Enzyme-free amperometric sensing of hydrogen peroxide and glucose at a hierarchical Cu2O modified electrode

In this paper, an enzyme-free amperometric electrochemical sensor was fabricated by casting Nafion-impregnated Cu₂O particles onto a glassy carbon electrode. A dual dependence of peak current on sweeping rate, which can be attributed for the accumulation of reaction products, was observed on the sensor. Electrochemical analysis of the particulate Cu₂O for detecting H₂O₂ and glucose is described, showing remarkable sensitivity in both cases. The estimated detection limits and sensitivities for H₂O₂ (0.0039 μM, 52.3 mA mM⁻¹ cm⁻²) and glucose (47.2 μM, 0.19 mA mM⁻¹ cm⁻²) suggest that the response for H₂O₂ detection was much higher than for glucose detection. Electron microscopy observation suggested that the hierarchical structures of Cu₂O resulting from self-assembly of nanocrystals are responsible for the specific electrochemical properties.     

Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion

Two different glucose biosensors for the amperometric determination of glucose, based on poly(p-chlorophenylamide) (PCPA) and bilayer film of PCPA and Nafion (PCPA/Nafion), are successfully developed. These two biosensors show linear amperometric responses to glucose ranging from 2.0×10⁻⁴ to 3.5×10⁻² mol l⁻¹ and 5.0×10⁻⁴ to 7.5×10⁻² mol l⁻¹, respectively, with the same correlation coefficient of 0.9988. Effects of polymerization potential and polymerization time on the performance of enzyme sensors are studied. It is found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Introduction of inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability.     

Sulfonated polyaniline network grafted multi-wall carbon nanotubes for enzyme immobilization, direct electrochemistry and biosensing of glucose

A new nanomaterial was prepared by grafting a layer of sulfonated polyaniline network (SPAN-NW) on to the surface of multi-walled carbon nanotube (MWNT) and effectively utilized for immobilization of an enzyme and for the fabrication of a biosensor. SPAN-NW was formed on the surface of MWNT by polymerizing a mixture of diphenyl amine 4-sulfonic acid (DPASA), 4-vinyl aniline (VA) and 2-acrylamido-2-methyl-1-propane sulfonic acid (APASA) in the presence of amine functionalized MWNT (MWNT-NH₂). The MWNT-g-SPAN-NW was immobilized with glucose oxidase (GOx) to fabricate the SPAN-NW/GOx biosensor. MWNT-g-SPAN-NW/GOx electrode showed direct electron transfer (DET) for GOx with a fast heterogeneous electron transfer rate constant (k_s) of 4.11 s^− 1. The amperometric current response of MWNT-g-SPAN-NW/GOx biosensor shows linearity up to 9 mM of glucose, with a correlation coefficient of 0.99 and a detection limit of 0.11 μM (S/N = 3). At a low applied potential of − 0.1 V, MWNT-g-SPAN-NW/GOx electrode possesses high sensitivity (4.34 μA mM^− 1) and reproducibility towards glucose.     

Selective and sensitive electrochemical detection of glucose in neutral solution using platinum-lead alloy nanoparticle/carbon nanotube nanocomposites

Electrodeposition of Pt-Pb nanoparticles (PtPbNPs) to multi-walled carbon nanotubes (MWCNTs) resulted in a stable PtPbNP/MWCNT nanocomposite with high electrocatalytic activity to glucose oxidation in either neutral or alkaline medium. More importantly, the nanocomposite electrode with a slight modification exhibited high sensitivity, high selectivity, and low detection limit in amperometric glucose sensing at physiological neutral pH (poised at a negative potential). At +0.30 V in neutral solution, the nanocomposite electrode exhibited linearity up to 11 mM of glucose with a sensitivity of 17.8 μA cm⁻² mM⁻¹ and a detection limit of 1.8 μM (S/N = 3). Electroactive ascorbic acid (0.1 mM), uric acid (0.1 mM) and fructose (0.3 mM) invoked only 23%, 14% and 9%, respectively, of the current response obtained for 3 mM glucose. At −0.15 V in neutral solution, the electrode responded linearly to glucose up to 5 mM with a detection limit of 0.16 mM (S/N = 3) and detection sensitivity of ∼18 μA cm⁻² mM⁻¹. At this negative potential, ascorbic acid, uric acid, and fructose were not electroactive, therefore, not interfering with glucose sensing. Modification of the nanocomposite electrode with Nafion coating followed by electrodeposition of a second layer of PtPbNPs on the Nafion coated PtPbNP/MWCNT nanocomposite produced a glucose sensor (poised at −0.15 V) with a lower detection limit (7.0 μM at S/N = 3) and comparable sensitivity, selectivity and linearity compared to the PtPbNP/MWCNT nanocomposite. The Nafion coating lowered the detection limit by reducing the background noise, while the second layer of PtPbNPs restored the sensitivity to the level before Nafion coating.     

