Fabrication of Active Horseradish Peroxidase Micropatterns with a High Resolution by Scanning Electrochemical Microscopy

We used a new reactive species OH^? to fabricate active horseradish peroxidase (HRP) micropatterns with a high resolution by scanning electrochemical microscopy (SECM) coupled with a carbon fiber disk electrode as the SECM tip. In this method, except for active HRP micropatterns predesigned other regions on a HRP‐immobilized substrate were deactivated by OH^? generated at the tip held at ?1.7 V in 1.0 mol/L KCl containing 2.0×10^?3 mol/L benzoquinone (BQ) (pH 8.0). The feedback mode of SECM with a tip potential of ?0.2 V was used to characterize the active HRP micropatterns in 1.0 mol/L KCl containing 2.0×10^?3 mol/L BQ and 2.0×10^?3 mol/L H₂O₂.     

Scanning Electrochemical Microscopy (SECM) Based Detection of Oligonucleotide Hybridization and Simultaneous Determination of the Surface Concentration of Immobilized Oligonucleotides on Gold

The direct mode of scanning electrochemical microscopy (SECM) was used for the local deposition of oligonucleotide (ODN) patterns on thin gold films and the generation‐collection (GC) mode was applied for the determining the amount of surface‐accessible oligonucleotides. The local deposition was achieved through the micrometer‐sized formation of a conducting polymer bearing 15^mer single‐stranded oligonucleotide strands. After the interaction of the oligonucleotide with its biotin‐labeled complimentary strand, streptavidin was bound. The molecular assembly was completed by linking biotin‐labeled β‐galactosidase from Escherichia coli to the streptavidin. The activity of the linked β‐galactosidase was mapped with SECM in the GC mode by monitoring the oxidation of p‐aminophenol (PAP) formed in the enzyme‐catalyzed hydrolysis of p‐aminophenyl‐β‐D ‐galactopyranoside. The feedback effect due to recycling of the reaction product at the gold surface was analyzed. It was shown experimentally that this effect becomes insignificant at ultramicroelectrode (UME)‐substrate distances larger than 3 UME radii. The flux of formed PAP allowed the determination the surface density of accessible oligonucleotide strands in the functionalized polymer. It was shown that that thicker pyrrole/ODN–Pyrrole polymer films do not lead to a significantly increased accessible ODN surface concentration.     

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.     

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.     

Evaluation of Thin Film Titanium Nitride Electrodes for Electroanalytical Applications

Titanium nitride is a hard and inert conducting material that has yet not been widely used as electrode material for electroanalytical applications although there are highly developed protocols available to produce well adherent micro and nanostructured electrodes. In this paper the possibilities of using titanium nitride thin films for electroanalytical applications is investigated. Scanning electrochemical microscope (SECM) was used for analysis of the redox kinetics of a selected fast redox couple at thin films of titanium nitride (TiN) in different thicknesses. The investigation was carried out by approaching an amperometric ultramicroelectrode (UME) to the TiN film while the soluble redox couple (ferrocenemethanol/ferrociniummethanol) served as mediator in a SECM configuration. The substrate was biased at a potential so that it rereduces the species being produced at the UME, thus controlling the feedback effect. Normalized current–distance curves were fitted to the theoretical model in order to find the apparent heterogeneous standard rate constant (k°) at the sample. The data are further supported by structural investigation of the TiN films using scanning force microscopy and X‐ray photoelectron spectroscopy. It was found that the kinetics are little influenced by prolonged storage in air. The heterogeneous standard rate constants in 2 mM ferrocenemethanol were (0.73±0.05)×10^?3 cm s^?1 for 20 nm TiN thin layer, (1.5±0.2)×10^?3 cm s^?1 for 100 nm TiN thin layer and (1.3±0.2)×10^?3 cm s^?1 for 300 nm TiN thin layer after prolonged storage in air. Oxidative surface treatment (in order to remove organic adsorbates) decreased the kinetics in agreement with a thicker oxide layer on the material. The results suggest that their direct use for amperometric detection of reversible redox systems in particular at miniaturized configurations may be advantageous.     

