A stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene)-single walled carbon nanotubes/ascorbate oxidase/nafion films for detection of L-ascorbic acid

In this paper, a stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene) (PEDOT)-single walled carbon nanotubes (SWCNT)/ascorbate oxidase (AO)/Nafion films for detection of l-ascorbic acid (AA) was successfully developed. PEDOT-SWCNT nanocomposite and Nafion films were used as inner and outer films, respectively. AO was immobilized between these two films. The PEDOT-SWCNT nanocomposite films were characterized by electrochemical impedance spectroscopy and scanning electron microscopy. The influence of detection potential and temperature on the biosensor performance was examined in detail. Despite the multilayer configuration, the biosensor exhibited a relatively fast response (less than 10 s) and a linear range from 1 μM to 18 mM (a correlation coefficient of 0.9974). The sensitivity of the biosensor was found to be 28.5 mA M⁻¹ cm⁻². Its experimental detection limit was 0.7 μM (S/N = 3) and the apparent Michaelis-Menten constant (K_m) was calculated to be 18.35 mM. Moreover, the biosensor exhibited good anti-interferent ability and excellent long-term stability. All the results showed that such sandwich-type PEDOT-SWCNT/AO/Nafion films could provide a promising platform for the biosensor designs for AA detection.     

A sensitive hydrazine electrochemical sensor based on electrodeposition of gold nanoparticles on choline film modified glassy carbon electrode

A highly sensitive hydrazine sensor was developed based on the electrodeposition of gold nanoparticles onto the choline film modified glassy carbon electrode (GNPs/Ch/GCE). The electrochemical experiments showed that the GNPs/Ch film exhibited a distinctly higher activity for the electro-oxidation of hydrazine than GNPs with 3.4-fold enhancement of peak current. The kinetic parameters such as the electron transfer coefficient (α) and the rate of electron exchange (k) for the oxidation of hydrazine were determined. The diffusion coefficient (D) of hydrazine in solution was also calculated by chronoamperometry. The sensor exhibited two wide linear ranges of 5.0 × 10⁻⁷-5.0 × 10⁻⁴ and 5.0 × 10⁻⁴-9.3 × 10⁻³ M with the detection limit of 1.0 × 10⁻⁷ M (s/n = 3). The proposed electrode presented excellent operational and storage stability for the determination of hydrazine. Moreover, the sensor showed outstanding sensitivity, selectivity and reproducibility properties. All the results indicated a good potential application of this sensor in the detection of hydrazine.     

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⁻.     

A novel amperometric biosensor for oxalate determination using multi-walled carbon nanotube-gold nanoparticle composite

An amperometric oxalate biosensor using nanohybrid film of multi-walled carbon nanotubes (MWCNTs) and gold colloidal nanoparticles (GNPs) via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM) has been prepared. The c-MWCNTs were immobilized on the gold (Au) electrode and characterized by FTIR. The morphologies of the c-MWCNT/Au and GNPs/MWCNT/Au electrodes were investigated by scanning electron microscopy (SEM) and the electrochemical performance of the Au, c-MWCNT/Au and GNPs/c-MWCNT/Au electrodes were also studied amperometrically. The Cl⁻ and NO₃⁻ insensitive oxalate oxidase from grain sorghum was finally immobilized on this electrode. The influence of pH, temperature and oxalate concentration on electrode activity was studied. The electrode showed optimum response within 7 s. The electrocatalytic response showed a linear dependence on the oxalic acid concentration ranging from 1 to 800 μM with a detection limit of 1 μM. The K_m value for the oxalic acid sensor was 444.44 μM. The enzyme electrode retained 30% of its initial activity after 5 months, when stored at 4 °C. The electrode was employed for measurement of oxalic acid in serum, urine and foodstuffs.     

Amperometric biosensor for catechol using electrochemical template process

A novel amperometric biosensor for the determination of catechol was developed accordingly to the electrochemical template procedure. The optimum fabricating conditions of the biosensor were studied. The resulting biosensor with the limit of less than 0.05 μM can be used for detection of catechol in the linear range of 2.5-140 μM. The maximum response current (I_max) and the Michaelis-Menten constant (k′_m) are 3.08 μA and 77.52 μM, respectively. The activation energy (E_a) of the polyphenol oxidase (PPO) catalytic reaction is 25.56 kJ mol⁻¹ in the B-R buffer. The stability of the PANI-CA biosensor fabricated with the electrochemical template process (retains 86% of the original activity after four months) is much higher than that fabricated with one-step and two-step processes (retains 75% of the original activity after four months). The effects of potential and pH on the response current of the biosensor are also described.     

