Detection and Quantification of Sulfonamide Antibiotic Residues in Milk Using Scanning Electrochemical Microscopy

The use of scanning electrochemical microscopy (SECM) for the qualitative and quantitative determination of sulfapyridine (SPY) in milk is described. A direct competitive immunoassay was performed involving an antibiotic horseradish peroxidase (HRP)‐labeled analog and using selective capture antibodies immobilized on the surface of Protein G‐modified glassy carbon plates. SECM detection was accomplished by means of the sample generator/tip collector (GC) mode involving the reduction of benzoquinone (BQ) generated upon the HRP‐catalyzed oxidation of hydroquinone (HQ) at the modified substrate surface in the presence of H₂O₂. The detection limit for SPY in milk samples was as low as 0.13 ng mL^?1.     

In Situ Self Assembly of Thiolated ortho‐Quinone Capped Electrocatalysts for Bioanalytical Applications

Thiolated o‐quinone‐capped electrocatalysts modeled on the naturally occurring o‐quinone cofactor pyrroloquinoline quinone (PQQ) were designed and synthesized for the development of biosensor devices. The o‐quinone‐capped electrocatalysts self assembled on gold electrodes through a thiolated phenyleneethynylene linkage to form a monolayer less than 2 nm in thickness. Cyclic voltammetric measurements demonstrated reversible electrochemical properties between the quinone and hydroquinone forms of the head group. In an amperometric sensing mode, the modified electrodes reproducibly detected ethanethiol at micromolar levels demonstrating their robust electrocatalytic activity toward thiols. Their redox cycling and electrocatalytic properties show promise for detection of biologically important thiols and other nucleophiles.     

Scanning electrochemical microscopy study of ion transfer process across water/2-nitrophenyloctylether interface supported by hydrophobic carbon ceramic electrode

A carbon ceramic electrode (CCE) modified with the redox probe—decamethylferrocene solution in hydrophobic organic solvent—2-nitrophenyloctyl ether and immersed into an aqueous solution was studied by scanning electrochemical microscopy (SECM). After the electrochemical oxidation of decamethylferrocene, its cations were detected near the electrode surface in the aqueous phase. This indicates that some fraction of the redox-active cations electrochemically produced in the organic phase is transferred across the liquid/liquid interface. They are reduced at the SECM tip and form a solid deposit. The amount of deposited decamethylferrocene was estimated by the anodic reaction at the tip. It is affected by the substrate–tip distance, deposition time, and electrolyte concentration. The SECM images of unmodified and modified CCEs are consistent with their heterogeneous structure.     

Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process.     

Scanning electrochemical microscopy: Visualization of local electrocatalytic activity of transition metals hexacyanoferrates

The redox competition mode of scanning electrochemical microscopy (SECM) was used to visualize differences in local electrocatalytic activity of Fe and Ni hexacyanoferrates (HCFs) in hydrogen peroxide reduction. The uniform round-shaped spots of electrocatalysts for the SECM measurements were electrochemically deposited using a scanning droplet cell. A negligible activity of NiHCF towards H₂O₂ reduction compared to Prussian Blue (PB) was observed. The dependence of local Prussian Blue activity on the applied potential was investigated. The proposed strategy explores the potential application of SECM as a rapid screening tool for HCF film activity within a single experiment.     

Electrochemical investigation of thin PEDOT film above an insulating substrate using scanning electrochemical microscopy

Thin layer of conducting polymer, poly(3,4-ethylenedioxythiophene) PEDOT, deposited on insulating substrates was electrochemically investigated. This study was performed through the reaction with a series of electrogenerated mediators at a microelectrode operating in the configuration of a scanning electrochemical microscope (SECM). The method proves to be a convenient tool for investigating redox properties of the electroactive materials onto insulating substrate and the occurrence of electron transfers across the modified substrate. The SECM results demonstrate the possibility of the regeneration of the mediator at the modified surface even if the used substrate is an insulator. The regeneration rate depends on the standard redox potential of the mediator, on the switching potential of the polymer and on its initial oxidation state. In addition, the obtained data could be analyzed through the construction of the steady state voltammograms allowing the extraction of the electrochemical properties of the thin organic layer deposited onto insulating surface.     

