Electrochemically monitoring the binding of concanavalin A and ovalbumin

To evaluate protein-protein interactions, a new voltammetric method was developed using a protein labeled with an electroactive compound. Concanavalin A (ConA), which is a lectin, recognizes α-mannose residues. Because the ConA was to be bound to ovalbumin (OVA), which has a high-mannose sugar chain, ConA labeled with daunomycin was prepared as the probe to monitor the binding. The binding to OVA was caused by the label modification of the ConA. As a result, the electrode response of the labeled ConA decreased as the OVA concentration increased. The electrode response of the labeled ConA was linearly over the range of 1.5 × 10⁻¹⁰ and 1.5 × 10⁻⁹ M OVA. The relative standard deviation of 1.5 × 10⁻⁸ M labeled ConA and 1.5 × 10⁻¹⁰ M OVA was 6.9% (n = 5). The labeled ConA-OVA binding could then be conveniently monitored based on the change in response. In contrast, interactions between the labeled ConA and a protein with no specific sugar chain also were investigated. Incubation scarcely influenced the peak current of the labeled ConA. When several concentrations of OVA were added to a serum, good recovery determined it. Consequently, this method could be applied to the measurement of protein-protein interactions.     

Voltammetric detection of ovalbumin using a peptide labeled with an electroactive compound

For this study, a new method was developed to electrochemically detect ovalbumin via its binding with the peptide-1(RNRCKGTDVQAW) in lysozymes. The peptide that exists at the C-terminal of a lysozyme was combined with ovalbumin. When an electroactive compound was introduced to the N-terminal side of the peptide through ethylene gycolbis(sulfosuccinimidyl succinate), the labeled peptide-1 served as a probe for the detection of ovalbumin. The electrode responses of labeled peptide-1 were measured after the labeled peptide-1 and ovalbumin were incubated in a 0.1 M phosphate buffer (pH 5.6). As a result, the electrode response decreased as the concentration of ovalbumin increased. The detection limit of ovalbumin was 2.3 × 10⁻¹¹ M as estimated at 3-fold the standard deviation (3σ) (n = 5). Because the steric structure of the peptide and some of the amino acid residues were related to the binding, we prepared a peptide-2, to which the N- and C-terminals of peptide-1 were alternated. The decrease in the response for the labeled peptide-2 was less than that for the labeled peptide-1. In addition, the peak current of a peptide-3, for which the D of peptide-1 was replaced with S, was hardly changed with or without ovalbumin. Therefore, it was clear that the binding was influenced by the steric factors and by the sequence of the peptide. However, a peptide-1 with bis(sulfosuccinimidyl) suberate was designed to investigate the hydrophobic influences on the probe. The change in the peak current was smaller than that of peptide-1 with ethylene gycolbis(sulfosuccinimidyl succinate), which was due to the hydrophobic properties of the alkyl chain between the peptide and the ovalbumin. The proposed method could be applied to the determination of ovalbumin in egg whites. Consequently, the concept becomes an electrochemical sensing method for proteins based on the protein–peptide interaction.     

Electrochemical sensing of concanavalin A using a non-ionic surfactant with a maltose moiety

To electrochemically detect concanavalin A (ConA), a new method was developed using mixed micelles between a non-ionic surfactant with a maltose moiety and electroactive daunomycin. The surfactants, in which the length of the alkyl chain was different, were n-decyl-β-d-maltoside, n-dodecyl-β-d-maltoside, and n-tetradecyl-β-d-maltoside. The measurement principle was due to the micelle breakdown caused by the binding between the ConA and maltose moieties. When ConA was combined with maltose moieties at a concentration of surfactant that was near the critical micelle concentration, the daunomycin that formed the micelles was moved to a solution from the micelles. As a result, the peak current of daunomycin increased as the concentration of ConA was increased. The mechanism was proposed using voltammetry, spectrometry, and gel filtration. The linear range using n-tetradecyl-β-d-maltoside was 2.0 × 10⁻⁹ to 8.0 × 10⁻⁸ M of ConA, and it was the most sensitive in the presence of the three surfactants. To examine whether selective binding took place, measurements with several proteins were carried out. The electrode responses of daunomycin were not influenced by the presence of 5.0 × 10⁻⁶ M protein. Furthermore, this method could be applied to the determination of ConA in a serum, and to the measurement of sugar chains that can be combined with ConA on the cell surface.     

