Application of amperometric sol-gel biosensor to flow injection determination of glucose

A glucose biosensor with enzyme immobilised by sol–gel technology was constructed and evaluated. The glucose biosensor reported is based on encapsulated GOX within a sol–gel glass, prepared with 3-aminopropyltriethoxy silane, 2-(3,4-epoxycyclohexyl)-ethyltrimetoxy silane and HCl. A flow system incorporating the amperometric biosensor constructed was developed for the determination of glucose in the 1×10⁻⁴–5×10⁻³ mol l⁻¹ range with a precision of 1.5%. The results obtained for the analysis of electrolytic solution for iv administration and human serum samples showed good agreement between the proposed method and the reference procedure, with relative error <5%.     

Low-potential stable detection of β-NADH at sol–gel derived carbon composite electrodes

Sol–gel derived carbon composite electrodes, prepared from different non-silicate metal alkoxide precursors, offer a substantial decrease in the overvoltage of the NADH oxidation reaction (compared to ordinary carbon electrodes). Such promotion is attributed to acceleration of the proton-transfer step by the metal-oxide component of the composite. Passivation problems, accrued by accumulation of reaction products, are also greatly minimized. Both titania–, zirconia–sol–gel carbon composite electrodes thus offer a highly sensitive and stable anodic detection of NADH at +0.2 V. Greatly improved retention of the redox mediator Meldola Blue within the sol–gel network permits convenient measurements at NADH at −0.1 V. These improvements indicate great promise for the design of dehydrogenase-based amperometic biosensors. An intrinsic activation action by the metal-oxide component is also reported towards the oxidation of hydrazine, hydrogen peroxide, ascorbic acid and catechol. Low-potential detection of NADH is also illustrated at microfabricated titania/carbon screen-printed electrodes.     

Poly(Neutral Red)/Cholesterol Oxidase Modified Carbon Film Electrode for Cholesterol Biosensing

New amperometric cholesterol oxidase (ChOx) based enzyme biosensors for cholesterol have been developed. The enzyme was immobilised with and without glutaraldehyde cross‐linking on top of carbon film electrodes modified with redox mediators. Mediators tested were: poly(neutral red) (PNR), Prussian blue and cobalt hexacyanoferrates. Amperometric detection of cholesterol showed that PNR/ChOx modified electrodes exhibited the best characteristics; under optimised conditions cholesterol was determined at ?0.4 V vs. SCE with a detection limit of 1.9 µM. The biosensors showed good reproducibility and stability and only a small influence from potential interferents in food. Analyses of cholesterol in egg yolk were successfully performed.     

Electrochemical behavior of methyl viologen at graphite electrodes modified with Nafion sol–gel composite

Electrodes were prepared by spin-coating spectroscopic graphite rods with a Nafion doped sol. Coating solutions consisting of Nafion:TEOS (tetraethoxysilane) ratios of 3:1 and 4:1 gave smooth films on the electrode surface. These modified electrodes were evaluated and compared with Nafion modified and bare spectroscopic graphite electrodes using methyl viologen (MV²⁺) as a representative cationic electroactive probe. Substantial partitioning of MV²⁺ into the Nafion:sol–gel matrix to the electrode surface was observed by cyclic voltammetry and square wave voltammetry. Cyclic voltammograms of MV²⁺ in 0.1 M NaCl at Nafion:sol–gel 4:1 modified electrodes showed a reversible reduction to MV⁺ with E^0′=−0.695 V vs. Ag/AgCl. Results of scan rate variation showed the wave to be characterized by semi-infinite diffusion for scan rates in the range 50–500 mV/s. Slowing the scan rate below 50 mV/s resulted in a transition to thin-layer behavior. MV²⁺ partitioned much more quickly into the sol–gel-Nafion modified electrodes compared to pure Nafion modified electrodes. Reversibility of the MV²⁺-loaded modified Nafion-doped sol–gel coatings on electrodes was obtained by soaking in 1 M NaCl solution. Concentration calibration plots for MV²⁺ at the sol–gel-Nafion modified electrodes were nonlinear. Substantial enhancement of current signal at low concentrations was observed by square wave voltammetry.     

