Speciation of chromium using chronoamperometric biosensors based on screen-printed electrodes

Chronoamperometric assays based on tyrosinase and glucose oxidase (GOx) inactivation have been developed for the monitoring of Cr(III) and Cr(VI). Tyrosinase was immobilized by crosslinking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as electron transfer mediator. The tyrosinase/SPC_TTFE response to pyrocatechol is inhibited by Cr(III). This process, that is not affected by Cr(VI), allows the determination of Cr(III) with a capability of detection of 2.0 ± 0.2 μM and a reproducibility of 5.5%. GOx modified screen-printed carbon platinised electrodes (SPC_PtEs) were developed for the selective determination of Cr(VI) using ferricyanide as redox mediator. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr(III) and to detect the toxic species Cr(VI). Chronoamperometric response of the biosensor towards glucose decreases with the presence of Cr(VI), with a capability of detection of 90.5 ± 7.6 nM and a reproducibility of 6.2%. A bipotentiostatic chronoamperometric biosensor was finally developed using a tyrosinase/SPC_TTFE and a GOx/SPC_PtE connected in array mode for the simultaneous determination of Cr(III) and Cr(VI) in spiked tap water and in waste water from a tannery factory samples.     

Characterization of a Disposable Electrochemical Biosensor Based on Putrescine Oxidase from Micrococcus rubens for the Determination of Putrescine

Putrescine oxidase (PUO) from Micrococcus rubens has been characterized, as a biocatalytic component for the development of electrochemical biosensors for the determination of putrescine. PUO was immobilized by cross‐linking on screen‐printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as mediator (TTF/SPCEs). Isothermal titration calorimetry was used for investigation of kinetic properties of immobilized PUO. These PUO/TTF/SPCEs showed a linear response range from 10 a 74 µM, a capability of detection of 10.1±0.6 µM (n=4, α=β=0.05) and a precision of 6.7 %. Octopus and zucchini samples have been analysed using HPLC as a reference technique.     

Amperometric Biosensors for Tyramine Determination Based on Graphene Oxide and Polyvinylferrocene Modified Screen‐printed Electrodes

A comparison of the analytical characteristics of two tyramine biosensors, based on graphene oxide (GRO) and polyvinylferrocene (PVF) modified screen‐printed carbon electrodes (SPCE), is reported. Diamine oxidase (DAOx) or monoamine oxidase (MAOx) was immobilized onto the PVF/GRO modified SPCE to fabricate the biosensors. Surface characteristics and electrochemical behaviour of the modified SPCEs were investigated by atomic force microscopy (AFM), scanning electron microscopy (SEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammetry (CV). Electrode surface composition and experimental variables such as pH and working potential were optimized in order to ensure a high performance. Under optimum experimental conditions, both DAOx/PVF/GRO/SPCE and MAOx/PVF/GRO/SPCE biosensors exhibited wide linear dynamic ranges for tyramine from 9.9×10^?7 to 1.2×10^?4 M and from 9.9×10^?7 to 1.1×10^?4 M, respectively. MAOx/PVF/GRO/SPCE biosensor showed higher sensitivity (11.98 μA mM^?1) for tyramine determination than the DAOx/PVF/GRO/SPCE biosensor (7.99 μA mM^?1). The substrate specifity of the biosensors to other biogenic amines namely histamine, putrescine, spermine, spermidine, tryptamine, β‐phenylethylamine and cadaverine was also investigated. The developed biosensors were successfully used for tyramine determination in cheese sample.     

Disposable Amperometric Immunosensor for the Determination of the E‐Cadherin Tumor Suppressor Protein in Cancer Cells and Human Tissues

This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H₂O₂/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL^?1) and a detection limit as low as 0.16 ng mL^?1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL^?1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated.     

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 μM and from 0.4 to 2.4 μM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18 ± 0.01 μM in the case of DAO/HRP and 0.40 ± 0.04 μM (α = 0.05 and β = 0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.     