The application of conducting polymer nanoparticle electrodes to the sensing of ascorbic acid

An ascorbic acid sensor was fabricated via the drop-casting of dodecylbenzene sulphonic acid (DBSA)-doped polyaniline nanoparticles onto a screen-printed carbon-paste electrode. The modified electrode was characterised with respect to the numbers of drop cast layers, optimum potential and operating pH. The sensor was found to be optimal at neutral pH and at 0 V vs. Ag/AgCl. Under these conditions, the sensor showed good selectivity and sensitivity in that it did not respond to a range of common interferents such as dopamine, acetaminophen, uric acid and citric acid, but was capable of the detection of ascorbic acid at a sensitivity of 0.76 μA mM⁻¹ or 10.75 μA mM⁻¹ cm⁻² across a range from 0.5 to 8 mM (r² = 0.996, n = 6), and a limit of detection of 8.3 μM (S/N = 3). The sensor was compared to a range of other conducting polymer-based ascorbate sensors and found to be comparable or superior in terms of analytical performance.     

Electrode modified with a composite film of ZnO nanorods and Ag nanoparticles as a sensor for hydrogen peroxide

A conducting fluorine-doped tin oxide (FTO) electrode, first modified with zinc oxide nanorods (ZnONRs) and subsequently attached with photosynthesized silver nanoparticles (AgNPs), designated as AgNPs/ZnONRs/FTO electrode, was used as an amperometric sensor for the determination of hydrogen peroxide. The first layer (ZnONRs) was obtained by chemical bath deposition (CBD), and was utilized simultaneously as the catalyst for the photoreduction of Ag ions under UV irradiation and as the matrix for the immobilization of AgNPs. The aspect ratio of ZnONRs to be deposited was optimized by controlling the number of their CBDs to render enough surface area for Ag deposition, and the amount of AgNPs to be attached was controlled by adjusting the UV-irradiation time. The immobilized AgNPs showed excellent electrocatalytic response to the reduction of hydrogen peroxide. The resultant amperometric sensor showed 10-fold enhanced sensitivity for the detection of H₂O₂, compared to that without AgNPs, i.e., only with a layer of ZnONRs. Amperometric determination of H₂O₂ at −0.55 V gave a limit of detection of 0.9 μM (S/N = 3) and a sensitivity of 152.1 mA M⁻¹ cm⁻² up to 0.983 mM, with a response time (steady-state, t₉₅) of 30-40 s. The selectivity of the sensor was investigated against ascorbic acid (AA) and uric acid (UA). Energy dispersive X-ray (EDX) analysis, transmission electron microscopic (TEM) image, X-ray diffraction (XRD) patterns, cyclic voltammetry (CV), and scanning electron microscopic (SEM) images were utilized to characterize the modified electrode. Sensing properties of the modified electrode were studied both by CV and amperometric analysis.     

On-line removal of redox-active interferents by a porous electrode before amperometric blood glucose determination

A porous reticulated vitreous carbon (RVC) electrode and a disk electrode coupled in tandem in an electrochemical flow cell has been used for electrolytic removal of interferents before amperometric glucose detection. The electrolytic efficiency at the upstream RVC electrode is 100% at a flow rate of 0.1 mL min⁻¹ or lower. Potential interferents such as acetaminophen, ascorbic acid, and uric acid can be completely eliminated by electrolysis at the RVC electrode. A mixed monolayer comprising glucose oxidase (GOD) and ferrocenyl-1-undecanethiol preformed at the downstream gold disk electrode was used as a mediator-based amperometric glucose sensor. The dependence of the amperometric current on the glucose concentration exhibits good linearity across over three orders of magnitude. The glucose measurements were also found to be reproducible (RSD < 3.5%) and accurate. Unlike the chemiluminescence method, this device obviates the use of carcinogenic substrates and the glucose sensor performance is independent of the oxygen present in sample. On the basis that the RVC electrode requires minimal cleanup and the GOD-modified electrode remains stable for a week, the electrochemical flow cell should be amenable for automated on-line removal of redox interferents for other types of enzyme-based biosensors.     

