Polydivinylbenzene/Ethylvinylbenzene Composite Membranes for the Optimization of a Whole Blood Glucose Sensor

A novel ultra thin polydivinylbenzene/ethylvinylbenzene composite membrane has been developed for use as the outer covering barrier in a model amperometric glucose oxidase enzyme electrode. The composite membrane was formed via the cathodic electropolymerization of divinylbenzene/ethylvinylbenzene at the surface of gold sputter coated host alumina membranes, (serving solely as a mechanical support for the thin polymer film). Permeability coefficients were determined for the enzyme substrates, O₂ and glucose, across composite membranes formed with a range of polymer thicknesses. Due to the highly substrate diffusion limiting nature of the composite membrane, it was found that anionic interferents present in blood (such as ascorbate), were effectively screened from the working electrode via a charge exclusion mechanism, in a manner similar to previous findings within our laboratory. The enzyme electrode showed an initial 32% signal drift when first exposed to whole human blood over a period of 2 hours, after which time enzyme electrode responses remained essentially stable. Whole blood patient glucose determinations yielded a correlation coefficient of r²=0.97 in comparison to standard hospital analyses.     

Glucose enzyme electrode with extended linearity: Application to undiluted blood measurements

An enzyme electrodes is described for glucose determination in unstirred, undiluted whole blood. The system comprises an H₂O₂-detecting electrode upon which is placed a membrane laminate incorporating glucose oxidase. The external membrane was pretreated with methyltrichlorosilane. The electrode response was linearly dependent on glucose concentration up to 50 mmol l^?1 glucose, it had a decreased dependence on dissolved oxygen concentrations and gave response times of 30–90 s. Whole blood glucose measurements correlated well with a routine spectrophotometric method.     

Preparation and application of a new glucose sensor based on iodide ion selective electrode

An electrode for glucose has been prepared by using an iodide selective electrode with the glucose oxidase enzyme. The iodide selective electrode used was prepared from 10% TDMAI and PVC according our previous study. The enzyme was immobilized on the iodide electrode by holding it at pH 7 phosphate buffer for 10 min at room temperature. The H₂O₂ formed from the reaction of glucose was determined from the decrease of iodide concentration that was present in the reaction cell. The iodide concentration was followed from the change of potential of iodide selective electrode. The potential change was linear in the 4×10⁻⁴ to 4×10⁻³ M glucose concentration (75-650 mg glucose/100ml blood) range. The slope of the linear portion was about 79 mV per decade change in glucose concentration. Glucose contents of some blood samples were determined with the new electrode and consistency was obtained with a colorimetric method. The effects of pH, iodide concentration, the amount of enzyme immobilized and the operating temperature were studied. No interference of ascorbic acid, uric acid, iron(III) and Cu(II) was observed. Since the iodide electrode used was not an AgI-Ag₂S electrode, there was no interference of common ions such as chloride present in biological fluids. The slope of the electrode did not change for about 65 days when used 3 times a day.     

An Air‐breathing Carbon Cloth‐based Screen‐printed Electrode for Applications in Enzymatic Biofuel Cells

We report a prototype air‐breathing carbon cloth‐based electrode that was fabricated starting from a commercially available screen‐printed electrode equipped with a transparent ITO working electrode (DropSens, ref. ITO10). The fabrication of the air‐breathing electrodes is straightforward, shows satisfactory reproducibility and a good electrochemical response as evaluated by means of [Fe(CN)₆]^3?/4? voltammetry. The gas‐diffusion electrodes were successfully modified with the O₂ reducing enzyme bilirubin oxidase from Myrothecium verrucaria in a direct electron transfer regime. The enzyme modified electrodes showed a remarkable high current density for O₂ reduction in passive air‐breathing mode of up to 5 mA cm^?2. Moreover, the enzyme modified electrodes were applied as O₂ reducing biocathodes in a glucose/air enzymatic biofuel cell in combination with a high current density glucose oxidase/redox polymer bioanode. The biofuel cell provides a high maximum power density of (0.34±0.02) mW cm^?2 at 0.25 V. The straightforward design, low cost and the high reproducibility of these electrodes are considered as basis for standardized measurements under gas‐breathing conditions and for high throughput screening of gas converting (bio‐)catalysts.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Preparation of Close‐Packed Silver Nanoparticles on Graphene to Improve the Enzyme Immobilization and Electron Transfer at Electrode in Glucose/O2 Biofuel Cell

