Oxidation-reduction dynamics in layer-by-layer self-assembled redox polyelectrolyte multilayer modified electrodes

The oxidation-reduction dynamics of layer-by-layer (LbL) self-assembled redox polyelectrolyte multilayer films on electrodes has been studied by cyclic voltammetry, chrono-amperometry, electrochemical quartz crystal microbalance (EQCM), ellipsometry, and Fourier transform reflection-absorption infrared spectroscopy (FT-IRRAS). Thin layer electrochemistry with fast electron transfer at the underlying metal-film interface and charge propagation by electron hopping between adjacent redox sites in the finite thin film has been observed. An almost ideal cyclic voltammetry for a fixed number of redox sites in the thin surface film suggests that the multilayer can be fully oxidized and reduced in the time scale of the experiment (RT/vF > or = 0.05 sec). The electron hopping diffusion coefficient 3 x 10(-10) cm2 s(-1) was obtained from the chronoamperometric current transient and the ellipsometric thickness. Both cyclic voltammetry and potential step yield a surface osmium bipyridyl redox concentration of gamma Os = 4 x 10(-10) mol x cm(-2) for (PAH-Os)5(PVS)4 film. Exchange of ions and solvent occur simultaneously to the charge injection as revealed by the EQCM mass change and the ellipsometric thickness change. From the end-to-end mass-to-charge linear relationship, the molar mass of the ionic and neutral species exchanged largely exceeds the molar mass of any ions or solvent which suggests an important flux of solvent during redox switching. An initial "break in" effect is observed for the first oxidation-reduction cycles when a newly self-assembled film equilibrates with the electrolyte as charge is injected during the electrochemical perturbation.     

Combination of the quartz crystal microbalance and the electrochemical technique for the characterization of self-assembled monolayers of octadecylmercaptan on gold surfaces

Quartz crystal microbalance (QCM) technique has been applied to the direct mass measurement of a self-assembled monolayer of octadecylmercaptan (OM) on a gold surface deposited onto a quartz-crystal. Repeated rinsing of the modified electrodes with used solvent was conducted to get self-assembled monolayers of OM. From the resonance frequency change, 2.5 nm molecular occupied area of OM on gold was obtained. The advantage of the combination of QCM and electrochemical methods was emphasized for the characterization of self-assembled monolayers.     

Selective electrochemical sensing of glycated hemoglobin (HbA1c) on thiophene-3-boronic acid self-assembled monolayer covered gold electrodes

We report a novel concept of sensing glycated hemoglobin, HbA 1c, which is now the most important index for a long-term average blood glucose level, by first selectively immobilizing it on the thiophene-3-boronic acid (T3BA) self-assembled monolayer (SAM)-covered gold electrode by a selective chemical reaction with boronic acid. HbA 1c thus immobilized is then detected by the label-free electrochemical impedance spectroscopic (EIS) measurements with a redox probe, an equimolar mixture of K 3Fe(CN) 6 and K 4Fe(CN) 6, present. The rate of charge transfer between the electrode and the redox probe is shown to be modulated by the amount of HbA 1c in the matrix hemoglobin solution due to the blocking effect caused by the binding of HbA 1c with boronic acid. Both the formation of a well-defined T3BA-SAM on the gold surface and the chemical binding of its boronic acid with HbA 1c in solution were confirmed by quartz crystal microbalance, atomic force microscopy, and EIS experiments.     

Structure and thickness dependence of "molecular wiring" in nanostructured enzyme multilayers

Supramolecular organized multilayers composed of glucose oxidase (GOx) and osmium-derivatized poly(allylamine) redox polymer have been self-assembled electrostatically from Os-polyelectrolyte solutions of variable pH (5.5-8.8) leading to a decrease of the linear charge density in the PAH-Os with increasing pH. The layer-by-layer enzyme multilayers were studied by ellipsometry, quartz crystal microbalance, AFM, cyclic voltammetry, and electrocatalytic oxidation of beta-D-glucose. At higher adsorption solution pH, an increase in the film thickness, enzyme loading, and redox charge was observed. While the electrocatalytic response increases with the increase of the adsorption solution pH (decrease of the polyelectrolyte linear charge), the FADH2 oxidation bimolecular rate constant has a maximum in the pH range 7.0-7.5 where a change in the film growth mechanism is observed.     

