Direct electron transfer of glucose oxidase at glassy carbon electrode modified with functionalized carbon nanotubes within a dihexadecylphosphate film

A glassy carbon electrode modified with functionalized multiwalled carbon nanotubes (CNTs) immobilized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) in a dihexadecylphosphate film was prepared and characterized by cyclic voltammetry and scanning electron microscopy. It was used as a support for FAD or glucose oxidase (GOx) immobilization with EDC/NHS crosslinking agents. Cyclic voltammetry of GOx immobilized onto the surface of CNTs showed a pair of well-defined redox peaks, which correspond to the direct electron transfer of GOx, with a formal potential of −0.418 V vs. Ag/AgCl (3 M KCl) in 0.1 M phosphate buffer solution (pH 7.0). An apparent heterogeneous electron transfer rate constant of 1.69 s⁻¹ was obtained. The dependence of half wave potential on pH indicated that the direct electron transfer reaction of GOx involves a two-electron, two-proton transfer. The determination of glucose was carried out by square wave voltammetry and the developed biosensor showed good reproducibility and stability. The proposed method could be easily extended to immobilize and evaluate the direct electron transfer of other redox enzymes or proteins.     

An electrochemical biosensor for determination of hydrogen peroxide using nanocomposite of poly(methylene blue) and FAD hybrid film

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) has been developed by the hybrid film of poly(methylene blue) and FAD (PMB/FAD). The PMB/FAD hybrid film was performed in PBS (pH 7) containing methylene blue and FAD by cyclic voltammetry. Repeatedly scanning potential range of −0.6-1.1 V, FAD was immobilized on the electrode surface by electrostatic interaction while methylene blue was electropolymerized on electrode surface. This modified electrode was found surface confined and pH dependence. It showed good electrocatalytic reduction for H₂O₂, KBrO₃, KIO₃, and NaClO as well as electrocatalytic oxidation for NADH. At an applied potential of −0.45 V vs. Ag/AgCl, the sensor showed a rapid and linear response to H₂O₂ over the range from 0.1 μM to 960 μM, with a detection limit of 0.1 μM and a significant sensitivity of 1109 μA mM⁻¹ cm⁻² (S/N = 3). It presented excellent stability at room temperature, with a variation of response current less than 5% over 30 days.     

Electrochemical studies of bovine serum albumin immobilization onto the poly-o-phenylenediamine and carbon-coated nickel composite film and its interaction with papaverine

A biosensor based on bovine serum albumin (BSA) and poly-o-phenylenediamine (PoPD)/carbon-coated nickel (C-Ni) nanobiocomposite film modified electrode has been developed to study the interaction of BSA with papaverine (PAP). The well-dispersed C-Ni nanoparticles were dripped onto the glassy carbon electrode (GCE) surface firstly, and PoPD films were subsequently electropolymerized by cyclic voltammetry (CV) to prepare PoPD/C-Ni/GCE. Finally, the BSA was easily immobilized on the PoPD films via electrostatic adsorption. The morphology and the electrochemical properties of the fabricated composite electrodes were examined by scanning electron microscope (SEM) and electrochemical impedance spectroscopy (EIS), respectively. The interaction of PAP with BSA was monitored by differential pulse voltammetry (DPV), using PoPD as the electrochemical indicator. The binding constant (K), obtained by DPV, was 1.7 × 10⁴ L/mol, which was consistent with the fluorescence analysis. This constructed biosensor also exhibited a fine linear correlation with PAP concentration range of 2.5 × 10⁻⁹-4.5 × 10⁻⁵ mol/L and a detection limit of 8.3 × 10⁻¹⁰ mol/L was achieved by DPV.     