Electrochemical Investigation of Cytochrome c Immobilized onto Self‐Assembled Monolayer of Captopril

The electrochemical behavior of cytochrome c (cyt‐c) that was electrostatically immobilized onto a self‐assembled monolayer (SAM) of captopril (capt) on a gold electrode has been investigated. Cyclic voltammetry, scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy were employed to evaluate the blocking property of the capt SAM. SECM was used to measure the bimolecular electron transfer (ET) kinetics (k_BI) between a solution‐based redox probe and the immobilized protein. In addition, the tunneling ET between the immobilized protein and the underlying gold electrode was calculated. A k_BI value of (5.0±0.6)×10⁸ mol^?1 cm³ s^?1 for the bimolecular ET and a standard tunneling rate constant (k⁰) of 46.4±0.2 s^?1 for the tunneling ET have been obtained.     

Scanning Electrochemical Microscopy for Electrochemical Detection of Single‐base Mismatches by Tagging Ferrocenecarboxylic Acid as a Redox Probe to DNA

Scanning electrochemical microscopy (SECM) was employed for sensitive detection of single base mismatches (SBMs) in a sandwiched dsDNA. Ferrocenecarboxylic acid (Fc), covalently conjugated to the dsDNA, was oxidized to Fc⁺ via the DNA‐mediated charge transfer from the underlying gold substrate, and reduced back to Fc by SECM tip generated ferrocyanide. The electrocatalytic oxidation of SECM tip‐generated ferrocyanide was sensitive to presence, as well as the type of SBMs. Apparent standard rate constants (k⁰_app) values for different SBMs, both near the electrode surface and far from it, were evaluated by SECM. The method can detect SBMs independent of their position in dsDNA.     

Study on Electrocatalytic Activity of Platinum for Hydrogen Oxidation in Acidic Solution by Scanning Electrochemical Microscopy

The electrocatalytic activity of platinum for hydrogen oxidation in 0.01 M H₂SO₄ + 0.1 MNa₂SO₄ solution has been investigated by scanning electrochemical microscopy (SECM) technique. The cyclic voltammogram (CV), approach curve, area scan imaging and chronoamperometric methods have been used. The results indicate that the imaging capability of the SECM feedback mode can be used more efficiently to visually identify materials' electrocatalytic activity, compared with the approach curve method for identification of the conductive or insulating nature of a surface. The SECM imaging method has demonstrated the effects of Pt substrate potential on the electrocatalytic oxidation of hydrogen under a constant tip potential. It is found that the more positive the Pt substrate potential, the lower the electrocatalytic activity of the Pt. Furthermore, the chronoamperometric results support the variation of the electrocatalytic activity with the Pt substrate potential as well.     

Probing TCNQ‐mediated Metal Reduction Reactions at Liquid‐liquid Interface with SECM

Metal reduction at the interface between two immiscible electrolyte solutions (ITIES) has been studied with scanning electrochemical microscopy (SECM). Metal cations in the aqueous phase are reduced by 7,7,8,8‐tetracyanoquinodimethane anion (TCNQ^?) residing in the oil phase, methyl isobutyl ketone (MIBK). TCNQ^? is formed at the SECM tip by reducing TCNQ, which results in a positive feedback loop between the tip and the ITIES when an electron is donated to a metal cation. The effect of the Galvani potential difference on the rate of the interfacial electron transfer was investigated, establishing the potential difference either by an additional substrate electrode in the aqueous phase or by an a common ion in both phases. It is shown that the Galvani potential difference as a driving force does enable TCNQ^? mediated Cu²⁺ reduction. Finite element method (FEM) simulations were run to provide information on the reaction kinetics and stoichiometry.     