Label-free detection of antibody-antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modified gold electrodes

Novel (R)-diaza-18-crown-6 has been prepared by a simple two-step synthetic method and characterized for its ability to form a uniform self-assembled monolayer (SAM) on gold as well as to immobilize proteins using atomic force microscopy, quartz crystal microbalance, and electrochemical impedance spectroscopy (EIS) experiments. The (R)-lipo-diaza-18-crown-6 was shown to form a well-defined SAM on gold, which subsequently captures the antibody (Ab) molecules that in turn capture the antigen (Ag) molecules. The Ab molecules studied include antibody C-reactive protein (Ab-CRP) and antibody ferritin (Ab-ferritin) along with their Ag's, i.e., CRP and ferritin. Quantitative detection of the Ab-Ag interactions was accomplished by EIS experiments with a Fe(CN)6(3-/4-) redox probe present. The ratios of the charge-transfer resistances for the redox probe on the SAM-antibody-covered electrode to those with the antigen molecules attached show an excellent linearity for log[Ag] with lower detection limits than those of other SAMs for the electrochemical sensing of proteins.     

Layer-by-layer assembly of poly(p-xylyleneviologen)–DNA multilayers and their electrochemical properties

Multilayers of poly(p-xylyleneviologen) (PXV) and calf thymus DNA were constructed on the gold surface by layer-by-layer (LBL) method. The assembly process was examined by quartz crystal microbalance (QCM) measurements. According to the frequency change, the average mass increase was estimated to be about 97 and 110 ng/cm² for each assembly of PXV and DNA layer, respectively. Cyclic voltammograms of the multilayer modified gold electrodes showed a couple of redox peaks, the potentials of which were closely dependent on the layer numbers and nature of the outmost layer. The alternatively assembled DNA layers could hinder formation of π-complex dimer of viologen groups due to the strong interlayer electrostatic interaction. The charge transfer process was discussed by the chronocoulometry method. The assembled PXV–DNA multilayers showed high long-term stability at ambient conditions and in electrolyte solution.     

Enzyme-modulated cleavage of dsDNA for supramolecular design of biosensors.

Supramolecular docking and immobilization of biotinylated dsDNA onto a self-assembled monolayer of avidin have been measured using impedance spectroscopy and quartz crystal microbalance technique. The formation of the serial assembly was first achieved by linearizing circular plasmid dsDNA using BamH I endonuclease enzyme. This was followed by a bisulfite-catalyzed transamination reaction in order to biotinylate the dsDNA. The reaction is single-strand specific, and it specifically targets unpaired cytosine bases generated during the enzyme cleavage. The biotinylated dsDNA was then used as a ligand at a gold electrode containing avidin. The process was monitored by ac impedance spectroscopy that was used to probe the changes in interfacial electron-transfer resistance upon binding and a microgravimetric quartz crystal microbalance that reflected in situ mass changes on the dsDNA-functionalized substrates. Our results demonstrated that this approach could be employed for the determination of small-molecular-weight organics such as cisplatin, daunomycin, bisphenol A, chlorinated phenols, and ethidium bromide. A detection limit in the magnitude of ca. 10 nM was achieved. This immobilization technique provides a generic approach for dsDNA-based sensor development and for the monitoring of DNA-analyte interactions.     

Electrochemical and morphological studies of an electroactive material derived from 3-hydroxyphenylacetic acid: a new matrix for oligonucleotide hybridization

This paper describes the formation of polymeric films derived from 3-hydroxyphenylacetic acid electropolymerized onto graphite electrodes through cyclic voltammetry. We observed the formation of an electroactive material over the electrode surface. The modified electrode showed significant blocking behavior to electron transfer reaction of the pair redox ferricyanide/ferrocyanide, indicating repulsion electrostatic with the negatively charged carboxylate groups of the polymer. The quasi-reversible behavior to Ru(NH₃)₆Cl₂ suggests electrostatic attraction, facilitating the charge transfer. The modified electrode was studied through electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, and atomic force microscopy. These analyses indicate modification of the graphite electrode. Surface analysis by AFM showed that the morphology of the modified electrode surface presents globular form, randomly distributed, and formed by lower globules with diameter near 100 nm. Immobilization and hybridization of oligonucleotide onto the modified electrode were successfully carried out by using both direct electrochemical oxidation of nitrogenated bases and the redox electroactive indicator methylene blue.     

