Highly Sensitive Choline Oxidase Enzyme Inhibition Biosensor for Lead Ions Based on Multiwalled Carbon Nanotube Modified Glassy Carbon Electrodes

The determination of lead ions by inhibition of choline oxidase enzyme has been evaluated for the first time using an amperometric choline biosensor. Choline oxidase (ChOx) was immobilized on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) through cross‐linking with glutaraldehyde. In the presence of ChOx, choline was enzymatically oxidized into betaine at –0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. The experimental conditions were optimised regarding applied potential, buffer pH, enzyme and substrate concentration and incubation time. Under the best conditions for measurement of the lowest concentrations of lead ions, the ChOx/MWCNT/GCE gave a linear response from 0.1 to 1.0 nM Pb²⁺ and a detection limit of 0.04 nM. The inhibition of ChOx by lead ions was also studied by electrochemical impedance spectroscopy, but had a narrower linear response range and low sensitivity. The inhibition biosensor exhibited high selectivity towards lead ions and was successfully applied to their determination in tap water samples.     

A Mediator‐Free Bienzyme Amperometric Biosensor Based on Horseradish Peroxidase and Glucose Oxidase Immobilized on Carbon Nanotube Modified Electrode

Multiwalled carbon nanotube (CNT) modified glassy carbon electrode immobilized with horseradish peroxidase (HRP) in Nafion coating showed direct electron transfer between HRP enzyme and the CNT‐modified electrode. A mediator‐free bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase was constructed. The bienzyme biosensor exhibited a high sensitivity for glucose detection at zero applied potential.     

Mathematical Modelling of a Non‐enzymatic Amperometric Electrochemical Biosensor for Cholesterol

Thickness of the electro‐polymerized layer grown on a substrate and used as the recognition element for the analyte is critical to measuring the response of a biosensor, with high sensitivity and accuracy. However, it is difficult to control the thickness during synthesis. A mathematical model is developed in this study that considers thickness of the electro‐polymerized layer in simulating the electrochemical response of a non‐enzymatic biosensor for cholesterol in blood. The model includes transient kinetics and one‐dimensional diffusion of the analyte in the poly‐methyl orange (PMO) recognition layer electrochemically grown on the electrode. The governing partial differential equations resulting from the species conservation balances in the PMO layer are numerically solved. Time and spatial concentration profiles of the analyte in the PMO layer are determined. Model predictions are calibrated with the experimental data for different PMO thicknesses. Interestingly, model predictions show a linear response over the calibrated concentration range of cholesterol for all PMO layer thicknesses. Based on the chronoamperometry measurements, the model predictions for the cholesterol concentrations measured in the laboratory samples were also found to be remarkably accurate. This is the first mathematical model developed to understand the transport and kinetics of an analyte in the electro‐polymerized layer used as the recognition element of a non‐enzymatic biosensor.     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Amperometric Biosensor for Choline Based on Gold Screen‐Printed Electrode Modified with Electrochemically‐Deposited Silica Biocomposite

A mediator‐free choline biosensor was developed using the electrochemically assisted sol‐gel deposition on gold screen‐printed electrodes. The addition of 12 mM of cationic surfactant CTAB in silica sol allowed enhancing the stability of the sensor. The modified electrode demonstrated catalytic activity and stable amperometric response to choline for over 3 weeks of exploitation with the sensitivity of 6 µA mM^?1 and LOD of 6 µM. The interference of ascorbic acid was reduced by pretreating the analyzed solution with MnO₂ powder. The application of the sensor with the purpose of identifying choline in the baby milk demonstrated satisfactory metrological characteristics.     

A Stable Enzyme Biosensor Using Concanavalin a Complex of Glucose Oxidase for Glucose Determination

Abstract

The thermal stability of an insoluble concanavalin A (ConA) complex of glucose oxidase (GOD) was researched. The thermal deactivation rate constants of the complexes were obtained. It was found that the GOD-ConA complexes were less sensitive to thermal inactivation than the native enzyme GOD. By using the complexes, ferrocene-mediated enzyme electrodes were constructed. The results suggested that the GOD-Con A complex electrodes had good thermal stability at room temperature.     

A Self‐Sampling‐and‐Flow Biosensor for Continuous Monitoring

A self‐sampling‐and‐flow biosensor was fabricated by sandwiching a nitrocellulose strip on the working electrode side of the double‐sided microporous gold electrodes and a wicking pad on the counter electrode side. The double‐sided microporous electrodes were formed by plasma sputtering of gold on a porous nylon substrate. Sample was taken up to the enzyme‐immobilized working electrode by the capillary action of the front nitrocellulose strip dipped into the sample solution, analyzed electrochemically at the enzyme‐immobilized electrode, and diffuses out to the backside wicking pad through the micropores of the electrodes, constituting a complete flow cell device with no mechanical liquid‐transporting device. Biosensor was formed by co‐immobilizing the glucose oxidase and electron transfer mediator (ferrocene acetic acid) on the thioctic acid self‐assembled monolayer‐modified working electrode. A typical response time of the biosensor was about 5 min with the sensitivity of 2.98 nA/mM glucose, providing linear response up to 22.5 mM. To demonstrate the use of self‐sampling‐and‐flow biosensor, the consumption rate of glucose in the presence of yeast was monitored for five days.     