Controllable Release and High‐Efficiency Collection of Hydrogen Peroxide: Application on the Quantitative Investigation of Biomolecule Oxidation Induced by Reactive Oxygen Species

Hydrogen peroxide and hydroxyl radical, both important members of the reactive oxygen species (ROS) family, can cause serious oxidative damages in biological systems. In order to proclaim and prevent oxidation stress, researches on the biomolecule oxidation induced by H₂O₂ or OH^. are in crucial need. However, due to the high reactivity of ROS, traditional methods are difficult to achieve the in situ quantitative investigations on those reactions involving ROS. In this work, using scanning electrochemical microscopy technique (SECM) in a tip generation‐substrate collection mode (TG‐SC), the controllable release and the high‐efficiency collection of electrogenerated H₂O₂ were achieved. Compared to ex situ fluorescence method, SECM improved the collection efficiency approximately two times larger. Based on it, SECM combined with surface plasmon resonance (SPR) was employed to in situ monitor the protein oxidation (taking Cu₁₂⁺? MT as a model) induced by H₂O₂. OH^., which was generated from the interaction between H₂O₂ and Cu₁₂⁺? MT, can attack the peptide chain and induced the unrepairable protein oxidation damage. The whole process was quantitatively characterized by SPR, and the linear relationship between SPR dip shift and the amounts of released H₂O₂ was successfully built. Our work proves that the combined SECM‐SPR technique can realize the in situ quantitative determinations of the biomolecule oxidation induced by ROS, which affords an avenue for further elucidation on the mechanisms of oxidation stress in organisms.     

Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Direct Nanomachining on Semiconductor Wafer By Scanning Electrochemical Microscopy

Scanning electrochemical microscopy (SECM) is one of the most important instrumental methods of modern electrochemistry due to its high spatial and temporal resolution. We introduced SECM into nanomachining by feeding the electrochemical modulations of the tip electrode back to the positioning system, and we demonstrated that SECM is a versatile nanomachining technique on semiconductor wafers using electrochemically induced chemical etching. The removal profile was correlated to the applied tip current when the tip was held stationary and when it was moving slowly (<20 μm s⁻¹), and it followed Faraday's law. Both regular and irregular nanopatterns were translated into a spatially distributed current by the homemade digitally controlled SECM instrument. The desired nanopatterns were “sculpted” directly on a semiconductor wafer by SECM direct-writing mode. The machining accuracy was controlled to the sub-micrometer and even nanometer scales. This advance is expected to play an important role in electrochemical nanomachining for 3D micro/nanostructures in the semiconductor industry.     

Local deposition and characterisation of K2Co[Fe(CN)6] and K2Ni[Fe(CN)6] by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is used to form local deposits of different Prussian blue analogs on macroscopic surfaces of gold and glassy carbon. Dissolution of Co and Ni sacrificial ultramicroelectrodes (UMEs) generates divalent cations in the gap between the UME and the macroscopic specimen electrode. Co²⁺ or Ni²⁺ precipitate with [Fe(CN)₆]^4– formed by reduction of [Fe(CN)₆]^3– at the macroelectrode. By moving the UME while generating Co²⁺ or Ni²⁺, lines can be "drawn" with a width of 130 μm. The line width can be adjusted by reagent concentration and translation speed of the UME. Different pulse programs allow the formation of ring-shaped structures. The deposited hexacyanoferrate microstructures show catalytic activity for the reduction of Fe³⁺ which was imaged in the feedback and generation-collection modes of the SECM. Electronic Publication     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

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.     

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.     

Local Activities of Hydroxide and Water Determine the Operation of Silver‐Based Oxygen Depolarized Cathodes

Local ion activity changes in close proximity to the surface of an oxygen depolarized cathode (ODC) were measured by scanning electrochemical microscopy (SECM). While the operating ODC produces OH^? ions and consumes O₂ and H₂O through the electrocatalytic oxygen reduction reaction (ORR), local changes in the activity of OH^? ions and H₂O are detected by means of a positioned Pt microelectrode serving as an SECM tip. Sensing at the Pt tip is based on the pH‐dependent reduction of PtO and obviates the need for prior electrode modification steps. It can be used to evaluate the coordination numbers of OH^? ions and H₂O, and the method was exploited as a novel approach of catalyst activity assessment. We show that the electrochemical reaction on highly active catalysts can have a drastic influence on the reaction environment.     