Heavy metal-free electrochemical detection of pyrophosphate ions by Nafion/carbon dots decorated screen printed carbon electrodes

We employ Nafion-mixed carbon dots (CDs) and low-cost screen-printed carbon electrodes (SPCEs) as foundation matrices for the fabrication of electrochemical biosensors. The  NH₂ and  COOH functional groups present on the SPCE surface after Nafion/CDs deposition allow for pyrophosphate ions (PPis) collection. Using Fe(CN)₆³⁻ as the electrochemical mediator, the SPCE-Nafion/CDs are applied to the detection of aqueous PPi by square wave voltammetry. Between 50 and 1 μM, a linear connection is established between the square wave voltammetry current and the PPi concentration. The limit of detection is determined to be 1.01 μM, and recoveries of 113% (±1.9%) and 108% (±3.9%) are achieved for human urine samples spiked with 6 and 3 μM of PPi, respectively. Furthermore, this PPi assay is suitable for the usage of complicated urine matrices without the inclusion of heavy metals. We anticipate that this unique approach will be beneficial for PPi level monitoring in urine during therapeutic treatments of illnesses and malignancies.     

CYP450 2B4 covalently attached to carbon and gold screen printed electrodes by diazonium salt and thiols monolayers

An easy covalent immobilization method used to develop enzyme biosensors based on carbon and gold screen printed electrodes (SPCEs and gold SPEs) is described. The linkage of biomolecules through 4-nitrobenzenediazonium tetrafluoroborate, mercaptopropionic acid and thioctic acid monolayers has been attempted using bare SPCEs and gold SPEs, as well as gold nanoparticles (AuNPs) modified SPCEs and gold SPEs. Direct covalent attachment of Cytochrome P450 2B4 (CYP450 2B4) to the transducer has been carried out by carbodiimide and hydroxysuccinimide. Experimental variables in the immobilization process and in the chronoamperometric determination of Phenobarbital (PB) have been optimized by the experimental design methodology. Reproducibility of the different biosensors has been checked under the optimum conditions, yielding values lower than 6%. Their performances have been shown by the determination of PB in pharmaceutical drugs.     

Comparison of Single Layer and Bilayer Biosensors Based on Crosslinking of Penicillinase for Potentiometric Detection of Penicillin in Milk and Antibiotics

The use of bilayers for fabrication of biosensors is advantageous for increasing enzyme loading. Substantial improvement in sensitivity is often achieved through immobilisation of the enzyme in both layers. In particular, the use of cross linking agents such as glutaraldehyde (GLA), bovine serum albumin (BSA) and polyvinyl alcohol (PVA) are beneficial for enhancing enzyme stability and, hence, for fabricating stable biosensors. The successful fabrication of a single layer BSA‐GLA‐P’nase biosensor for potentiometric detection of penicillin is described. Subsequently, the three crosslinking agents were employed with two polymers, polypyrrole (PPy) and polytyramine (PTy), together with penicillinase (P’nase) for fabrication of PPy‐NO₃‐P’nase/BSA‐PVA‐P’nase and PTy‐NO₃‐P’nase/BSA‐GLA‐P’nase bilayer biosensors. The analytical performances of the bilayer biosensors were then compared with the single layer BSA‐GLA‐P’nase biosensor for the determination of penicillin in milk and amoxycillin tablets. While the determination of penicillin in milk was somewhat problematic, its determination in amoxicillin tablets proved to be successful, with recoveries of 102±15 % obtained with the PPy‐NO₃‐P’nase/BSA‐PVA‐P’nase biosensor, 100±19 % with PTy‐NO₃‐P’nase/BSA‐GLA‐P’nase biosensor and 103±5 % with BSA‐GLA‐P’nase biosensor. Notably, the results of the latter agreed favourably with those obtained through a reference titrimetric method.     