A Reagentless Tyrosinase Biosensor Based on 1,6‐Hexanedithiol and Nano‐Au Self‐Assembled Monolayers

An amperometric tyrosinase biosensor was developed via a simple and effective immobilization method using the self‐assembled monolayers (SAMs) technique. The organic monolayer film was first formed by the spontaneous assembly of thiolor sulfur compound (1,6‐hexanedithiol, HDT) from solution onto gold electrode. When these thiol‐rich surfaces were exposed to Au colloid, the sulfurs form strong bonds to gold nanoparticles, anchoring the clusters to the electrode substrate. After the assembly of gold nanoparticles layer, a new nano‐Au surface was obtained. Thus, the tyrosinase could be immobilized onto the electrode. The tyrosinase retained its activity well in such an immobilization matrix. The various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady‐state current within 10 s, and the trend in the sensitivity of different phenolic compounds was as follows: catechol>phenol>p‐cresol. In addition, the apparent Michaelis–Menten constant (K and the stability of the enzyme electrode were estimated.     

Facile Fabrication of a Graphene‐based Electrochemical Biosensor for Glucose Detection

A simple and effective glucose biosensor based on immobilization of glucose oxidase (GOD) in graphene (GR)/Nafion film was constructed. The results indicated that the immobilized GOD can maintain its native structure and bioactivity, and the GR/Nafion film provides a favorable microenvironment for GOD immobilization and promotes the direct electron transfer between the electrode substrate and the redox center of GOD. The electrode reaction of the immobilized GOD shows a reversible and surface‐controlled process with the large electron transfer rate constant (k_s) of 3.42±0.08 s^?1. Based on the oxygen consumption during the oxidation process of glucose catalyzed by the immobilized GOD, the as‐prepared GOD/GR/Nafion/GCE electrode exhibits a linear range from 0.5 to 14 mmol·L^?1 with a detection limit of 0.03 mmol·L^?1. Moreover, it displays a good reproducibility and long‐term stability.     

Avidin and Glucose Oxidase‐non‐covalently Functionalized Multi‐walled Carbon Nanotubes: A New Analytical Tool for Building a Bienzymatic Glucose Biosensor

We report an innovative supramolecular architecture for bienzymatic glucose biosensing based on the non‐covalently functionalization of multi‐walled carbon nanotubes (MWCNTs) with two proteins, glucose oxidase (GOx) (to recognize glucose) and avidin (to allow the specific anchoring of biotinylated horseradish peroxidase (b‐HRP)). The optimum functionalization was obtained by sonicating for 10 min 0.50 mg mL^?1 MWCNTs in a solution of 2.00 mg mL^?1 GOx+1.00 mg mL^?1avidin prepared in 50 : 50 v/v ethanol/water. The sensitivity to glucose for glassy carbon electrodes (GCE) modified with MWCNTs‐GOx‐avidin dispersion and b‐HRP (GCE/MWCNTs‐GOx‐avidin/b‐HRP), obtained from amperometric experiments performed at ?0.100 V in the presence of 5.0×10^?4 M hydroquinone, was (4.8±0.3) μA mM^?1 (r²=0.9986) and the detection limit was 1.2 μM. The reproducibility for 5 electrodes using the same MWCNTs/GOx‐avidin dispersion was 4.0 %, while the reproducibility for 3 different dispersions and 9 electrodes was 6.0 %. The GCE/MWCNT‐GOx‐avidin/b‐HRP was successfully used for the quantification of glucose in a pharmaceutical product and milk.     

Multi‐walled Carbon Nanotubes Non‐covalently Functionalized with Polyarginine: A New Alternative for the Construction of Reagentless NAD+/Dehydrogenase‐based Ethanol Biosensor

This work reports for the first time the development of a reagentless enzymatic amperometric biosensor for ethanol based on the use of a glassy carbon electrode (GCE) modified with multi‐walled carbon nanotubes (MWCNTs) non‐covalently functionalized with polyarginine (Polyarg) as platform for the robust immobilization of alcohol dehydrogenase (ADH) and NAD⁺. The new strategy allows to obtain an integrated GCE/MWCNTs‐Polyarg/NAD⁺‐ADH ethanol biosensor with important advantages compared to the existing ethanol biosensors: avoids the external addition of the cofactor for each measurement, ensures a fast and sensitive quantification of ethanol due to the intimate interaction of the components, and allows the detection at considerably lower potentials due to the catalytic activity of the carbon nanostructures. These unique properties have made possible a very efficient ethanol quantification with a sensitivity of (1487±6) μA M^?1, detection limit of 0.65 μM, response time of 8 s, and reproducibility of 5.5 % with a very successful application for the quantification of ethanol in different commercial beverages.     