Impedance spectroscopy and affinity measurement of specific antibody–antigen interaction

We use impedance spectroscopy technique to develop a rapid and sensitive antigen biosensor. The biosensor is based on the immobilization of antibodies onto an 11-Mercaptoundecanoic acid self-assembled monolayer. The high insulating properties of the thiol can be measured with cyclic voltammetry and impedance spectroscopy with a redox couple. The, the modified gold electrode is activated with 1-ethyl-3 (3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) ester for antibody immobilisation. The affinity interaction of the antigen–antibody can be measured with quartz crystal microbalance technique. Dissociation and association constant are determined by applying a Fortran program fitting for the experimental data.     

石英晶体微天平质量检测量研究

基于石英晶体微天平质量检测原理,通过分析石英谐振器表面的振动位移,得出决定其电极表面每一点处质量灵敏度的物理量为该点处的振动加速度。在此基础上,基于石英谐振器的巴特沃斯-范·戴克等效电路模型提出一种计算石英晶体谐振器表面振荡幅度的方法,最终得到石英晶体微天平在整个检测过程中能检测到的平均质量。用10MHz的石英晶体微天平进行了数值验证,计算值与其理论上能达到的pg级检测限基本相吻合。     

Copper(II) nanosensor based on a gold cysteamine self-assembled monolayer functionalized with salicylaldehyde

Fabrication and electrochemical characterization of a novel nanosensor for determination of Cu2+ in subnanomolar concentrations is described. The sensor is based on gold cysteamine self-assembled monolayer functionalized with salicylaldehyde by means of Schiff's base formation. Cyclic voltammetry, Electrochemical impedance spectroscopy (EIS), and electrochemical quartz crystal microbalance were used to probe the fabrication and characterization of the modified electrode. The sensor was used for quantitative determination of Cu2+ by the EIS in the presence of parabenzoquinone in comparison with stripping Osteryoung square wave voltammetry (OSWV). The attractive ability of the sensor to efficiently preconcentrate trace amounts of Cu2+ allowed a simple and reproducible method for copper determination. A wide range linear calibration curve was observed, 5.0 x 10(-11)-5.0 x 10(-6) and 5.0 x 10(-10)-5.0 x 10(-6) M Cu2+, by using the EIS and OSWV, respectively. Moreover, the sensor presented excellent stability with lower than 10% change in the response, as tested for more than three months daily experiments, and a high repeatability with relative standard deviations of 6.1 and 4.6% obtained for a series of eight successive measurements in 5.0 x 10(-7) M Cu2+ solution, by the EIS and OSWV, respectively.     

Study of nickel passivation in a borate medium

The adsorption of borate ions at the nickel and/or nickel oxide-electrolyte electrochemical interface was studied at various concentrations and pH values in lithium and borate solutions. First, the passivation range of nickel was estimated using cyclic voltammetry. The nickel passive layer formation kinetics (transfer resistance, capacitance of passive film formed, adsorption capacitance), as well as the semiconducting properties of this oxide layer, were studied using electrochemical impedance spectroscopy (E.I.S.). These electrochemical techniques were used in conjunction with adsorption measurements performed with an electrochemical quartz crystal microbalance (E.Q.C.M.) and with surface analyses (Auger spectroscopy). The nickel oxide showed type p semiconducting properties and was depleted at, corrosion potential. Moreover, very little borate adsorption was observed during the different tests. This may have been the result of the negative surface charge, in the pH and potential conditions applied.     

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.     

Preparation of low-crystalline apatite nanoparticles and their coating onto quartz substrates

We prepared low-crystalline apatite nanoparticles and coated them onto a surface of a Au/Cr-plated quartz substrate by the electrophoretic deposition (EPD) method or by using a self-assembled monolayer of 11-mercaptoundecanoic acid (SAM method). Low-crystalline apatite nanoparticles around 10?nm in size with extremely low contents of undesirable residual products were obtained by adding (NH(4))(2)HPO(4) aqueous droplets into a modified synthetic body fluid solution that contained Ca(CH(3)COO)(2). The apatite nanoparticles were successfully coated by either the EPD method or the SAM method; the nanoparticle coating achieved by the SAM method was more uniform than that achieved by the EPD method. The present SAM method is expected to be a promising technique for obtaining a quartz substrate coated with apatite nanoparticles, which can be used as a quartz crystal microbalance device.     

