Migration of small molecules through the structure of hemoglobin: evidence for gating in a protein electron-transfer reaction

It has previously been shown that the rates and activation energies for migration molecules of different sizes through myoglobin are very similar. The results were interpreted in terms of conformational changes in the protein structure that facilitate the passage of the different molecules to a similar extent. Here we ask whether the quaternary structural changes that accompany the binding of ligands (O2 or CO) to hemoglobin might influence the migration rate from the solution into the protein's binding site. As a model for the R state of hemoglobin, we used the protein in which the Fe protoporphyrin (FePP) in the alpha subunit was substituted by Zn protoporphyrin (ZnPP) and the oxidized heme was ligated by CN-. The T state of hemoglobin was represented by the protein in which all four FePP groups were substituted by ZnPP. The quenching rate of the excited ZnPP triplet state within the hemoglobin by oxygen, methyl viologen, and anthraquinonesulfonate served as a measure of the migration rate through the protein into the binding site. It was found that the activation energies for all three quenchers were very similar and closely resembled those in myoglobin, suggesting that the migration rates are determined by the subunit structure only and that the quaternary configurational changes do not influence the quenching rates. The implications of the results for electron transfer in proteins are briefly discussed.     

Protoporphyrin IX sensitized titanium oxide gel electrode

Titanium oxide (Ti(O)) xerogel films functionalized by protoporphyrin IX (PPIX) and ferrocene carboxylic acid (FCA) were deposited on indium tin oxide (ITO) electrodes following a sol-gel synthesis. PPIX and FCA were first complexed to titanium oxide precursors, which were then subjected to hydrolysis to obtain a homogenous Ti(O) polymeric network gel doped with PPIX and FCA. The Ti(O) film cast on the ITO electrode has been characterized by UV-Vis absorption, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy and cyclic voltammetry. Illumination of the PPIX doped Ti(O) films on the ITO electrode immersed in aqueous electrolytes onsets photoinduced electron transfer reactions, and a cathodic photocurrent was observed in most cases. This photocurrent response was investigated in detail using a kinetic model. Preliminary investigations of oxygen reduction, lithium and proton insertion into the Ti(O) film have also been carried out.     

Biomimetic oxidase sensor based on functionalized surface of carbon nanotubes and iron prophyrins for catechol detection

A novel and highly stable biomimetic oxidase sensor system was designed for catehol detection. FePP used as biomimetic horseradish peroxidase (HRP) was immobilized onto modified multi-walled carbon nanotubes (MWCNTs). Functional groups such as –OH, –NH₂ and –COOH were introduced onto the surface of MWCNTs to provide biomimetic microenvironment for iron porphyrins (FePP). Stable biomimetic enzyme electrode has been developed to detect catechol as a simple, economical and efficient method. At optimal condition, the detection limit of OH-MWCNTs/FePP/Nafion was 3.754 × 10^− 6 M. After stored at − 4 °C for 35 days, the oxidation current value still maintained 98.3% of initial activity. In repetitive nature test, relative standard deviation (RSD) of oxidation current remained within 1.0% after ten consecutive measurements in the same concentration of catechol solution, while most of reported oxidase sensor was within 2.0% under the same condition.

     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.     

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

Urea biosensor based on PANi(urease)-Nafion/Au composite electrode

The polyaniline (PANi)-Nafion composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion/Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 microA in 60 mg dl(-1) NH4Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl(-1) LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 microA in the 60 mg dl(-1) NH4Cl buffer solution when the cycle number for preparing PANi-Nafion/Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion/Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 microA (mg dl(-1))(-1)cm(-2), as well as 6 and 0.3 mg dl(-1), respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion network, respectively.     

