Amperometric Enzyme Biosensor Research Articles

Development of Amperometric Enzyme Biosensor for Determination of Heavy Metal Ions in aqueous samples

An amperometric urease inhibition-based biosensor was developed to detect Pb2+ and Cr3+ ions in water matrix. The modified GCE/ZnO/urease electrode was developed by immobilizing ZnO nanoparticles and urease using gelatin and glutaraldehyde as crosslinking agent. With urea as a substrate, ZnO catalytic activity was examined through cyclic voltammetry. Working conditions of the biosensing system were optimized in terms of current generated in the presence of substrate (urea) as a function of different variables. Optimal sensor activity (current response) was obtained with a urease loading of 4 µl and ZnONPs concentration of 1.5 mg/L. For solution parameters, optimum pH was in the range of 7.0-7.5, optimum temperature was 35oC, optimal substrate concentration 40 µl. Evaluation of some key biosensing properties revealed relatively stable biosensor activity within the first 7 days, good reproducibility with an RSD of 3.61%. Limits of detection (LOD) for the biosensor ranged from 0.04 - 0.07 while limits of quantitation (LOQ) ranged from 0.22- 0.13 ppm. Applying the developed biosensor to real samples was evaluated by analysing water samples from Kasuwangada River in Mubi North Local Government Area. The system showed good percentage recovery of the added heavy metal standard (0.05 – 0.25 ppm). Percentage recovery within the acceptable range of 98.00% to 103.00% was observed for Pb2+, 92.00% - 102.00 % recovery for Cr3+. Analysis of real samples and assessment of obtained results against a standard method (AAS) showed minimal differences. The developed biosensor showed sufficient sensitivity to the analysed sample and gave quantitative information on the Pb2+ and Cr3+content of the sample. Keywords: Biosensor, Heavy Metals, Urease enzymes, Inhibition studies, Enzyme activity

Shield, Anchor, and Adhesive Roles of Methylene Blue in Tyrosinase Adsorbed on Carbon Felt for a Flow Injection Amperometric Enzyme Biosensor for Phenolic Substrates and Inhibitors.

Methylene blue (MB) acted as a stabilizer for preventing surface-induced denaturation of tyrosinase (TYR) adsorbed on a carbon felt (CF) surface, which is based on shield and anchor roles preventing the unfavorable conformational change of TYR on the hydrophobic CF surface. Furthermore, MB acted as an effective adhesive for TYR immobilization on CF. The resulting TYR and MB coadsorbed CF (TYR/MB-CF) worked as an excellent working electrode unit in an electrochemical detector in a flow injection amperometric biosensor, which allowed highly sensitive consecutive determination of not only TYR substrates but also competitive inhibitors. Simultaneous adsorption of TYR and MB from their mixed solution was much useful as compared with step-wise separated adsorption of TYR on the MB-adsorbed CF, which suggests that the binding interaction of MB with TYR in the solution phase is important for this phenomenon. Fluorescence and UV-vis spectroscopy revealed that not only electrostatic forces between the cationic MB and anionic amino acid residues of TYR but also hydrophobic interactions via the phenothiazine ring of MB play a principal binding driving force of MB with TYR at the surface of the TYR molecules. Synchronous fluorescence, three-dimensional fluorescence, and circular dichroism (CD) spectroscopy clarified that the conformation and the secondary structure of TYR slightly changed upon the MB binding, implying that MB binding leads to the modification of the original intramolecular bonding in part.

The Development of an Amperometric Enzyme Biosensor Based on a Polyaniline‐Multiwalled Carbon Nanocomposite for the Detection of a Chemotherapeutic Agent in Serum Samples from Patients

Polyaniline was electrochemically polymerized onto the platinum electrode modified with a mixture of Nafion and multiwalled carbon nanotube (PANI/MWCNT/Nf/Pt) to detect ammonium ion. The nanobiosensor (ASNase/PANI/MWCNT/Nf/Pt) was then prepared by immobilizing L‐asparaginase (L‐ASNase) on the PANI/MWCNT/Nf nanocomposite. The prepared nanobiosensor was used for the rapid and sensitive detection of serum concentration of the anticancer agent L‐asparagine (L‐Asp) during chemotherapy. The nanobiosensor has dynamic ranges of zero to 180 μM. The sensitivity of the nanobiosensor was 0.829 μA μM−1 cm−2, and the response time was less than 30 s. The detection limit was 140 nM of L‐Asp. The Michaelis–Menten constant (Km) was measured to be 36.2 mM. The nanobiosensor was successfully applied for the determination of L‐Asp in the blood samples of leukemia patients.

