A nano self-assembled artificial peroxidase: spectroscopic and electrochemical investigations

A nano-micelle with highly efficient peroxide activity was constructed by self-assembly of sodium dodecyl sulfate micellar, histidine and hematin in 50 mM phosphate buffer at 25 °C. UV–Vis spectrometry methods were utilized for characterization of the nanostructured material or artificial peroxidase (AP). The Michaelis–Menten (K _m) and catalytic rate (k _cat) constants of the AP were obtained to be 5.5 μM and 0.06 s^?1, respectively, in 50 mM phosphate buffer solution at pH 8.0. The catalytic efficiency of AP was evaluated to be 0.011 μM^?1 s^?1. The AP was also immobilized on a functional multi-wall carbon nanotubes-gold nanoparticles (AuNPs) nano-complex modified glassy carbon electrode (GCE). The transmission electron microscopy method was utilized for the characterization of the nano-materials. The electron-transfer rate constant (k _s) and the apparent Michaelis–Menten constant K _m ^app of the AP modified GCE were evaluated to be 1.36 s^?1 and 0.19 μM, respectively. For a biosensor without a redox protein, the properties of the AP modified GCE were significant and will further benefit from additional studies and improvement.     

Cloning and expression of NDM-1 metallo-β-lactamase gene and study of the catalytic properties of the recombinant enzyme

A gene-expression system has been designed to express the NDM-1 metallo-β-lactamase gene in E. coli cells. This system enables the synthesis of the recombinant protein in a soluble and active form. A method for the isolation and purification of the recombinant enzyme has been developed. The yield of the homogeneous protein preparation was 10–15 mg per liter of E. coli culture medium. The catalytic parameters of the recombinant NDM-1 β-lactamase were measured for ampicillin (K_m = 185 μM and k_cat = 585 s^–1) and meropenem (K_m = 85 μM and k_cat = 160 s^–1). These values correlate well with the literature data. The catalytic parameters for the chromogenic CENTA substrate (K_m = 14 μM and k_cat = 290 s^–1) were obtained for the first time.     

NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

Self-assembling of Prussian blue nanocubic particles on nanoporous glassy carbon and its use in the electrocatalytic reduction of hydrogen peroxide

In this paper, self-assembled Prussian blue nanocubic particles on nanoporous glassy carbon was developed. The morphology of the PBNP-modified porous glassy carbon was characterized by scanning electron microscopy. The PBNP-GCE-red film-modified electrode was used for the sensitive detection of hydrogen peroxide. The electrochemical behavior of the resulting sensor was investigated using cyclic voltammetry and chronoamperometry. The value of α, k _cat, and D was calculated as 0.35, 1.7 × 10⁵ cm³ mol^?1 s^?1, and 2.6 × 10^?5 cm² s^?1, respectively. The calibration curve for hydrogen peroxide determination was linear over 0–600 μM with a detection limit (S/N = 3) of 0.51 μM.     

Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4～20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K _M was 210 μM and k _cat was 3.4 s^?1. Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4～40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Resveratrol and arctigenin production from polydatin and arctiin respectively by a thermostable β-glucosidase from Thermotoga maritima

Abstract

For preparation of resveratrol and arctigenin from peanut hulls and arctium lappa fruits, respectively, a recombinant β-glucosidase (TmBglA) from hyperthermophile Thermotoga maritima was purified and characterized. The hydrolytic activity was the highest at 90?°C and pH 6.2 for arctiin with K_m of 1.61?mM and k_cat of 197.4?s^?1, and 90?°C and 5.8 for polydatin with K_m of 0.38?mM and k_cat of 47.6?s^?1. The enzyme produced 215.4?mg L^?1 resveratrol and 355.7?mg L^?1 arctigenin from 400?mg L^?1 polydatin and 540?mg L^?1 arctiin after 60?min of incubation at 80?°C, with capable of hydrolyzing up to 92.1 and 94.9% of polydatin and arctiin, respectively. The enzymatic hydrolysis of peanut hulls and fructus arctii displayed a conversion yield of 3.8 and 0.33?mg resveratrol and arctigenin per gram of substrate material flour, respectively. Of the reported β-glucosidase, TmBglA exhibited the highest thermostability, k_cat, k_cat/K_m, and conversion productivity for hydrolyzing polydatin and arctiin, and has great potential applications in functional food and medicine production.     

