Electrochemical characterization of a superoxide biosensor based on the co-immobilization of cytochrome c and XOD on SAM-modified gold electrodes and application to garlic samples

This paper describes the characterization and optimization of an amperometric cytochrome c (cyt c)-based sensor for the determination of the antioxidant capacity of pure substances and natural samples. The cyt c and the xanthine oxidase (XOD) enzyme were co-immobilized on the electrode using the combination of several long-chain thiols. The self-assembled monolayer (SAM) was optimized in terms of composition and ratio between thiols. The immobilization protocol for both cyt c and XOD and the SAM formation time were evaluated through electrochemical methods, such as cyclic voltammetry (CV), square wave voltammetry (SWV), chronoamperometry (CA) and impedance spectroscopy (IS). Finally, the biosensor was applied to the determination of the antioxidant capacity of pure alliin and two compounds extracted from garlic bulbs.     

Voltammetry of immobilized cytochrome c on novel binary self-assembled monolayers of thioctic acid and thioctic amide modified gold electrodes

Horse heart cytochrome c (cyt c) was adsorbed on the binary self-assembled monolayers (SAMs) composed of thioctic acid (T-COOH) and thioctic amide (T-NH₂) at gold electrodes via electrostatic interaction. The cyt c adsorbed on the modified gold electrode exhibited well-defined reversible electrochemical behavior in 10 mM phosphate buffer solution (PBS, pH 7.0). The surface concentration (Γ) of electroactive species, cyt c, on the binary SAMs was higher than that in single-component SAMs of T-COOH, and reached a maximum value of 9.2 × 10⁻¹² mol cm⁻² when the ratio of T-COOH to T-NH₂ in adsorption solution was of 3:2, and the formal potential (E^0′=(E_pa+E_pc)/2) of cyt c was −0.032 V (vs. Ag|AgCl (3 M NaCl)) in a 10 mM PBS. The interaction between cyt c and the binary SAMs made the E^0′ shift negatively when compared with that of cyt c in solution (+0.258 V vs. NHE, i.e., +0.058 V vs. Ag|AgCl (3 M NaCl)). The fractional coverage of bound cyt c was a 0.64 theoretical monolayer. The standard electron transfer rate constant of cyt c immobilized on the binary SAMs was also higher than that on single-component SAMs of T-COOH, and the maximum value of 15.8 ± 0.6 s⁻¹ was obtained when the ratio of T-COOH to T-NH₂ in adsorption solution was at 3:2. The results suggest that the electrode modified with the binary SAMs functions better than the electrode modified with single-component SAMs of T-COOH.     

Microscale Crystals of Cytochrome c and Calixarene on Electrodes: Interprotein Electron Transfer between Defined Sites

The assembly of redox proteins on electrodes is an important step in biosensor development. Recently, p‐sulfonato‐calix[4]arene was shown to act as “molecular glue” for the assembly and crystallization of cytochrome c (cyt c). Electrochemical data are presented for microscale cyt c–calixarene crystals grown on self‐assembled monolayers (SAM)‐modified Au electrodes. The crystals were characterized by cyclic voltammetry and exceptionally high concentrations of electroactive cyt c were obtained. The peak currents were found to increase linearly with the square root of the scan rate, thus allowing an evaluation of the rate constant for electron self‐exchange. This study revealed high electroactivity accompanied by fast interprotein electron transfer in crystals, which may have implications for the construction of novel bioelectronic devices.     

A study of the antioxidative behavior of phenolic acids, in aqueous herb extracts, using a dsDNA biosensor

Electrochemical DNA biosensors are promising tools for the fast, inexpensive and simple in vitro analysis for the determination of free radicals and antioxidants. High concentrations of antioxidants in such compounds as phenolic acids and plant extracts, act as free radical terminators which reduce the effect of the oxidative dam-age on DNA. The electrochemical behavior of three representative phenolic acids, caffeic acid, gallic acid and trolox were studied by cyclic voltammetry. Moreover, the determination of the above antioxidants under the optimized conditions (scan rate, deposition potential and time) using differential pulse voltammetry was also investigated. In vitro studies focused on their antioxidative effect were performed by adsorptive transfer stripping voltammetry and dsDNA biosensor. Using Fenton’s system, with FeSO₄ and H₂O₂ was chosen as a strong oxidative system. This biosensor was applied as a screening antioxidant test in order to estimate the antioxidant capacity of aqueous herb extracts.      