An L‐Lactate Amperometric Enzyme Electrode Based on L‐Lactate Oxidase Immobilized in a Laponite Gel on a Glassy Carbon Electrode. Application to Dairy Products and Red Wine

A biosensor based on the immobilization of Lactate oxidase in laponite–organosilasesquioxane films on glassy carbon electrode for the quantification of L ‐lactate in wine and dairy products is presented. The bioelectrode showed a very high sensitivity (0.33±0.01) A cm^?2 M^?1 and a short time response (10 s) for less than 1 U of enzyme. No significant interferences, including ascorbic acid, were detected. For red wine, matrix effects assigned to polyphenols and anthocyanins were observed, which ware easily overcome by sample dilution. Our L ‐lactate determinations were in good agreement with those of two standard methods.     

Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H₂O₂ and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM^?1 cm^?2) and stability (80% remains after 30 days).     

A Novel Glucose Biosensor Based on Glucose Oxidase Immobilized on AuPt Nanoparticle – Carbon Nanotube – Ionic Liquid Hybrid Coated Electrode

A novel amperometric glucose biosensor is presented in this article, which is based on the adsorption of glucose oxidase on gold‐platinum nanoparticle (AuPt NP)‐multiwalled carbon nanotube (MWNT) – ionic liquid (i.e., 1‐octyl‐3‐methylimidazolium hexafluorophosphate, [OMIM]PF₆) composite. The gold‐platinum nanoparticles is prepared through direct electrodeposition. Owing to the synergistic action of AuPt nanoparticle, MWNT and [OMIM]PF₆, the biosensor shows good response to glucose, with wide linear range (0.01 to 9.49 mM), short response time (3 s), and high sensitivity (3.47 μA mM⁻¹). With the biosensor the determination of glucose in human serum is performed.     

4‐Aminothiophenol Self‐Assembled Monolayer for the Development of a DNA Biosensor Aiming the Detection of Cylindrospermopsin Producing Cyanobacteria

The development of a DNA biosensor for the detection of cylindrospermopsin, based on self‐assembled monolayers (SAMs) of 4‐aminothiophenol, is investigated. SAMs were characterized by electrochemical reductive desorption. Detection of probe immobilization and hybridization has been achieved by cyclic and square‐wave voltammetry (SWV), using methylene blue (MB) as electroactive indicator. The SWV data obtained in phosphate buffer, with and without NaCl, after MB accumulation, revealed an increase of the redox indicator current peaks after the hybridization step. This behavior is consistent with MB intercalation into DNA, for high ionic strength media and attributed to electrostatic interactions in the absence of salt. Evidence for surface modification is also provided by atomic force microscopy and ellipsometry.     

Reduced Graphene Oxide/Pt Nanoparticles/Zn‐MOF‐74 Nanomaterial for a Glucose Biosensor Construction

In this study, a new glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on platinum nanoparticles (Pt NPs) decorated reduced graphene oxide (rGO)/Zn‐MOF‐74 hybrid nanomaterial. Herein, the biosensor fused the advantages of rGO with those of porous Zn‐MOF and conductive Pt NPs. This has not only enlarged the surface area and porosity for the efficient GOx immobilization and faster mass transport, but also provided favorable electrochemical features such as high current density, remarkable electron mobility through metal nanoparticles, and improved electron transfer between the components. The GOx‐rGO/Pt NPs@Zn‐MOF‐74 coated electrode displayed a linear measurement range for glucose from 0.006 to 6 mM, with a detection limit of 1.8 μM (S/N: 3) and sensitivity of 64.51 μA mM^?1 cm^?2. The amperometric response of the enzyme biosensor demonstrated the typical behavior of Michaelis‐Menten kinetics. The obtained satisfying sensitivity and measurement range enabled fast and accurate glucose measurement in cherry juice using the fabricated biosensor. The water‐stable Zn‐MOF‐74 demonstrated higher enzyme loading capacity and can be potent supporting material for biosensor construction.     

Detection of DNA and Protein Molecules Using an FET‐Type Biosensor with Gold as a Gate Metal

In this study, a field effect transistor (FET)‐type biosensor based on 0.5 μm standard complementary metal oxide semiconductor (CMOS) technology is proposed and its feasibility for detecting deoxyribonucleic acid (DNA) and protein molecules is investigated. Au, which has a chemical affinity with thiol by forming a self‐assembled monolayer (SAM), was used as the gate metal in order to immobilize DNA and protein molecules. A Pt pseudo‐reference electrode was employed for the detection of biomolecules. The sensor was fabricated as a p‐channel (P)MOSFET‐type because PMOSFET with positive surface potential is useful for detecting negatively charged biomolecules from the view point of its high sensitivity and fast response time. DNA and protein molecules were detected by measuring the variation of the drain current due to the variation of biomolecular charge and capacitance. DNA and protein molecules used in the experiment were 15mer–oligonucleotide probe and streptavidin‐biotin protein complexes, respectively. DNA was detected by both in situ and ex situ measurements. Additionally, to verify the interactions among SAM, streptavidin, and biotin, surface plasmon resonance (SPR) measurement was performed.