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An amperometric biosensor for the determination of glycated hemoglobin in human whole blood is proposed. The principle is based on the electrochemical measurement of ferroceneboronic acid (FcBA) that has been specifically bound to the glycated N-terminus. Hemoglobin is immobilized on a zirconium dioxide nanoparticle modified pyrolytic graphite electrode (PGE) in the presence of didodecyldimethylammonium bromide (DDAB). The incubation of this sensor in FcBA solution leads to the formation of an FcBA-modified surface due to the affinity interaction between boronate and the glycated sites of the hemoglobin. The binding of FcBA results in well-defined redox peaks with an E-0' of 0.299 V versus Ag/AgCl (1 M KCl). The square wave voltammetric response of the bound FcBA reflects the amount of glycated hemoglobin at the surface. This signal increases linearily with the degree of glycated hemoglobin from 6.8 to 14.0% of total immobilized hemoglobin. The scheme was applied to the determination of the fraction of glycated hemoglobin in whole blood samples.
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by similar to 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold
The binding of acetylcholinesterase (AChE) to a propidium-modified piezoelectric quartz crystal and its surface enzymatic activity have been investigated. Propidium binds to a site remote to the active center of AChE - the peripheral anionic site (PAS) - which is located on the rim of the gorge to the active site. The gold electrodes of the quartz crystal were first modified with 11-mercaptoundecanoic acid to which propidium was coupled. AChE binding was monitored by a quartz crystal nanobalance (QCN), followed by amperometric activity evaluation of the AChE loaded on the sensor. Interestingly, the binding is strong but does not inhibit AChE. However, an excess of propidium in solution inhibits the immobilized enzyme. The surface enzymatic activities observed depend on the amount of enzyme and differ according to the type and species, i.e. number of enzyme subunits (Electrophorus electricus tetrameric, Drosophila melanogaster mono- and dimeric form - DmAChE). The operational stability and regeneration, effect of propidium in solution and detection limit for substrate for various AChEs were investigated amperometrically.
Binding or catalysis? Both can be distinguished with a molecularly imprinted polymer (MIP) by the different patterns of heat generation. The catalytically active sites, like in the corresponding enzyme, generate a steady-state temperature increase. Thus, enzyme-like catalysis and antibody-analogue binding are analyzed simultaneously in a bifunctional MIP for the first time (see scheme).
A piezoelectric biosensor has been developed on the basis of the reversible acetylcholinesterase (AChE) inhibitor propidium. The propidium cation was bound to a 11-mercaptoundecanoic acid monolayer on gold-coated quartz crystals. The immobilization was done via activation of carboxyl groups by 1,3-dicyclohexylcarbodiimide (DCC). Different types of cholinesterases (acetyl- and butyryl-ChE) from different origins were tested for their binding ability towards the immobilized propidium. Binding Studies were performed in a flow system, Furthermore, catalytically active and organophosphate-inhibited enzyme were compared re-aiding their binding capability. The binding constants were derived by using an one to one binding model and a refined model also including rebinding effects. It was shown that organophosphorylation of the active site hardly influences the affinity of AChE towards propidium. Furthermore the propidium-based biosensor provides equal sensitivity as compared with piezolelectric sensors with immobilized paraoxon- an active site ligand of AChE. (c) 2005 Elsevier B.V. All rights reserved
We summarize here the development of various piezoelectric biosensors utilizing cholinesterase (ChE) as the recognition element. In our work we studied the interaction between cholinesterase and its ligands (propidium, carnitine, benzylgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) and paraoxon). The sensor modification was based on a self-assembled monolayer (SAM) of a thiol compound (11-mercaptoundecanoic acid) on the gold electrode and the subsequent covalent coupling of the cholinesterase ligand to this SAM. The ligand-modified piezoelectric sensors were placed in a flow system to allow the on-line monitoring of cholinesterase binding and the enzymatic activity quantification by amperometry. Cholinesterases from different species-acetylcholinesterase (AChE) from Electrophorus electricus , AChE from Drosophila melanogaster , and butyrylcholinesterase (BChE) of human origin-were tested on the various immobilized ligands. Our research allowed the development of a competitive assay for the detection of organophosphates in river water samples using the BZE-DADOO-modified piezosensor. Another direction of research was pointed on the characterization of the interactions between ChE and its ligands. The kinetic binding constants were derived using a one- to-one binding model
A tailor-made horseradish peroxidase (HRP) bulk composite electrode was developed on the basis of pyrolyzed cobalt tetramethoxyphenylporphyrin (CoTMPP) by modifying pore size and surface area of the porous carbon material through varying amounts of iron oxalate and sulfur prior to pyrolyzation. The materials were used to immobilize horseradish peroxidase (HRP). These electrodes were characterized in terms of their efficiency to reduce hydrogen peroxide. The heterogeneous electron transfer rate constants of different materials were determined with the rotating disk electrode method and a k(S) (401 +/- 61 s(-1)) exceeding previously reported values for native HRP was found.
The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltarnmetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an EO' of +200 mV versus Ag/AgC1 (1 M KC1). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.