An Amperometric Biosensor for trans‐Resveratrol Determination in Aqueous Solutions by Means of Carbon Paste Electrodes Modified with Peroxidase Basic Isoenzymes from Brassica Napus

The catalytic properties of peroxidase basic isoenzymes (PBI's) from Brassica napus towards trans‐resveratrol (t‐Res) oxidation were demonstrated by the first time by conventional UV‐visible spectroscopic measurements. The enzymatic reaction rate was studied under different experimental conditions and the kinetics parameters were determined. An amperometric biosensor based on Brassica napus PBI's to determine t‐Res is also proposed by the first time. The method employs a dialysis membrane covered, PBI's entrapped and ferrocene (Fc)‐embedded carbon paste electrode (PBI's‐Fc‐CP) and is based on the fact that the decreased amount of H₂O₂ produced by the action of PBI's is proportional to the oxidised amount of t‐Res in the solution. Comparative amperometric experiments showed that, in spite of PBI's activity was much lower than commercial horseradish peroxidase (HRP) activity, t‐Res was a much better substrate for PBI's biosensors than those biosensors constructed by using HRP. The PBI's‐Fc‐CP biosensors showed a very good stability during at least twenty days. The reproducibility and the repeatability were 4.5% and 8.3%, respectively, showing a good biosensor performance. The calibration curve was linear in the t‐Res concentration (c_t‐Res) range from 1×10^?6 to 2.5×10^?5 M, with a sensibility of (2.31±0.05)×10⁶ nA M^?1. The lowest c_t‐Res value measured experimentally for a signal to noise ratio of 3 : 1 was 0.83 μM.     

Novel Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Azide Covalently Immobilized on Ethynyl‐Modified Screen‐Printed Carbon Electrode via Click Chemistry

A novel, simple and versatile protocol for covalent immobilization of horseradish peroxidase (HRP) on screen‐printed carbon electrode (SPCE) based on the combination of diazonium salt electrografting and click chemistry has been successfully developed. The ethynyl‐terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacted efficiently and rapidly with horseradish peroxidase bearing an azide function (azido‐HRP), thus forming a covalent 1,2,3‐triazole linkage by means of click chemistry. All the experimental results suggested that HRP was immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP showed a fast electrocatalytic reduction for H₂O₂. A linear range from 5.0 to 50.0 µM in a phosphate buffer (pH 5.5) with detection limit of 0.50 µM and sensitivity of 0.23 nA/µM were obtained. The heterogeneous electron transfer rate constant K_ct was 1.52±0.22 s^?1 and the apparent Michaelis? Menten constant was calculated to be 0.028 mM. The HRP‐functionalized electrode demonstrated a good reproducibility and long‐term stability.     

Simultaneous Determination of Cr(III) and Cr(VI) by Differential Pulse Voltammetry Using Modified Screen‐Printed Carbon Electrodes in Array Mode

A new procedure for the speciation of chromium by means of differential pulse voltammetry using screen‐printed carbon electrodes (SPCEs) has been proposed. Two different modified carbon working, a Ag/AgCl reference and a carbon counter screen‐printed electrodes have been connected in array mode for the simultaneous determination of Cr(III) and Cr(VI). Mercury films or gold nanoparticles have been ground onto the SPCEs in order to improve their selectivity to each chromium species. The quantification of the peak currents observed at ?1.25 V in Hg‐SPCE and ?0.1 V in AuNPs‐SPCE were carried out. The method has been applied to the speciation of chromium in waste water from a tannery factory.     

Hot Electron Induced Cathodic Electrochemiluminescence at Disposable Screen Printed Carbon Electrodes

Hot electron induced cathodic electrochemiluminescence (ECL) was observed at screen printed carbon electrodes (SPCEs) during pulse polarization. The thin insulating film resulted from the printing inks was found to be suitable for generating hot electrons, which can further be converted to hydrated electrons and induce the subsequent luminescence. Compared with disposable Al/Al₂O₃ electrode, SPCEs show more stable and reproducible ECL in a wider pH range without background emission. A sensitive ECL method for determination of quercetin is proposed. The detection limit is 8.0×10^?10 mol L^?1(S/N=3), which is two magnitudes lower than that of common ECL method.     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.     