Quantitative Concentration Mapping with AFM-SECM

Efficient transport of reaction intermediates between active sites can improve the efficiency of multi-site reactions.1–3 Experimental detection of these nanometer length scale transport mechanisms is enabled by Scanning Electrochemical Microscopy (SECM). Electrochemical measurements can allow for concentration profile mapping to elucidate the transport processes in experimental systems. SECM can correlate position dependent responses to properties of the system. There is a significant impact on the response profile due to the SECM tip’s presence at the nanoscale. Finite Element Analysis (FEA) has been used to understand and remove these in situ tip effects.The calculated concentration, using current-flux relations, can be complex near sites generating flux through reactions and/or transport. Additionally, uncertainties such as tip positioning in relation to the interface site can further complicate the response. Quantitative concentration fields can more readily be interpreted through FEA, by matching the experimental tip response.Electrodeposited platinum nanoparticles were imaged topographically and electrochemically with a Bruker AFM-SECM (Figure 1). The positive response over the nanoparticles vs. the planar HOPG sites allows for kinetic response imaging. The model is parameterized through approach curves and verified though SECM mapping correlation.We have previously applied FEA to correlate experimental in situ results to in operando conditions through a non-linear regression of approach curve responses. Here, we use this approach to generate corrected concentration profiles. We use Pt nanoparticle catalysts deposited on HOPG, to develop surface property correlations. In this way, a comparison can be drawn between in situ and in operando approaches. References (1) Schuhmann, W. Amperometric Enzyme Biosensors Based on Optimised Electron-Transfer Pathways and Non-Manual Immobilisation Procedures. Rev. Mol. Biotechnol. 2002, 82 (4), 425–441.(2) Wheeldon, I.; Minteer, S. D.; Banta, S.; Barton, S. C.; Atanassov, P.; Sigman, M. Substrate Channelling as an Approach to Cascade Reactions. Nat. Chem. 2016, 8 (4), 299–309.(3) Liu, Y.; Hickey, D. P.; Guo, J. Y.; Earl, E.; Abdellaoui, S.; Milton, R. D.; Sigman, M. S.; Minteer, S. D.; Calabrese Barton, S. Substrate Channeling in an Artificial Metabolon: A Molecular Dynamics Blueprint for an Experimental Peptide Bridge. ACS Catal. 2017, 7 (4), 2486–2493. Figure 1

Quantitative Concentration Mapping with AFM-SECM

Efficient transport of reaction intermediates between active sites can improve the efficiency of multi-site reactions1–3. Experimental detection of these nanometer length scale transport mechanisms is enabled by Scanning Electrochemical Microscopy (SECM). Electrochemical measurements can allow for concentration profile mapping to elucidate the transport processes in experimental systems. SECM can correlate position dependent responses to properties of the system. There is a significant impact on the response profile due to the SECM tip’s presence at the nanoscale. Finite Element Analysis (FEA) has been used to understand and remove these in situ tip effects.The calculated concentration, using current-flux relations, can be complex near sites generating flux through reactions and/or transport. Additionally, uncertainties such as tip positioning in relation to the interface site can further complicate the response. Quantitative concentration fields can more readily be interpreted through FEA, by matching the experimental tip response.Electrodeposited platinum nanoparticles were imaged topographically and electrochemically with a Bruker AFM-SECM (Figure 1). The positive response over the nanoparticles vs. the planar HOPG sites allows for kinetic response imaging. The model is parameterized through approach curves and verified though SECM mapping correlation.We have previously applied FEA to correlate experimental in situ results to in operando conditions. Here, we use this approach to generate corrected concentration profiles. We use Pt nanoparticle catalysts deposited on HOPG, to develop surface property correlations. In this way, a comparison can be drawn between in situ and in operando approaches. References (1) Schuhmann, W. Amperometric Enzyme Biosensors Based on Optimised Electron-Transfer Pathways and Non-Manual Immobilisation Procedures. Rev. Mol. Biotechnol. 2002, 82 (4), 425–441.(2) Wheeldon, I.; Minteer, S. D.; Banta, S.; Barton, S. C.; Atanassov, P.; Sigman, M. Substrate Channelling as an Approach to Cascade Reactions. Nat. Chem. 2016, 8 (4), 299–309.(3) Liu, Y.; Hickey, D. P.; Guo, J. Y.; Earl, E.; Abdellaoui, S.; Milton, R. D.; Sigman, M. S.; Minteer, S. D.; Calabrese Barton, S. Substrate Channeling in an Artificial Metabolon: A Molecular Dynamics Blueprint for an Experimental Peptide Bridge. ACS Catal. 2017, 7 (4), 2486–2493. Figure 1