Design of an electroactive peptide probe for sensing of a protein

We designed a new electroactive peptide probe that has a molecular recognition function for the sensing of a protein. Ovalbumin (OVA) was the model protein, and when RNRCKGTDVQAW interacted with OVA, it conjugated with a tyrosine-rich peptide (Y₄C). This peptide is electroactive, has a high degree of biocompatibility, and offers the possibility of gene expression. To measure the effect of a number of the tyrosine residues, voltammetric measurements were conducted using a series of tyrosine-rich peptides (Y_nC, n = 3–7) with sensitivities that ranged from 10⁻⁹ to 10⁻⁸ M. The electrode response of Y₅C was the maximum value in the series. However, the peak current did not increase when the number of tyrosine residues was increased in a linear fashion. This may have been due to the micelles that are formed by a tyrosine-rich surfactant peptide. Thus, Y₄C was suitable as an electroactive label for the construction of the peptide probe. The electrode response of Y₄CRNRCKGTDVQAW obtained by a glassy carbon electrode was 100-fold that of tyrosine alone. The measurement of OVA via the peptide probe resulted in a detection on the order of 10⁻¹² M. In contrast, the sensitivity of OVA using RCKGTDVQAWY₄C probe was at the 10⁻¹¹ M level, because the hydrophobic moiety gave it a molecular recognition function. The recoveries of the OVA using Y₄CRNRCKGTDVQAW in a solution containing fetal bovine serum ranged between 98 and 101%. Consequently, the combination of a specific peptide and an electroactive element could be a powerful probe for the sensing of proteins.     

A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti-walled carbon nanotubes in polypyrrole

A novel sensitive electrochemical biosensor based on magnetite nanoparticle for monitoring DNA hybridization by using MWNT-COOH/ppy-modified glassy carbon electrode is described. In this new detection system, mercapatoacetic acid (RSH)-coated magnetite nanoparticles, capped with 5′-(NH₂) oligonucleotide, is used as DNA probe to complex 29-base polynucleotide target (a piece of human porphobilinogen deaminase PBGD promoter from 170 to 142). Target sequence hybridized with the probe results in the decrease of the reduction peak current of daunomycin connected with probe. The response of non-complementary sequence was almost the same as the blank, and the response of three-base mismatched sequence within 29-base polynucleotide was obviously distinguished from complementary sequence, which can easily identify point mutation of DNA. The equation of calibration plot is i_p (μA) = 0.8255 − 0.0847c_{target oligonucleotide} × 10¹³ in the range of 6.9 × 10⁻¹⁴ to 8.6 × 10⁻¹³ mol/L, and correlation coefficient is 0.9974. The detective limit is 2.3 × 10⁻¹⁴ mol/L of target oligonucleotide. This device can be optimized for the detection of complex sequence.     

Synthesis of nano-sized arsenic-imprinted polymer and its use as As selective ionophore in a potentiometric membrane electrode: Part 1

In this study, a new strategy was proposed for the preparation of As (III)-imprinted polymer by using arsenic (methacrylate)₃ as template. Precipitation polymerization was utilized to synthesize nano-sized As (III)-imprinted polymer. Methacrylic acid and ethylene glycol dimethacrylate were used as the functional monomer and cross-linking agent, respectively. In order to assembly functional monomers around As (III) ion, sodium arsenite and methacrylic acid were heated in the presence of hydroquinone, leading to arsenic (methacrylate)₃. The nano-sized As (III) selective polymer was characterized by FT-IR and scanning electron microscopy techniques (SEM). It was demonstrated that arsenic was recognized as As³⁺ by the selective cavities of the synthesized IIP. Based on the prepared polymer, the first arsenic cation selective membrane electrode was introduced. Membrane electrode was constructed by dispersion of As (III)-imprinted polymer nanoparticles in poly(vinyl chloride), plasticized with di-nonylphthalate. The IIP-modified electrode exhibited a Nernstian response (20.4 ± 0.5 mV decade⁻¹) to arsenic ion over a wide concentration range (7.0 × 10⁻⁷ to 1.0 × 10⁻¹ mol L⁻¹) with a lower detection limit of 5.0 × 10⁻⁷ mol L⁻¹. Unlike this, the non-imprinted polymer (NIP)-based membrane electrode was not sensitive to arsenic in aqueous solution. The selectivity of the developed sensor to As (III) was shown to be satisfactory. The sensor was used for arsenic determination in some real samples.     