Glucose Biosensors Based on Electrodes Modified with Ferrocene Derivatives Intercalated into Mg/Al Layered Double Hydroxides

Two amperometric biosensors based on glassy carbon electrodes (GC) modified with Mg/Al layered double hydroxides (LDHs) containing ferrocene‐carboxylate (Fc? CO₂H) or ferrocene‐sulfonate (Fc? SO₃H), as interlayer anions, and glucose oxidase (GOx) are presented. Amperometric detection of glucose involves the electrochemical oxidation of H₂O₂ mediated by the ferrocene derivative. Optimization of the biosensors construction and of the operative conditions was investigated and is discussed herein. The performances of the two biosensors were evaluated by chronoamperometry, working at low anodic potentials (+0.400 V for Fc? CO₂H and +0.500 V for Fc? SO₃H vs. SCE). The linearity extended up to 1.5 mM and 10.0 mM in batch and in flow conditions, respectively, for both biosensors, whereas the sensitivity was higher for the one based on Fc? CO₂H (4.8±0.3 versus 2.0±0.3 μA mM^?1cm^?2 in batch mode, and 63.9±0.4 versus 25.8±0.4 μA mM^?1cm^?2 in flow mode). The biosensors display rapid response time (less than 20 s), good reproducibility, as well as good operational stability. Glucose can be accurately determined in the presence of the most common interferences that coexist in blood serum if an oxidative membrane containing nanoparticles of MnO₂ is applied on the biosensors' surface.     

Pt Nanoparticle‐modified Carbon Fiber Microelectrode for Selective Electrochemical Sensing of Hydrogen Peroxide

We report a simple approach to the production of carbon fiber‐based amperometric microbiosensors for selective detection of hydrogen peroxide (H₂O₂), which was achieved by electrometallization of carbon fiber microelectrodes (CFMs) by electrodeposition of Pt nanoparticles. The Pt‐carbon hybrid sensing interface provided a sensitivity of 7711±587 μA ? mM^?1 ? cm^?2, a detection limit of 0.53±0.16 μM (S/N=3), a linear range of 0.8 μM–8.6 mM, and a response time of <2 sec. The morphologies of the Pt nanoparticle‐modified CFMs were characterized by scanning electron microscopy. To achieve selectivity, permseletive layers, polyphenylenediamine (PPD) and Nafion, were deposited resulting in exclusion of the anionic and cationic interferents, ascorbic acid and dopamine, respectively, at their physiologically relevant concentrations. The resultant sensors displayed a sensitivity to hydrogen peroxide of 1381±72 μA ? mM^?1 ? cm^?2, and a detection limit of 0.86±0.19 μM (S/N=3). This simple and rapid metallization method converts carbon fiber microelectrodes, which are readily accessible, to microscale Pt electrodes in 2 min, providing a platform for oxidase‐based amperometric biosensors with improved spatial resolution over more commonly used platinum electrode array microprobes.     

Amperometric Biosensor for Evaluation of Competitive Cholinesterase Inhibition by the Reactivator HI‐6

Abstract

Amperometric biosensors containing enzymes butyrylcholinesterase or acetylcholinesterase were prepared. The biosensors were employed for studying of cholinesterase reactivator: HI‐6. Competitions between HI‐6 and acetylthiocholine as enzyme substrate were used for determination of IC₅₀ value. Biosensors with butyrylcholinesterase from human serum determined IC₅₀ as (1.00±0.02)×10^?6 M; the biosensor with acetylcholinesterase from human erythrocytes performance provided IC₅₀ (3.31±0.13)×10^?6 M, the one with human recombinant acetylcholinesterase (2.00±0.06)×10^?6 M and finally biosensor with acetylcholinesterase from electric eel (6.17±0.17)×10^?6 M when 5 mM acetylthiocholine as substrate was used. We are encouraged to consider presented biosensors as a very useful for evaluation of newly prepared cholinesterase reactivators.     

