Immobilization of catalase via adsorption onto metal-chelated affinity cryogels

A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe³⁺ ions. This immobilized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 μmol Fe³⁺ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)-CB-Fe³⁺ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)-CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K_m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V_max value of free catalase (2.0 × 10³ U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10³ U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)-CB-Fe³⁺ cryogel.     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide–tetrazolium coupling reactions for quantitative l-lactate analysis

In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2⁻⁶ U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD⁺ (2 μl, 1.5 × 10⁻⁵ M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

The antioxidant effect of the mesoionic compound SYD-1 in mitochondria

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75 μmol mg⁻¹ protein) inhibited the lipoperoxidation induced by Fe³⁺/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0 μmol mg⁻¹ protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α′-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25–1.0 μmol mg⁻¹ protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9–19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25–1.0 μmol mg⁻¹ protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca²⁺/phosphate by approximately 30% (1.0 μmol mg⁻¹ protein). When Ca²⁺/H₂O₂ were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0 μmol mg⁻¹ of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Nafion/lead nitroprusside nanoparticles modified carbon ceramic electrode as a novel amperometric sensor for l-cysteine

This work describes the electrochemical and electrocatalytic properties of carbon ceramic electrode (CCE) modified with lead nitroprusside (PbNP) nanoparticles as a new electrocatalyst material. The structure of deposited film on the CCE was characterized by energy dispersive X-ray (EDX), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM). The cyclic voltammogram (CV) of the PbNP modified CCE showed two well-defined redox couples due to [Fe(CN)₅NO]³⁻/[Fe(CN)₅NO]²⁻ and Pb^IV/Pb^II redox reactions. The modified electrode showed electrocatalytic activity toward the oxidation of l-cysteine and was used as an amperometric sensor. Also, to reduce the fouling effect of l-cysteine and its oxidation products on the modified electrode, a thin film of Nafion was coated on the electrode surface. The sensor response was linearly changed with l-cysteine concentration in the range of 1 × 10⁻⁶ to 6.72 × 10⁻⁵ mol L⁻¹ with a detection limit (signal/noise ratio [S/N] = 3) of 0.46 μM. The sensor sensitivity was 0.17 μA (μM)⁻¹, and some important advantages such as simple preparation, fast response, good stability, interference-free signals, antifouling properties, and reproducibility of the sensor for amperometric determination of l-cysteine were achieved.     

One-step "green" preparation of graphene nanosheets and carbon nanospheres mixture by electrolyzing graphite rob and its application for glucose biosensing

The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO₃ solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (E_pa) and −0.509 V (E_pc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s⁻¹ and the surface coverage of HRP was 3.18 × 10⁻¹⁰ mol cm⁻². The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.     

ZnO–CuxO/polypyrrole nanocomposite modified electrode for simultaneous determination of ascorbic acid,dopamine, and uric acid

Novel zinc oxide (ZnO) nanosheets and copper oxide (Cu_xO, CuO, and Cu₂O) decorated polypyrrole (PPy) nanofibers (ZnO–Cu_xO–PPy) have been successfully fabricated for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The morphology and structure of ZnO–Cu_xO–PPy nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. Compared with the bare glassy carbon electrode (GCE), PPy/GCE, Cu_xO–PPy/GCE, and ZnO–PPy/GCE, ZnO–Cu_xO–PPy/GCE exhibits much higher electrocatalytic activities toward the oxidation of AA, DA, and UA with increasing peak currents and decreasing oxidation overpotentials. Cyclic voltammetry (CV) results show that AA, DA, and UA could be detected selectively and sensitively at ZnO–Cu_xO–PPy/GCE with peak-to-peak separation of 150 and 154 mV for AA–DA and DA–UA, respectively. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.2 to 1.0 mM, 0.1 to 130.0 μM, and 0.5 to 70.0 μM, respectively. The lowest detection limits (signal/noise = 3) were 25.0, 0.04, and 0.2 μM for AA, DA, and UA, respectively. With good selectivity and sensitivity, the current method was applied to the determination of DA in injectable medicine and UA in urine samples.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Formation and decay of P680 (PD1–PD2)PheoD1 radical ion pair in photosystem II core complexes

