alcohol dehydrogenase for the development of biosensors for aldehydes and ethanol, respectively.

Effects of competitive binding on the amperometric determination of copper with electrodes modified with chromotrope 2B.

Electrodes modified with Chromotrope 2B incorporated by ion-exchange into a polycationic film of electropolymerized [Ru(v-bpy)(3)](2+) (v-bpy = 4-vinyl-4'-methyl-2,2'-bipyridyl) have been employed in the amperometric determination of copper in solution and exhibit very high sensitivity as well as linear calibration curves in the concentration range 7 x 10(-8)-1 x 10(-4)M. The effects of competing ligands, including chloride, bromide, oxalate, ammonia, acetate, citrate, borate, humic and fulvic acids, or the presence of competing metal ions such as cobalt or nickel on the uptake of copper by the modified electrodes have also been studied. The presence of competing ligands or metal ions decreases the analytical signal due to copper incorporation. The magnitude of this effect is dependent on the relative strength of coordination of the competing ligands for copper ions or of Chromotrope 2B for the competing metals, and also on the concentration of the interferents. The relevance of this work to speciation studies is discussed.

Mechanical control of spin states in spin-1 molecules and the underscreened Kondo effect.

The ability to make electrical contact to single molecules creates opportunities to examine fundamental processes governing electron flow on the smallest possible length scales. We report experiments in which we controllably stretched individual cobalt complexes having spin S = 1, while simultaneously measuring current flow through the molecule. The molecule's spin states and magnetic anisotropy were manipulated in the absence of a magnetic field by modification of the molecular symmetry. This control enabled quantitative studies of the underscreened Kondo effect, in which conduction electrons only partially compensate the molecular spin. Our findings demonstrate a mechanism of spin control in single-molecule devices and establish that they can serve as model systems for making precision tests of correlated-electron theories.

Single-mismatch position-sensitive detection of DNA based on a bifunctional ruthenium complex.

A ruthenium complex, pentaamine ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] (which we refer to as RuL in the text) generated in situ has been used as a sensitive and selective electrochemical indicator in DNA sensing. The complex incorporates dual functionalities with the Ru center providing a redox probe and the ligand (L) providing a fluorescent tag. The presence of the aromatic groups in the ligand endows the complex with an intercalative character and makes it capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Combining spectroscopic and electrochemical techniques, we have elucidated the nature of the interactions. From these data we conclude that the binding mode is fundamentally intercalative. The ligand-based fluorescence allows characterization of the complex formation as well as for melting experiments to be carried out. The metal-based redox center is employed as an electrochemical indicator to detect the hybridization event in a DNA biosensor. The biosensor has been developed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. With the use of this approach, complementary target sequences of Helicobacter can be quantified over the range of 106 to 708 pmol with a detection limit of 92+/-0.4 pmol and a linear correlation coefficient of 0.995. In addition, this approach allows the detection, without the need for a hybridization suppressor in solution, such as formamide, of not only a single mismatch but also its position in a specific sequence of H. pylori, due to the selective interaction of this bifunctional ruthenium complex with dsDNA.

Tuning the Kondo effect with a mechanically controllable break junction.

We study electron transport through C(60) molecules in the Kondo regime using a mechanically controllable break junction. By varying the electrode spacing, we are able to change both the width and the height of the Kondo resonance, indicating modification of the Kondo temperature and the relative strength of coupling to the two electrodes. The linear conductance as a function of T/T(K) agrees with the scaling function expected for the spin-1/2 Kondo problem. We are also able to tune finite-bias Kondo features which appear at the energy of the first C(60) intracage vibrational mode.

Comprehensive study of bioanalytical platforms: xanthine oxidase.