Molecularly imprinted polymer based potentiometric sensor for the determination of hydroxyzine in tablets and biological fluids

Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. In this work, a biomimetic potentiometric sensor, based on a non-covalent imprinted polymer, was fabricated for the recognition and determination of hydroxyzine in tablets and biological fluids. The molecularly imprinted polymer (MIP) was synthesized by precipitation polymerization, using hydroxyzine dihydrochloride as a template molecule, methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensor showed a high selectivity and a sensitive response to the template in aqueous system. The MIP-modified electrode exhibited a Nernstian response (29.4 ± 1.0 mV decade⁻¹) in a wide concentration range of 1.0 × 10⁻⁶ to 1.0 × 10⁻¹ M with a lower detection limit of 7.0 × 10⁻⁷ M. The electrode demonstrated a response time of ∼15 s, a high performance and a satisfactory long-term stability (more than 5 months). The method has the requisite accuracy, sensitivity and precision to assay hydroxyzine in tablets and biological fluids.     

Electrochemical detection of DNA hybridization based on polypyrrole/ss-DNA/multi-wall carbon nanotubes paste electrode

A sensitive electrochemical detection of DNA hybridization using a paste electrode assembled by multi-wall carbon nanotubes (MWNT) and immobilizing DNA probe within electropolymerized polypyrrole (ppy) was developed. The detection approach relied on entrapping of DNA probe within electropolymerized ppy film on the MWNT paste electrode and monitoring the current change generated from an electroactive intercalator of ethidium bromide (EB) after DNA hybridization. As a consequence of DNA hybridization, significant changes in the current of EB intercalated with double-stranded DNA (ds-DNA) on the MWNT paste electrode were observed. Based on the response of EB, only the complementary DNA sequence gave an obvious current signal compared with the five-point mismatched and non-complementary sequences. The oxidation peak current was linearly related to the logarithm of the concentration of the complementary DNA sequence from 1.0 × 10⁻¹⁰ to 1.0 × 10⁻⁸ M with a detection limit of 8.5 × 10⁻¹¹ M. This work demonstrates that the incorporation of MWNT paste electrode with electropolymerization is a promising strategy of functional interfaces for the immobilization of biological recognition elements.     

Enoxacin-Tb complex as an environmentally friendly fluorescence probe for DNA and its application

The fluorescence intensity of the enoxacin (ENX)-Tb³⁺ complex enhanced by DNA was studied. On the basis of this study, an environmentally friendly fluorescence probe of enoxacin-Tb³⁺ for the determination of single-stranded and double-stranded DNA was developed. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of DNA in the range of 2.0 × 10⁻⁸ to 2.0 × 10⁻⁶ g mL⁻¹ for hsDNA, 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ g mL⁻¹ for ctDNA and 5.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g mL⁻¹ for thermally denatured ctDNA. The detection limits (S/N = 3) were 5.0, 9.0 and 3.0 ng mL⁻¹, respectively. The interaction modes between ENX-Tb³⁺ and DNA and the mechanism of the fluorescence enhancement were also discussed in details. The experimental results from UV absorption spectra, fluorescence spectra and the competing combination tests between the ENX-Tb³⁺ complex and EB probe indicated that the possible interaction modes between enoxacin-Tb³⁺ complex and DNA had at least two different binding modes: the electrostatic binding and the intercalation binding. Additionally, this fluorescence probe was used to study the interaction between heavy metals and DNA.     

Application of a new potentiometric method for determination of phosphate based on a surfactant-modified zeolite carbon-paste electrode (SMZ-CPE)

A phosphate-selective electrode based on surfactant-modified zeolite (SMZ) particles into carbon-paste has been proposed (SMZ-CPE). The electrode was fully characterized in terms of composition, response time, ionic strength, thermal stability and usable pH range. The electrode containing 20% SMZ exhibited linear response range to phosphate species in the range of 1.58 × 10⁻⁵ to 1.00 × 10⁻² M with a detection limit of 1.28 × 10⁻⁵ M and a Nernstian slope of 29.9 ± 0.9 mV per decade of phosphate concentration. The electrode response to phosphate remains constant in the pH range of 4-12 and in the presence of 1 × 10⁻⁴ to 4 × 10⁻³ M NaNO₃. The response of the electrode reaches equilibrium within several seconds after immersing the electrode in phosphate solution. Common anions such as Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻ and Cr₂O₇²⁻ have little effect on the determination of phosphate but AsO₄³⁻ shows some interference. A successful application of the electrode for determination of phosphate in a fertilizer, using direct potentiometry, is presented. The electrode was also used for the potentiometric titration of phosphate. The validation of the obtained results in each case was proved by statistical methods.     