PANI–TiC nanocomposite film for the direct electron transfer of hemoglobin and its application for biosensing

A novel polyaniline and titanium carbide (PANI–TiC) nanocomposite was synthesized by an in situ chemical oxidative polymerization method, and a hydrogen peroxide (H₂O₂) biosensor was fabricated by PANI–TiC with hemoglobin (Hb)-modified glassy carbon electrode (GCE). Scanning electron microscope and energy dispersive X-ray spectroscopy showed the morphology and ingredient of PANI–TiC. Electrochemical investigation of the biosensor showed a pair of well-defined, quasi-reversible redox peaks with E _pa?=??0.318 V and E _pc?=??0.356 V (vs SCE) in 0.1 M, pH 7.0 sodium phosphate-buffered saline at the scan rate of 150 mV s^?1. Transfer rate constant (k _s) was 2.01 s^?1. The Hb/PANI–TiC/GCE showed a good electrochemical catalytic response for the reduction of H₂O₂ with the linear range from 0.5 to 285.5 μM and the detection limit of 0.2 μM (S/N?=?3). The apparent Michaelis–Menten constant (K _m) was estimated to be 1.21 μM. Therefore, the PANI–TiC as a novel matrix opened up a further possibility for study on the design of enzymatic biosensors with potential applications.     

Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k _cat/K _m 4NPA/k _cat/K _m 4NPX was ～7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k _cat/K _m muA/k _cat/K _m muX was ～5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K _i of 6.8?±?0.62 mM and xylose K _i of 76?±?8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t _1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k _non) the enzyme at pH 5.3, which lies in the pH-independent region for k _cat and k _non. Rate enhancements (k _cat/k _non) for 4NPX, 4NPA, and X2 are 4.3?×?10¹¹, 2.4?×?10⁹, and 3.7?×?10¹², respectively, at 25 °C and increase with decreasing temperature. Relative parameters k _cat ^4NPX/k _cat ^4NPA, k _cat ^4NPX/k _cat ^X2, and (k _cat/K _m)^4NPX/(k _cat/K _m)^X2 increase and (k _cat/K _m)^4NPX/(k _cat/K _m)^4NPA, (1/K _m)^4NPX/(1/K _m)^4NPA, and (1/K _m)^4NPX/(1/K _m)^X2 decrease with increasing temperature.     

Phenomenological patterns in enzymatic catalysis

A statistical analysis of rate constants of limiting reaction steps and of Michaelis constants is carried out. The densities of parameter distributions follow the normal logarithmic equations with parameter values, k_cat = 55 s⁻¹ and K_M = 1 × 10⁻⁴M. The efficiency of catalysis with enzymes, and the mean and limiting k_cat and K_M values are discussed. The correlations between the limiting rate constants and the Michaelis constants are analyzed. Strong correlations between the parameters are observed in a number of cases, with slopes in logarithmic coordinates of −1, 0, 1, 2, infinity. The possible molecular mechanisms explaining the observed phenomenological patterns are discussed.     

Enzyme assay for chitinase catalyzed hydrolysis of tetra-N-acetylchitotetraose by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has been used to observe the chitinase-catalyzed hydrolysis of tetra-N-acetylchitotetraose. Enzymatic hydrolysis of tetra-N-acetylchitotetraose by chitinase B from Serratia marcescens produces exclusively two molecules of di-N-acetylchitobiose allowing for the determination of a single glycosidic bond hydrolysis heat that was used to monitor the rate of the enzymatic reaction. The change in heat rate with respect to time (dQ/dt) was translated to the reaction rate, and the total heat produced was related to substrate concentration throughout the reaction. Reaction rates versus substrates concentration were fit to Michaelis-Menten plots, yielding a k_cat of 40.9 ± 0.5 s⁻¹ and a K_m of 54 ± 2 μM.     