Chronic gastroduodenal pathology in children with atopic dermatitis complicated by mycotic infection

Objective: To identify the features of chronic gastroduodenal pathology in children with atopic dermatitis (AD) complicated by secondary mycotic infection.Methods: The examination of mucous membrane micro-biocenosis of stomach and duodenum was performed in 64 children aged 3–16 years with diseases of gastrointestinal tract and AD complicated by fungal infection. 45 out of 64 patients, having been under medical supervision, had a combined damage of skin and gastrointestinal tract with Candida fungi. In-depth clinical and mycological study was performed represented by esophagogastroduodenoscopy with biopsy of intestine mucous membrane. The detection of circulating candida antigens in blood serum was conducted with method of amperometric enzyme biosensor. Helicobacter pylori were detected using urease test.Results: Children with AD combined with secondary mycotic infection had a damage of the upper parts of gastrointestinal tract and skin with Candida fungi in 70.3% of cases. Gastroduodenitis accounted for 66.7%; peptic ulcer disease was 33.3% in the structure of gastrodudenal pathology. Candida albicans fungi were plated in 80 % of cases; all children had a candida antigen in the blood serum.Conclusions: Children with AD, complicated by mycotic infection, and chronic gastrodudenal pathology should be examined for candidiasis of upper parts of gastrointestinal tract. If there is a candidiasis they should be prescribed with systemic antimycotics and dietary adjustment.

Controlling the Network Properties of Polymer Matrices for Improvement of Amperometric Enzyme Biosensors: Contribution of Positron Annihilation

Controlling the Network Properties of Polymer Matrices for Improvement of Amperometric Enzyme Biosensors: Contribution of Positron Annihilation

Amperometric sarcosine biosensor with strong anti-interference capabilities based on mesoporous organic-inorganic hybrid materials

Amperometric enzyme biosensors are some of the simplest and cheapest types of medical devices used in the rapid detection of biomarkers that have been developed in the past fifty years. When the concentrations of biomarkers are at micromoles per liter, such as for sarcosine, which was recently discovered as a biomarker for prostate cancer, the response signal of the interferences is huge, and the biosensor is hard to satisfy the requirements of practical applications. In this manuscript, we describe a strategy for synthesizing a surface electronegative organic-inorganic hybrid mesoporous material, which could reduce the interference signal much better than Nafion and Chitosan. We verify that the surface potential of the carrier nanomaterial plays an important role in excluding anionic interferences. We also prepare a sensitive (16.35 μA mM−1), low LOD (0.13 μM) and wide linear range (1–70 μM) amperometric sarcosine biosensor with excellent anti-interference properties. This mesoporous material provides a bio-composite platform for the development of simple amperometric biosensors for detecting micromoles per liter of analytes in serum or urine.