Inhibition biosensor for determination of nicotine

An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in tobacco sample. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1,4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Choline oxidase was immobilized on the carbon paste electrode through cross-linking with bovine serum albumin (BSA) by glutaraldehyde. In the presence of choline oxidase and its endogenous cofactor flavin-ademine dinneleotide (FAD), choline was oxidized into betaine while FAD was reduced to FADH₂ which subsequently reduced 1,4-benzoquinone into hydroquinone. The later was finally oxidized at a relatively low potential of +450 mV versus saturated calomel electrode (SCE). Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to choline within 1.25×10⁻⁴ to 1.25×10⁻³ mol l⁻¹. The nicotine measurements were carried out in 0.067 mol l⁻¹phosphate buffer of pH 7.4 at an applied potential of 450 mV versus SCE. The electrode provided a linear response to nicotine over a concentration range of 2.0×10⁻⁵ to 9.2×10⁻⁴ mol l⁻¹ with a detection limit of 1.0×10⁻⁵ mol l⁻¹. The system was applied to the determination of nicotine in tobacco samples.     

Determination of Cd by electrothermal atomic absorption spectrometry after electrodeposition on a graphite probe modified with palladium

Traces of Cd were determined by electrothermal atomic absorption spectrometry after electrochemical preconcentration on a commercial graphite ridge probe modified with Pd. The Pd electrochemically deposited on the probe surface served not only as the modifier but it also protected the graphite surface. Cd was electrodeposited at a controlled potential − 1.2 V (vs. saturated calomel electrode) using the Pd-modified graphite probe as a working electrode. The sensitivity of Cd determination remained unchanged for 300 electrodeposition and atomization cycles. The detection limit (3σ_blank) was improved with increasing time of electrolysis and was 1.2 ng l^− 1 for a 10 min electrolysis time in the presence of 0.1 mol l^− 1 NaNO₃. The procedure was applied for the determination of Cd in (1 + 1) diluted seawater and in (1 + 1) diluted urine samples using the standard addition method.     

Electrochemical DNA biosensors based on platinum nanoparticles combined carbon nanotubes

Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.0 × 10⁻¹¹ mol l⁻¹.     

Nanostructured rough gold electrodes as platforms to enhance the sensitivity of electrochemical genosensors

An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5′ end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg μL⁻¹ and 10 pg μL⁻¹ were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.     

Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

Slow release from gels in various solvents: a fluorescence study

A novel in situ steady state fluorescence experiment was performed for studying slow release processes in gels formed by free radical cross-linking copolymerization of methyl methacrylate and ethylene glycol dimethacrylate. Gels were prepared at 75 °C with pyrene (P_y) as a fluorescence probe. After drying these gels, slow release experiments were performed in various solvent with different molar-volume, V and solubility parameters, δ at room temperature by real-time monitoring of the P_y fluorescence intensity. Slow release diffusion coefficients (D) were measured and found to be in between 0.23×10⁻⁶ and 2.06×10⁻⁶ cm² s⁻¹ depending on the solvent used.     

Quartz crystal microbalance biosensor for recombinant human interferon-beta detection based on antisense peptide approach

Quartz crystal microbalance (QCM) biosensors for recombinant human interferon-β (rhIFN-β) were constructed by utilizing antisense peptides adhering to the QCM gold surfaces. Two antisense peptides, both corresponding to the N-terminal fragment 1-14 of rhIFN-β, were used in this study. Antisense peptide AS-1 was the original antisense peptide and AS-2 was the modified antisense peptide based on the antisense peptide degeneracy. Both antisense peptides were immobilized on the gold electrodes of piezoelectric crystals, respectively, via a self-assembling monolayer of 1,2-ethanedithiol. The binding affinity between rhIFN-β and each immobilized antisense peptide in solution was evaluated using a quartz crystal microbalance-flow injection analysis (QCM-FIA) system. The dissociation constant of rhIFN-β on the antisense peptide AS-1 and AS-2 biosensor was (1.89 ± 0.101) × 10⁻⁴ and (1.22 ± 0.0479) ×10⁻⁵ mol L⁻¹, respectively. The results suggested that AS-2 had a higher binding affinity to rhIFN-β than AS-1. The detection for rhIFN-β using each biosensor was precise and reproducible. The linear response ranges of rhIFN-β binding to both biosensors were same with a concentration range of 0.12-0.96 mg mL⁻¹. The results demonstrated the successful construction of highly selective QCM biosensors using antisense peptide approach, and also confirmed the feasibility of increasing antisense peptide binding affinity by appropriate sequence modification.     