Kinetics and Mechanism of the Interaction of Di-μ-hydroxobis(1,10-phenanthroline)dipalladium(II) Perchlorate with Thioglycolic Acid and Glutathione in Aqueous Solution

The kinetics of interaction between di-μ-hydroxobis(1,10-phenanthroline)dipalladium(II) perchlorate and thioglycolic acid and with glutathione has been studied spectrophotometrically in aqueous medium as a function of the complex concentration as well as the ligand concentrations, pH, and temperature at constant ionic strength. The observed pseudo-first-order rate constants k _obs (s^?1) obeyed the equation k _obs = k ₁[Nu] (Nu = nucleophile). At pH = 6.5, the interaction with thioglycolic acid shows two distinct consecutive steps and both steps are dependent on the concentration of thioglycolic acid. The rate constants for the process are: k ₁ ≈ 10^?5 s^?1 and k ₂ ≈ 10^?3 dm³ · mol^?1 · s^?1. The association equilibrium constant (K _E) for the outer sphere complex formation has been evaluated together with the rate constants for the two subsequent steps. The other bio-active ligand, glutathione, showed a single step reaction depending on [ligand] with a second-order anation rate constant: the 10² (k ₂) values are (61.72, 79.20, 109.24 and 154.33) dm³ · mol^?1 · s^?1 at 20, 25, 30 and 35 °C, respectively. On the basis of the kinetic observations and evaluated activation parameters, plausible associative mechanisms are proposed for both interaction processes.     

The hemoglobin-modified electrode with chitosan/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanocomposite for the detection of trichloroacetic acid

A hemoglobin (Hb)-modified electrode based on chitosan/Fe₃O₄ nanocomposite coated glassy carbon has been constructed for trichloroacetic acid (TCA) detection. The structure of chitosan/Fe₃O₄ nanocomposite was investigated using energy-dispersive X-ray analysis (EDS) and X-ray diffraction (XRD) patterns. The electron transfer rate constant (k _s) of Hb was estimated for as high as 3.12 s^?1. The immobilized Hb exhibited excellent electro-catalytic activity toward the reduction of TCA. The response current regressed to the concentration of TCA within the range of 5.70 μM to 205 μM with a detection limit of 1.9 μM (S/N = 3).     

Electrochemical evaluation of Abelson tyrosine-protein kinase 1 activity and inhibition by imatinib mesylate and danusertib

Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis–Menten constant for abltide binding K_M ∼ 4.5 μM, turnover number k_cat ∼ 11 s⁻¹ and enzyme efficiency k_cat/K_M ∼ 2.3 s⁻¹ μM⁻¹ were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 μM determined.     

Characteristics of a High Maltose-Forming, Acid-Stable, and Ca2+-Independent α-amylase of the Acidophilic Bacillus acidicola

The purified acidic α-amylase of Bacillus acidicola is a monomer of 66.0 kDa, optimally active at pH 4.0 and 60 °C. The enzyme is Ca²⁺ independent with T _1/2 for 18 min at 80 °C. The K _m, V _max, and catalytic efficiency (k _cat/K _m) of the enzyme are 1.6 mg mL^?1, 23.8 μmol mg^?1 min^?1, and 981 μmol s^?1, respectively. Among detergents, Tween 20, 40, and 80 stimulated enzyme activity, whereas sodium dodecyl sulfate and Triton X-100 inhibited even at low concentration. EGTA has not affected the activity, whereas EDTA β-mercaptoethanol, iodoacetic acid, and Dithiothreitol exhibited a slight inhibitory action. Phenylmethanesulfonyl fluoride, N-bromosuccinimide, and Hg²⁺ strongly inhibited enzyme activity. The experimental activation energy and temperature quotient are 50.12 kJ mol^?1 and 1.37. When thermodynamic parameters (ΔH and ΔS) of the enzyme have been determined at different temperatures, ΔG is positive suggesting that the enzyme is thermostable. The enzyme hydrolyzes raw starches, and therefore, the enzyme finds application in raw starch saccharification at sub-gelatinization temperatures that saves energy needed for gelatinization of raw starch at 105 °C.     