A novel electrochemical immunoassay based on the multiple affinity labeling of the indicator antibody with an electro-active tag is presented. The concept is illustrated for the determination of the glycated hemoglobin HbA1c in hemoglobin samples. Hemoglobin is adsorbed to the surfactant-modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance is used to validate the total Hb binding, the HbA1c on the sensor surface is recognized by an antibody and quantified electrochemically after the sugar moieties of the antibody have been labeled in-situ with ferroceneboronic acid. The sensitivity of this sensor is about threefold higher than the sensitivity of a hemoglobin sensor, where the ferroceneboronic acid is bound directly to HbA1c.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
The layer-by-layer adsorption technique based on the consecutive deposition of oppositely charged species is for the preparation of protein multilayers with fully electro-active protein molecules. The methodology was established with cytochrome c and the polyelectrolyte sulfonated polyaniline (PASA). The technique is also useful for the construction of bi-protein architectures confining protein-protein communication to an electrode. Following natural examples of protein complexes with defined signal transfer, cytochrome c was arranged with enzymes such as xanthine oxidase, bilirubin oxidase, laccase, and sulfite oxidase in self-assembled multilayer architectures. Thus, biomimetic signal chains from the enzyme substrate via the enzyme and cytochrome c towards the electrode can be established. Communication between proteins immobilised in multiple layers on the electrode can be achieved by in situ generation of small shuttle molecules or more advantageously by direct interprotein electron transfer. This allows the construction of new sensing electrodes, the properties of which can be tuned by the number of deposited protein layers. The mechanism of electron transfer within such protein assemblies on gold electrodes will be discussed.
Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase
(2009)
In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples (OsHRP)-H-III/(OsHRP)-H-IV, (OsHRP)-H-IV/(OsHRP)-H-V and (OsHRP)-H-V/ (OsHRP)-H-VI. The midpoint potentials differ dependent on the electrode material used with E-1/2 (Os-III/IV) of -0.4 V (ITO) and -0.25 V (GC), E-1/2 (Os-IV/V) of -0.16 V (ITO) and +0.10 V (GC), and E-1/2 (Os-V/VI)of +018 V (ITO), respectively Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
A biosensor, based on a redoxactive osmium polymer and sulfite oxidase on screen-printed electrodes, is presented here as a promising method for the detection of sulfite. A catalytic oxidative current was generated when a sample containing sulfite was pumped over the carbon screen-printed electrode modified with osmium redox polymer wired sulfite oxidase. A stationary value was reached after approximately 50 s and a complete measurement lasted no more than 3 min. The electrode polarized at -0.1 V (vs. Ag vertical bar AgCl 1M KCl) permits minimizing the influence of interfering substances, since these compounds can be unspecific oxidized at higher potentials. Because of the good stability of the protein film on the electrode surface, a well functioning biosensor-flow system was possible to construct. The working stability and reproducibility were further enhanced by the addition of bovine serum albumin generating a more long-term stable and biocompatible protein environment. The optimized biosensor showed a stable signal for more than a week of operation and a coefficient of variation of 4.8% for 12 successive measurements. The lower limit of detection of the sensor was 0.5 mu M sulfite and the response was linear until 100 mu M. The high sensitivity permitted a 1:500 dilution of wine samples. The immobilization procedure and the operational conditions granted minimized interferences. Additionally, repeating the immobilization procedure to form several layers of wired SO further increased the sensitivity of such a sensor. Finally. the applicability of the developed sulfite biosensor was tested on real samples, such as white and red wines.
Stable immobilization and reversible electrochemistry of cytochrome c in a tranparent indium tin oxide film with a well-defined mesoporosity (mpITO) is demonstrated. the transparency and good conductivity, in combination with the large surface area of mpITO, allow the incorporation of a high amount of elelctroactive biomolecules and their electrochemical and spectroscopic investigation. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry are employed for the characterization of cytochrome c immobilized in the mpITO and reveal no perturbant of the structural of the integrity of the redox protein. The potential of this modified material as a biosensor detection of superoxide anions is also demonstrated.
Focus on bioanalysis
(2010)
Three different sizes of chitosan-capped Au nanoparticles were synthesized and were used to incorporate Agrocybe aegerita peroxygenase (AaeAPO) onto the surface of glassy carbon electrode. The direct electron transfer of AaeAPO was achieved in all films. The highest amount of electroactive enzyme and highest electron transfer rate constant k(s) of AaeAPO were obtained in the film with the smallest size of chitosan-capped Au nanoparticles.
In anaerobic solutions, quasi-reversible oxidation and reduction are obtained with a formal potential of -0.280V vs. Ag/AgCl 1 M KCl in 100 mM (pH 7.0) PBS at scan rate of 1 V s(-1). Bioelectrocatalytic reduction currents can be obtained with the AaeAPO-modified electrode on addition of hydrogen peroxide. This reaction was suppressed when sodium azide, an inhibitor of AaeAPO, was present. Furthermore, the peroxide-dependent conversion of aniline was characterized and it was found that a polymer product via p-aminophenol is formed. And the AaeAPO biosensor was applied to determine aniline and p-aminophenol.
Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11
(2011)
Microperoxidase-11 (MR-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline. 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-II and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.