Dependence of operational parameters of laccase-based biosensors on structure of photocross-linked polymers as holding matrixes

Laccase-based amperometric enzyme biosensors of the third generation for analysis of phenol derivates have been constructed using graphite rods (type RW001) as working electrodes and the photocross-linked polymers as a matrix. Such matrix consisted of epoxidized linseed oil (ELO), bisphenol A diglycidyl ether (RD) as reactive diluent and 50% mixture of triarylsulfonium hexafluorophosphate in propylene carbonate (PI) as photoinitiator. The synthesis was made by the reaction of ELO and 10 mol.% or 30 mol.% of RD, using 3 mol.% of PI (ELO/10RD and ELO/30RD, respectively). The holding matrixes were used for an immobilization of commercial laccase from the fungus Trametes versicolor. The network properties of the polymer matrixes, holding biosensing element, were studied by means of swelling and positron annihilation lifetime spectroscopy measurements. The amperometric enzyme biosensor parameters were evaluated using cyclic voltamperometry and chronoamperometric analysis. A correlation between the constructed biosensor parameters and microscopical free volume of the biosensor holding matrixes was established.

An electrospun nanofiber matrix based on organo-clay for biosensors: PVA/PAMAM-Montmorillonite

Diagnostic techniques based on biomolecules have huge a potential to be applied in the application in various areas such as food/beverage industries, diseases diagnostics, monitoring of bio-processes and environmental pollutants. Immobilization of biomolecules on a transducer is the key parameter to being able to prepare a highly stable diagnostic tests. Electrospun nanofibers are a good alternative to immobilize biomolecules. Here, electrospun nanofibers based on an organoclay were used to design the first generation amperometric enzyme biosensor. PAMAM G2 dendrimers were used to intercalate montmorillonite clay (Mt) and then the modification of Mt by PAMAM was characterized using FTIR, XRD, TGA and zeta potential measurements. After that nanofibers were prepared by electrospinning Mt and PAMAM-Mt using poly(vinyl) alcohol (PVA) as an auxiliary polymer and the formed PVA/PAMAM-Mt electrospun nanofibers were proved by SEM, TEM and AFM techniques. Finally, pyranose oxidases (PyOx) were immobilized on a glassy carbon electrode surface, which was modified using the PVA/PAMAM-Mt electrospun nanofibers. Amperometric measurements were carried out using buffer solution at −0.7 V under stirring conditions. The linear response for glucose was from 0.005 mM to 0.25 mM using PVA/Mt/PyOx and PVA/PAMAM-Mt/PyOx biosensors. The limit of detection was 0.7 µM glucose with PVA/PAMAM-Mt/PyOx biosensor. To detect glucose in real sample, measurements were carried out using soft drink cola as a substrate instead of glucose.

Electrochemical Study of Trametes Versicolor Laccase Compatibility to Different Polyphenolic Substrates

The aim of this electrochemical study was to ascertain which position of hydroxy groups on a benzene ring provides electroactive products after enzymatic oxidation by laccase originating from the Trametes versicolor mushroom, exhibiting intense redox signals that are exploitable for their amperometric determination. The electrochemical properties of phenol together with all isomers of benzenediol and cresol at the bare carbon paste electrode (CPE) and CPE modified with enzyme laccase (CPE/Laccase) were investigated using cyclic voltammetry at various scan rates. Comparison of resulting redox signals and their differences confirmed the suitability of classes of polyphenolic compounds as substrates for Trametes versicolor laccase and their potential use as suitable biological components in the development of amperometric enzyme biosensors for the determination of such species. The feasibility of the proposed approach was verified by electrochemical assays of the enzymatic oxidation of polyphenolic analogues of simple phenols, e.g., gentisic acid, caffeic acid, resveratrol, and others.

Highly sensitive amperometric enzyme biosensor for detection of superoxide based on conducting polymer/CNT modified electrodes and superoxide dismutase

A novel highly sensitive electrochemical biosensor for the direct determination of the reactive oxygen species superoxide, O2•−, using superoxide dismutase has been developed, by incorporating multiwalled carbon nanotubes (MWCNT) together with the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) in different configurations. After characterisation, the experimental conditions have been optimized and the analytical parameters of superoxide dismutase biosensors based on PEDOT/CNT or CNT/PEDOT modified glassy carbon electrodes, as well as those with one component only (MWCNT, PEDOT), have been determined. The biosensor with CNT on top of PEDOT presented the best analytical performance due to synergistic effects, with fast, and selective response to O2•−, a high sensitivity of ∼1115μAcm−2mM−1 and a low detection limit of 1μM, and was applied to the determination of the antioxidant capacity of beverages. The biosensor exhibited outstanding stability over a period of 2 months, with a slight increase in the initial sensitivity.