Voltammetric studies of the interaction of rutin with DNA and its analytical applications on the MWNTs-COOH/Fe3O4 modified electrode

Multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH)/iron oxide (Fe₃O₄) modified glassy carbon electrode (MWNTs-COOH/Fe₃O₄/GCE) and DNA/MWNTs-COOH/Fe₃O₄/GCE were prepared. The electrochemical behaviors of rutin (RU) were investigated on MWNTs-COOH/Fe₃O₄/GCE by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in britton-robinson buffer solution (B-R). The interaction of RU with DNA was also explored. Dramatic decrease of peak current without obvious peak potential shift were observed in both cases of DNA in the solution and immobilized on the electrode surface. In addition, the electron transfer coefficient (α) and the rate constant (k_s) kept unchanged in the absence and presence of DNA. So interaction of DNA with RU formed a non-electroactive complex. The binding constant and binding ratio was obtained in the process. The interaction was also confirmed by UV-visible spectroscopy. The reduction peak current was linear with the concentration of RU in the range of 2.50 × 10⁻⁸ to 1.37 × 10⁻⁶ M, with a detection limit of 7.5 nM. The MWNTs-COOH/Fe₃O₄/GCE showed comparatively low detection limit, rapid response, simplicity for the determination of RU.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized into poly (lactic-co-glycolic acid)/room temperature ionic liquid composite film

A promising material of poly(lactic-co-glycolic acid) (PLGA) and, room temperature ionic liquid (ILs) (1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF₄) was firstly used as an immobilization matrix to entrap proteins and its bioelectrochemical properties were studied. Direct electrochemistry and electrocatalytic behaviors of hemoglobin (Hb) entrapped in the PLGA/ILs composite film on the surface of glass carbon electrode were investigated. UV-vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well-defined redox peaks of Hb was obtained at the Hb/PLGA/ILs composite film-modified GC electrode through direct electron transfer between the protein and the underlying electrode. The proposed biosensor showed good reproducibility and high sensitivity to H₂O₂ with the detection limit of 2.37 × 10⁻⁷ M (S/N = 3). In the range of 5.0 × 10⁻⁶ to 8.05 × 10⁻³ M, the catalytic reduction current of H₂O₂ was proportional to its concentration. The apparent Michaelis-Menten constant of Hb in the PLGA/ILs composite film was estimated to be 0.069 mM, showing its high affinity.     

Development of enzyme immobilized monolith micro-reactors integrated with microfluidic electrochemical cell for the evaluation of enzyme kinetics

This paper describes a simple and efficient method for producing an on-chip enzyme immobilized monolith micro-reactor that integrates a microfluidic electrochemical cell for rapid characterization of enzymatic kinetics. The monolith was generated using a sol–gel method, followed by PEI functionalization and enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. Using the proposed immobilization strategy, a glucose oxidase (GOD) immobilized monolith micro-reactor has been produced with the controllable porosity that gives better enzyme kinetics compared to previously reported devices. This can be attributed to a favourable enzyme-substrate affinity in which more than 98% of the immobilized enzyme remains in an active conformation. The kinetic studies conducted have identified that a similar value of the k _cat is obtained for immobilized GOD (13.4 s⁻¹) and GOD free in solution (14 s⁻¹) whilst the immobilized Michaelis constant K _m(app) (7.2 mM) is ~4 times lower than GOD in solution (25 mM). In addition, the immobilized GOD exhibits increased stability, retaining at least 95% of the initial activity when stored of 30 days at 4°C, compared to only 60% for GOD in solution. Furthermore, the same enzyme immobilization strategy has been used for choline oxidase immobilization and similar kinetics to choline oxidase in solution were observed, once again indicating better maintenance of the enzyme conformation provided by the proposed method.     

Electrochemical performances of B doped and undoped diamond-like carbon (DLC) films deposited by femtosecond pulsed laser ablation for heavy metal detection using square wave anodic stripping voltammetric (SWASV) technique

Pure diamond-like carbon (DLC) thin films and boron-doped DLC thin films have been deposited on silicon substrates using femtosecond pulsed laser. The amorphous carbon materials (DLC), have been deposited at room temperature by ablating graphite targets with an amplified Ti:sapphire laser of 800 nm wavelength and a pulse duration of 150 fs in high vacuum conditions. Doping with boron has been performed by ablating alternatively graphite and boron targets.In this study, the DLC films were used as working electrodes for the electrochemical detection of trace heavy metals namely, Cd²⁺, Pb²⁺, Ni²⁺ and Hg²⁺, by using square wave anodic stripping voltammetry (SWASV) technique. Four metals were detected at −1.3 V deposition potential, and 90 s deposition time. The DLC films have been characterized by multiwavelength Raman spectrometry and high resolution scanning electron microscopy. The effect of the boron doping on the electrochemical behavior has been shown. The a-C:B 8%/Si₃N₄ electrode gives the more sensitive detection. The four metals are detected simultaneously with a detection limit of 1 μg/L or 2 μg/L and a dynamic range from 1 or 2 to 25 μg/L for every metal, as presented in third table of this article. The different sensitivities obtained are 6.2, 20.0, 1.2 and 6.6 μA/ppb or μA μg⁻¹ L for Cd²⁺, Pb²⁺, Ni²⁺ and Hg²⁺, respectively.     