Glyphosate and glufosinate detection at electrogenerated NiAl-LDH thin films

An amperometric sensor based on Ni_1−xAl_x(OH)₂NO_3x·nH₂O layered double hydroxide (LDH) has been developed for the electrochemical analysis in one step of two herbicides: glyphosate (N-(phosphonomethyl)glycine, Glyp) and glufosinate ((DL-homoalanine-4-yl)-methylphosphinic acid, Gluf). NiAl-LDH was prepared by coprecipitation or by electrodeposition at the Pt electrode surface. Inorganic films were fully characterized by X-ray diffraction, Raman spectroscopy and scanning electron microscopy. Adsorption isotherms of Glyp onto this inorganic lamellar material have been established. Electrocatalytic oxidation of Glyp and Gluf is possible at the Ni³⁺ centres of the structure. The electrochemical responses of the NiAl-LDH modified electrode were obtained by cyclic voltammetry and chronoamperometry at 0.49 V/SCE as a function of herbicide concentration in 0.1 M NaOH solution. The electrocatalytic response showed a linear dependence on the Glyp concentration ranging between 0.01 and 0.9 mM with a detection limit of 1 μM and sensitivity 287 mA/M cm². The sensitivity found for Gluf was lower (178 mA/M cm²).     

Optimized multilayer oxalate biosensor

The optimization of a biosensor prepared by the immobilization of oxalate oxidase (OOX) with a cross-linking agent onto a multilayer inorganic/organic modified electrode, is presented. A very thin Prussian Blue (PB) film covered by a self-doped polyaniline (SPAN) layer acts as very sensitive amperometric sensor for the H₂O₂ formed by the enzymatic reaction. The electrode allows the very reliable and sensitive oxalate detection in the 0.08 to 0.45 mmol l⁻¹ concentration range. The observed sensitivity was 131.3 μA mmol⁻¹ cm⁻² at the operation potential of 0.05 V versus Ag/AgCl in a succinate buffer solution (pH=3.8). The bilayer Prussian blue/SPAN leads to a very stable, sensitive and selective system that not only minimizes the interference caused by ascorbic and uric acids but also forms a very adherent sensing film that allows repetitive successive determinations.     

An electrochemical ascorbic acid sensor based on palladium nanoparticles supported on graphene oxide

In this study, an electrochemical ascorbic acid (AA) sensor was constructed based on a glassy carbon electrode modified with palladium nanoparticles supported on graphene oxide (PdNPs-GO). PdNPs with a mean diameter of 2.6 nm were homogeneously deposited on GO sheets by the redox reaction between PdCl₄²⁻ and GO. Cyclic voltammetry and amperometric methods were used to evaluate the electrocatalytic activity towards the oxidation of AA in neutral media. Compared to a bare GC or a Pd electrode, the anodic peak potential of AA (0.006 V) at PdNPs-GO modified electrode was shifted negatively, and the large anodic peak potential separation (0.172 V) of AA and dopamine (DA), which could contribute to the synergistic effect of GO and PdNPs, was investigated. A further amperometric experiment proved that the proposed sensor was capable of sensitive and selective sensing of AA even in the presence of DA and uric acid. The modified electrode exhibited a rapid response to AA within 5 s and the amperometric signal showed a good linear correlation to AA concentration in a broad range from 20 μM to 2.28 mM with a correlation coefficient of R = 0.9991. Moreover, the proposed sensor was applied to the determination of AA in vitamin C tablet samples. The satisfactory results obtained indicated that the proposed sensor was promising for the development of novel electrochemical sensing for AA determination.     