Non‐destructive Patterning of Carbon Electrodes by Using the Direct Mode of Scanning Electrochemical Microscopy

Patterning of glassy carbon surfaces grafted with a layer of nitrophenyl moieties was achieved by using the direct mode of scanning electrochemical microscopy (SECM) to locally reduce the nitro groups to hydroxylamine and amino functionalities. SECM and atomic force microscopy (AFM) revealed that potentiostatic pulses applied to the working electrode lead to local destruction of the glassy carbon surface, most likely caused by etchants generated at the positioned SECM tip used as the counter electrode. By applying galvanostatic pulses, and thus, limiting the current during structuring, corrosion of the carbon surface was substantially suppressed. After galvanostatic patterning, unambiguous proof of the formation of the anticipated amino moieties was possible by modulation of the pH value during the feedback mode of SECM imaging. This patterning strategy is suitable for the further bio‐modification of microstructured surfaces. Alkaline phosphatase, as a model enzyme, was locally bound to the modified areas, thus showing that the technique can be used for the development of protein microarrays.     

Investigation of Active and Inactivated Yeast Cells by Scanning Electrochemical Impedance Microscopy

Scanning electrochemical microscopy (SECM), electrochemical impedance spectroscopy (EIS) and scanning electrochemical impedance microscopy (SEIM) were used to investigate electrochemical activity of active and inactivated yeast Saccharomyces cerevisiae cells. SEIM experiment was performed using a unique electrochemical impedance spectrometer with a fast Fourier transform (FFT‐EIS) function, which enabled simultaneously perturb/evaluate electrochemical system at 50 frequencies. This allowed very quick observing the differences between impedance spectra, which were taken every few seconds. Therefore, we were able to apply SEIM for relatively fast determination of electrochemical impedance dependence on the distance between ultramicroelectrode (UME) and surface modified by immobilized yeast cells. It was determined that electrochemical activity and ‘breathing’ (a consumption of dissolved oxygen) of yeast can be electrochemically observed when the distance between UME and surface of yeast cells is in the range from 0 μm to 25 μm. Therefore, 25 μm is the maximum distance suitable for efficient investigation of yeast cell activity when experiments are performed in FFT‐SEIM mode. Charge transfer resistance of active and inactivated yeast cells was determined using EIS. It was calculated that charge transfer resistance of active yeast cells is 1.5 times lower than that of inactivated yeast cells. Lipophilic vitamin K₃ (Vit‐K₃) and hydrophilic vitamin K₁ (Vit‐K₁) were mixtured and used as redox mediators for charge transfer from yeast cells.     

A Comparative Study of Functionalized High‐Purity Carbon Nanotubes towards the V(IV)/V(V) Redox Reaction Using Cyclic Voltammetry and Scanning Electrochemical Microscopy

Functionalized high purity carbon nanotubes (CNTs) with various amounts of oxygen containing surface groups were investigated towards the relevant redox reactions of the all‐vanadium redox flow battery. The quinone/hydroquinone redox peaks between 0.0 and 0.7 V vs. Ag|AgCl|KCl_sat. were used to quantifying the degree of functionalization and correlated to XPS results. Cyclic voltammetry in vanadyl sulfate‐containing 3 M H₂SO₄ as a common supporting electrolyte showed no influence of the amount of surface groups on the V(IV)/V(V) redox system. In contrast, the reactions occurring at the negative electrode (V(II)/V(III) and V(III)/V(IV)) are strongly affected by oxygen surface groups. However, under modified experimental conditions, SECM experiments detecting the consumption of VO²⁺ molecules by CNT thin films in pH=2 solution show improved onset potentials with increased surface oxygen content up to ∼ 3 at%. Further increase in surface oxygen up to 8 at% led to minor improvement. These dissimilar results under different experimental conditions are rationalized by suggesting that oxygen functional groups do not form the active site for the V(IV)/V(V) reaction but wetting of the catalyst layer is of high importance.     

Localized Deposition of Au Nanoparticles by Direct Electron Transfer through Cellobiose Dehydrogenase

Cellobiose dehydrogenase (CDH) is a fascinating extracellular fungal enzyme that consists of two domains, one carrying a flavin adenine dinucleotide (FAD) and the other a cytochrome‐type heme b group as cofactors. The two domains are interconnected by a linker and electrons can shuttle from the FAD to the heme group by intramolecular electron transfer. Electron transfer between CDH and an electrode can occur by direct electron transfer (DET) and by mediated electron transfer (MET). This characteristic makes CDH an interesting candidate for integration in systems such as biosensors and biofuel cells. Moreover, it makes CDH an alternative for the reduction of metal ions through DET and MET. In this work we have explored the localized deposition of gold on Pd substrates by CDH through DET and MET. For this purpose we exploited the advantage of scanning electrochemical microscopy (SECM) as a patterning tool. We first demonstrated that gold nanoparticles can be formed in homogenous solution. Then we showed that Au nanoparticles can also be locally formed and deposited on surfaces through DET at low pH and by MET at neutral pH using benzoquinone/hydroquinone as mediator.     

