Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC(R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface.     

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H₂O₂ takes place at more negative potentials (0.1-0.4 V versus Ag/AgCl). Moreover, H₂O₂ oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.     

Electroanalytical applications of Prussian Blue and its analogs

The applications of transition metal hexacyanoferrates in electroanalysis are surveyed. Prussian Blue (ferric hexacyanoferrate) is recognized as the most promising low-potential transducer for hydrogen peroxide reduction among all known systems. The advantages of Prussian Blue over platinum or peroxidase electrodes for hydrogen peroxide detection are discussed. Various types of biosensors based on transition metal hexacyanoferrates and oxidase enzymes are considered. Amperometric biosensors based on Prussian Blue-modified electrodes allow the detection of glucose and glutamate down to 10^–7 mol L^–1 in the flow-injection mode. The future prospects of Prussian Blue-modified electrodes in analytical chemistry for the monitoring of chemical toxic agents, in clinical diagnostics, and in food control are outlined.     

Physicochemical Characteristics of Polypyrrole/(Glucose oxidase)/(Prussian Blue)‐based Biosensor Modified with Ni‐ and Co‐Hexacyanoferrates

In this work, three types of electrodes suitable for amperometric glucose biosensors were designed. One type of electrode was based on bio‐selective layer of polypyrrole/(glucose oxidase)/(Prussian Blue) (Ppy/GOx/PB) and it was used as a control electrode regarding to which electrochemical properties of two other types of electrodes were compared. During the formation of Prussian blue layers graphite electrodes were additionally modified by Ni‐hexacyanoferrate (NiHCF) and by Co‐hexacyanoferrate (CoHCF) in order to design Ppy/GOx/PB‐NiHCF and Ppy/GOx/PB‐CoHCF electrodes, respectively. Some physicochemical characteristics of all three types of electrodes were evaluated and compared. The Ppy/GOx/PB‐NiHCF electrode showed wider linear range of the calibration curve than Ppy/GOx/PB and Ppy/GOx/PB‐CoHCF electrodes. The effect of temperature on analytical performance of the Ppy/GOx/PB‐NiHCF based biosensor has been evaluated and activation energy of enzyme catalysed reaction has been calculated within the temperature range of 15 °C to 30 °C.     

Investigation of the Effect of Different Glassy Carbon Materials on the Performance of Prussian Blue Based Sensors for Hydrogen Peroxide

Three different kinds of glassy carbon (GC‐R, GC‐K, GC‐G) were equally pretreated, further modified with electrochemically deposited Prussian Blue and used as sensors for hydrogen peroxide at an applied potential of ?50 mV (vs. Ag|AgCl). Their performance was evaluated with respect to the following parameters: the coverage and electrochemistry of the electrodeposited Prussian Blue, the sensitivity and the lower limit of detection for hydrogen peroxide, and the operational stability of the sensors. GC‐R showed the best behavior concerning the surface coverage and the operational stability of the electrodeposited Prussian Blue. For this electrode the sensitivity for hydrogen peroxide (10 μM) was 0.25 A/M cm² and the detection limit was 0.1 μM. Scanning electron microscopy was used to study the surfaces of the three electrodes before and after the electrodeposition of Prussian Blue and to search for the reason for the three different behaviors between the different glassy carbon materials. The Prussian Blue modified GC‐R was also used for the construction of a glucose biosensor based on immobilizing glucose oxidase in Nafion membranes on top of electrodeposited Prussian Blue layer. The operational stability of the glucose biosensors was studied in the flow injection mode at an applied potential of ?50 mV (vs. Ag|AgCl) and alternatively injecting standard solutions of hydrogen peroxide (10 μM) and glucose (1 mM) for 3 h. For the GC‐R based biosensor a 2.8% decrease of the initial glucose response was observed.     