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~ 11 ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (Pheo_D1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺Pheo_DI⁻. The subsequent electron transfer from Pheo_D1⁻ to primary plastoquinone electron acceptor (Q_A) was accompanied by relaxation of the 460-nm band and occurred within ~ 250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo_DI⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo_DI⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo_D1. According to previous measurements in the femtosecond–picosecond time range this Chl-670 was ascribed to Chl_D1 [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45–50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~ 1 ps and ~ 14 ps. These components appear to reflect formation of P680⁺Chl_D1⁻ and P680⁺Pheo_D1⁻, respectively, as found earlier. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Synthesis, spectroscopy, electrochemistry, and photochemistry of cyano-bridged mixed-valence coordination compounds containing two different types of intervalent charge-transfer bands

The tetranuclear and pentanuclear mixed-valence coordination compounds Na[(NC)₅Fe^II-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅], or FePtFeRu, and [Ru^III(NH₃)₅-μ(NC)-Fe^II(CN)₄-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅](OSO₂CF₃)₂, or RuFePtFeRu, were synthesized and characterized by IR and UV-Vis spectroscopy, electron microprobe analysis (EPMA), inductively coupled plasma (ICP), and cyclic voltammetry (CV). Both molecules exhibit Fe^II → Pt^IV intervalent charge transfer (IVCT) absorptions in the 400-450 nm range and Fe^II → Ru^III transition(s) between 750 and 950 nm. The energies, intensities, and half-widths of these transitions correspond well with those of model compounds. The cyclic voltammogram of FePtFeRu between 0.00 and 0.90 V versus SCE exhibits two quasi-reversible Fe waves at 0.56 and 0.74 V versus SCE, while that for RuFePtFeRu has only one Fe redox event at 0.72 V versus SCE. When the potential of the working electrode is scanned negative of −0.38 V versus SCE, however, both complexes undergo an ECE (electrochemical-chemical-electrochemical) mechanism whereby the electrochemical reduction of Ru(III) is followed by a double electron transfer to reduce Pt(IV) to Pt(II). Upon reduction to Pt(II), the cyanide bridges break and the complexes dissociate into smaller fragments. Irradiation of the Fe^II → Pt^IV IVCT transition in both compounds leads to a photolysis solution that contains dissociated Fe(II)-Ru(III) as one of its products. Irradiation of the Fe^II → Ru^III IVCT transition yields a similar UV-Vis spectrum, suggesting that the same intermediate is common to both photolysis mechanisms. The implications of this research within the larger context of multiple electron transfer are also discussed.     

Electrolyte effects on the intervalence transition within discrete binuclear cyano-bridged complexes. An estimation of activation free energy from static, optical and electrochemical data

A study of the metal-to-metal charge-transfer (MMCT) transition within the binuclear cyano-bridged complexes cis-[L¹³Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹³ = 12-methyl-1,4,7,10-tetraazacyclotridecan-12-amine), trans-[L¹⁴Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹⁴ = 6-methyl-1,4,8,11-tetraazacyclotetradecan-6-amine) and trans-[L¹⁵Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹⁵ = 10-methyl-1,4,8,12-tetraazacyclopentadecan-10-amine) has been carried out in electrolyte solutions at varying concentrations. Using these data, as well as the reaction free energies obtained from electrochemical measurements, the reorganisation and activation free energies for the forward and reverse thermal electron-transfer processes have been estimated. The changes of these parameters with the electrolyte concentration, as well as those of the energy of the maximum MMCT band and the reaction free energy, are mainly due to ion-pairing effects.     

Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide

The CAT/PANi/ITO bioelectrode has been prepared as a catalase biosensor and shows response for monitoring not only of H₂O₂ but also azide. The sensor exhibited an excellent response to the H₂O₂ and azide. The linear range of H₂O₂ was 0.064∼1 mM and for azide 0.125∼4 mM, respectively. Catalase biosensor was based on the principle of the measurements as the decrease in the differentiation of oxygen level, which has been caused by the inhibition of catalase in the bioactive layer of the biosensor by azide. The repeatability experiments were done in triplicate. The logarithm response of the biosensor to H₂O₂ (r² = 0.99), as well as, for azide (r² = 0.90) were reported, respectively. The bioelectrode was characterized by CV and AFM. The proposed biosensor would be applied for the determination of H₂O₂ and azide in various biological samples.     

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i₀ and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 °C for 30 min, a quantitative analysis could be achieved in the range of 10⁻⁴-10⁻² units/μl of DNase I.     

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.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.