A novel amperometric sensor for the detection of difenidol hydrochloride based on the modification of Ru(bpy)3 on a glassy carbon electrode

An amperometric sensor for the detection of difenidol, a tertiary amine-containing analyte, was proposed. Ruthenium(II) tris(bipyridine)/multi-walled carbon nanotubes/Nafion composite film was suggested to modify the glassy carbon electrode. The modified electrode was shown to be an excellent amperometric sensor for the detection of difenidol hydrochloride. The linear range is from 1.0 × 10⁻⁶ to 3.3 × 10⁻⁵ M with a correlation coefficient of 0.998. The limit of detection was 5 × 10⁻⁷ M, which was obtained through experimental determination based on a signal-to-noise ratio of three. The sensor was employed to the determination of the active ingredients in the tablets containing difenidol hydrochloride.     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

Non-enzymatic electrochemical CuO nanoflowers sensor for hydrogen peroxide detection

The electrocatalytic activity of a CuO flower-like nanostructured electrode was investigated in terms of its application to enzyme-less amperometric H₂O₂ sensors. The CuO nanoflowers film was directly formed by chemical oxidation of copper foil under hydrothermal condition and then used as active electrode material of non-enzymatic electrochemical sensors for H₂O₂ detection under alkaline conditions. The sensitivity of the sensor with CuO nanoflowers electrode was 88.4 μA/mM cm² with a linear response in the range from 4.25 × 10⁻⁵ to 4 × 10⁻² M and a detection limit of 0.167 μM (S/N = 3). Excellent electrocatalytic activity, large surface-to-volume ratio and efficient electron transport property of CuO nanoflowers electrode have enabled stable and highly sensitive performance for the non-enzymatic H₂O₂ sensor.     

Electrochemical determination of L-phenylalanine at polyaniline modified carbon electrode based on β-cyclodextrin incorporated carbon nanotube composite material and imprinted sol-gel film

A sensitive and selective electrochemical sensor based on a polyaniline modified carbon electrode for the determination of l-phenylalanine has been proposed by utilizing β-cyclodextrin (β-CD) incorporated multi-walled carbon nanotube (MWNT) and imprinted sol-gel film. The electrochemical behavior of the sensor towards l-phenylalanine was investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometric i-t curve. The surface morphologies of layer-by-layer assembly electrodes were displayed by scanning electron microscope (SEM). The response mechanism of the imprinted sensor for l-phenylalanine was based on the inclusion interaction of β-CD and molecular recognition capacity of the imprinted film for l-phenylalanine. A linear calibration plot was obtained covering the concentration range from 5.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.0 × 10⁻⁹ mol L⁻¹. With excellent sensitivity, selectivity, stability, reproducibility and recovery, the electrochemical imprinted sensor was used to detect l-phenylalanine in blood plasma samples successfully.     

Electrochemical study of mono-6-thio-β-cyclodextrin/ferrocene capped on gold nanoparticles: Characterization and application to the design of glucose amperometric biosensor

A novel amperometric glucose sensor based on inclusion complex of mono-6-thio-β-cyclodextrin/ferrocene capped on gold nanoparticles (GNPs/CD-Fc) and glucose oxidase (GOD) was described. The inclusion complex of mono-6-thio-β-cyclodextrin/ferrocene capped on gold nanoparticles played an effective role of an electron shuttle and allowed the detection of glucose at 0.25 V (versus SCE), with dramatically reduced interference from easily oxidizable constituents. The sensor (GNPs/CD-Fc/GOD) showed a relatively fast response time (5 s), low detection limit (15 μM, S/N = 3), and high sensitivity (ca. 18.2 mA M⁻¹ cm⁻²) with a linear range of 0.08-11.5 mM of glucose. The excellent sensitivity was possibly attributed to the presence of the GNPs/CD-Fc film that can provide a convenient electron tunneling between the protein and the electrode. In addition, the biosensor demonstrated high anti-interference ability, stability and natural life. The good stability and natural life can be attributed to the following two aspects: on the one hand, the fabrication process was mild and no damage was made on the enzyme molecule, on the other hand, the GNPs possessed good biocompatibility that could retain the bioactivity of the enzyme molecules immobilized on the electrode.     