A Simple Fabrication Method for Three‐Dimensional Gold Nanoparticle Electrodes and Their Application to the Study of the Direct Electrochemistry of Cytochrome c

A simple method for constructing gold nanoparticle‐modified electrodes with three‐dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate‐reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area‐to‐volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self‐assembled monolayer of 11‐mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.     

Characterization of the Cytochrome c Membrane‐Binding Site Using Cardiolipin‐Containing Bicelles with NMR

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL‐containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A‐site, the CXXCH motif, and the N‐ and C‐terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane‐binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.     

Electroanalytical properties of cytochrome c by direct electrochemistry on multi-walled carbon nanotubes incorporated with DNA biocomposite film

A novel conductive biocomposite film (MWCNTs–DNA–cyt c) which contains multi-walled carbon nanotubes (MWCNTs) along with the incorporation of DNA and cytochrome c (cyt c) has been synthesized on glassy carbon electrode (GCE), gold (Au), indium tin oxide (ITO) and screen printed carbon electrode (SPCE) by potentiostatic methods. The presence of both MWCNTs and DNA in the biocomposite film enhances the surface coverage concentration (Γ), increases the electron transfer rate constant (K_s) up to 21% and decreases the degradation of cyt c during the cycling. The biocomposite film also exhibits a promising enhanced electrocatalytic activity towards the reduction of halogen oxyanions and oxidation of biochemical compounds such as ascorbic acid and l-cysteine. The cyclic voltammetry has been used for the measurement of electroanalytical properties of analytes by means of biocomposite film modified GCEs. The sensitivity of MWCNTs–DNA–cyt c modified GCE possess higher values than the values obtained for DNA–cyt c film modified GCE. Further, the reduction potentials of halogen oxyanions E_pc, clearly shows that the activity of the biocomposite is dependent on the electronegativity of halogen oxyanions. Electrochemical quartz crystal microbalance studies revealed the enhancements in the functional properties of MWCNTs, DNA and cyt c. We have studied the surface morphology of the biocomposite films using scanning electron microscopy and atomic force microscopy, which revealed that DNA and cyt c have been incorporated on MWCNTs. Finally, the flow injection analysis has been used for the amperometric detection of analytes at MWCNTs–DNA–cyt c film modified SPCE.     

Investigating the Role of Adducts in Protein Supercharging with Sulfolane

The supercharging effect of sulfolane on cytochrome c (cyt c) during electrospray ionization mass spectrometry (ESI-MS) in the absence of conformational effects was investigated. The addition of sulfolane on the order of 1 mM or greater to denaturing solutions of cyt c results in supercharging independent of protein concentration over the range of 0.1 to 10 μM. While supercharging was observed in the positive mode, no change in the charge state distribution was observed in the negative mode, ruling out polarity-independent factors such as conformational changes or surface tension effects. A series of sulfolane adducts observed with increasing intensity concurrent with increasing charge state suggests that a direct interaction between sulfolane and the charged sites of cyt c plays an important role in supercharging. We propose that charge delocalization occurring through large-scale dipole reordering of the highly polar supercharging reagent reduces the electrostatic barrier for proximal charging along the cyt c amino acid chain. Supporting this claim, supercharging was shown to increase with increasing dipole moment for several supercharging reagents structurally related to sulfolane.     

Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

Immune recognition of cytochrome c I. — Molecular requirements for antibody recognition and immune response stimulation studied in vitro with synthetic peptides

The epitope specificity of antibodies to horse cytochrome c (cyt.c) in the primary and secondary immune response of C57BL mice was studied by means of the ELISA technique with synthetic peptides of cyt.c. It was found that, in the early primary response, N- and C-end fragments of cyt.c (peptides 2–13, 14–22 and 92–104) were preferentially recognized. In the secondary response, more antibodies to the epitopes of the central part of the molecule (peptides 61–69 and 46–56) were found. This was presumed to be due to the mode of cyt.c processing and presentation in the course of immune response: at first, cyt.c was recognized in the native form and then in the processed one. The capacity of cyt.c peptides to stimulate the formation of cyt.c-specific antibody-secreting cells (ASC) was studied in splenocyte culture of C57BL mice. Peptides stimulated more ASC than cyt.c did, but larger molar doses of peptides were required. Comparison of the capacity of related peptides (1–13 and 2–13, 61–69, 61–77 and 57–77) to be recognized by antibodies produced to native cyt.cin vivo and to stimulate anti-cyt.c ASC in vitro suggested certain molecular requirements for cyt.c epitope and agretope formation. These were partially confirmed by computer analysis.     

Functionalization of gold electrode surface with heterobifunctional poly(ethylene oxide)s having both mercapto and aldehyde groups

We synthesized heterobifunctional poly(ethylene oxide) (PEO) (α‐formyl‐ω‐mercapto‐PEO; CHO‐PEO₄₀₀‐SH, average molecular weight of PEO part being 400), which had both an aldehyde group as a binding site with amino group of protein and a mercapto group for gold electrode surface. The CHO‐PEO₄₀₀‐SH was adsorbed on a gold electrode surface and cytochrome c (cyt.c) was fixed on this modified electrode. The redox response of covalently immobilized cyt.c was observed on the cyclic voltammetry measurement, showing that CHO‐PEO₄₀₀‐SH can be used as a linker to fix cyt.c on an electrode. Another type of heterobifunctional PEO (α‐formyl‐ω‐(2‐pyridyldithio)‐PEO; CHO‐PEO₃₀₀‐SS‐Py), which had an aldehyde group and a 2‐pyridinethiol (2‐Py) through disulfide bond, was synthesized to form co‐adsorbed monolayer of PEO chain and 2‐Py on an electrode surface. It was expected, due to the spacer with shorter PEO chain and lower surface density, that better redox response of the fixed cyt.c was obtained. However, the redox response of fixed cyt.c was not detected on the CHO‐PEO₃₀₀‐SS‐Py modified gold electrode. Instead, this heterobifunctional PEO was found to function as a good promoter for cyt.c dissolved in phosphate buffer solution. Copyright © 2003 John Wiley & Sons, Ltd.     

Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method

Cytochrome c was immobilized on a mixed-thiol (mercaptoundecanoic acid/mercaptoundecanol) modified gold electrode (MUA:MU/cyt c electrode). Characterization of the cyt c electrode showed a quasi-reversible, electrochemical redox behavior with a formal potential of −13±5 mV (versus Ag/AgCl) for the surface adsorbed protein and 3±5 mV for covalently immobilized cyt c. The heterogeneous electron transfer rate constants were determined to be about 70 and 40 s⁻¹ for both states of the protein, respectively. They were found to be significantly higher than those of pure MUA-modified cyt c electrodes (MUA/cyt c electrodes). The interaction of superoxide radicals (O₂⁻) with the (MUA:MU)/cyt c electrode was characterized and used for an amperometric O₂⁻ detection. The influence of H₂O₂ and uric acid on the sensor signal was investigated. The sensitivity of the (MUA:MU)/cyt c electrode to O₂⁻ was significantly improved compared with that of the MUA/cyt c electrode. Based on a kinetic model for the superoxide detection system, a new calibration method was established. This simple and fast method used the spontaneous dismutation of KO₂ and was compared with the enzymatic superoxide generation system using xanthine oxidase.     