Enhancing the sensitivity and stability of HRP/PANI/Pt electrode by implanted bovine serum albumin

Polyaniline (PANI) is considered as one of the most fascinating conductive polymers in fabricating enzyme-based biosensors. Nevertheless, to improve both sensitivity and stability of the PANI-modified biosensor has been and continues to be a technical challenge. In this study, we have electrochemically synthesized the PANI film on a platinum (Pt) electrode and then used this electrode to construct a horseradish peroxidase (HRP)-based biosensor for the detection of hydrogen peroxide (H(2)O(2)). The electrochemical and structural properties of electrodes were characterized with scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Fourier-transform infrared (FTIR) spectrophotometer, and cyclic voltammetry (CV). It was interestingly found that the PANI film synthesized in the presence of bovine serum albumin (BSA) has provided the electrode with enhanced sensitivity and excellent stability. Our results suggested that the embedded BSA might serve as an initial template for aniline polymerization and stabilized the microstructure of the PANI film significantly. The constructed HRP/PANI(BSA)/Pt electrode also exhibited a fine linear correlation with H(2)O(2) concentration. This approach by implanted BSA was useful for improving the sensitivity and stability of PANI-modified biosensor.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

Photoelectrocatalytic treatment of pentachlorophenol in aqueous solution using a rutile nanotube-like TiO2/Ti electrode.

Taking pentachlorophenol (PCP) as a reference, we investigated the photoelectrocatalytic degradation of organic pollutants using a rutile nanotube-like TiO(2)/Ti film electrode. The nanotube-like TiO2 electrode was prepared by first oxidizing the surface of a titanium sheet to form rutile TiO2 and then treating it to form the tubular structure in NaOH aqueous solution. The occurrence of PCP degradation was indicated by the decrease in pH, concentration of PCP and TOC, and by the formation of chloride ions. The photoelectrochemical (PEC) efficiency of the nanotube-like TiO2/Ti electrode has been determined in terms of degradation of PCP and the incident photo-to-current conversion efficiency (IPCE). The experimental results showed that PCP could be degraded more efficiently by a photoelectrocatalytic process than by a photocatalytic or electrochemical oxidation alone. A significant photoelectrochemical synergetic effect was observed. The kinetic constant of PEC degradation of PCP using a nanotube-like TiO2 electrode was over 60% higher than that using a TiO2 film electrode. It is noted that chloride ion and hydrogen ion concentration increased with irradiation time in the PEC degradation of PCP; PCP was gradually mineralized and the complete minimization of PCP took more time than its degradation.     

Optical-transparent and flexible glucose sensor with ITO electrode

The glucose sensor was constructed by immobilizing glucose oxidase (GOD) with glutaraldehyde solution onto the sensitive area of the transparent oxygen electrode. The oxygen electrode was fabricated by sealing KCl electrolyte solution including the Indium-Tin Oxide (ITO)-electrode with both metal-weldable film and gas-permeable membrane coated with Ag/AgCl electrode. The sensor behavior was evaluated using standard glucose solutions in a batch measurement system with a computer-controlled potentiostat at a reduction potential of -900 mV. The sensor device has flexible structure and good optical transparency (less than 0.6 abs) at the visible wavelength from 400 to 700 nm. The sensor was possible to be used for measuring glucose from 0.06 to 1.24 mmol/l (correlation coefficient: 0.999), including the reported concentration of tear glucose in normal (0.14 mmol/l), with good reproducibility.     

Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF(+)ClO(4)(-) film was coated on a Pt foil at +0.70 V by electrooxidation of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF(+)HRP(-) was prepared by anion-exchange in a solution of HRP(-) in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy was used to identify PVF, PVF(+)ClO(4)(-), and PVF(+)HRP(-). The immobilized amount of the enzyme in the film was determined by UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined. Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide.     

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 microM.     

