Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM⁻¹ (RSD = 6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02–0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7 h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4 °C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.     

A novel microbial biosensor based on cells of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> for the selective determination of 1,3-propanediol in the presence of glycerol and its application to bioprocess monitoring

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards <2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h⁻¹) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R ² = 0.999).     

Electrochemical polymerization of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole as a novel immobilization platform for microbial sensing

Two types of bacterial biosensor were constructed by immobilization of Gluconobacter oxydans and Pseudomonas fluorescens cells on graphite electrodes modified with the conducting polymer; poly(1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole) [SNS(NO₂)]. The measurement was based on the respiratory activity of cells estimated by the oxygen consumption at − 0.7 V due to the metabolic activity in the presence of substrate. As well as analytical characterization, the linear detection ranges, effects of electropolymerization time, pH and cell amount were examined by using glucose as the substrate. The linear relationships were observed in the range of 0.25–4.0 mM and 0.2–1.0 mM for G. oxydans and P. fluorescens based sensors, respectively.     

Amperometric Glucose Biosensors Based on Integration of Glucose Oxidase onto Prussian Blue/Carbon Nanotubes Nanocomposite Electrodes

Nanosized Prussian blue (PB) particles were synthesized with a chemical reduction method and then the PB nanoparticles were assembled on the surface of multiwall carbon nanotubes modified glassy carbon electrode (PB/MWNTs/GCE). The results showed that the PB/MWNTs nanocomposite exhibits a remarkably improved catalytic activity towards the reduction of hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the PB/MWNTs platform by an electrochemically polymerized o‐phenylenediamine (OPD) film to construct an amperometric glucose biosensor. The biosensor exhibited a wide linear response up to 8 mM with a low detection limit of 12.7 μM (S/N=3). The Michaelis–Menten constant K_m and the maximum current i_max of the biosensor were 18.0 mM and 4.68 μA, respectively. The selectivity and stability of the biosensor were also investigated.     

The Construction of Glucose Biosensor Based on Platinum Nanoclusters—Multiwalled Carbon Nanotubes Nanocomposites

One-step synthesis method was proposed to obtain the nanocomposites of platinum nanoclusters and multiwalled carbon nanotubes (PtNCs–MWNTs), which were used as a novel immobilization matrix for the enzyme to fabricate glucose biosensor. The fabrication process of the biosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, atomic force microscopy and scanning electron microscope. Due to the favorable characteristic of PtNCs–MWNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0 μM–12.1 mM), low detection limit (1.0 μM), high sensitivity (12.8 μA mM⁻¹), rapid response time (within 6 s). The apparent Michaelis–Menten constant ( K_m^\textapp K_m^{\text{app}} ) is 2.1 mM. The performance of the resulting biosensor is more prominent than that of most of the reported glucose biosensors. Furthermore, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples.     

Multioperational Oxidase Biosensors Based on Carbosilane Dendrimers with Interacting Ferrocenes

The bioelectrocatalytical properties and kinetic characteristics of new oxidase biosensors based on two different carbosilane dendrimers are described. The best glucose biosensor developed displayed, in an ascorbate interference free work potential interval, a strictly linear range from 0 to 4.0 mM, a detection limit of 40,6 μM and a response time less than 3 s. The lactate biosensor displayed a linear range from 0 to 0.8 mM, a detection limit of 0.73 µM and a response time less than 2 s. The apparent Michaelis–Menten constants were calculated to be 4.39 mM and 2.08 mM respectively, according to Lineweaver–Burk equation.     

Amperometric Biosensor for Hydrogen Peroxide,Using Supramolecularly Immobilized Horseradish Peroxidase on the β‐Cyclodextrin‐Coated Gold Electrode

Horseradish peroxidase, previously modified with 1‐adamantane moieties, was supramolecularly immobilized on gold electrodes coated with perthiolated β‐cyclodextrin. The functionalized electrode was employed for the construction of an amperometric biosensor device for hydrogen peroxide using 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (6 s) and a good linear response toward H₂O₂ concentration between 12 μM and 450 μM. The biosensor showed a sensitivity of 1.02 mA/M cm², and a very low detection limit of 5 μM. The electrode retained 97% of its initial electrocatalytic activity after 30 days of storage at 4 ⁰C in 50 mM sodium phosphate buffer, pH 7.0.     