Reduced graphene oxide-carboxymethylcellulose layered with platinum nanoparticles/PAMAM dendrimer/magnetic nanoparticles hybrids. Application to the preparation of enzyme electrochemical biosensors

The assembly of a novel layer-by-layer biosensor architecture using hybrid nanomaterials is explored for the construction of an amperometric enzyme biosensors. The nanostructured sensing interface was prepared with poly(dopamine)-modified magnetic nanoparticles which were covalently coated with four-generation ethylenediamine core polyamidoamine G-4 dendrimers and further decorated with platinum nanoparticles. This nanohybrid was fully characterized and further layered on glassy carbon electrodes coated with a graphene oxide-carboxymethylcellulose hybrid nanomaterial through electrostatic interactions. The nanostructured surface was then employed as scaffold for the covalent immobilization of the enzyme xanthine oxidase through a glutaraldehyde-mediated cross-linking. The enzyme electrode allowed the amperometric detection of xanthine in the 50nM–12μM range, with a high sensitivity of 140mA/Mcm2 and low detection limit of 13nM. The biosensor exhibited high reproducibility and repeatability, and was successfully tested for the quantification of xanthine in fish samples.

Mechanism of amperometric biosensor with electronic-type-controlled carbon nanotube

An amperometric enzyme biosensor with electronic-type-controlled (metallic and semiconducting) single-walled carbon nanotubes (CNTs) is presented. In this research, we investigate how the electronic types of CNTs influence the amperometric response of enzyme biosensors and what their working mechanisms are. The biosensor of interest is for glucose detection using enzyme glucose oxidase (GOD). In the presence of oxygen, the response of a metallic CNT-GOD electrode was 2.5 times more sensitive than that of a semiconducting CNT-GOD electrode. In contrast, in the absence of oxygen, the response of the semiconducting CNT-GOD electrode was retained, whereas that of the metallic CNT-GOD electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting CNT-GOD electrode, whereas the metallic CNT-GOD electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. Electrochemical impedance spectroscopy was used to show that the semiconducting CNT network has less resistance for electron transfer than the metallic CNT network. The optimized glucose biosensor revealed a sensitivity of 5.6 µA mM−1 cm−2 at +0.6 V vs Ag/AgCl, a linear dynamic range of 0.025–1.4 mM, and a response time of 8 s.

Novel reduced graphene oxide-glycol chitosan nanohybrid for the assembly of an amperometric enzyme biosensor for phenols.

A novel water-soluble graphene derivative was prepared from graphene oxide via a two-step modification approach. Graphene oxide was first functionalised with reactive epoxy groups by covalent modification with (3-glycidyloxypropyl)trimethoxysilane and further cross-linked with glycol chitosan. This graphene derivative was characterized using different microscopy and physicochemical methods and employed as a coating material for a glassy carbon electrode. The nanostructured surface was used as a support for the covalent immobilization of the enzyme laccase through cross-linking with glutaraldehyde. The enzyme electrode was tested for the amperometric detection of different phenolic compounds, which displayed excellent analytical behaviour toward catechol with a linear range of response from 200 nM to 15 μM, sensitivity of 93 mA M(-1) cm(-2), and low detection limit of 76 nM. The enzyme biosensor showed high stability when stored at 4 °C under dry conditions and was successfully employed to quantify the total phenolic compounds in commercial herbal tea samples.

Enzyme Biosensor for Detection of Formaldehyde on Carbon Nanotube-Screen Printed Electrodes Modified with Pyrenyl Groups

Formaldehyde has been recognized as a marker in diseases associated with kidneys, nerves and lungs. Our objective is to develop a novel, highly sensitive electrochemical amperometric enzyme biosensor for detection of low levels of formaldehyde. Our biosensor design involves CNTs-modified screen-printed electrodes (SPEs) with immobilized formaldehyde dehydrogenase (FDH). CNT-coated electrodes modified with non-covalently attached pyrenebutyric acid linkers were used to attach the FDH enzyme and achieve enhanced electron transfer and biosensing properties. The FDH catalyzed oxidation of formaldehyde, using NAD+ as the electron acceptor, yields formic acid and NADH. This was detected electrochemically using an electrochemical mediator in solution. Such electrochemical sensors upon further optimization are expected to be useful for diagnosing clinically relevant levels of small molecule disease markers and environment pollutants. Acknowledgements: We are grateful for the financial support by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R15DK103386.