Reaction of ferricytochrome c with the iron nitrosyl complex {Fe2[S(CH2)2NH3]2(NO)4}SO4 • 2.5H2O

By an example of cysteamine iron nitrosyl complex {Fe₂[S(CH₂)₂NH₃]₂(NO)₄}SO₄ ? 2.5H₂O (CAC) it was shown for the first time that the NO donor hydrolysis in the presence of ferricytochrome c (cyt c³⁺) affords the iron nitrosyl complex NO—cyt c³⁺. It was found that cyt c³⁺ can serve as a depot for NO evolved during the hydrolysis of CAC. In the presence of CAC, the rate of NO—cyt c³⁺ complex decomposition to NO and cyt c³⁺ depends on the molar ratio [cyt c³⁺]: [CAC] and at [cyt c³⁺]: [CAC] = 0.3 it was found to be lower than that in decomposition of CAC in the absence of cyt c³⁺. As a result, the total NO evolving process becomes 5.6 times more prolonged. The number of NO groups evolved from CAC can be determined by the reaction of CAC with cyt c³⁺ in the presence of ferricyanide: at most one NO group is evolved to a solution in the spontaneous hydrolysis of CAC (pH 7.0), and no less than three of them are evolved from oxidized CAC.     

Lichen-based biosensor for the determination of benzene and 2-chlorophenol: microcalorimetric and amperometric investigations

Preliminary microcalorimetric studies have been performed to analyse the response of a whole epiphytic lichen tissue (Evernia prunastri) to 2-chlorophenol (2Cl-), a pollutant of oil mill waste-water, in order to evaluate whether the tissue might be used to assess the toxic characteristics of polluted waters. The obtained results (lichen viability expressed in hours, enthalpy variations for the 2Cl-/lichen interactions) were used to create a lichen-based biosensor that uses an amperometric oxygen electrode (a Clark electrode) as a transducer. The lichen catalyses aromatic ring cleavage (via pyrocatechase enzymes present in the lichen), and transforms aromatic substances like 2Cl- into muconic acid (C₆H₆O₄). Following a full electroanalytical characterisation, the performance of the proposed lichen biosensor was compared to that of a biosensor based on Pseudomonas putida cells, which was originally constructed to monitor benzene in different matrices (water, air, petrol and oil) and was tested in our laboratory previously.     

Determination of Cytochrome C with Cellulose – DNA Modified Carbon Paste Electrodes

A sensor for cytochrome c (cyt c) was developed using a carbon paste electrode (CPE) modified with cellulose‐DNA. Cyt c was adsorbed on the cellulose‐DNA modified CPE through the electrostatic interaction between them. Owing to this process, a pair of well‐defined peaks appeared at +48 mV/85 mV (E_pc/E_pa vs. Ag/AgCl). This property of the cellulose‐DNA modified CPE was utilized for the analysis of cyt c in a biological sample. The optimum experimental conditions for analysis were investigated and a calibration plot was obtained between 1.0×10^?4 M and 1.0×10^?6 M (±5% at n=5) at the optimized condition. The detection limit for cyt c at the optimized experimental condition was 5.0×10^?7 M (S/N=3) for 30 min of deposition time with differential pulse voltammetry (DPV). The real sample analysis was carried out with the standard addition method to evaluate the developed method. The content of cyt c in total proteins of 80.0 mg/mL in rat mitocondria fractions was determined to be 0.12 (±0.02) mg/mL.     