High Sensitive Microsensor Based on Organic‐Inorganic Composite for Two‐Dimensional Mapping of H2O2 by SECM

The present work describes the development of a selective, sensitive and stable sensing microsensor for scanning electrochemical microscopy (SECM) to measure H₂O₂ during electrochemical reduction of oxygen. The microsensor is based on graphene and Poly(3,4‐ethylenedioxythiophene) composite as support to iron (III) hexacyanoferrate (II) (PEDOT/graphene/Fe^III₄[Fe^II(CN)₆]₃ microsensor). The electrochemical properties of the PEDOT/graphene/Fe^III₄[Fe^II(CN)₆]₃ microsensor were investigated by cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM). The PEDOT/graphene/Fe^III₄[Fe^II(CN)₆]₃ microsensor showed an excellent electrocatalytic activity toward hydrogen peroxide (H₂O₂) reduction with a diminution of the overpotential of about 500 mV in comparison to the process at a bare gold microelectrode. The microsensor presented excellent performance for two dimensional mapping of H₂O₂ by SECM in 0.1 mol L^?1 phosphate buffer solution (pH 7.0). Under optimized conditions, a linear response range from 1 up to 1000 µmol L^?1 was obtained with a sensitivity of 0.08 nA L µmol^?1 and limit of detection of 0.5 µmol L^?1.     

Electro‐oxidation and reduction of H2 on platinum studied by scanning electrochemical microscopy for the purpose of local detection of H2 evolution

Electrochemical detection of H₂ using scanning electrochemical microscopy (SECM) has shown to hold great promise as a sensitive characterization method with high spatial resolution for active surfaces generating H₂. Herein, the factors contributing to the current that is measured by SECM in generation/collection mode for H₂ detection are studied. In particular, the concentration gradient of H₂ at the substrate, the H₂/H⁺ recycling between the SECM tip and substrate and hemispherical profile of H₂ diffusion has been discussed. It was postulated that H₂/H⁺ recycling plays a dominant role in the oxidative current measured in generation/collection mode of SECM when the microelectrode is positioned in close vicinity of substrate. Copyright © 2015 John Wiley & Sons, Ltd.     

Scanning Electrochemical Microscopy as an In Vitro Technique for Measuring Convective Flow Rates Across Dentine and the Efficacy of Surface Blocking Treatments

Scanning electrochemical microscopy (SECM) is shown to be a powerful technique for both the measurement of local solution velocities through human dentine slices, in vitro, and for assessing quantitatively the effect of surface treatments on the flow process. SECM employs a small ultramicroelectrode (micron dimensions) as an imaging probe to provide information on the topography and transport characteristics of dentine, with high spatial resolution. In these studies the dentine sample is a membrane in a two compartment cell, which contains solutions of identical composition, including a redox active mediator (Fe(CN) . In the absence of an applied pressure, the transport‐limited current response at the probe electrode is due to diffusion of Fe(CN) to the UME, which depends on the probe to sample separation. Under an applied hydrostatic pressure, hydrodynamic flow across the sample enhances mass transport to the UME. With this methodology it was possible to accurately measure effective fluid velocities, by recording tip currents with and without pressure, and assess the efficacy of potential flow retarding agents for the treatment of dentinal hypersensitivity. For native dentine, the solution velocity was found to vary dramatically with location on the sample. The application of a glycerol monooleate ‐ base paste treatment to the surface of dentine was found to lower local flow velocities significantly. This electroanalytical methodology is simple to implement and is generally applicable to assessing the efficacy and mode of action of a wide variety of potential fluid flow retarding agents.