Amperometric sensor for nitrite using a glassy carbon electrode modified with alternating layers of iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin and cobalt(II) tetrasulfonated phthalocyanine

The development of a highly sensitive amperometric sensor for nitrite using a glassy carbon electrode modified with alternated layers of iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin (FeT4MPyP) and cobalt(II) tetrasulfonated phthalocyanine (CoTSPc) is described. The modified electrode showed an excellent catalytic activity and stability for the nitrite oxidation decreasing the peak potentials about 200 mV toward less positive values and presenting much higher peak currents than those obtained on the bare GC electrode. A linear response range of 0.2-8.6 μmol l⁻¹, with a sensitivity of 0.37 μA l μmol⁻¹ and detection limit of 0.04 μmol l⁻¹ were obtained with this sensor. The repeatability of the proposed sensor, evaluated in term of relative standard deviation, was verified to be 1.4% for 10 measurements of 0.2 μmol l⁻¹ nitrite solution. Interference caused by common ions has been investigated in simulated mixtures containing high concentration level of interfering ions and the sensor was found to be tolerant against these ions. The developed sensor was applied for the nitrite determination in water samples and the results were in agreement with those obtained by a comparative method described in the literature. The average recovery for these samples was 100.1 (±0.7)%.     

Preparation and characterization of Prussian blue nanowire array and bioapplication for glucose biosensing

Prussian blue nanowire array (PBNWA) was prepared via electrochemical deposition with polycarbonate membrane template for effective modification of glassy carbon electrode. The PBNWA electrode thus obtained was demonstrated to have high-catalytic activity for the electrochemical reduction of hydrogen peroxide in neutral media. This enabled the PBNWA electrode to show rapid response to H₂O₂ at a low potential of −0.1 V over a wide range of concentrations from 1 × 10⁻⁷ M to 5 × 10⁻² M with a high sensitivity of 183 μA mM⁻¹ cm⁻². Such a low-working potential also substantially improved the selectivity of the PBNWA electrode against most electroactive species such as ascorbic acid and uric acid in physiological media. A detection limit of 5 × 10⁻⁸ M was obtained using the PBNWA electrode for H₂O₂, which compared favorably with most electroanalysis procedures for H₂O₂. A biosensor toward glucose was then constructed with the PBNWA electrode as the basic electrode by crosslinking glucose oxidase (GOx). The glucose biosensor allowed rapid, selective and sensitive determination of glucose at −0.1 V. The amperometric response exhibited a linear correlation to glucose concentration through an expanded range from 2 × 10⁻⁶ M to 1 × 10⁻² M, and the response time and detection limit were determined to be 3 s and 1 μM, respectively.     

Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents

The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC₅₀, under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC₅₀ = 3.7 × 10⁻⁶ M, K_i = 8.6 × 10⁻⁷ M), ascorbic acid (IC₅₀ = 1.2 × 10⁻⁵ M), benzoic acid (IC₅₀ = 7.2 × 10⁻⁵ M, K_i = 2.0 × 10⁻⁵ M) and azelaic acid (IC₅₀ = 1.3 × 10⁻⁴ M, K_i = 4.2 × 10⁻⁵ M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.     

Flow electrochemical biosensors based on enzymatic porous reactor and tubular detector of silver solid amalgam

A flow amperometric enzymatic biosensor for the determination of glucose was constructed. The biosensor consists of a flow reactor based on porous silver solid amalgam (AgSA) and a flow tubular detector based on compact AgSA. The preparation of the sensor and the determination of glucose occurred in three steps. First, a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) was formed at the porous surface of the reactor. Second, enzyme glucose oxidase (GOx) was covalently immobilized at MUA-layer using N-ethyl-N′-(3-dimethylaminopropyl) carboimide and N-hydroxysuccinimide chemistry. Finally, a decrease of oxygen concentration (directly proportional to the concentration of glucose) during enzymatic reaction was amperometrically measured on the tubular detector under flow injection conditions. The following parameters of glucose determination were optimized with respect to amperometric response: composition of the mobile phase, its concentration, the potential of detection and the flow rate. The calibration curve of glucose was linear in the concentration range of 0.02–0.80 mmol L⁻¹ with detection limit of 0.01 mmol L⁻¹. The content of glucose in the sample of honey was determined as 35.5 ± 1.0 mass % (number of the repeated measurements n = 7; standard deviation SD = 1.2%; relative standard deviation RSD = 3.2%) which corresponds well with the declared values. The tested biosensor proved good long-term stability (77% of the current response of glucose was retained after 35 days).     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.