Screen-printed enzyme electrodes for the detection of marker analytes during winemaking

Biosensors for malic acid and glucose have been developed, using screen-printed electrodes and two different classes of enzymes: NAD(P)⁺-dependent dehydrogenases and oxidases. The active surface of the electrodes was modified using Meldola Blue (malic acid) and Prussian Blue (glucose) and in this way sensitive, low cost and reliable NAD(P)H and H₂O₂ probes were obtained. Fixed potential amperometry was used for the detection of substrates in small volumes of sample (50 μl). Immobilization of the enzymes in a polyethylenimine-glutaraldehyde cross-linking membrane allowed sensors to be obtained with sufficient operational stability. The detection limits were of 10⁻⁵ M for malic acid and 10⁻⁶ M for glucose. The sensors were applied in the analysis of different samples of wine.     

Screen-printed biosensors for the control of wine quality based on lactate and acetaldehyde determination

Biosensors for d-lactate and acetaldehyde were developed, based on screen-printed electrodes and NAD⁺-dependent dehydrogenases. Modification of screen-printed electrodes with the mediator Meldola Blue or with Meldola Blue-Reinecke salt resulted in sensitive, low cost and reliable NADH detectors. The biosensors were realised in two configurations, as disposable and reusable devices. Single-use sensors were obtained by simple deposition of enzyme and cofactor on the surface of mediator-modified electrodes. Chronoamperometry was used for the detection of substrates in small volumes of samples (25 μl). Immobilisation of dehydrogenases by entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) allowed sensors to be obtained with sufficient operational stability. Amperometry in stirred solutions was the detection technique with biosensors for multiple use. The 3σ detection limits for acetaldehyde were 1 μM by amperometry and 6 μM by chronoamperometry and for d-lactate-0.03 μM and 0.05 μM for reusable and disposable biosensors respectively. The biosensors were applied in the analysis of some French and Romanian wines.     

Prussian-Blue-based amperometric biosensors in flow-injection analysis

Optimisation of the electrodeposition of Prussian Blue onto mirrored glassy carbon electrodes yielded a modified electrode practically insensitive to oxygen reduction. At the same time the electrode activity towards hydrogen peroxide reduction was extremely high. This allowed the detection of hydrogen peroxide by electroreduction over a wide potential range. Flow-injection investigations of this electrode inserted into a flowthrough electrochemical cell of the confined wall-jet type showed that the response for hydrogen peroxide is limited by diffusion. Glucose and alcohol biosensors were made by immobilisation of glucose oxidase and alcohol oxidase respectively, within a Nafion layer, onto the top of the Prussian-Blue-modified electrodes. By increasing the density of Nafion and decreasing the measuring potential the glucose biosensor was made completely insensitive to both ascorbate and acetominophes.     

Polypyrrole/copper hexacyanoferrate hybrid as redox mediator for glucose biosensors

A copper containing Prussian Blue analogue was incorporated into a conducting polypyrrole film. The modified electrode was synthesized through an electrochemical two-step methodology leading to very stable and homogeneous hybrid films. These electrodes were proved to show excellent catalytic properties towards H₂O₂ detection, with a performance higher than those observed for Prussian Blue and other analogues. Electrochemical impedance spectroscopy experiments demonstrated that the excellent performance of these hybrid films is strongly related to the electronic conductivity of the polymeric matrix that is wiring the copper hexacyanoferrate sites. A glucose biosensor was built-up by the immobilization of glucose oxidase; the sensitivity obtained being higher than other biosensors reported in the literature even in Na⁺ containing electrolytes.     

Size dependence of SiO2 particles enhanced glucose biosensor

A wide size range of SiO₂ particles were synthesized and were used as enzyme immobilization carriers to fabricate glucose biosensors. The size of the particles was in the range of 17-520 nm. These biosensors could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.2). Particle size could affect the performance of SiO₂ modified glucose biosensors drastically. The smaller particles had higher performance. The smallest SiO₂ modified biosensor could work well in the glucose concentration range of 0.02-10 mM with a correlation coefficient of 0.9993. Its sensitivity was 2.08 μA/mM and the detection limit was 1.5 μM glucose.     

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.     

Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5 mM, with detection limits 60 μM, and pretreatment of the carbon film electrode at a fixed potential of +0.9 V versus SCE for 5 min was found to be the most beneficial. Michaelis-Menten constants between 5 mM and 10 mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.     