Reduction of the NO-mediated response in the rat aorta by metalloporphyrins

Metalloporphyrins (MPs) have been found to affect the production of carbon monoxide (CO) and nitric oxide (NO). Unlike that for CO, little is known about the mechanism of action of MPs on the NO system. We determined the in vitro ability of ferrous protoporphyrin (heme, FePP), zinc protoporphyrin (ZnPP), and bilirubin (BR) to scavenge NO. Heme and ZnPP were studied in the rat aortic ring system for their ability to affect phenylephrine-induced contraction and methacholine-stimulated relaxation. Heme was found to be a good NO scavenger with a ks = 0.53 +/- 0.19 x 10(4) M(-1)xs(-1) (n = 6). ZnPP and BR did not scavenge NO. Neither heme nor ZnPP treatment affected the phenylephrine response as measured by -logEC50 and the maximal effect. However, heme and ZnPP treatments decreased the -logEC50 and the maximal effects of methacholine, therefore decreasing vasorelaxation. We conclude that when ZnPP is administered in vivo blood pressure should be carefully monitored.     

Determination of dopamine in the presence of ascorbic acid using poly(3,5-dihydroxy benzoic acid) film modified electrode

A polymerized film of 3,5-dihydroxy benzoic acid (DBA) was prepared on the surface of a glassy carbon electrode (GCE) in neutral solution by cyclic voltammetry (CV). The poly(DBA) film-coated GCE exhibited excellent electrocatalytic activity toward the oxidation of dopamine (DA). A linear range of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M and a detection limit of 6.0 × 10⁻⁸ M were observed in pH 7.4 phosphate buffer solutions. Moreover, the interference of ascorbic acid (AA) was effectively eliminated. This work provides a simple and easy approach to selective detection of DA in the presence of AA.     

Overoxidized polypyrrole film directed single-walled carbon nanotubes immobilization on glassy carbon electrode and its sensing applications

In this paper, the films of overoxidized polypyrrole (PPyox) directed single-walled carbon nanotubes (SWNTs) have been electrochemically coated onto glassy carbon electrode (GCE). Electroactive monomer pyrrole was added into the solution containing sodium dodecyl sulfate (SDS) and SWNTs. Then, electropolymerization was proceeded at the surface of GCE, and a novel kind of conducting polymer/carbon nanotubes (CNTs) composite film with the orientation of CNTs were obtained correspondingly. Finally, this obtained polypyrrole (PPy)/SWNTs film modified GCE was oxidized at a potential of +1.8 V. It can be found that this proposed PPyox/SWNTs composite film modified GCE exhibited excellent electrocatalytic properties for some species such as nitrite, ascorbic acid (AA), dopamine (DA) and uric acid (UA), and could be used as a new sensor for practical applications. Compared with previous CNTs modified electrodes, SWNTs were oriented towards the outside of modified layer by PPyox and SDS, which made the film easily conductive. Moreover, this proposed film modified electrode was more stable, selective and applicable.     

An amperometric uric acid biosensor based on modified Ir-C electrode

The level of uric acid (UA) has a high relationship with gout, hyperuricemia and Lesch-Nyan syndrome. The determination of UA is an important indicator for clinics and diagnoses of kidney failure. An amperometric UA biosensor based on an Ir-modified carbon (Ir-C) working electrode with immobilizing uricase (EC 1.7.3.3) was developed by thick film screen printing technique. This is the first time to report the utilization of an uricase/Ir-C electrode for the determination of UA by using chronoamperometric (CA) method. The high selectivity of UA biosensor was achieved due to the reduction of H(2)O(2) oxidation potential based on Ir-C electrode. Using uricase/Ir-C as the sensing electrode, the interference from the electroactive biological species, such as ascorbic acid (AA) and UA (might be directly oxidized on the sensing electrode) was slight at the sensing potential of 0.25 V (versus Ag/AgCl). UA was detected amperometrically based on uricase/Ir-C electrode with a sensitivity of 16.60 microAmM(-1) over the concentration range of 0.1-0.8 mMUA, which was within the normal range in blood. The detection limit of UA biosensor was 0.01 mM (S/N=6.18) in pH 7 phosphate buffer solution (PBS) at 37 degrees C. The effects of pH, temperature, and enzymatic loading on the sensing characteristics of the UA biosensor were also investigated in this study.     