Development of Novel Glucose and Pyruvate Biosensors at Poly(Neutral Red) Modified Carbon Film Electrodes. Application to Natural Samples

Amperometric biosensors based on the corresponding oxidase enzyme with poly(neutral red) redox mediator have been developed for the determination of glucose and pyruvate. The enzymes have been immobilized on top of poly(neutral red) modified carbon film electrodes with glutaraldehyde as the cross‐linking agent. The biosensors were characterized by cyclic voltammetry and by electrochemical impedance spectroscopy. The glucose biosensor exhibited a linear response in the range 90 μM to 1.8 mM with a detection limit of 22 μM and the pyruvate biosensor in the range 90 to 600 μM with a detection limit of 34 μM. The relative standard deviations were found to be 2.1% (n=3) and 2.8% (n=4) respectively. The interference effects of various compounds were also studied. The glucose content of several types of wine and the amount of pyruvate in onion and garlic were determined and the results were compared with those obtained by standard spectrophotometric methods.     

Urea potentiometric biosensor based on urease immobilized on chitosan membranes

Potentiometric biosensors based on urease (E.C. 3.5.1.5.) immobilized on chitosan membranes coupled to all-solid-state nonactin ammonium ion selective electrodes are described. The enzyme was immobilized on the chitosan membranes by four procedures: (A) adsorption; (B) adsorption followed by reticulation with dilute aqueous glutaraldehyde solution; (C) activation with glutaraldehyde followed by contact with the enzyme solution; and (D) activation with glutaraldehyde, contact with the enzyme solution and reduction of the Schiff base with sodium borohydride. The response characteristics of the biosensors obtained with these enzymatic membranes were determined and compared. The biosensor with best response characteristics, obtained by procedure (B), showed the following characteristics of response to urea: (i) linearity in the 10(-4) to 10(-2) M range; (ii) slope of up to 56 mV per decade; (iii) response time between 30 s and 2 min; and (iv) lifetime of 2 months. This biosensor was tested in the determination of urea in blood serum samples.     

Enzyme immobilisation on electroactive nanostructured membranes (ENM): optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H₂O₂, the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 μM and an apparent Michaelis–Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples.     

Application of Some Room Temperature Ionic Liquids in the Development of Biosensors at Carbon Film Electrodes

Two room temperature ionic liquids, 1‐butyl‐3‐methylimidazolium bistriflimide and 1‐butyl‐3‐methylimidazolium nitrate, were employed for enzyme immobilization in a new sol‐gel matrix and, for the first time, were successfully applied as electrolyte carriers in a biosensing system. The new sol‐gel matrix, based on 3‐aminopropyltrimethoxysilane and 1‐butyl‐3‐methylimidazolium bistriflimide mixtures, did not crack even after several weeks when kept dry, and exhibited similar analytical properties to aqueous sol‐gel based glucose biosensors. The linear range was up to 1.1 mM of glucose, sensitivity was 62 nA mM^?1 and the limit of detection was 28.8 μM. The optimum ionic liquid electrolyte carrier was found to be 1‐butyl‐3‐methylimidazolium nitrate, where the biosensor was made by electrodeposition of the redox mediator, poly(neutral red), and the enzyme was immobilized by cross‐linking with glutaraldehyde. The results showed that application of room temperature ionic liquids to biosensors is very promising and can be further exploited.     

Characterisation of poly(neutral red) modified carbon film electrodes; application as a redox mediator for biosensors

The polymer redox mediator, poly(neutral red) (PNR), has been synthesised and characterised electrochemically to investigate the best electropolymerisation and mediation conditions for application in enzyme biosensors and to clarify the mechanism of action. Neutral red was electropolymerised by potential cycling on carbon film electrode substrates by allowing the monomer to be oxidised during the full 20 cycles of polymerisation or reducing the positive limit of the potential window after the first 2 cycles to impede monomer oxidation with a view to obtaining longer polymer chains and a lesser degree of branching. Comparison was made with glassy carbon substrates. The PNR films on carbon film electrodes were characterised using cyclic voltammetry and electrochemical impedance spectroscopy, as well as in glucose biosensors prepared with PNR. Glucose oxidase enzyme was immobilised by encapsulation in silica sol-gel and compared with that obtained by cross-linking with glutaraldehyde. The biosensors were evaluated by chronoamperometry in 0.1 M phosphate buffer saline solution, pH 7.0, and showed evidence of electron transfer between the enzyme cofactor flavin adenine dinucleotide and PNR dissolved in the enzyme layer competing with PNR-mediated electrochemical degradation of H₂O₂ formed during the enzymatic process. This paper is dedicated to Professor Dr. Algirdas Vaskelis on the occasion of his 70th birthday.     