In this study, chemical reduced graphene‐silver nanoparticles hybrid (AgNPs @CR‐GO ) with close‐packed AgNPs structure was used as a conductive matrix to adsorb enzyme and facilitate the electron transfer between immobilized enzyme and electrode. A facile route to prepare AgNPs @CR‐GO was designed involving in β ‐cyclodextrin (β ‐CD ) as reducing and stabilizing agent. The morphologies of AgNPs were regulated and controlled by various experimental factors. To fabricate the bioelectrode, AgNPs @CR‐GO was modified on glassy carbon electrode followed by immobilization of glucose oxidase (GOx ) or laccase. It was demonstrated by electrochemical testing that the electrode with close‐packed AgNPs provided high GOx loading (Γ =4.80 × 10⁻¹⁰ mol•cm⁻²) and fast electron transfer rate (k _s=5.76 s⁻¹). By employing GOx based‐electrode as anode and laccase based‐electrode as cathode, the assembled enzymatic biofuel cell exhibited a maximum power density of 77.437 μW •cm⁻² and an open‐circuit voltage of 0.705 V.     

Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H₂O₂ needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H₂O₂ concentration in solution has on HRP activity. The H₂O₂ in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L^?1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10^?3 mol L^?1 glucose, and 0.1 mol L^?1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p‐phenylenediamine, 2‐aminophenol, o‐phenylenediamine, m‐phenylenediamine, 1‐naphthylamine, o‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.     

On-line glucose monitoring by using microdialysis sampling and amperometric detection based on ‘wired’ glucose oxidase in carbon paste

In-vitro on-line glucose monitoring is described, based on microdialysis sampling and amperometric detection operated in a flow-injection system. Samples were injected into a two-electrode microcell containing an Ag/AgCl quasi-reference electrode and a glucose enzyme electrode as the working electrode, operated at + 0.15 Vvs. Ag/AgCl. The enzyme electrode is constructed by mixing the wired glucose oxidase into carbon paste. {Poly[1-vinylimidazole osmium(4,4-dimethylbipyridine)₂Cl)]}^+/2+ was used to wire the enzyme. The non-coated electrodes, cross-linked with poly(ethylene glycol) diglycidyl ether, responded linearly to glucose concentrations up to 60 mM, and were characterized by a sensitivity of 0.23 A mM^–1 cm^–2, when operated in flow injection mode and of 5.4 AmM ^–1 cm^–2 in steady-state conditions. This sensitivity of the resulting enzyme electrode was 50% lower than that of similarly prepared but non-cross-linked electrodes. However, the cross-linked electrodes showed superior operational and storage stabilities, which were further improved by coating the electrodes with a negatively charged Eastman AQ film. An in-house designed microdialysis probe, equipped with a polysulphone cylindrical dialysis membrane, yielded a relative recovery of 50–60% at a perfusion rate of 2.5 l/min^–1 in a well stirred glucose solution. The on-line set up effectively rejected common interferences such as ascorbic acid and 4-acetaminophen when present at their physiological concentrations.     

Guanine/Ionic Liquid Derived Ordered Mesoporous Carbon Decorated with AuNPs as Efficient NADH Biosensor and Suitable Platform for Enzymes Immobilization and Biofuel Cell Design

Guanine‐ionic liquid derived ordered mesoporous carbon (GIOMC) decorated with gold nanoparticles was used as electrocatalyste for NADH oxidation and electrochemical platform for immobilization of glucose dehydrogenase (GDH) enzyme. The resulting GIOMC/AuNPs on the glassy carbon electrode can be used as novel redox‐mediator free for NADH sensing and this integrated system (GIOMC/AuNPs/GDH) shows excellent electrocatalytic activity toward glucose oxidation. Furthermore, the ionic liquid derived ordered mesoporous carbon derivate with Ph‐SO₃H (IOMC‐PhSO₃H) decorated with AuNPs has been developed to bilirubin oxidase enzyme (BOD) immobilization and the GC/IOMC‐PhSO₃H/BOD integrated system shows excellent bioelectrocatalytic activity toward oxygen reduction reaction. The proposed mesostructured platforms decorated by AuNPs have been developed to enhance mass transfer and charge transfer from biocatalyst to electrode, leading these bioanode and biocathode used for biofuel cell assembly. Integration designed bioanode and biocathode yielding a membrane‐less glucose/O₂ biofuel cell with power density of 33 (mW.cm⁻²) at 257 mV. The open circuit voltage of this biofuel cell and maximum produced current density were 508 mV and 0.252 (mA.cm⁻²) respectively.     

Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications

Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes–nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm⁻² and oxygen reduction current exceeded 0.6 mA cm⁻². The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 μW cm⁻² without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface.     

Direct Electron Transfer of Glucose Oxidase and Glucose Biosensor Based on Nano-structural Attapulgite Clay Matrix

The direct electrochemistry of glucose oxidase (GOD) was achieved based on the immobilization of GOD on a natural nano‐structural attapulgite (ATP) clay film modified glassy carbon (GC) electrode. The immobilized GOD displayed a pair of well‐defined quasi‐reversible redox peaks with a formal potential (E^0′) of ?457.5 mV (vs. SCE) in 0.1 mol·L^?1 pH 7.0 phosphate buffer solution. The peak current was linearly dependent on the scan rate, indicating that the direct electrochemistry of GOD in that case was a surface‐controlled process. The immobilized glucose oxidase could retain bioactivity and catalyze the oxidation of glucose in the presence of ferrocene monocarboxylic acid (FMCA) as a mediator with the apparent Michaelis‐Menten constant K^app_m of 1.16 mmol·L^?1. The electrocatalytic response showed a linear dependence on the glucose concentration ranging widely from 5.0×10^?6 to 6.0×10^?4 mol·L^?1 (with correlation coefficient of 0.9960). This work demonstrated that the nano‐structural attapulgite clay was a good candidate material for the direct electrochemistry of the redox‐active enzyme and the construction of the related enzyme biosensors. The proposed biosensors were applied to determine the glucose in blood and urine samples with satisfactory results.     

Integrated Microcentrifuge Carbon Entrapped Glucose Oxidase Poly (N-Isopropylacrylamide) (pNIPAm) Microgels for Glucose Amperometric Detection

This study demonstrates a miniaturized integrated glucose biosensor based on a carbon microbeads entrapped by glucose oxidase (GOx) immobilized on poly (N-isopropylacrylamide) (pNIPAm) microgels. Determined by the Lowry protein assay, the pNIPAm microgel possesses a high enzyme loading capacity of 31?mg/g. The pNIPAm GOx loaded on the microgel was found to maintain a high activity of approximately 0.140?U determined using the 4-aminoantipyrine colorimetric method. The integrated microelectrochemical cell was constructed using a microcentrifuge vial housing packed with (1:1, w/w) carbon entrapped by pNIPAm GOx microgels, which played the dual role of the microbioreactor and the working electrode. The microcentrifuge vial cover was used as a miniaturized reference electrode and an auxiliary electrode holder. The device can work as biosensor, effectively converting glucose to H₂O₂, with subsequent amperometric detection at an applied potential of ?0.4?V. The microelectrochemical biosensor was used to detect glucose in wide linear range from 30?µM to 8.0?mM, a low detection limit of 10?µM, a good linear regression coefficient (R²) of 0.994, and a calibration sensitivity of 0.0388?µA/mM. The surface coverage of active GOx, electron transfer rate constant (ks), and Michaelis–Menten constant (K_M^app) of the immobilized GOx were 4.0?×?10^?11?mol/cm², 5.4?s^?1, and 0.086?mM, respectively. To demonstrate the applicability and robustness of the biosensor for analysis of high sample matrix environment, glucose was analyzed in root beer. The microelectrochemical device was demonstrated for analysis of small sample (<50?µL), while affording high precision and fast signal measurement (≤5?s).     

Entrapment of glucose oxidase in non-porous poly(vinyl chloride)

We have used solvent casting techniques to immobilise glucose oxidase (GOD) within unplasticised and plasticised poly(vinyl chloride) (PVC) matrices. The plasticisers studied were the cationic surfactant, tricaprylmethylammonium chloride (Aliquat 336s), the anionic surfactant bis(2-ethylhexyl) hydrogenphosphate (BEP) and the lipid, isopropylmyristate (IPM). The activity of the enzyme-membrane was tested by amperometric electrode. Changes in enzyme-membrane electrode response are rationalised on the basis of membrane permselective properties. The Aliquat and IPM modified PVC membranes gave amplified signals due to better retention and subsequent concentration of the H₂O₂ signal species. Effectively, less was being lost to the bulk solution. In the case of the BEP-modified membrane, while there was a linear step change in response up to 50 mM, at higher concentrations, responses did not reach steady-state; they were characterised by an upward drift in response of 0.050 nA/min. This characteristic is thought to be due to a build up of gluconic acid resulting in a pH reduction in the membrane microenvironment and hydrogen bonding between neighbouring BEP molecules. Under these conditions, we have previously shown that the membrane permeability to hydrophilic species is attenuated and it is tentatively suggested that the upward drift due to the build up of H₂O₂ on the electrode side with less permeating through the acidified membrane into bulk solution.The results were compared against using variously plasticised PVC (but no enzyme entrapped) as an outer membrane of a classical dual-membrane glucose enzyme electrode construct. In the latter case, the enzyme was chemically crosslinked between the membranes using glutaraldehyde.     