Direct electrochemistry and bioelectrocatalysis of a class II non-symbiotic plant haemoglobin immobilised on screen-printed carbon electrodes

In this study, direct electron transfer (ET) has been achieved between an immobilised non-symbiotic plant haemoglobin class II from Beta vulgaris (nsBvHb2) and three different screen-printed carbon electrodes based on graphite (SPCE), multi-walled carbon nanotubes (MWCNT-SPCE), and single-walled carbon nanotubes (SWCNT-SPCE) without the aid of any electron mediator. The nsBvHb2 modified electrodes were studied with cyclic voltammetry (CV) and also when placed in a wall-jet flow through cell for their electrocatalytic properties for reduction of H₂O₂. The immobilised nsBvHb2 displayed a couple of stable and well-defined redox peaks with a formal potential (E°′) of ?33.5 mV (vs. Ag|AgCl|3 M KCl) at pH 7.4. The ET rate constant of nsBvHb2, k _s, was also determined at the surface of the three types of electrodes in phosphate buffer solution pH 7.4, and was found to be 0.50 s^?1 on SPCE, 2.78 s^?1 on MWCNT-SPCE and 4.06 s^?1 on SWCNT-SPCE, respectively. The average surface coverage of electrochemically active nsBvHb2 immobilised on the SPCEs, MWCNT-SPCEs and SWCNT-SPCEs obtained was 2.85?×?10^?10 mol cm^?2, 4.13?×?10^?10 mol cm^?2 and 5.20?×?10^?10 mol cm^?2. During the experiments the immobilised nsBvHb2 was stable and kept its electrochemical and catalytic activities. The nsBvHb2 modified electrodes also displayed an excellent response to the reduction of hydrogen peroxide (H₂O₂) with a linear detection range from 1 μM to 1000 μM on the surface of SPCEs, from 0.5 μM to 1000 μM on MWCNT-SPCEs, and from 0.1 μM to 1000 μM on SWCNT-SPCEs. The lower limit of detection was 0.8 μM, 0.4 μM and 0.1 μM at 3σ at the SPCEs, the MWCNT-SPCEs, and the SWCNT-SPCEs, respectively, and the apparent Michaelis–Menten constant, $ {\hbox{K}}_{\rm{M}}^{\rm{app}} $ , for the H₂O₂ sensors was estimated to be 0.32 mM , 0.29 mM and 0.27 mM, respectively.     

Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris

The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K _m, k _cat, and V _max values of the purified PACD were calculated to be 40.86 μM, 0.566 s^?1 and 0.693 U?mg^?1 min^?1. The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg²⁺ had an activating effect on the enzyme, while Mn²⁺, Ca²⁺, Zn²⁺, and Cu²⁺ had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass–sugars.     

Comparison of antibody and albumin catalyzed hydrolysis of steroidalp-nitrophenylcarbonates

A monoclonal antibody (MAb) was produced against thep-nitrophenylphosphate derivative of 3α,5β-lithocholic acid, a transition-state analog for hydrolysis of a steroidalp-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants k_cat = 4.0 × 10^-2min and K_m = 3.3 μM at pH 9.0 and 35°C. The Ab also hydrolyzed the isomericp-nitrophenylcarbonate of 3β,5β-lithocholic acid with k_cat = 8.4 × 10^-2/min and K_m = 1.0 μM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 μM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC₅₀ ca. 40 μM), the Ab-catalyzed reaction was completely inhibited at less than 1 μM of the TSA. The relative rates and efficiencies of the MAbcatalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.     

Oxidation of dopamine on multi-walled carbon nanotubes

Novel films consisting of multi-walled carbon nanotubes (MWCNTs) were fabricated by means of the chemical vapor deposition technique with decomposition of either acetonitrile (ACN) or benzene (BZ) in the presence of ferrocene (FeCp₂) which served as catalyst. The electrochemical response of the two different kinds of MWCNT-based films, further referred to as MWCNT-ACN and MWCNT-BZ, towards the oxidation of dopamine (DA) to dopamine-o-quinone (DAQ) was tested by means of cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. Both MWCNT-based films exhibit quasi-reversible response towards DA/DAQ with some slight kinetic differences; specifically, the charge-transfer process was found to be faster on MWCNT-ACN (k _s?=?35.3?×?10^?3 cm s^?1) compared to MWCNT-BZ (k _s?=?6.55?×?10^?3 cm s^?1). The detection limit of MWCNT-BZ for DA (0.30 μM) appears to be poorer compared to that of MWCNT-ACN (0.03 μM), but nevertheless, both MWCNT-based films exhibit greater detection ability compared to other electrodes reported in the literature. The sensitivities of MWCNT-ACN and MWCNT-BZ towards DA/DAQ were determined as 0.65 and 0.22?A M^?1 cm^?2, respectively. The findings suggest that the fabricated MWCNT-based electrodes can be successfully applied for the detection of molecules with biological interest.