Study of an amperometric glucose sensor based on Pd-Ni/SiNW electrode

An amperometric glucose sensor based on Pd-Ni/SiNW electrode has been investigated. The silicon nanowire (SiNW) electrodes were first fabricated by chemical etching, and then nickel and palladium particles were deposited onto the surfaces of SiNWs via electroless co-plating technique followed by annealing in nitrogen atmosphere at 350 °C for 300 s. The morphology of Pd-Ni/SiNW electrode was characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). The sensor performance was characterized by cyclic voltammetry (CV) and fixed potential amperometry techniques. In 0.1 M KOH alkaline medium with different glucose concentrations, the sensor shows an excellent sensitivity of 190.72 μA mM⁻¹ cm⁻² with the detection limit (S/N ratio = 3) of 2.88 μM. And it also exhibits superior anti-interference properties to the species including ascorbic acid (AA), uric acid (UA) and 4-acetamidophenol (AP). All results demonstrate that this Pd-Ni/SiNW electrode is a candidate with great potential for glucose detection.     

New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum

A new amperometric immunosensor for alpha-fetoprotein (AFP), based on nanobiocomposite substrate and with response enhanced by polyamidoaminic (PAMAM) dendrimers was developed and characterized. The nanostructurated substrate obtained by electrochemical deposition of 100 nm-sized gold nanoparticles on glassy carbon electrodes (GCE) was functionalized by deposition of a SAM of 2-aminoethanethiol (AET), used as linker for the subsequent immobilization of polyamidoaminic dendrimers (PAMAM G.1.5). Two different modes were investigated for the reading of the assay: cyclic voltammetry (CV) or Double Step ChronoAmperometry (DSCA). Satisfying results in terms of response range and precision were reached with both methods. Immunosensors were tested and validated for AFP determination in human serum, showing a limit of detection of 3 ng/mL and a limit of quantitation of 15 ng/mL. The enhanced immunosensor has proved an attractive diagnostic tool able to match the needs of clinical monitoring purposes for AFP quantification in human serum at levels useful both for prognosis of pregnancy progression, and for the identification of the occurrence of neoplastic diseases.     

An amperometric ethanol sensor based on a Pd–Ni/SiNWs electrode

A new amperometric ethanol sensor has been developed. The sensor uses the silicon nanowires covered with co-deposited palladium–nickel (Pd–Ni/SiNWs) as the working electrode. The detection of ethanol concentration is based on the response currents resulted from the electro-catalytic oxidation of ethanol. The performance of the sensor was characterized by cyclic voltammetry and fixed potential amperometry techniques. In 1 M KOH solution containing different ethanol concentrations, the sensor shows a good sensitivity of 7.48 mA mM⁻¹ cm⁻² and the corresponding detection limit (signal-to-noise ratio = 3) of 6 μM for cyclic voltammetry. Meanwhile, it also displays a sensitivity of 0.76 mA mM⁻¹ cm⁻² and the corresponding detection limit of 10 μM for fixed potential amperometry. The results demonstrate that the Pd–Ni/SiNWs electrodes are potential as the electrochemical integrated sensors for ethanol detection.     

Impedimetric immunosensor for electronegative low density lipoprotein (LDL) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL⁻) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL⁻ electrochemical biosensor was developed by adsorption of anti-LDL⁻ MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL⁻. The interaction between MAb-LDL⁻ leads to a blockage in the electron transfer of the [Fe(CN)₆]⁴⁻/K₄[Fe(CN)₆]³⁻ redox couple, which may could result in high change in the electron transfer resistance (R_CT) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R_CT, and the charge transferring rate constant κ⁰ decreases from 18.2 × 10⁻⁶ m/s to 4.6 × 10⁻⁶ m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL⁻, which could be used as a biosensor to quantify plasmatic levels of LDL⁻.     