Fabrication of Glucose Biosensor Based on Encapsulation of Glucose‐Oxidase on Sol‐Gel Composite at the Surface of Glassy Carbon Electrode Modified with Carbon Nanotubes and Celestine Blue

A simple procedure was developed to prepare a glassy carbon electrode modified with multi walled carbon nanotubes (MWCNTs) and Celestin blue. Cyclic voltammograms of the modified electrode show stable and a well defined redox couple with surface confined characteristic at wide pH range (2–12). The formal potential of redox couple (E′) shifts linearly toward the negative direction with increasing solution pH. The surface coverage of Celestine blue immobilized on CNTs glassy carbon electrode was approximately 1.95×10^?10 mol cm^?2. The charge transfer coefficient (α) and heterogeneous electron transfer rate constants (k_s) for GC/MWCNTs/Celestine blue were 0.43 and 1.26 s^?1, respectively. The modified electrode show strong catalytic effect for reduction of hydrogen peroxide and oxygen at reduced overpotential. The glucose biosensor was fabricated by covering a thin film of sol‐gel composite containing glucose oxides (GOx) on the surface of Celestine blue /MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 0.3 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. The accuracy of the biosensor for glucose detection was evaluated by detection of glucose in a serum sample, using standard addition protocol. In addition biosensor can reach 90% of steady currents in about 3.0 sec and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) was eliminated. Furthermore, the apparent Michaelis–Menten constant 2.4 mM, of GOx on the nano composite exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility of redox couple, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this procedure for modification of glucose biosensor.     

Direct Electrochemistry of Glucose Oxidase Immobilized on Titanium Carbide‐Au Nanoparticles‐Fullerene C60 Composite Film and Its Biosensing for Glucose

The direct electrochemistry of glucose oxidase (GOD) immobilized on the designed titanium carbide‐Au nanoparticles‐fullerene C₆₀ composite film modified glassy carbon electrode (TiC‐AuNPs‐C₆₀/GCE) and its biosensing for glucose were investigated. UV‐visible and Fourier‐transform infrared spectra of the resulting GOD/TiC‐AuNPs‐C₆₀ composite film suggested that the immobilized GOD retained its original structure. The direct electron transfer behaviors of immobilized GOD at the GOD/TiC‐AuNPs‐C₆₀/GCE were investigated by cyclic voltammetry in which a pair of well‐defined, quasi‐reversible redox peaks with the formal potential (E^0′) of ‐0.484 V (vs. SCE) in phosphate buffer solution (0.05 M, pH 7.0) at the scan rate of 100 mV·s^?1 were obtained. The proposed GOD modified electrode exhibited an excellent electrocatalytic activity to the reduction of glucose, and the currents of glucose reduction peak were linearly related to glucose concentration in a wider linearity range from 5.0 × 10^?6 to 1.6 × 10^?4 M with a correlation coefficient of 0.9965 and a detection limit of 2.0 × 10^?6 M (S/N = 3). The sensitivity and the apparent Michaelis‐Menten constant (K_M^app) were determined to be 149.3 μA·mM^?1·cm^?2 and 6.2 × 10^?5 M, respectively. Thus, the protocol will have potential application in studying the electron transfer of enzyme and the design of novel electrochemical biosensors.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Real‐time Selective Detection of Hydrogen Peroxide Based on a Tantalum Deposited Pencil Lead Electrode for Evaluation of Enzyme Activities

In this study, we have carried out electrodeposition of tantalum (Ta) nanostructures on pencil lead electrode in non‐aqueous media at room temperature by applying a constant potential. The deposited Ta on pencil lead was examined for the catalytic effect regarding hydrogen peroxide (H₂O₂) reduction with voltammetry and amperometry. Ta/pencil lead electrode exhibited amperometric sensitivity of 0.317 μA mM⁻¹ cm⁻² and fast response time of 0.75 s, where selective detection of H₂O₂ was fulfilled without interruption from common electroactive biomaterials such as O₂, uric acid, ascorbic acid, dopamine, acetamidophenol, and glucose. For practical applications, the dynamic concentration changes of H₂O₂ during catalase and glucose oxidase‐involved reactions, either eliminating or producing H₂O₂, were successfully traced in real time with as‐prepared electrode. From the kinetics study for catalase and glucose oxidase, we evaluated Michaelis constants (K _m^app) as 7.8 mM for catalase and 37 mM for glucose oxidase, respectively.     