Development of Silver Nanowires Based Highly Sensitive Amperometric Glucose Biosensor

The fabrication of a highly sensitive amperometric glucose biosensor based on silver nanowires (AgNWs) is presented. The electrochemical behavior of glassy carbon electrode modified by Ag NWs exhibits remarkable catalytic performance towards hydrogen peroxide (H₂O₂) and glucose detection. The biosensor could detect glucose in the linear range from 0.005 mM to 10 mM, with a detection limit of 50 µM (S/N=3). The glucose biosensor shows high and reproducible sensitivity of 175.49 µA cm^?2 mM and good stability. In addition, the biosensor exhibits a good anti‐interference ability and favorable stability over relatively long‐term storage (more than 21 days).     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

A new third‐generation biosensor for H₂O₂ assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

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.     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

Amperometric Detection of Glucose with Glucose Oxidase Immobilized in Layered Double Hydroxides

A novel cheap and simple amperometric biosensor, based on the immobilization of glucose oxidase (GOD) into anionic clay; layered double hydroxides (LDHs) [Zn₃‐Al‐Cl] is presented. GOD can be entrapped in the LDHs gel via electrostatic interaction. Amperometric detection of glucose with an unmediated sensor at 0.6 V (vs. SCE) results in a rapid response (5 s), a wide linear range of 0.001–12 mM, as well as good operational stability. The low detection limit was 0.1 μM at 3σ. The apparent Michaelis‐Menten constant (K is 4.4 mM. The general interferences that coexisted in blood serum do not affect glucose determination, except for uric acid. In addition, optimization of the biosensor construction and the effects of the applied potential on the amperometric response of the sensor were investigated and discussed herein.     

Novel Enzymatic Rhodium Modified Poly(styrene‐g‐oleic amide) Film Electrode for Hydrogen Peroxide Detection

Newly synthesized poly(styrene‐g‐oleic amide) was coated onto a rhodium nanoparticle modified glassy carbon (GC) surface for the fabrication of horseradish peroxidase based biosensor used for hydrogen peroxide detection. The rhodium modifed electrode presented ten times higher signal than unmodified electrode even at low elecrtroactive enzyme quantity by enhancing the electron transfer rate at the applied potential of −0.65 V. The biosensor designed by under the optimized rhodium electrodeposition time exhibited a fast response less than 5 s, an excellent operational stability with a relative standard deviation of 0.6 % (n=6), an accuracy of 96 % and a large linear range between 50 μM and 120 mM for hydrogen peroxide. Detection limit and the sensitivity parameters were calculated to be 44 μM and 57 μA mM⁻¹ cm⁻², respectively by preserving its entire initial response up to the 15 days, while only 20 % of its initial response was lost at the end of one month.     

Amperometric Lactate Biosensor Based on Carbon Paste Electrode Modified with Benzo[c]cinnoline and Multiwalled Carbon Nanotubes

Amperometric lactate biosensor based on a carbon paste electrode modified with benzo[c]cinnoline and multiwalled carbon nanotubes is reported. Incorporation of benzo[c]cinnoline acting as a mediator and multiwalled carbon nanotubes providing a conduction pathway to accelerate electron transfer due to their excellent conductivity into carbon paste matrix resulted in a high performance lactate biosensor. The resulting biosensor exhibited a fast response, high selectivity, good repeatability and storage stability. Under the optimal conditions, the enzyme electrode showed the detection limit of 7.0×10^?8 M with a linear range of 2.0×10^?7 M–1.1×10^?4 M. The usefulness of the biosensor was demonstrated in serum samples.     

Amperometric Biosensor Coupled to Capillary Electrophoresis for Glucose Determination

A novel glucose biosensor has been fabricated and employed as the amperometric detector of a capillary electrophoresis (CE) system. (±)-1-Ferrocenylethylamine and chitosan were successively modified on a 500-µm diameter disc platinum electrode by dip-coating. The modified electrode was subsequently immersed in glucose oxidase (GOx) solution to entrap the enzyme in the chitosan membrane. The primary amino groups of 1-ferrocenylethylamine, GOx, and chitosan were cross-linked by glutaraldehyde to obtain a biosensing membrane so as to reduce leaching of 1-ferrocenylethylamine and GOx. The electrochemical behavior of the target biosensor was investigated. It was demonstrated that the investigated biosensor features fast response, high stability, long lifetime, and ideal compatibility with the CE system. When CE was employed to introduce a glucose plug into the surface of the biosensor, the current response was linear to the glucose concentration in the range of 0.0025 to 2.5 mM with a detection limit of 1.2 µM (S/N = 3) at a working potential of +0.6 V (vs. SCE). The CE-biosensor system was applied to the determination of the glucose level in human serum. The results were satisfactory and in good agreement with the hospital assay results.     