A comprehensive study of a general bioanalytical platform for biosensor applications is presented using xanthine oxidase (XnOx) as a case study within the framework of developing approaches of broad applicability. In this context, emphasis is placed on amperometric biosensors based on XnOx, which has been immobilized by covalent binding to gold electrodes modified with dithiobis-N-succinimidyl propionate. The immobilized XnOx layers have been characterized using atomic force microscopy under liquid conditions and quartz crystal microbalance techniques. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy. Redox dyes of phenothiazine derivatives, specifically, thionine and methylene blue, have been found to work well as electron acceptors for reduced XnOx. The kinetic parameters and equilibrium constants of the mediated enzymatic oxidation of xanthine in the presence of the above-mentioned redox dyes have been calculated. The response of the enzymatic electrode to varying xanthine concentrations has been obtained in the presence of thionine or methylene blue as redox mediator in solution. Under these conditions, xanthine could be determined amperometrically at +0.2 V versus SSCE.

Interactions of benzyl viologen with surface-bound single- and double-stranded DNA.

The interactions of benzyl viologen (BV) with single- and double-stranded calf-thymus DNA immobilized onto gold electrodes have been studied by electrochemical methods. Benzyl viologen interacts electrostatically with both double-stranded (ds) and single-stranded (ss) DNA, and the strength of the interactions is dependent on ionic strength (mu). The dicationic form (BV2+) binds to dsDNA 9 times more strongly than the singly reduced form, BV*+, in a pH 7.4 Tris-HCl buffer solution at mu = 8.4 mM. BV2+ binds to ssDNA 5 times more strongly than the BV*+ form. From measurements at mu = 8.4 mM, a binding constant (K2+) of 2.0 (+/-0.2) x 10(4) M(-1) and a binding site size (s) of 1 base pair were obtained, respectively, for dsDNA. For ssDNA, at the same ionic strength, the values obtained for K and s were 3.6 (+/-0.4) x10(4) M(-1) and 2 nucleotides, respectively. The amount of BV bound, whether to dsDNA or ssDNA, decreased with increasing ionic strength. Whereas the binding rate of BV to both dsDNA and ssDNA immobilized onto gold electrodes is relatively low, once immobilized, it dissociates rapidly away from the electrode surface. The electron-transfer rate constant for BV is moderately fast at both dsDNA- and ssDNA-modified gold electrodes. The application of benzyl viologen as an electroactive indicator capable of differentiating between surface-immobilized single- and double-stranded DNA in denaturation/regeneration cycles has been explored.

Micromethod for the investigation of the interactions between DNA and redox-active molecules.

A novel microscale and surface-based method for the study of the interactions of DNA with other redox-active molecules using DNA-modified electrodes is described. The method is simple, convenient, reliable, reagent-saving, and applicable for DNA studies, especially those involving microsamples. Information such as binding site size (s, in base pairs), binding constant (K), ratio (K0x/KRed) of the binding constants for the oxidized and reduced forms of a bound species, binding free energy (delta Gb), and interaction mode, including changes in the mode of interaction, and "limiting" ratio K0x0/KRed0 at zero ionic strength can be obtained using only 3-15 micrograms of DNA samples. The method was developed using [Co(Phen)3]3+/2+ (Phen = 1,10-phenanthroline)/double-stranded DNA (dsDNA)-modified gold electrodes and [Co(bpy)3]3+/2+ (2,2'-bipyridyl)/dsDNA-modified gold electrodes as model systems. For the [Co(Phen)3]3+/2+/dsDNA-modified gold electrode system, a K2+ of (2.5 +/- 0.3) x 10(5) M-1 and an s of 5 bp were obtained in 5 mM pH 7.1 Tris-HCl buffer solution containing 50 mM NaCl. For [Co(bpy)3]3+/2+/dsDNA-modified gold electrodes, K3+ and s values of (1.3 +/- 0.3) x 10(5) M-1 and 3 bp, respectively, were obtained. While the s values are consistent with those reported in the literature obtained by solution methods, the K values are almost an order of magnitude larger. A transition in the nature of the interaction between dsDNA and [Co(Phen)3]3+/2+, from electrostatic to intercalative with increasing ionic strength, was found in our studies. Negative values of delta E0' for [Co(bpy)3]3+/2+ bound to dsDNA suggest that its interaction with dsDNA is predominantly electrostatic over the ionic strength range of 5-105 mM. The "limiting" ratio K3+0/K2+0 of 22 obtained for [Co(Phen)3]3+/2+ bound to dsDNA at zero ionic strength suggests that electrostatic interactions are predominant over intercalative ones under these limiting conditions. The ratio for [Co(bpy)3]3+/2+ of 16 also indicates that the 3+ form binds to dsDNA more strongly than the 2+ form at zero ionic strength. For [Co(Phen)3]3+/2+/single-stranded DNA (ssDNA)-modified gold electrodes, the nonuniformity of the surface structure of ssDNA-modified gold electrodes greatly complicates the analysis. A system consisting of a dsDNA-modified gold electrode and [Co(tppz)2]3+/2+ (tppz = tetra-2-pyridyl-1,4-pyrazine) was studied by this method, with a K2+ value of (5 +/- 1) x 10(5) M-1 and an 8 value of 7 bp being obtained.