New biosensing platforms based on the layer-by-layer self-assembling of polyelectrolytes on Nafion/carbon nanotubes-coated glassy carbon electrodes

We are proposing for the first time the use of a Nafion/multi-walled carbon nanotubes dispersion deposited on glassy carbon electrodes (GCE) as a new platform for developing enzymatic biosensors based on the self-assembling of a chitosan derivative and different oxidases. The electrodes are obtained by deposition of a layer of Nafion/multi-wall carbon nanotubes dispersion on glassy carbon electrodes, followed by the adsorption of a chitosan derivative as polycation and glucose oxidase, l-aminoacid oxidase or polyphenol oxidase, as polyanions and biorecognition elements. The optimum configuration for glucose biosensors has allowed a highly sensitive (sensitivity = (0.28 ± 0.02) μA mM⁻¹, r = 0.997), fast (4 s in reaching the maximum response), and highly selective (0% interference of ascorbic acid and uric acid at maximum physiological levels) glucose quantification at 0.700 V with detection and quantification limits of 0.035 and 0.107 mM, respectively. The repetitivity for 10 measurements was 5.5%, while the reproducibility was 8.4% for eight electrodes. The potentiality of the new platform was clearly demonstrated by using the carbon nanotubes/Nafion layer as a platform for the self-assembling of l-aminoacid oxidase and polyphenol oxidase. Therefore, the platform we are proposing here, that combines the advantages of nanostructured materials with those of the layer-by-layer self-assembling of polyelectrolytes, opens the doors to new and exciting possibilities for the development of enzymatic and affinity biosensors using different transdution modes.     

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.     

Optical biosensors based on Prussian Blue films

Optical biosensing schemes based on enzymatically modified inorganic/organic transparent films predominately composed of Prussian Blue are demonstrated. The composite film, which is non-electrochemically deposited on a non-conducting support. is used as an optical transducer for flow-through biosensors based on hydrolases and oxidases. Urease and glucose oxidase are utilized as model enzymes. Action of the urea biosensor is based on optical pH sensitivity of Prussian Blue indicator. The glucose biosensor is acting as first-generation optical biosensor based on in situ generated Prussian White transducer for hydrogen peroxide. These simple, single-pass transmission optical biosensors exhibit sensitivity in the millimolar range of concentration. The biosensors are very stable owing to presence of a poly(pyrrolylbenzoic acid) network in the composite material. This organic polymer plays a dual role as a binding agent for inorganic material and as a functionalized support for strong covalent immobilization of enzyme molecules.     

A novel procedure for rapid surface functionalisation and mediator loading of screen-printed carbon electrodes

We report a simple and rapid procedure that leads to incorporation of mediator and introduction of amine functionality onto the surface of screen-printed carbon electrodes (SPCE). The electrodes were doped with cobalt phthalocyanine (CoPc) by enhanced adsorption in a process that uses minimal amounts of this redox mediator as compared with CoPc loaded inks. The CoPc-doped SPCE showed a substantially increased sensitivity to hydrogen peroxide and thiocholine as compared to unmodified electrodes. This greatly facilitated their use as transducers for the construction of amperometric biosensors based on enzymes producing oxidizable products such as hydrogen peroxide or thiols. Immobilisation of enzymes including glucose oxidase, acetylcholinesterase and choline oxidase was achieved through their multi-contact electrostatic interaction with polyethyleneimine (PEI) which was electrodeposited on the surface of CoPc-doped electrodes in one step from ethanolic solution. The efficiency of enzyme immobilisation was shown to depend on the molecular weight of the PEI used, reaching a maximum for 25 kDa PEI. The biosensors shown sensitivity to glucose at 130 nA mM⁻¹ (LOD 0.15 mM) and to acetylcholine at 70 nA mM⁻¹ (LOD 0.10 mM) under +0.6 V. Detection of glucose has been demonstrated at +0.4 V with the sensitivity of 60 nA mM⁻¹ and LOD of 0.33 mM. Possibility of the inhibition analysis of pesticides has been shown for acetylcholinesterase-based sensors.     