Correlation between cell growth rate and glucose consumption determined by electrochemical monitoring

The electrochemical monitoring of glucose consumption is relevant for cell biology studies because of its wide detection range, high sensitivity and easy implementation. Whereas the glucose consumption and cell growth rate can be tightly correlated, they should also be cell population density dependent. In this work, we fabricated high sensitive enzyme electrodes for accurate monitoring of glucose consumption of cells in different growth stages. The performance of the fabricated device was firstly evaluated by cyclic voltammetry (CV) with p-benzoquinone (PBQ) as redox mediator, showing a linear response over a wide detection range (0.3-60 mM), a high sensitivity (1.61 ± 0.10 μA mM⁻¹ mm⁻² (n = 5)) and a low detection limit (80 μM). Then, daily glucose consumptions of NIH 3T3 cells in 24-well plates were determined for a period of 7 days. The results could be compared to the cell population growth curve, showing a close correlation but different behavior. We found that the increase of the glucose consumption took place prior the cell number increase but the glucose consumption per cell decreases linearly in the exponential growth stage of cells.     

Electroanalytical detection of Pb, Cd and traces of Cr at micro/nano-structured bismuth film electrodes

A micro/nanoparticle (μ-NP) bismuth film electrode (BiFE) has been developed for the determination of lead and cadmium by anodic stripping voltammetry (ASV) and trace levels of chromium by adsorptive stripping voltammetry (AdSV). Chromium was detected in a flow cell. Bismuth was electrodeposited on a hydrated aluminum oxide template, which was previously coated on a glassy-carbon (GC) electrode. Then, the template was selectively removed by soaking the electrode in a 0.1 M NaOH solution for 30 min, leaving a dispersed bismuth film covering the electrode surface; such electrodeposits had a particulate appearance, which was observed by scanning electron microscopy (SEM). The voltammetric analyses from a chromium solution on a μ-NPs/BiFE provided a limit of detection (LOD) equal to 0.12 ng L⁻¹ (n = 6) and a slope of 0.27 μC/ng L⁻¹ (R² = 0.9903). The signals registered were more than 50 times higher than the peaks obtained from a conventional BiFE. The analysis of aqueous solutions of Cd and Pb gave also lower LOD and higher sensitivity against the conventional BiFe experiments.     

Conductometric glucose biosensor made with cellulose and tin oxide hybrid nanocomposite

This paper reports the feasibility study of glucose oxidase (GO_x) immobilized cellulose-tin oxide (SnO₂) hybrid nanocomposite as a glucose biosensor. Porous SnO₂ layer was grown on regenerated cellulose films via liquid phase deposition technique with varying deposition time. Tin oxide was crystallized in the solution and formed nanocrystal coatings on the cellulose films. Enzyme (GO_x) was immobilized into cellulose-SnO₂ hybrid nanocomposite by physical absorption method. X-ray photoelectron spectroscopy analysis revealed the successful immobilization of GO_x into the cellulose-SnO₂ hybrid nanocomposite via covalent bonding between GO_x and SnO₂. The glucose biosensor under study is displayed linear response in the range of 0.5-12 mM with correlation coefficient of 0.96, which can cover the clinical region of glucose concentration. These results indicate that the cellulose-SnO₂ hybrid nanocomposite can be an inexpensive, flexible and disposable glucose biosensor.     