Precise adjustment of nanometric-scale diffusion layers within a redox dendrimer molecule by ultrafast cyclic voltammetry: an electrochemical nanometric microtome.

Performing cyclic voltammetry at scan rates into the megavolt per second range allows the exploration of the nanosecond time scale as well as the creation of nanometric diffusion layers adjacent to the electrode surface. This latter property is used here to adjust precisely the diffusion layer width within the outer shell of a fourth-generation dendrimer molecule decorated by 64 [Ru(II)(tpy)2] redox centers (tpy = terpyridine). Thus the shape of the dendrimer molecule adsorbed onto the ultramicroelectrode surface can be explored voltammetrically in a way reminiscent of an analysis with a nanometric microtome. The quantitative analysis developed here applied to the experimental voltammograms demonstrates that in agreement with previous scanning tunneling microscopy (STM) studies the adsorbed dendrimer molecules are no more spherical as they are in solution but resemble more closely hemispheres resting onto the electrode surface on their diametrical planes. The same quantitative analysis gives access to the apparent diffusion coefficient featuring electron hopping between the [Ru(II)/ Ru(III)(tpy)2] redox centers distributed on the dendrimer surface. Based on the electron hopping rate constant thus measured and on a Smoluchowski-type model developed here to take into account viscosity effects during the displacement of the [Ru(II)/Ru(III)(tpy)2] redox centers around their equilibrium positions, it is shown that the [Ru(II)/Ru(III)(tpy)2] redox centers are extremely labile in their potential wells so that they may cross-talk considerably more easily than they would do in solution at an equivalent concentration.

Mechanistic Studies of the Electrocatalytic Oxidation of NADH and Ascorbate at Glassy Carbon Electrodes Modified with Electrodeposited Films Derived from 3,4-Dihydroxybenzaldehyde.

Studies of the electrocatalytic oxidation of ß-nicotinamide adenine dinucleotide (NADH) at glassy carbon rotated disk electrodes modified with electrodeposited films derived from 3,4-dihydroxybenzaldehyde (3,4-DHB) indicate that the mechanism of such electrooxidation proceeds via the formation of an intermediate complex. The reaction also appears to be strongly influenced by the presence of Ca(2+) and Mg(2+) ions as well as by pH. Ascorbate can also be electrocatalytically oxidized at these modified electrodes, giving rise to an electrochemical response very similar to that obtained for NADH. Due to this similarity, the presence of ascorbate in NADH determinations presents a severe interference that cannot be mitigated on the basis of electrochemical responses alone. However, this interference effect can be virtually suppressed by the presence of ascorbate oxidase in solution or immobilized on a nylon mesh which, in turn, is in contact with the electrode modified with the film of 3,4-DHB. Using this approach, we describe the construction of an alcohol biosensor based on alcohol dehydrogenase and which is, furthermore, free from interference effects due to ascorbate.

Enantiomerically pure chiral coordination polymers: synthesis, spectroscopy, and electrochemistry in solution and on surfaces.