Graphene/Poly(vinylferrocene) Composite Based Amperometric Biosensor for L‐lysine Determination

A novel and sensitive amperometric biosensor for L‐lysine determination based on a glassy carbon electrode (GCE) modified with graphene (GR) and redox polymer poly(vinylferrocene) (PVF) was constructed. L‐lysine‐α‐oxidase was immobilized onto the modified GCE by a glutaraldehyde/bovine serum albumin cross‐linking procedure. SEM, CV and EIS were used for the characterization of the surface morphology and stepwise fabrication processes of PVF/GR composite. Optimal composition of the biosensor and experimental conditions that affect the performance of the biosensor are discussed. The effect of buffer pH on biosensor response was studied in detail over a wide pH range. L‐lysine biosensor displayed a linear range of 9.9×10⁻⁷ ‐ 3.1×10⁻⁴ M with a low detection limit of 2.3×10⁻⁷ M and K_M ^app value of 0.4 mM. The L‐lysine biosensor was tested using pharmaceutical sample and cheese with satisfactory results.     

Hemoglobin‐Titanate Composite Based Biosensor for the Amperometric Determination of Hydrogen Peroxide in Acidic Medium

A hemoglobin‐titanate composite based biosensor was chosen for determination of H₂O₂ in an acidic medium. CV results of the Hb‐titanate modified pyrolytic graphite electrode showed a pair of well‐defined, quasi‐reversible redox peaks centered at ?246 mV (vs. Ag/AgCl) in a pH 5.0 HAc‐NaAc buffer solution. The modified electrode exhibited good electrocatalytic response for monitoring H₂O₂ and had a large linear detection range from 20 μM to 3.2 mM with a detection limit of 8 μM (S/N=3) and a sensitivity of 29.7 mA M^?1 cm^?2 in the pH 5.0 solution. The biosensor also possessed good long term storage stability.     

Electrocatalytic Oxidation of Glucose by the Glucose Oxidase Immobilized in Graphene‐Au‐Nafion Biocomposite

Graphene was successfully prepared and well separated to individual sheets by introducing  SO₃⁻. XRD and TEM were employed to characterize the graphene. UV‐visible absorption spectra indicated that glucose oxidase (GOx) could keep bioactivity well in the graphene‐Au biocomposite. To construct a novel glucose biosensor, graphene, Au and GOx were co‐immobilized in Nafion to further modify a glassy carbon electrode (GCE). Electrochemical measurements were carried out to investigate the catalytic performance of the proposed biosensor. Cyclic voltammograms (CV) showed the biosensor had a typical catalytic oxidation response to glucose. At the applied potential +0.4 V, the biosensor responded rapidly upon the addition of glucose and reached the steady state current in 5 s, with the present of hydroquinone. The linear range is from 15 μM to 5.8 mM, with a detection limit 5 μM (based on the S/N=3). The Michaelis‐Menten constant was calculated to be 4.4 mM according to Lineweaver–Burk equation. In addition, the biosensor exhibits good reproducibility and long‐term stability. Such impressive properties could be ascribed to the synergistic effect of graphene‐Au integration and good biocompatibility of the hybrid material.     

In situ synthesis of thulium(III) hexacyanoferrate(II) nanoparticles and its application for glucose detection

Thulium hexacyanoferrate (TmHCF) nanoparticles (NPs) were in situ synthesized within the chitosan film on the electrode surface by a biocatalyzed reaction. The properties of the obtained nanoparticles are characterized with scanning electron microscope (SEM) and energy-dispersive X-ray (EDX). The optimized conditions for the formation of TmHCF NPs were 16 mM Fe(CN)₆³⁻ and 1.5 mM Tm³⁺ with an accumulation time of 20 min. Based on process of in situ synthesis of TmHCF NPs, a novel biosensor for glucose was designed, and there is a linear relationship between the current response of TmHCF NPs and glucose concentration. The linear range for glucose detection was 0.02–0.4 mM (r = 0.9975, n = 5) and 0.4–13.6 mM (r = 0.9935, n = 10) and the detection limit was 6 μM at a signal-to-noise ratio of 3.     

Direct Electrochemistry of Hemoglobin Immobilized on Carbon‐Coated Iron Nanoparticles for Amperometric Detection of Hydrogen Peroxide

A novel method for preparation of hydrogen peroxide biosensor was presented based on immobilization of hemoglobin (Hb) on carbon‐coated iron nanoparticles (CIN). CIN was firstly dispersed in a chitosan solution and cast onto a glassy carbon electrode to form a CIN/chitosan composite film modified electrode. Hb was then immobilized onto the composite film with the cross‐linking of glutaraldehyde. The immobilized Hb displayed a pair of stable and quasireversible redox peaks and excellent electrocatalytic reduction of hydrogen peroxide (H₂O₂), which leading to an unmediated biosensor for H₂O₂. The electrocatalytic response exhibited a linear dependence on H₂O₂ concentration in a wide range from 3.1 μM to 4.0 mM with a detection limit of 1.2 μM (S/N=3). The designed biosensor exhibited acceptable stability, long‐term life and good reproducibility.