Electrochemical deoxyribonucleic acid biosensor based on carboxyl functionalized graphene oxide and poly-l-lysine modified electrode for the detection of tlh gene sequence related to vibrio parahaemolyticus

A carboxyl functionalized graphene oxide (GO-COOH) and electropolymerized ploy-l-lysine (PLLy) modified glassy carbon electrode (GCE) was fabricated and used for the construction of an electrochemical deoxyribonucleic acid (DNA) biosensor. The NH₂ modified probe ssDNA sequences were immobilized on the surface of GO-COOH/PLLy/GCE by covalent linking with the formation of amide bonds, which was stable and furthur hybridized with the target ssDNA sequence. Differential pulse voltammetry (DPV) was used to monitor the hybridization events with methylene blue as electrochemical indicator, which gave a sensitive reduction peak at −0.287 V (vs. SCE). Under the optimal conditions the reduction peak current was proportional to the concentration of tlh gene sequence in the range from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ mol L⁻¹ with a detection limit as 1.69 × 10⁻¹³ mol L⁻¹ (3σ). The polymerase chain reaction products of tlh gene from oyster samples were detected with satisfactory results, indicating the potential application of this electrochemical DNA sensor.     

Preparation of a new solid state fluoride ion selective electrode and application

A new solid state fluoride ion selective electrode composed of 70% Ag₂S, 10% Cu₂S and 20% CaF₂ has been developed. An analytically useful potential change occurred, from 1 × 10⁻⁶ to 1 × 10⁻¹ M fluoride ion. The slope of the linear portion (1 × 10⁻¹-1 × 10⁻⁵ M) was about 26 ± 2 mV/10-fold concentration changes in fluoride. It was found that pH change between 1 and 8 had no effect on the potential of the electrode. There was no interference of most common cations such as K^+, Na⁺, Ca²⁺ and Mg²⁺ and anions such as Cl⁻, NO₃⁻, SO₄²⁻ and PO₄³⁻. The lifetime of the electrode was more than 2 years, when used at least 4-5 times a day, and the response time was about 60 s.The measurements were made at constant ionic strength (0.1 M NaNO₃) and at room temperature. This electrode has been used for the determination of fluoride ion in Ankara city tap water and in bottled spring water using standard addition method. The validation of the electrode has been made with a commercial fluoride ion selective electrode (Orion) and high consistency was obtained.     

Zeolite A functionalized with copper nanoparticles and graphene oxide for simultaneous electrochemical determination of dopamine and ascorbic acid

A novel Cu-zeolite A/graphene modified glassy carbon electrode for the simultaneous electrochemical determination of dopamine (DA) and ascorbic acid (AA) has been described. The Cu-zeolite A/graphene composites were prepared using Cu²⁺ functionalized zeolite A and graphene oxide as the precursor, and subsequently reduced by chemical agents. The composites were characterized by X-ray diffraction, Fourier transform infrared spectra and scanning electron microscopy. Based on the Cu-zeolite A/graphene-modified electrode, the potential difference between the oxidation peaks of DA and AA was over 200 mV, which was adequate for the simultaneous electrochemical determination of DA and AA. Also the proposed Cu-zeolite/graphene-modified electrode showed higher electrocatalytic performance than zeolite/graphene electrode or graphene-modified electrode. The electrocatalytic oxidation currents of DA and AA were linearly related to the corresponding concentration in the range of 1.0 × 10⁻⁷–1.9 × 10⁻⁵ M for DA and 2.0 × 10⁻⁵–2.0 × 10⁻⁴ M for AA. Detection limits (<!-- no-mfc -->S/N<!-- /no-mfc --> = 3) were estimated to be 4.1 × 10⁻⁸ M for DA and 1.1 × 10⁻⁵ M for AA, respectively.