Electrochemical behavior of polyphenol oxidase immobilized in self-assembled structures layer by layer with cationic polyallylamine

We report here a novel bioelectrode based on self-assembled multilayers of polyphenol oxidase intercalated with cationic polyallylamine built up on a thiol-modified gold surface. We use an immobilization strategy previously described by Hodak J. et al. (Langmuir 1997, 13, 2708-2716) Quartz crystal microbalance with electroacustic impedance experiments were carried out to follow quantitatively the multilayer film formation. The response of the self-assembly polyphenol oxidase-polyallylamine electrodes toward different metabolically related catecholamines was studied, to evaluate enzyme kinetics. For the analyzed compounds, only dopamine and its metabolite Dopac gave catalytic currents at applied potential close to 0 V. These responses were proportional to the number of polyphenol oxidase-immobilized layers and were also controlled by the enzymatic reaction. The combination of microgravimetric and electrochemical techniques allowed us to determine the kinetic enzymatic constants, showing that the decomposition rate for the enzyme-substrate complex is slower than the enzymatic reoxidation step.     

Electrochemical quartz crystal impedance study of redox hydrogel mediators for amperometric enzyme electrodes

Quartz crystal impedance around the resonant frequency at 10 MHz of a composite quartz crystal resonator has been studied simultaneously with cyclic voltammetry. A modified quartz crystal with a redox hydrogel (poly(allylamine)-ferrocene cross-linked with glucose oxidase) and immersed in liquid electrolyte was used. Impedance parameters (R(f) and X(L)((f))) of the surface redox gel film were obtained by fitting the resonator transfer function |V(o)/V(i)| vs ω to a BVD equivalent circuit and analyzed with the multiple nonpiezoelectric layer model of Martin. Two limiting hydrogel layers of the same composition were studied while oxidizing and reducing the ferrocene/ferricenium moieties attached to the swollen polymer backbone: thin and thick redox hydrogel films. For the thin films, the Sauerbrey approximation was valid. The mass/thickness and film viscosity changes that resulted from the anion and water exchange were evaluated while redox switching the polymer on the assumption of negligible storage modulus G' and a density of 1. For thick gel layers, on the other hand, the penetration depth of the acoustic wave was far less than the film thickness, and a liquid-like behavior was apparent. Film storage modulus and film loss modulus were simultaneously evaluated with the cyclic voltammetry.     

Contributions of amplitude measurement in QCM sensors

The characteristics of the amplifier and the feedback loop of a quartz crystal series oscillating circuit are investigated. The fact that the change of the vibration amplitude of the quartz crystal is proportional to the change of its motional resistance is derived. The concept of a characteristic damping coefficient is introduced and the behavior of a quartz crystal vibrating in liquids is analyzed. The experiment shows that the effect of mass loading can be distinguished from that of the liquid damping of a quartz crystal microbalance (QCM) sensor in liquids by simultaneously measuring the amplitude and the frequency change     

Small mass-change detectable quartz crystal microbalance and its application to enzymatic one-base elongation on DNA

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.     

A biotinylated conducting polypyrrole for the spatially controlled construction of an amperometric biosensor.

A new biotin derivative functionalized by an electropolymerizable pyrrole group has been synthesized. The electrooxidation of this biotin pyrrole has allowed the formation of biotinylated conducting polypyrrole films in organic electrolyte. Gravimetric measurements based on a quartz crystal microbalance, modified by the biotinylated polymer, revealed an avidin-biotin-specific binding at the interface of polymer-solution. The estimated mass increase corresponded to the anchoring of 1.5 avidin monolayers on the polypyrrole surface. In addition, the subsequent grafting of biotinylated glucose oxidase was corroborated by electrochemical permeation studies. Enzyme multilayers composed of glucose oxidase or polyphenol oxidase were elaborated on the electrode surface modified by the biotinylated polypyrrole film. The amperometric response of the resulting biosensors to glucose or catechol has been studied at +0.6 or -0.2 V vs SCE, respectively.     

Performances and limits of a parallel oscillator for electrochemical quartz crystal microbalances

This paper describes a driving circuit for an electrochemical quartz crystal microbalance (EQCM) adapted to a wide range of applications. The oscillator is a Miller-type parallel oscillator using an operational transconductance amplifier (OTA). A theoretical study of the oscillating circuit led to the analytical expression of the microbalance frequency as well as to an overestimation of the error on the mass measurement. The reliability of the EQCM was then experimentally verified through electrochemical copper deposition and dissolution. The limit of operation of the EQCM was also investigated, both analytically and experimentally. This work shows that parallel oscillators using few electronic components allow a very reliable EQCM to be obtained for mass measurements on metallic films, even if they are highly damped.