有机涂层／金属界面腐蚀的微区电化学

以扫描开尔文探针，局部交流阻抗和扫描电化学显微镜为代表的微区电化学技术以其高精度、高空间解析度的定域分析优势被广泛应用。本文简要介绍了这三种技术的测量原理和关键问题，并着重归纳了这些技术应用于有机涂层／金属界面腐蚀行为的研究成果，包括界面微区表观形貌的获取，不同界面腐蚀机制的推理验证及其影响因素的作用机理等，并对三种技...     

Evaluation of Redox Activity of Human Myocardium‐derived Mesenchymal Stem Cells by Scanning Electrochemical Microscopy

In this study the redox activity of human myocardium‐derived mesenchymal stem cells (hmMSC) were investigated by redox‐competition (RC‐SECM) and generation‐collection (GC‐SECM) modes of scanning electrochemical microscopy (SECM), using 2‐methylnaphthalene‐1,4‐dione (menadione, MD) as a redox mediator. The redox activity of human healthy and dilated hmMSCs was evaluated by measuring reduction of MD. Measurements were performed by approaching and retracting the UME from the surface of growing hmMSC cells. The current study shows that the RC‐SECM mode can be applied to investigate integrity of cell membranes, whereas the most promising results were observed by using the GC‐SECM mode and applying the Hill's equation for the calculation/fitting of dependencies of electrical current vs menadione concentration. The calculated apparent Michaelis constant (K_M) for the production of menadiol (MDH₂) in the pathological hmMSC cells was 14.4 folds higher compared to that of the healthy hmMSC revealing the lover redox activity of pathological cells. Moreover, the calculated Hill's coefficient n shows a negative cooperative binding between MD and healthy hmMSC and positive cooperative binding between MD and pathological hmMSC. It means that healthy hmMSC is of lower affinity to MD, which is also related to the better membrane integrity of healthy cells. Data of this study demonstrate that SECM can be applied to investigate intracellular redox and membrane changes ongoing in human dilated myocardium‐derived hmMSC in order to improve their functioning and further regenerative potential.     

Dielectrophoretic micropatterning with microparticle monolayers covalently linked to glass surfaces

Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 mum) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (DSS) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity.     

Scanning electrochemical microscopy for the direct patterning of a gold surface with organic moities derived from iodonium salt

The scanning electrochemical microscope (SECM) is used in the direct mode to draw patterns of a thin passivating organic layer on a gold electrode surface and to image them. The patterning is ensured by the local electrografting of the organic moieties obtained by reduction of an aryliodonium salt, as evidenced by XPS and SECM line scans. The resolution of the writing process is controlled by the charge injected.     

Electrodeposition of Catechol on Glassy Carbon Electrode and Its Electrocatalytic Activity Toward NADH Oxidation

Catechol can be oxidized electrochemically to its corresponding o‐benzoquinone. The electrogenerated quinone can be deposited by cycling the potential at the surface of glassy carbon electrodes. We have studied the electrochemical features of films derived from catechol by cyclic voltammetry. The electrodeposited film shows stable reversible redox response, dependent on pH as anticipated for quinone/catechol functionalities. Glassy carbon electrodes covered with a film derived from catechol exhibit catalytic activity in the electrooxidation of NADH at a low potential. The catalytic current is proportional to the concentration of NADH over the range 0.02–0.34 mM.     

Visualization of the Local Catalytic Activity of Electrodeposited Pt–Ag Catalysts for Oxygen Reduction by means of SECM

Pt–Ag nanoparticle co‐deposits with different Pt–Ag ratios were prepared on a glassy carbon (GC) surface by pulsed electrodeposition and investigated for their catalytic activity in electrocatalytic oxygen reduction by using cyclic voltammetry (CV), rotating disc electrode (RDE) and scanning electrochemical microscopy (SECM) in 0.1 M phosphate buffer (pH 7.0). The atomic composition of the Pt–Ag co‐deposits was studied by means of energy‐dispersive X‐ray analysis (EDAX). In combination with X‐ray diffraction (XRD), the presence of partly alloyed Pt and Ag on the GC surface was confirmed. Scanning electron microscopy (SEM) images indicate that the prepared Pt–Ag catalyst particles are homogenously dispersed over the GC surface. Their size and morphology depend on their composition. The electrocatalytic activity of Pt–Ag deposits with high Pt content was the highest, exceeding even that of electrodeposited Pt as evaluated by quantitative RDE analysis. The redox competition mode of scanning electrochemical microscopy (RC‐SECM) was successfully used to visualize the local catalytic activity of the deposited Pt–Ag particles. Semi‐quantitative assessment of the SECM results confirmed the same order of activity of the different catalysts as the RDE investigations.     

