Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements.     

Characterization and manipulation of microscopic biochemically active regions by scanning electrochemical microscopy (SECM).

Preparation and characterization of microscopic biochemically active regions are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. Scanning electrochemical microscopy (SECM) has emerged as an ideal tool for prototyping such systems. The technique is based on electrochemical conversions of dissolved species at a micrometer-sized probe electrode. It offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activities. The working modes of the techniques are illustrated by recent studies from this laboratory for the design and characterization of patterned enzyme layers covalently linked to gold surfaces via thiol self-assembly chemistry.     

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 of enzymes immobilized on structured glass-gold substrates

Scanning electrochemical microscopy (SECM) was used to characterize enzyme-modified glass-gold specimens. The exposed gold surface was functionalized with an aminothiol and reacted with carbodiimide-activated glucose oxidase. The specimen surface was examined with SECM, using a 25 μm platinum electrode. Images were acquired showing the topography, electric conductivity, and enzymatic activity of the composite surface. It was found that the hydroxy-groups of the glass surface are as likely to bind to the activated enzyme as the amino-groups on the gold surface.     

Localized Deposition of Chitosan as Matrix for Enzyme Immobilization

A local electrodeposition method was developed for chitosan by exploiting a pH gradient between a macroscopic electrode (the support) and a much smaller counter electrode. The deposition was confined either by using the direct mode of scanning electrochemical microscopy (SECM) or by performing the deposition in channels of a microfluidic network. The roughness was characterized by noncontact scanning force microscopy. The availability of amino groups at the surface of the microstructures was visualized after labeling by confocal laser scanning microscopy. The enzyme glucose oxidase could be entrapped during the electrochemical deposition and showed activity as seen by SECM images.     

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.     

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.     

Tip current/positioning close-loop mode of scanning electrochemical microscopy for electrochemical micromachining

Scanning electrochemical microscopy (SECM) has been approved as a prospective electrochemical micromachining (ECMM) technique soon after its birth. However, it still remains challenge for SECM to fabricate arbitrary three-dimensional (3D) microstructures because of the limitation of positioning system. To solve this problem, we proposed a tip current signal/positioning close-loop mode in which the tip current signal is fed back to the positioning system in order to program the motion trial of SECM tip. Both the triedge-cone and sinusoidal microstructures were obtained by the close-loop positioning mode. The static-state etching process was demonstrated not to be disturbed by the slow motion rate of SECM tip. The unique positioning mode would be significant for both ECMM and electrochemical imaging.     

Scanning electrochemical microscopy for biosurface imaging

Scanning electrochemical microscopy (SECM) is a powerful technique that can provide chemical identity, quantification, and spatiotemporal information on biosurfaces. The ability of SECM for noninvasive and high-resolution electrochemical imaging has made it valuable for the study of cell phenotypes and functions. This review focuses on the latest advances of SECM technique for the biosurface imaging. The SECM measurements of different biomarkers, including oxygen consumption rate and enzyme activity of cell aggregates, redox state of cardiomyocytes, and bacterial metabolic activity, are introduced. The applicability of SECM on membrane permeability measurements, neurotransmitter measurements, and intracellular measurements is discussed.     

Localized Visualization of Chemical Cross-Talk in Microsensor Arrays by Using Scanning Electrochemical Microscopy

 An investigation of an array of four Pt microband electrodes 25 μm wide and spaced 25 μm apart was performed with the scanning electrochemical microscope (SECM). Where possible the SECM measurements were confirmed with conventional electrochemical measurements. It is shown how the sensiti- vity of the SECM recycling current to the activity of the underlying surface can be used to probe the homogeneity of enzyme-modified microelectrodes. The diffusion of H₂O₂ between these micro enzyme- electrodes and unmodified electrodes was investigated and it was demonstrated how the SECM can be a powerful tool in the elucidation of the properties of these electrodes. Received June 8, 1998. Revision November 12, 1998.     

Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope

Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator.     

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.     

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.     

Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope

Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator.     

Chemical imaging of biological systems with the scanning electrochemical microscope

A brief overview on recent advances in the application of scanning electrochemical microscopy (SECM) to the investigation of biological systems is presented. Special emphasis is given to the mapping of local enzyme activity by SECM, which is exemplified by relevant original systems.     

Immobilization of Glucose Oxidase on Cellulose／Cellulose Acetate Membrane and its Detection by Scanning Electrochemical Microscope （SECM）

Cellulose/cellulose acetate membranes were prepared and functionalized by introducingamino group on it, and then immobilized the glucose oxidase (Gox) on the functionalizd membrane.SECM was applied for the detection of enzyme activity immobilized on the membrane.Immobilized biomolecules on such membranes was combined with analysis apparatus and can beused in bioassays.     

Investigation of ion-bombarded conducting polymer films by scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) was used to investigate the effect of ion bombardment on thin films of the conducting polymers poly[3-ethoxy-thiophene] (PEOT) and poly[ethylenedioxy-thiophene] (PEDT). Bombardment with Ar+-ions converts the topmost 30 nm thick layer to an essentially insulating material. SECM approach curves as well as two dimensional scans prove the existence of regions of different conductivity within the irradiated regions that did not show a significant dependence on ion dosage. PEDT layers patterned by ion bombardment through microscopic masks are investigated as prototypes of miniaturized printed circuit boards that can be formed by galvanic copper deposition onto conducting PEDT. Defects in conducting polymer patterns were analyzed by SECM imaging before any deposition of copper. Appropriate representations of SECM images for the evaluation of this technologically important question are discussed.     

Development of Solid Contact Micropipette Zn-Ion Selective Electrode for Corrosion Studies

Micron-size ion selective micropipettes can be used in scanning electrochemical microscopy (SECM). They can provide excellent spatial resolution. Unfortunately the resistance of these small sensors is high. Their application needs special shielding and slow scanning rates. Usually their lifetime hardly exceeds a few days.

Zinc layer or dispersed zinc particles containing films are often used for providing cathodic protection against corrosion in case of metal surfaces. Therefore, in corrosion studies, measurements of local zinc ion concentration can give important information about the nature of the process. For corrosion studies we needed SECM measuring tips for imaging concentration profiles of Zn²⁺ions involved in surface processes. Based on our earlier experience, solid contact micropipettes for selective measurements of Zn²⁺ion concentration were prepared with a tip size of a few micrometers. The properties of the micropipettes were investigated. They were also used in SECM imaging. In this paper, details of Zn²⁺ion selective microelectrode preparation are described. Data about their properties, lifetime, resistance, and ion activity response are shown. Preliminary findings in SECM imaging of zinc ion concentration profiles are shown. The improvement of the scanning rate achieved by lowering tip resistance is a main advantage in potentiometric SECM.     

Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.     

Scanning electrochemical microscopy in the 21st century. Update 1: Five years after

This Perspective is an update to our more extensive survey of scanning electrochemical microscopy (SECM) published in 2007. During this time, the SECM field retained its momentum by expanding into new areas and meeting the emerging scientific and technological challenges. Here we focus on most prominent developments such as high-resolution imaging, investigation of structures and processes on the nanoscale, alternative energy applications, and new approaches to solving "real world" problems. The fabrication of novel SECM probes and related theoretical advances are also discussed.