A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements

A new formaldehyde-selective biosensor was constructed using NAD⁺- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilised on the surface of Si/SiO₂/Si₃N₄ structure. Sensor's response to formaldehyde was evaluated by capacitance measurements. The calibration curves obtained for formaldehyde concentration range from 10 μM to 20 mM showed a broad linear response with a sensitivity of 31 mV/decade and a detection limit about 10 μM. It has been shown that the output signal decreases with the increase of borate buffer concentration and the best sensitivity is observed in 2.5 mM borate buffer, pH 8.40. The response of the created formaldehyde-sensitive biosensor has also been examined in 2.5 mM Tris–HCl buffer, and the shift to the positive bias of the C(V) curves along with the potential axis has been observed, but the sensitivity of the biosensor in this buffer is decreased dramatically to the value of 2.4 mV/decade.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO₂ layer to increase the surface area available for immobilization.

The biosensor enabled the detection of 2.5 μM paraoxon, and 10 μM DFP and parathion, respectively. Very short response time of 30 s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 °C, the waveguide retained reasonable activity for greater than 45 days.     

Fabrication of a miniature CMOS-based optical biosensor

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm × 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H₂O₂ and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 μL. Both optical H₂O₂ and glucose biosensors have an optimal operation temperature and pH of 20–25 °C and pH 8.4, respectively. The linear dynamic range of optical H₂O₂ and glucose biosensors is 0.05–20 mM H₂O₂ and 0.5–20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

Development of amperometric horseradish peroxidase based biosensors for clozapine and for the screening of thiol compounds

Two amperometric biosensors with immobilized horseradish peroxidase (HRP) were developed for the investigation of the clozapine drug oxidation and for thiols screening based on biosensor signal inhibition. The HRP was retained either in magnetized nanoporous silica microparticles (MMPs) or in a carbon paste (CP). The latter served for the carbon paste electrode while the MMPs were attracted in close proximity of a magnetized carbon electrode. The potential use of these configurations for drug oxidation and inhibition studies was illustrated by the enzymatic oxidation of clozapine (CLZ) in the presence of hydrogen peroxide. The biosensor signal corresponded to the electro-reduction of CLZ oxidation products namely a nitrenium ion (CLZox) generated by the enzyme HRP. Several thiols reactive towards CLZox were investigated and the biosensor signal inhibition (IC(50)) was comparatively determined. A protective effect of the nanoporous silica and carbon paste matrices towards HRP inactivation was inferred by comparing the biosensor inhibition results with those obtained with the free enzyme in solution.     

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody–enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 μm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 μm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody–poly-HRP immunoconjugates contained an estimated number of 10⁵ HRP/streptavidin microparticle and 10⁶ HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 μm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5–8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.     

Effect of melanin on netilmicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

It is known that various drugs form complexes with melanins and that melanins are abundant constituents of the inner ear. In this study, we determined whether the aminoglycoside antibiotic, netilmicin, interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. The obtained results indicate that netilmicin forms stable complexes with melanin characterized by the association constants K₁  10⁶ M⁻¹ and K₂  10³ M⁻¹. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for aminoglycoside-induced inhibition of collagen biosynthesis. We found that netilmicin strongly induced inhibition of prolidase activity (IC₅₀ < 5 μM) and collagen biosynthesis (IC₅₀  10 μM). At 10 μM concentration of netilmicin, prolidase activity in human skin fibroblasts was inhibited by about 80% and DNA biosynthesis—only by about 25%. Melanin at 100 μg/mL produced about 30% inhibition of collagen biosynthesis and about 30% inhibition of prolidase activity in cultured fibroblasts. However, the addition of melanin (100 μg/mL) to netilmicin-treated cells (10 μM) restored the prolidase activity in fibroblasts to almost 100% of control values and partially reversed the inhibitory action of the drug on collagen and DNA biosynthesis. The data suggest that the ability of netilmicin to form stable complexes with melanin may prevent its toxicity on prolidase activity and collagen biosynthesis.     

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H₂O₂ in a choline solution at +0.6 V. The other one modified with ChO/HRP utilized the reduction process of H₂O₂ in a choline solution at −0.2 V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0×10⁻⁶ to 8.0×10⁻⁵ M and the other based on ChO/CPME from 1.0×10⁻⁶ to 5.0×10⁻⁵ M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0×10⁻⁷ and 4.0×10⁻⁷ M, respectively. The response time of sensors was less than 5 s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

Enzyme immunosensing of pepsinogens 1 and 2 by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) was applied to a dual enzyme immunoassay for the detection of pepsinogen 1 (PG1) and pepsinogen 2 (PG2). Sandwich-type immunocomplexes labeled with horseradish peroxidase (HRP) were constructed on microspots consisting of anti-PG1 IgG antibody and anti-PG2 IgG antibody. These microspots were fabricated on a hydrophobic glass substrate using a capillary microspotting technique. In the presence of H₂O₂ and ferrocenemethanol (FcOH; used as an electron mediator), the labeled HRP catalyzed the oxidation of FcOH by H₂O₂ to generate the oxidized form of FcOH (Fc⁺OH) at localized areas corresponding to microspots containing both immunocomplexes. The enzymatically generated Fc⁺OH was reduced and detected with a SECM probe (0.05 V versus Ag/AgCl), and the substrate surface was mapped to generate SECM images of the PG1 and PG2 spots. Relationships between the reduction current in the SECM images and the concentrations of PG1 and PG2 were obtained in the range 1.6–60.3 ng/ml protein. Dual imaging of PG1 and PG2 was achieved using microspots containing PG1 and PG2 immunocomplexes separated by a 200 μm physical barrier on the substrate. Pyramidal hole arrays with 100 μm × 100 μm openings on the silicon wafer were utilized to fabricate spots using antibodies on poly(dimethylsiloxane) (PDMS) membranes. Current responses obtained from microspots fabricated with pyramidal holes are significantly sharper compared to the responses obtained from spots fabricated using the capillary method.