Voltammetry of Organic Microparticles

 Solid microparticles of several different insoluble organic compounds were mechanically immobilized on the surface of graphite electrodes and immersed into a liquid electrolyte in order to study their electrochemical reactions. Cyclic staircase voltammetry and square-wave voltammetry were used. Quinhydrone was found to be a stable intermediate in the reversible redox reaction of solid quinone and hydroquinone on the electrode surface. The reaction occurs on the surface of the solid particle which is in contact with water. Indigo can be reduced to leucoindigo and oxidized to dehydroindigo in two separate reversible redox reactions. In strongly basic medium indigo dissolves in water upon electroreduction. A hydroacridine radical was detected as a stable intermediate in the electrochemically irreversible redox reaction of acridine and dihydroacridine. Famotidine can be electrooxidized and the product of this reaction can be electroreduced in two separate chemically irreversible reactions. Probucol is oxidized to a semiquinone radical which can be re-reduced in an electrochemically irreversible redox reaction. Propyl- thiouracil can be also oxidized to an unknown product which can be re-reduced in a chemically reversible, but slow solid state surface redox reaction. Reductions of solid thionicotinoylanilide and nicotinoylanilide are totally irreversible. Received September 22, 1998. Revision March 19, 1999.     

Synthesis and characterization of an electroactive surface that releases gamma-aminobutyric acid (GABA)

We report the synthesis and characterization of a new electroactive surface capable of releasing the neurotransmitter gamma-aminobutyric acid (GABA) upon reduction. The GABA was anchored to an alkanethiol via electrochemically active quinone (abbreviation, TM-GABA). The quinone unit, upon reduction to the hydroquinone, cyclizes to release GABA into solution. The half-life is 99 s. The self-assembled monolayer (SAM) of TM-GABA on gold was prepared and characterized with several surface sensitive techniques. X-ray photoelectron spectroscopy (XPS) explored the SAM formation of TM-GABA on Au surfaces. Cyclic voltammograms showed the ability to electrochemically control the quinone unit at the distal end of the chain. GABA was selectively released upon electrochemical reduction at a potential of -700 mV. The functional GABA terminal group was detected by surface plasmon resonance measurements of anti-GABA antibody binding.     

Label‐Free Electrochemical Imaging of Latent Fingerprints on Metal Surfaces

A label‐free electrochemical method based on scanning electrochemical microscopy (SECM) has been developed to image latent fingerprints with high resolution on five kinds of metal surfaces (platinum, gold, silver, copper and stainless steel), as it could measure the minor conductivity differences of the substrate surface and avoid the interference of the background‐color. The images of sebaceous fingerprints on clean metals were revealed by SECM with ferrocene methanol acting as a redox mediator to detect the topology of the fingerprint deposits in constant‐height feedback mode. Inhibition of electrochemical processes on areas of the surface masked by the insulating fingerprint residues generated a negative image of the fingerprint.     

Evaluation of Enzymatic Kinetics of GOx‐based Electrodes by Scanning Electrochemical Microscopy at Redox Competition Mode

Glucose oxidase (GOx) is an enzyme, which is used for the development of enzymatic biofuel cells. Therefore in this research redox competition mode of scanning electrochemical microscopy (RC‐SECM) was applied for the investigation of glucose oxidase (GOx) catalyzed reaction kinetics. The GOx was immobilized by glutaraldehyde on substrates of different electrical conductivity: (i) gold covered glass was used as conducting substrate and (ii) plastic poly(methyl methacrylate) was used as non‐conducting substrate. Current vs distance dependencies were registered by SECM at different concentrations of glucose in the absence of redox mediator. The potential of −750 mV vs Ag/AgCl(3 M KCl) was applied to the microelectrode (ME), which was used as a probe in SECM, in order to register oxygen reduction current. Consumption of oxygen by the GOx based layer was evaluated according to principles determined by Michaelis‐Menten kinetics. Apparent Michaelis constants K _{M (app.)} were calculated from the dependencies of current vs glucose concentration. In both these cases the K _{M (app.)} value increased when the distance between ME and enzyme modified surface was increasing from 10 to 30 μm, while the K _{M (app.)} value decreased by increasing the distance from 30 to 60 μm.