Detection of Adrenaline Based on Substrate Recycling Amplification

An amperometric enzyme biosensor has been applied for the detection of adrenaline. The adrenaline biosensor has been prepared by modification of an oxygen electrode with the enzyme laccase that operates at a broad pH range between pH 3.5 to pH 8. The enzyme molecules were immobilized via cross-linking with glutaraldehyde. The sensitivity of the developed adrenaline biosensor in different pH buffer solutions has been studied.

Surface nitrogen-enriched carbon nanotubes for uniform dispersion of platinum nanoparticles and their electrochemical biosensing property

Nitrogen containing carbon layer-coated carbon nanotubes (CNx-CNTs) was prepared as the support for platinum nanoparticles (Pt NPs) and their enhanced properties for electrochemical biosensor has been demonstrated in this paper. The CNx-CNTs were obtained from pyrolysis of polydopamine-wrapped CNTs, which were synthesized by a single deposition process based on the oxidative self-polymerization of dopamine on CNTs. It is found that Pt NPs are deposited on the surface of the CNx-CNTs (Pt/CNx-CNTs) with highly dispersion and small particle size (with an average diameter of 1.7±0.3nm). Compared to the nitrogen-free CNTs supported Pt NP composite (Pt/CNTs), the Pt/CNx-CNTs modified glassy carbon (GC) electrode exhibits superior electrocatalytic performance towards the oxidation of hydrogen peroxide (increase by about 55% of response current). Taking glucose oxidase (GOD) as the model, the proposed amperometric enzyme biosensor based on the Pt/CNx-CNTs shows excellent analytical characteristics to glucose detection, such as excellent sustainability in large range of pH values, high sensitivity (66.51μA (mmoldm−3)−1cm−2), wide linear range (0.01-6.1mmoldm−3) and low detection limit (0.4μmoldm−3).

Ionic Liquid of a Gold Nanocluster: A Versatile Matrix for Electrochemical Biosensors

Ionic liquids are room-temperature molten salts that are increasingly used in electrochemical devices, such as batteries, fuel cells, and sensors, where their intrinsic ionic conductivity is exploited. Here we demonstrate that combining anionic, redox-active Au25 clusters with imidazolium cations leads to a stable ionic liquid possessing both ionic and electronic conductivity. The Au25 ionic liquid was found to act as a versatile matrix for amperometric enzyme biosensors toward the detection of glucose. Enzyme electrodes prepared by incorporating glucose oxidase in the Au25 ionic liquid show high electrocatalytic activity and substrate affinity. Au25 clusters in the electrode were found to act as effective redox mediators as well as electronic conductors determining the detection sensitivity. With the unique electrochemical properties and almost unlimited structural tunability, the ionic liquids of quantum-sized gold clusters may serve as versatile matrices for a variety of electrochemical biosensors.

Geometrical effects of nanowire electrodes for amperometric enzyme biosensors

Enzymatic biosensor reactions follow the Michaelis–Menten kinetics, coupled with diffusion. The diffusion reaction processes for amperometric enzyme biosensors have been simulated to explore the geometrical effects of nanowire array electrodes (NWAEs) and nanowire array stack electrodes (NWASEs) from the viewpoint of enhanced mass transport and increased reaction surface area in two limiting cases. For practical analysis considering sensor fabrication, most samples are assumed to have the same unit square (1cm×1cm) footprint. In the reaction-controlled case, the surface area increment improves the sensitivity regardless of electrode geometry. However, in the diffusion-controlled case, well-controlled NWAE or NWASE geometries as well as the increased surface area improve the sensitivity when the peak current at an early stage of the reaction is measured. Peak current engineering by adjusting the geometric parameters of NWAEs and NWASEs will result in a highly sensitive amperometric enzyme biosensor in the diffusion-controlled case. In contrast to previous micro- and nanoelectrode array studies, we investigated NWASEs representing entangled nanowire network electrodes, and report significant improvements in both limiting cases.