Determination of L-dopa using electropolymerized 3,3',3',3'-tetraaminophthalocyanatonickel(II) film on glassy carbon electrode

Electropolymerized film of 3,3',3',3'-tetraaminophthalocyanatonickel(II) (p-Ni(II)TAPc) on glassy carbon (GC) electrode was used for the selective and stable determination of 3,4-dihydroxy-L-phenylalanine (L-dopa) in acetate buffer (pH 4.0) solution. Bare GC electrode fails to determine the concentration of L-dopa accurately in acetate buffer solution due to the cyclization reaction of dopaquinone to cyclodopa in solution. On the other hand, p-Ni(II)TAPc electrode successfully determines the concentration of L-dopa accurately because the cyclization reaction was prevented at this electrode. It was found that the electrochemical reaction of L-dopa at the modified electrode is faster than that at the bare GC electrode. This was confirmed from the higher heterogeneous electron transfer rate constant (k(0)) of L-dopa at p-Ni(II)TAPc electrode (3.35 x 10(-2) cms(-1)) when compared to that at the bare GC electrode (5.18 x 10(-3) cms(-1)). Further, it was found that p-Ni(II)TAPc electrode separates the signals of ascorbic acid (AA) and L-dopa in a mixture with a peak separation of 220 mV. Lowest detection limit of 100 nM was achieved at the modified electrode using amperometric method. Common physiological interferents like uric acid, glucose and urea does not show any interference within the potential window of L-dopa oxidation. The present electrode system was also successfully applied to estimate the concentration of L-dopa in the commercially available tablets.     

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Enzyme electrode composed of the pyruvate oxidase from pediococcus species coupled to an oxygen electrode for measurements of pyruvate in biological media

Pyruvate oxidase from Pediococcus species was immobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto the tip of an oxygen electrode. The enzyme electrode was specific for pyruvate measurements. This electrode was sensitive to 0.1 mM and could be used up to a final pyruvate concentration of 2 mM.At each step of the enzymatic film preparation and assay 0.7 mM thiamine pyrophosphate, 10 μM flavin adenine dinucleotide, 5mM Mg²⁺ and 10 mM phosphate buffer were necessary.A computerized probe allowed successive measurements every 3 min for more than 20 h with the same enzymatic film. The reproducibillty for the same pyruvate concentration was 2% during 400 assays without special optimization.This enzyme electrode has many applications in basic (metabolism, enzymology) and applied (blood, yoghurt) research. Results obtained from assays carried out in yoghurt are presented.     

Highly selective and sensitive determination of dopamine using a Nafion/carbon nanotubes coated poly(3-methylthiophene) modified electrode

A poly(3-methylthiophene) modified glassy carbon electrode coated with Nafion/single-walled carbon nanotubes film was fabricated and used for highly selective and sensitive determination of dopamine. The hybrid film surface of the modified electrode was characterized by scanning electrochemical microscopy (SECM) and the results indicated that the carbon nanotubes were dispersed uniformly on the conductive polymer. The experimental results suggest that the hybrid film modified electrode combining the advantages of poly(3-methylthiophene), carbon nanotubes with Nafion exhibits dramatic electrocatalytic effect on the oxidation of dopamine (DA) and results in a marked enhancement of the current response. In 0.1M phosphate buffer solution (PBS) of pH 7.0, the differential pulse voltammetric (DPV) peak heights are linear with DA concentration in three intervals, viz. 0.020-0.10 microM, 0.10-1.0 microM and 1.0-6.0 microM, with correlation coefficients of 0.9993, 0.9996 and 0.9993, respectively. The detection limit of 5.0 nM DA could be estimated (S/N=3). Moreover, the interferences of ascorbic acid (AA) and uric acid (UC) are effectively diminished. This hybrid film modified electrode can be applied to the determination of DA contents in dopamine hydrochloride injection and human serum. These attractive features provide a potential application for either in vitro measurement of DA in the presence of excess AA and UA or as detectors in flow injection analysis (FIA) and high performance liquid chromatography (HPLC).