Synergistic effect of mediator–carbon nanotube composites for dehydrogenases and peroxidases based biosensors

Very sensitive, low cost and reliable NADH and H₂O₂ sensors were realised and used for development of enzyme based biosensors. The active surface of the electrodes was modified with a nanocomposite obtained by modification of SWNT with a proper mediator: Meldola Blue (for NADH) and Prussian Blue (for H₂O₂). Low applied potential of − 50 mV vs. Ag/AgCl reference electrode proved the synergistic effect of nanocomposite materials towards NADH and H₂O₂ detection. Biosensors for malic acid and alkylphenols have been developed, using mediator-functionalised-SWNT-based electrodes and two different classes of enzymes: NAD⁺-dependent dehydrogenases and peroxidases. Immobilization of the enzymes was realised using a series of different procedures — adsorption, Nafion membrane, sol–gel and glutaraldehyde, in order to find the best configuration for a good operational stability. A higher sensitivity comparing with other reported biosensors of about 12.41 mA/M·cm² was obtained for l-malic acid biosensor with enzyme immobilised in Nafion membrane. Phenol, 4-t-octylphenol and 4-n-nonylphenol were used as standard compounds for HRP based biosensor. Fast biosensor response and comparable detection limit with HPLC methods were achieved.     

Microbiosensor for acetylcholine and choline based on electropolymerization/sol–gel derived composite membrane

Amperometric enzyme biosensors for the determination of acetylcholine (ACh) and choline (Ch) have been described. For the fabrication of the biosensors, N-acetylaniline (nAN) was first electropolymerized on a Pt electrode surface to be served as a permselective layer to reject interferences. Bovine serum albumin (BSA) and choline oxidase (CHOD) were co-immobilized in a zinc oxide (ZnO) sol–gel membrane on the above modified Pt electrode for a Ch sensor, or CHOD, acetylcholinesterase (AChE) and BSA immobilized together for an ACh/Ch sensor. The poly (N-acetylaniline) (pnAN) film was the first time used for an ACh/Ch sensor and found to have excellent anti-interference ability, and the BSA in the sol–gel can improve the stability and activity of the enzymes. Amperometric detection of ACh and Ch were realized at an applied potential of +0.6 V versus SCE. The resulting sensors were characterized by fast response, expanded linear range and low interference from endogenous electroactive species. Temperature and pH dependence and stability of the sensor were investigated. The optimal ACh/Ch sensor gave a linear response range of 1.0 × 10⁻⁶ to 1.5 × 10⁻³ M to ACh with a detection limit (S/N = 3) of 6.0 × 10⁻⁷ M and a linear response range up to 1.6 × 10⁻³ M to Ch with a detection limit of 5.0 × 10⁻⁷ M. The biosensor demonstrated a 95% response within less than 10 s.     

Carbon film electrodes for oxidase-based enzyme sensors in food analysis

Carbon film resistor electrodes have been evaluated as transducers for the development of multiple oxidase-based enzyme electrode biosensors. The resistor electrodes were first modified with Prussian Blue (PB) and then covered by a layer of covalently immobilized enzyme. Electrochemical impedance spectroscopy was used to characterize the interfacial behaviour of the Prussian Blue modified and enzyme electrodes; the spectra demonstrated that the access of the substrates is essentially unaltered by application of the enzyme layer. These enzyme electrodes were used to detect the substrate of the oxidase (glucose, ethanol, lactate, glutamate) via reduction of hydrogen peroxide at +50 mV versus Ag/AgCl in the low micromolar range. Response times were 1-2 min. Finally, the glucose, ethanol and lactate electrochemical biosensors were used to analyse complex food matrices—must, wine and yoghurt. Data obtained by the single standard addition method were compared with a spectrophotometric reference method and showed good correlation, indicating that the electrodes are suitable for food analysis.     