Fabrication of Active Horseradish Peroxidase Micropatterns with a High Resolution by Scanning Electrochemical Microscopy

We used a new reactive species OH^? to fabricate active horseradish peroxidase (HRP) micropatterns with a high resolution by scanning electrochemical microscopy (SECM) coupled with a carbon fiber disk electrode as the SECM tip. In this method, except for active HRP micropatterns predesigned other regions on a HRP‐immobilized substrate were deactivated by OH^? generated at the tip held at ?1.7 V in 1.0 mol/L KCl containing 2.0×10^?3 mol/L benzoquinone (BQ) (pH 8.0). The feedback mode of SECM with a tip potential of ?0.2 V was used to characterize the active HRP micropatterns in 1.0 mol/L KCl containing 2.0×10^?3 mol/L BQ and 2.0×10^?3 mol/L H₂O₂.     

Determination of Xenoestrogenic Compounds Using a Nanostructured Biosensing Device

A ternary composite material based on Prussian blue, single‐walled carbon nanotubes and 1‐butyl‐3‐methylimidazolium hexafluorophosphate was prepared and tested for electrochemical detection of H₂O₂. The sensor allows amperometric detection of H₂O₂ at ?0.05 V, with a sensitivity of 137 mA M^?1?cm^?2. The nanocomposite provides a favorable microenvironment for immobilization of horseradish peroxidase (HRP). Determination of xenoestrogenic compounds was performed by enzymatic oxidation at the surface of modified screen printed biosensor in the presence of H₂O₂. The developed electrochemical biosensors exhibited high sensitivity, low detection limits, good operational and storage stability, for detection of 4‐t‐butylphenol, 4‐t‐octylphenol, 4‐n‐nonylphenol and 4‐n‐nonylphenol ethoxylate.     

Natural Melanin Nanoparticle‐decorated Screen‐printed Carbon Electrode: Performance Test for Amperometric Determination of Hexavalent Chromium as Model Trace

A biosensor was prepared with natural melanin nanoparticles (MNP) decorated on a screen‐printed carbon electrode (SPCE). Hexavalent chromium was selected as a well‐known heavy metal ion to be detected for testing the performance of novel biosensor. Natural MNP was extracted from cuttlefish (Sepia officinalis) ink. Surface decoration of SPCEs with MNP was performed by two different methods. The first one was layer‐by‐layer assembly (LBL‐A) for different cycle times(n). In the second one, plasma treatment of SPCE incorporated with evaporation‐induced self‐assembly (EI‐SA) techniques including different incubation times in MNP solutions. The performance of both modified SPCEs were tested for amperometric detection of Cr(VI) in various water samples, and peak reduction of Cr(VI) was determined at 0.33 V. Amperometric results showed wide linear ranges of 0.1–2 μM and 0.1–5 μM of Cr(VI) for SPCEs modified with 14n‐LBL‐A and 12h‐EI‐SA, respectively. The sensitivities of SPCEs modified with 14n‐LBL‐A and 12h‐EI‐SA techniques were 0.27 μA μM^?1 and 0.52 μA μM^?1, respectively. In addition, both modified SPCEs selectively detected Cr(VI) in a model aqueous system composed of certain other heavy metals and minerals, and tap and lake water samples. The LOD and LOQ values for 12h‐EI‐SA were 0.03 μM and 0.1 μM, respectively. This showed that MNP‐modified‐SPCEs generated via EI‐SA techniques have the potential to be an alternative to conventional detection methods such as ICP‐MS.     

The Modification of Screen‐Printed Carbon Electrodes with Amino Group and Its Application to Construct a H2O2 Biosensor

Electrooxidation of thionine on screen‐printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen‐printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H₂O₂ biosensor exhibited a fast response (2 s) and low detection limit (0.5 μM).