A Novel Enzymatic Glucose Biosensor and Nonenzymatic Hydrogen Peroxide Sensor Based on (3‐Aminopropyl) Triethoxysilane Functionalized Reduced Graphene Oxide

In the present study, a novel enzymatic glucose biosensor using glucose oxidase (GOx) immobilized into (3‐aminopropyl) triethoxysilane (APTES) functionalized reduced graphene oxide (rGO‐APTES) and hydrogen peroxide sensor based on rGO‐APTES modified glassy carbon (GC) electrode were fabricated. Nafion (Nf) was used as a protective membrane. For the characterization of the composites, Fourier transform infrared spectroscopy (FTIR), X‐ray powder diffractometer (XRD), and transmission electron microscopy (TEM) were used. The electrochemical properties of the modified electrodes were investigated using electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry. The resulting Nf/rGO‐APTES/GOx/GC and Nf/rGO‐APTES/GC composites showed good electrocatalytical activity toward glucose and H₂O₂, respectively. The Nf/rGO‐APTES/GC electrode exhibited a linear range of H₂O₂ concentration from 0.05 to 15.25 mM with a detection limit (LOD) of 0.017 mM and sensitivity of 124.87 μA mM⁻¹ cm⁻². The Nf/rGO‐APTES/GOx/GC electrode showed a linear range of glucose from 0.02 to 4.340 mM with a LOD of 9 μM and sensitivity of 75.26 μA mM⁻¹ cm⁻². Also, the sensor and biosensor had notable selectivity, repeatability, reproducibility, and storage stability.     

Investigation of a Biosensor Based on Phenylalanine Dehydrogenase Immobilized on a Polymer‐Blend Film for Phenylketonuria Diagnosis

We investigated a L ‐phenylalanine (L ‐phe) biosensor, functionalized through enzyme immobilization on a polymer‐blend film. The electron mediator 3,4‐dihydroxybenzaldehyde (3,4‐DHB) was employed at the electrode surface to improve direct oxidation of NADH to NAD⁺ and no additional reagents is required to be added to the sample solution. The bioactivated electrode was coated with a semi‐permeable cellulose acetate membrane in order to prevent dissolution of biofunctionalized polymer‐blend film. This constructed enzyme electrode is the first selective biosensor for phenylketonuria (PKU) detection. The sensitivity of the enzyme electrode was determined as 12.014 mA/M cm². The Michaelis–Menten and current responses as well as sensitivity of the electrode showed improved values than those of previous works. This selective biosensor presented an excellent electroanalytical response for L ‐phe, with a high steady‐state current being obtained after 20 s. The sensitivity of our biodevice is quite sufficient for the purpose of PKU detection because the reference range of clinical concern for L ‐phenylalanine concentration is C_{L ‐phe}>0.5 mM. This surface‐bioactivated enzyme electrode retained more than 80 % of its electrocatalytic activity after 16 days.     

Electrochemical Fabrication of Cobalt Oxides/Nanoporous Gold Composite Electrode and its Nonenzymatic Glucose Sensing Performance

A nonenzymatic glucose sensor was successfully established by electrochemically decorating cobalt oxides (CoO_x) on a nanoporous gold electrode (NPG) using cobalt hexacyanoferrate (CoHCF) as a precursor. It exhibited high sensitivity and long‐term stability as well as satisfactory quantification of glucose concentration in human serum samples. The morphology and surface analysis of the resulting CoO_x/NPG were carefully characterized. Two detection methods, cyclic voltammetry and amperometry, were employed to evaluate the performance of CoO_x/NPG towards glucose sensing in alkaline solution. Using cyclic voltammetry, at ?0.5 V, the glucose partial oxidation peak current is linear to the glucose concentration up to 14 mM with a sensitivity of 283.7 µA mM^?1 cm^?2. A linear amperometric response at 0.55 V was obtained in the glucose concentration range from 2 µM to 2 mM with a sensitivity of 2025 µA mM^?1 cm^?2 and a response time <3 s.