A comparative study of immobilization methods of a tyrosinase enzyme on electrodes and their application to the detection of dichlorvos organophosphorus insecticide

The use of several designs of amperometric enzymatic biosensors based on the immobilized tyrosinase enzyme (Tyr) for determining dichlorvos organophosphate pesticide are described. The biosensors are based on the reversible inhibition of the enzyme and the chronocoulometric measurement of the charge due to the charge-transfer mediator 1,2-naphthoquinone-4-sulfonate (NQS). Tyr becomes active when reducing the quinone form of the mediator molecule (NQS) to the reactive o-diol form substrate of Tyr (H₂NQS) at the working electrode, thus permitting modulation of the catalytic activity of the enzyme and measurement of the inhibition produced by the pesticide. The full activity of the enzyme reversibly recovers after removal of the pesticide and re-oxidation of H₂NQS.Tyr was immobilized onto electrodes using different procedures: (i) entrapment within electropolymerized conducting and non-conducting polymers, (ii) covalent attachment to self-assembled monolayers (SAM), (iii) cross-linking with glutaraldehyde (and nafion covering) and (iv) dispersion within carbon-paste electrodes. The mediator was co-immobilized onto the working electrode next to the enzyme and reagentless biosensors were subsequently constructed. In the SAM design (ii) NQS was added to the solution. The analytical properties of the different biosensors based on the competitive inhibition produced by dichlorvos were then compared. A detection limit of about 0.06 μM was obtained for dichlorvos with entrapment of NQS and Tyr within electropolymerized poly(o-phenylenediamine) polymer (oPPD), which was the design that proved to have the best analytical performance.     

Electrochemical Glucose Biosensors: Whole Cell Microbial and Enzymatic Determination Based on 10-(4H-Dithieno[3,2-b:2′,3′-d]Pyrrol-4-yl)Decan-1-Amine Interfaced Glassy Carbon Electrodes

The fabrication of amperometric biosensors based on whole cell Gluconobacter oxydans DSMZ 2343 (G. oxydans) and glucose oxidase (GOx) was performed for the detection of glucose. Glassy carbon electrodes (GCE) were coated with a 10-(4H-dithiyeno [3,2-b:2’,3’-d]pyroll-4-il)decan-1-amine (DTP-alkyl-NH₂) polymer using an electropolymerization method and the formed interface was used to connect the bacteria and the enzyme to the electrode. The transfer of electrons from enzyme to electrode was successfully demonstrated by the biocatalytic activity and unique morphology of the conducting polymer. Characterization of the biosensors was assessed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM) analyses. The detection limits of the enzyme and microbial based biosensors for glucose were 0.022 and 0.081?mM, respectively. The broad linear dynamic ranges of the GOx and G. oxydans biosensors were observed to be 0.045–50.0 and 0.19–50.0?mM, respectively. The analytical performances of biosensors were compared according to the following figures of merit: detection limits, limits of quantification, pH and current response time. In addition, to demonstrate the applicability of the biosensors, real-time measurements and recovery studies were evaluated.     

Amperometric Glucose Biosensors Based on Glassy Carbon and SWCNT‐Modified Glassy Carbon Electrodes

Different carbonaceous materials, such as single‐walled carbon nanotubes (SWCNTs) and glassy carbon submitted to an electrochemical activation at +1.80 V (vs. SCE) for 900 s, have been used with the aim of comparing their performances in the development of enzyme electrodes. Commercial SWCNTs have been pretreated with 2.2 M HNO₃ for 20 h prior to use. The utility of activated GC as promising material for amperometric oxidase‐based biosensors has been confirmed. With glucose oxidase (GOx) as a model enzyme, glucose was efficiently detected up to 1 mM without the use of a mediator. Both electrodes operated in stirred solutions of 0.1 M phosphate buffer (pH 5.5), containing dissolved oxygen, at a potential of ?0.40 V vs. SCE. Although the performances of the two carbonaceous materials were comparable, the biosensors based on activated GC were characterized by a practically unchanged response 40 days after the fabrication, a better signal to noise ratio, and a little worse sensitivity. In addition, the preparation procedure of such biosensors was more simple, rapid and reproducible.     