Electrochemical polymerization of N-substituted pyrrols for the development of novel lactate biosensor

Lactate oxidase from the species Pediococcus is immobilized in a conducting polymer film on the surface of planar electrodes modified with Prussian blue. Polypyrrole ammonium is electropolymerized to obtain the conducting polymer. The analytical characteristics of the resulting biosensor are as follows: a sensitivity of 190 ± 14 mA M⁻¹ cm⁻², a linear dynamic range of 5 × 10⁻⁷ to 5 × l0⁻⁴ M, and high operational stability. The applicability of a lactate biosensor for food quality control (for example, quality control of kvass) is shown. Effective and inexpensive biosensors for lactate analysis may be applied in clinical diagnostics, sports medicine, quality control of food and farm products, as well as for biotechnology processes.     

Amperometric enzyme electrodes of glucose and lactate based on poly(diallyldimethylammonium)-alginate-metal ion-enzyme biocomposites

Sodium alginate (AlgNa) and poly(diallyldimethylammonium chloride) (PDDA) were mixed to obtain an interpenetrating polymer composite via electrostatic interaction and then cast on an Au electrode surface, followed by incorporation of metal ions (e.g. Fe³⁺ or Ca²⁺, to form AlgFe or AlgCa hydrogel) and glucose oxidase (GOx) (or lactate oxidase (LOx)), to prepare amperometric enzyme electrodes. The interactions of PDDA, Alg, and Fe³⁺ are studied by visual inspection as well as microscopic and electrochemical methods. Under optimized conditions, the PDDA-AlgFe-enzyme/Au and PDDA-AlgCa-enzyme/Au electrodes can give good analytical performance (e.g. nM-scale limit of detection of glucose or lactate, and sensitivities > 50 μA cm⁻² mM⁻¹) in the first-generation biosensing mode, which are better than the reported analogs using typical polysaccharide biopolymers as enzyme-immobilization matrices. The enzyme electrodes also worked well in the second-generation biosensing mode in the coexistence of p-benzoquione or ferrocene monocarboxylic acid artificial mediator. Biofuel cells (BFCs) with the enzyme electrodes as the bioanodes and glucose (or lactate) as the biofuel were also fabricated with satisfactory results. The proposed protocols for preparation of high performance Alg-based biocomposites may find wide applications in bioanalysis.     

Application of Amperometric Enzyme Biosensors for Wine and Must Analysis

The work presents application of amperometric biosensors based on platinum printed electrodes and immobilized enzymes alcohol oxidase, glucose oxidase and lactate oxidase for wine analysis. Created devices demonstrate wide dynamic range of work, good stability and high selectivity to the substrate, and are successfully applied for analysis of such complex mixtures as wine and must. Good correlation of the results of analysis of different wines and must obtained by amperometric biosensors and chromatography method is shown. Created biosensors can be used as a basis of commercial device for express analysis of ethanol, glucose and lactate in wine and must during its fermentation.     

Detection of carbamic and organophosphorous pesticides in water samples using a cholinesterase biosensor based on Prussian Blue-modified screen-printed electrode

In the present paper, a comparative study using Co-phthalocyanine and Prussian Blue-modified screen-printed electrodes, has been performed. Both the electrodes have demonstrated an easiness of preparation together with high sensitivity towards thicoholine (LOD = 5 × 10⁻⁷ and 5 × 10⁻⁶ M for Co-phthalocyanine and Prussian Blue, respectively) with high potentialities for pesticide measurement. Prussian Blue-modified screen-printed electrodes were then selected for successive enzyme immobilization due to their higher operative stability demonstrated in previous works. AChE and BChE enzymes were used and inhibition effect of different pesticides was studied with both the enzymes. AChE-based biosensors have demonstrated a higher sensitivity towards aldicarb (50% inhibition with 50 ppb) and carbaryl (50% inhibition with 85 ppb) while BChE biosensors have shown a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with 1 ppb). Real samples were also tested in order to evaluate the matrix effect and recovery values comprised between 79 and 123% were obtained.