Label-free electrochemical immunosensor for sensitive detection of kanamycin

A novel label-free electrochemical immunosensor for sensitive detection of kanamycin based on water-soluble graphene sheet (WGS)/prussian blue-chitosan (PB-CTS)/nanoporous gold (NPG) composited film has been reported. PB was selected as an electron transfer mediator, and was modified onto the electrode together with WGS through electrostatic adsorption. Then NPG was immobilized onto the as-prepared film for biomolecules anchoring. The electroactivity of PB was greatly enhanced in the presence of WGS and NPG. It could mainly be ascribed to the fact that the good conductivity of WGS and NPG promoted electron transfer and enhanced the sensitivity. kanamycin antibody, as a model, was immobilized onto the composite film for the detection of kanamycin. Under optimum conditions, the amperometric signal of PB decreased linearly with kanamycin concentration (0.02-14 ng mL⁻¹), a linear calibration plot (y = 1.3817 + 4.7544x, r = 0.9993), resulting in a low limit of detection (6.31 pg mL⁻¹). The novel immunosensor for the detection of kanamycin in real sample with satisfactory results has been proved. In addition, this method would be easily adapted for the detection of other residual antibiotics in animal derived foods.     

Sensitive electrochemical determination of acetaminophen in pharmaceutical formulations at multiwalled carbon nanotube-alumina-coated silica nanocomposite modified electrode

A highly sensitive electrochemical sensor for the determination of acetaminophen at the multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS) nanocomposite modified glassy carbon electrode is reported. The morphology of the MWCNT-ACS nanocomposite was characterized by field emission scanning electron microscopy. The electrocatalytic properties of the MWCNT-ACS nanocomposite modified glassy carbon electrode were characterized by cyclic voltammetry and square-wave voltammetry in the presence of acetaminophen. The MWCNT-ACS nanocomposite modified glassy carbon electrode exhibited the abilities to raise the current response and to decrease the electrooxidation potential. In cyclic voltammetric responses, the oxidation peak current of acetaminophen obtained at the MWCNT-ACS modified glassy carbon electrode was 100 times greater than that of bare glassy carbon electrode. The MWCNT-ACS nanocomposite modified glassy carbon electrode for the determination of acetaminophen displayed a sensitivity of 376.5 A M⁻¹ cm⁻² and a detection limit of 0.05 μM using square-wave voltammetry. The analytical applicability of the developed method was achieved by analyzing the content of acetaminophen in five commercial drugs without pretreatment.     

Development of molecularly imprinted electrochemical sensor with titanium oxide and gold nanomaterials enhanced technique for determination of 4-nonylphenol

4-Nonylphenol (4-NP) was reported to affect the health of wildlife and humans through altering endocrine function. A novel electrochemical sensor for sensitive and fast determination of 4-NP was developed. Titanium oxide (TiO₂) nanoparticles and gold nanoparticles (AuNPs) were introduced for the enhancement of electron conduction and sensitivity. 4-NP-imprinted functionalized AuNPs composites with specific binding sites for 4-NP was modified on electrode. The resulting electrodes were characterized by cyclic voltammetry (CV). Rebinding experiments were carried out to determine the specific binding capacity and selective recognition. The linear range was over the range from 4.80 × 10⁻⁴ to 9.50 × 10⁻⁷ mol L⁻¹, with the detection limit of 3.20 × 10⁻⁷ mol L⁻¹ (S/N = 3). The sensor was successfully employed to detect 4-NP in real samples.     

SPR biosensor by using E. coli outer membrane layer with autodisplayed Z-domains

In this work, the Z-domain of protein A with IgG-binding activity was expressed on the outer membrane (OM) of E. coli as a fusion protein of AIDA-1 by using “autodisplay technology”. The OM of E. coli with the autodisplayed Z-domains was isolated and then the OM with the Z-domains was layered on the gold surface of SPR biosensor in order to prepare the orientation-controlled antibody layer. The properties of the OM layer were analyzed by cyclic voltammetry, impedance spectroscopy and AFM. The immunoassay by using the OM layer with Z-domains showed that the limit of detection was improved as much as 50-folds by the orientation-control effect of the Z-domains. The sensitivity of assay (y-axis) at the each analyte concentration (x-axis) was also observed to be far improved by the orientation-control. The OM layer was then prepared on the SPR biosensor, and the far improved sensitivity from the orientation-control effect by the Z-domains was presented in comparison to the randomly oriented antibody layer by using hIgG as an analyte. For the regeneration of the SPR biosensor with the antibody layer immobilized to the Z-domains of OM layer, a cross-linker called glutaraldehyde was used for the covalent immobilization of the antibodies, and the feasibility of this method was demonstrated by performing repeated regeneration processes.