Biotinylated alginate immobilization matrix in the construction of an amperometric biosensor: application for the determination of glucose

The use of biotinylated alginate as an immobilization matrix of enzymes on the surface of the amperometric transducer is described herein. The model used is that of the well-established glucose detection. Several types of immobilization protocols were tested. In the exception of one protocol, biotin labeled glucose oxidase was shown to first require conjugation with avidin, before its immobilization onto a biotin-alginate gel matrix. The response of the biosensors to incremental additions of glucose, was measured by potentiostating the modified electrodes at 0.6 V/SCE. The permeability of the modified electrodes was thereafter measured by using rotating disk electrode (RDE) voltammetry with ferrocenemonocarboxylic acid as the electroactive probe.     

A rapid and easy procedure of biosensor fabrication by micro-encapsulation of enzyme in hydrophilic synthetic latex films. Application to the amperometric determination of glucose

Novel enzyme electrodes based on synthetic hydrophilic latex matrices are described for the detection of glucose. Glucose oxidase was immobilised through micro-encapsulation, by the simple adsorption of enzyme–latex suspensions on the surface of a platinum electrode. Two latex films functionalised by a hydroxy or a gluconamide group were used. The response of these biosensors to glucose additions was measured by potentiostating the modified electrodes at 0.6 V/SCE in order to oxidise the hydrogen peroxide generated by the enzymatic oxidation of glucose in the presence of dioxygen. The response of such electrodes was evaluated as a function of film thickness and temperature. The sensitivity for a two-layer latex-based biosensor was found to be 38.78 mA M⁻¹ cm⁻² with a response time of 3–5 s. Moreover, a marked improvement of the thermal stability of the biosensor was observed. Only at temperatures higher than 65°C the enzyme started to be denatured and being inactive.     

Electron-transfer properties of different carbon nanotube materials,and their use in glucose biosensors

Different types of carbon nanotube material (single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) of different internal diameter) have been used for preparation of CNT-modified glassy-carbon electrodes. Redox reactions involving ferricyanide and hydrogen peroxide were examined at the CNT-modified electrodes. Electrodes modified with SWCNTs usually had better electron-transfer properties than MWCNT-modified electrodes. Glucose biosensors were also prepared with electropolymerized polyphenylenediamine films, CNT materials, and glucose oxidase. Amperometric behavior in glucose determination was examined. SWCNT-modified glucose biosensors usually had a wider dynamic range (from 0.1 to 5.5 mmol L⁻¹) and greater sensitivity in glucose determination. The detection limit was estimated to be 0.05 mmol L⁻¹.     

Computer simulation of the steady state currents at enzyme doped carbon paste electrodes

The plate-gap model of porous enzyme doped electrode has been proposed and analyzed. It was suggested that reaction diffusion conditions in pores of bulk electrode resemble particular conditions in thin gap between parallel conducting plates. The model is based on the diffusion equations containing a nonlinear term related to the Michaelis–Menten kinetic of the enzymatic reaction inside gap. Steady state current was calculated for the wide range of given parameters and substrate concentrations. All dependences of current on substrate concentration were approximated by hyperbolas in order to obtain “apparent” parameters (maximal currents and apparent Michaelis constants) of modelled biosensors. Simple approximate relationships between given and apparent parameters were derived. The applicability of theoretical plate-gap model was tested for the case of carbon paste electrodes which were doped with PQQ – dependent glucose dehydrogenase. It was found, that soluble glucose dehydrogenase based biosensors exhibit characteristic features of the theoretical plate-gap biosensors.     

Coupled enzyme reaction microarrays based on pin-printing of sol–gel derived biomaterials

We report on the development of a new class of protein microarrays based on the co-immobilization of multiple components within a single pin-printed sol–gel array element. In the first case, the microarraying of a coupled two enzyme reaction involving glucose oxidase and horseradish peroxidase along with the fluorogenic reagent Amplex Red is demonstrated to allow “reagentless” fluorimetric detection of glucose. The second system involved the detection of urea using co-immobilized urease and fluorescein dextran, which works on the basis of a pH induced change in fluorescein emission intensity upon production of ammonium carbonate owing to hydrolysis of urea. In both the cases, it is demonstrated that the changes in intensity from the array are time-dependent, consistent with the enzyme catalyzed reaction, showing that such arrays can be used for kinetic studies. The rate of intensity change was also found to be dependent on the concentration of analyte added to the array, showing that such arrays could be useful for quantitative multianalyte biosensing. Inhibition of urease by the competitive inhibitor thiourea is also demonstrated on a microarray, demonstrating that sol–gel-based microarrays may find use in high-throughput drug-screening applications.