The two enantiomerically pure bridging ligands (+/-)-[ctpy-x-ctpy] have been prepared employing a two-fold stereospecific alkylation reaction of the enantiomerically pure, chiral terpyridyl-type ligands (+/-)-ctpy. The reaction of each of the enantiomerically pure bridging ligands with Fe(2+) gives rise to chiral coordination polymers whose formation and stoichiometry were followed spectrophotometrically. An assignment of the absolute configuration of the formed helical polymeric structures was carried out on the basis of circular dichroism studies. Highly ordered domains (as determined from STM imaging) of the enantiomerically pure chiral redox polymers could be prepared via the interfacial reaction, over an HOPG substrate, of the bridging ligand in CH(2)Cl(2) and FeSO(4) in water. The degree of polymerization was estimated to be up to 60 from analysis of the STM images of the highly ordered domains on HOPG. The helicality of the domains was compared to the configuration obtained from the circular dichroism studies. The electrochemical properties of the polymers were investigated using cyclic voltammetry and the results compared to those of the respective monomeric complexes. The redox behavior of the iron centers in the polymer was comparable to that of the monomeric complex [Fe((-)-ctpy)(2)](PF(6))(2) as well as to that of [Fe(tpy)(2)](PF(6))(2). The polymeric materials undergo electrodeposition following the two-electron reduction of each bridging ligand unit (one electron per terpyridine group). No ligand-mediated metal-metal interactions were evident from the cyclic voltammetric measurements, suggesting that the metal centers act independently. Moreover, oxidation of the metal centers within the polymeric materials did not give rise to electrodeposition.

Electrocatalytic reduction of nitric oxide at electrodes modified with electropolymerized films of [Cr(v-tpy)2]3+ and their application to cellular NO determinations.

Nitric oxide can be electrocatalytically reduced at electrodes modified with electropolymerized films of [Cr(v-tpy)2]3+. Upon further modification with a thin film of Nafion (to prevent interferences from anions, especially nitrite), these electrodes can be employed as NO sensors in solution with submicromolar detection limits and fast response. We have carried out preliminary studies of cellular NO release from Rhodobacter sphaeroides bacterial cells with excellent results.

Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium "hedysari" strain HCNT1.

Rhizobium "hedysari" HCNT1 is an unclassified rhizobium which contains a nitric oxide-producing nitrite reductase but is apparently incapable of coupling the reduction of nitrite to energy conservation. The gene encoding the nitrite reductase, nirK, has been cloned and sequenced and was found to encode a protein closely related to the copper-containing family of nitrite reductases. Unlike other members of this family, nirK expression in HCNT1 is not dependent on the presence of nitrogen oxides, being dependent only on oxygen concentration. Oxygen respiration of microaerobically grown Nir-deficient cells is not affected by concentrations of nitrite that completely inhibit oxygen respiration in wild-type cells. This loss of sensitivity suggests that the product of nitrite reductase, nitric oxide, is responsible for inhibition of oxygen respiration. By using a newly developed chemically modified electrode to detect nitric oxide, it was found that nitrite reduction by HCNT1 produces significantly higher nitric oxide concentrations than are observed in true denitrifiers. This indicates that nitrite reductase is the only nitrogen oxide reductase active in HCNT1. The capacity to generate such large concentrations of freely diffusible nitric oxide as a consequence of nitrite respiration makes HCNT1 unique among bacteria.

Dithiobissuccinimidyl propionate as an anchor for assembling peroxidases at electrodes surfaces and its application in a H2O2 biosensor.