Synthesis and reactions of 2,3-dimethylfuro[3,2-c]pyridines

Summary A number of substituted 2,3-dimethylfuro[3,2-c]pyridines was synthesized. 3-(4,5-Dimethyl-2-furyl)propenoic acid (1) was converted to the acid azide2, which in turn was cyclized to give 2,3-dimethyl-5H-furo[3,2-c]pyridine-4-one (3) by heating at 240°C in Dowtherm. The pyridone3 was chlorinated with phosphorus oxychloride to give4, which was reduced with zinc and acetic acid to 2,3-dimethylfuro[3,2-c]pyridine (5). Treatment of4 with several secondary heterocyclic amines led to compounds6a–6c. Reaction of pyridone3 with phosphorus pentasulfide rendered the thione7, which was methylated to8a. The 4-methoxy derivative8b was obtained from4 with sodium methoxide. 2,3,5-Trimethylfuro[3,2-c]pyridine-4-one (9) was obtained by reaction of3 with methyl iodide.Dedicated to Professor Dr.Fritz Sauter on the occasion of his 65th birthday     

A 3.3 Å-Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc₁ complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc₁ complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N-terminus of cyt. c₁ was found to greatly enhance the interaction between cyt. b and cyt. c₁, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc₁ complex in an extreme thermal environment.     

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS-(CH₂)₃-PO₃H₂, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and -PO₃H₂ groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the -PO₃H₂ terminated SAMs with short chain is nearly reversible. Its formal potential (E⁰′ = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E⁰′ = 18-22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on -PO₃H₂ terminated SAMs is relatively slow as compared to -SO₃H and -COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.     

Synthesis and reactions of 2-[3-(trifluoromethyl)phenyl]furo[3,2-<Emphasis Type="Italic">c</Emphasis>]pyridine

(E)-3-{5-[3-(Trifluoromethyl)phenyl]furan-2-yl}propenoic acid (I) was prepared from 5-[3-(tri-fluoromethyl)phenyl]furan-2-carbaldehyde under the Doebner’s conditions. The obtained acid was converted to the corresponding azide II, which was cyclized by heating in diphenyl ether to 2-[3-(trifluoromethyl)phenyl]-4,5-dihydrofuro[3,2-c]pyridin-4-one (III). This compound was aromatized with phosphorus oxychloride to chloroderivative IV which was reduced with H₂NNH₂-Pd/C to the title compound V. 2-[3-(Trifluoromethyl)phenyl]furo[3,2-c]pyridin-5-oxide (VI) was synthesized by reaction of V with 3-chloroperoxybenzoic acid in dichloromethane. On treatment of VI with benzoyl chloride and potassium cyanide (Reissert-Henze reaction), corresponding 2-[3-(trifluoromethyl)phenyl]furo[3,2-c]pyridine-1-carbonitrile (VII) resulted. 5-Amino-2-[3-(trifluoromethyl)phenyl]furo[3,2-c]pyridin-5-ium-4-methylbenzene sulfonate (VIII) was prepared by direct N-amination of the title compound V with 1-[(aminooxy)sulfonyl]-4-methylbenzene in dichloromethane. Then, VIII was transformed to a non-isolated zwitterionic N-imid IX which afforded the corresponding furo[3,2-c]pyrazolo[1,5-a]pyridine carboxylic acid esters X, XI by 1,3-dipolar cycloaddition reactions with dimethyl but-2-ynedionate (DBD) or ethyl propiolate. The structures of all compounds were confirmed by their IR and NMR spectra. Presented at the 1st International Conference “Applied Natural Sciences” on the occasion of 10th anniversary of the University of St. Cyril and Methodius, Trnava, 7–9 November 2007.     

Development of a Xanthine Oxidase Modified Amperometric Electrode for the Determination of the Antioxidant Capacity

This paper describes the development of a xanthine oxidase/poly‐m‐phenylenediamine (XOD‐PPD)‐modified electrode. The biosensor was constructed by encapsulating XOD in a sol‐gel matrix deposited onto a platinum based screen‐printed electrode functionalized with a permselective PPD membrane. The hydrogen peroxide generated as a final product of the enzymatic reaction between the hypoxanthine and the XOD or by the spontaneous dismutation of superoxide radicals was selectively monitored at +700 mV. The use of a highly selective PPD layer blocked the nonspecific oxidation of other oxidizable molecules. Finally the biosensor was applied to the determination of the antioxidant capacity of acetylsalicylic acid.