Exposure of gold surfaces to solutions of dithiobis N-succinimidyl propionate (DTSP) gives rise to the modification of the surface with N-succinimidyl-3-thiopropionate (NSTP) which can, in turn, react with amino groups allowing for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The coverage of NSTP has been estimated to be of the order of 1.3 x 10(-10) from the charge consumed during its reductive desorption. The binding reaction of HRP with NSTP modified gold surfaces has been studied with the quartz crystal microbalance, and the results suggest that the immobilization process involves two steps in which the first (faster) appears to correspond to the rapid incorporation of the enzyme whereas the second is likely due to the slow incorporation of additional enzyme and/or reorganization of the immobilized layer. Spectrophotometric and electrochemical assays indicate that the immobilized HRP retains its enzymatic activity after immobilization onto the DTSP modified gold surface. The amount of immobilized (and active) HRP was estimated from QCM and spectrophotometric measurements to be of the order of 1.5 x 10(-11) mol/cm2. A peroxide biosensor was developed making use of a gold surface modified with DTSP and HRP employing Os and Ru complexes of 1,10-phenanthroline 5,6-dione (phen-dione) of the type [M(phendione)x(L)3-x]+2 (where L = 1,10-phenanthroline or 2,2'-bipyridine, x = 1-3) as mediators with the quinone moieties being the active component. The efficiency of the mediators increased with increasing number of phendione ligands.

Electrocatalytic reduction of S-nitrosoglutathione at electrodes modified with an electropolymerized film of a pyrrole-derived viologen system and their application to cellular S-nitrosoglutathione determinations.

The preparation, electrochemical characterization, and analytical applications of glassy carbon (GC) electrodes modified with electropolymerized films of the cation N,N'-di(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (DPPB) are described. Electropolymerized films of DPPB on GC electrodes exhibit two one-electron redox processes centered at -0.45 and -0.85 V, respectively. S-Nitrosoglutathione (GSNO) can be electrocatalytically reduced at electrodes modified with electropolymerized films of DPPB at approximately -0.4 V vs sodium-saturated calomel electrode, which represents a dramatic diminution of about 600 mV in the overpotential in comparison with the reaction carried out at a bare GC electrode. The kinetics of the catalytic reaction have been characterized using cyclic voltammetry and rotated disk electrode techniques from which a value of (1.3 +/- 0.2) x 10(3)M-1 s-1 was obtained. Using electrodes modified with an electropolymerized film of DPPB we have carried out preliminary studies of the determination of intracellular GSNO concentrations in two strains of the bacterium Rhodobacter sphaeroides.

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.

A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole-derived bipyridinium.

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBP-Nir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 +/- 0.5) x 10(3) M-1 S-1 was obtained for the rate constant.

Cobaltocenium-functionalized poly(propylene imine) dendrimers: redox and electromicrogravimetric studies and AFM imaging.

The first four generations of cobaltocenium-functionalized, diaminobutane-based poly(propylene imine) dendrimers DAB-dend-Cb,(PFb)x (x = 4, 8, 16, and 32; Cb=[Co(eta5-C5H4CONH)(eta5-C5H5)] (1-4) have been synthesized and characterized. The redox activity of the cobaltocenium centers in 1-4 has been characterized by using cyclic voltammetry and the electrochemical quartz-crystal microbalance (EQCM). All of the dendrimers exhibit reversible redox chemistry associated with the cobaltocenium/cobaltocene redox couple. Upon reduction. the dendrimers exhibit a tendency to electrodeposit onto the electrode surface, which is more pronounced for the higher generations. Pt and glassy carbon electrodes could be modified with films derived from 1-4,exhibiting a well-defined and persistent electrochemical response. EQCM measurements show that the dendrimers adsorb, at open circuit, onto platinum surfaces at monolayer or submonolayer coverage. Cathodic potential scanning past -0.75 V at which the cobaltocenium sites are reduced, gave rise to the electrodeposition of multilayer equivalents of the dendrimers. The additional material gradually desorbs upon re-oxidation so that only a monolayer equivalent remains on the electrode surface. Changes in film morphology as a function of dendrimer generation and surface coverage were studied by using admittance measurements of the quartz-crystal resonator on the basis of its electrical equivalent circuit, especially in terms of its resistance parameter. In general, we find that films of the lower dendrimer generation 1 behave rigidly, whereas those of the higher generation 4 exhibit viscoelastic behavior with an intermediate behavior being exhibited by 2 and 3. Using tapping-mode atomic force microscopy (AFM). we have been able to obtain molecularly resolved images of dendrimer 4 adsorbed on a Pt(111) electrode.