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.     

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.     

Electrochemical behaviors and determination of isoniazid at ordered mesoporous carbon modified electrode

In this work, an electrochemical sensor based on ordered mesoporous carbon (OMC) for the amperometric detection of isoniazid was developed. OMC was dispersed in a solution of Nafion, and the suspension was modified onto the surface of glassy carbon (GC) electrode. Cyclic voltammetry and amperometry were used to investigate the electrochemical behaviors of isoniazid on Nafion-OMC modified electrode (Nafion-OMC/GC). The results indicate that OMC can facilitate the electrochemical oxidation of isoniazid with a great decrease of overpotential in pH 7.0 phosphate buffer solution. The proposed biosensor provides excellent performance towards the determination of isoniazid with a high sensitivity of 0.031 μA/μM, a low detection limit of 8.4 × 10⁻⁸ M and wide linear range from 1.0 × 10⁻⁷ M to 3.7 × 10⁻⁴ M at +0.20 V vs. Ag/AgCl. The method was successfully applied to the determination of isoniazid tablets with satisfying results. All the results suggest that Nafion-OMC/GC electrode is a potential candidate for a stable and efficient electrochemical sensor to detect isoniazid.     

Sensitive amperometric determination of chemical oxygen demand using Ti/Sb-SnO2/PbO2 composite electrode

A novel Ti/Sb-SnO₂/PbO₂ composite electrode was fabricated for COD determination. The new electrode configuration improved the sensitivity of the amperometric method apparently. Effects of common experimental parameters, such as applied potential, pH and concentration of the electrolyte on its analytical performance were investigated. A linear range of 0.5-200 mg L⁻¹ COD and a detection limit (a signal-to-noise ratio of 3) of 0.3 mg L⁻¹ were achieved under optimized conditions. The experiments for detecting COD in model samples and real samples were carried out to evaluate the electrode's performance. The obtained results were in good agreement with those determined by the standard dichromate method, with a relative error less than 12%.     

An amperometric glucose biosensor based on layer-by-layer GOx-SWCNT conjugate/redox polymer multilayer on a screen-printed carbon electrode

An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0 mM, a high sensitivity of 32 μA mM⁻¹ cm⁻², and a response time of less than 5 s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose.     

Hybridization biosensor based on the covalent immobilization of probe DNA on chitosan-mutiwalled carbon nanotubes nanocomposite by using glutaraldehyde as an arm linker

A label-free DNA biosensor for hybridization detection of short DNA species related to the transgenic plants gene fragment of cauliflower mosaic virus (CaMV) 35S promoter was developed in this paper. The nanocomposite containing chitosan (CS) and mutiwalled carbon nanotubes (MWNTs) was first coated on a glassy carbon electrode. Then a highly reactive dialdehyde reagent of glutaraldehyde (GTD) was applied as an arm linker to covalently graft the 5′-amino modified probe DNA to the CS-MWNTs surface via the facile aldehyde-ammonia condensation reaction. The hybridization capacity of the developed biosensor was monitored with electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as an indicating probe, and the experimental results showed that the biosensor had fast hybridization rate and low background interference. A wide dynamic detection range (1.0 × 10⁻¹³-5 × 10⁻¹⁰ M) and a low detection limit (8.5 × 10⁻¹⁴ M) were achieved for the complementary sequence. In addition, the hybridization specificity experiments showed that the sensing system can accurately discriminate complementary sequence from mismatch and noncomplementary sequences.     

A dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase

Neurotoxic organophosphorus (OP) compounds are commonly used as chemical warfare agents and pesticides. Due to their high toxicity, rapid and sensitive field detection of these compounds has been an ongoing topic of interest. Biosensors made with organophosphate hydrolase enzyme (OPH) are generally designed to either amperometrically detect an electroactive leaving group produced following enzymatic cleavage, or to potenitometrically detect the pH change that occurs during cleavage. Since OPs are more likely to have phenolic leaving groups as compared to electroactive leaving groups, we have developed a new amperometric dual enzyme electrochemical assay that enables the detection of a broad class of OP compounds using the OPH enzyme combined with horseradish peroxidase (HRP). The assay has been applied to the detection of dichlofenthion, which does not have an electroactive leaving group and is not a commonly investigated OPH substrate. Using reverse phase HPLC, we have determined the Michaelis-Menten kinetic parameters of an engineered OPH enzyme to be K_M = 0.11 ± 0.02 mM and k_cat = 0.046 ± 0.003 s⁻¹ with dichlofenthion as the substrate. Detection of the phenolic leaving group from the OPH enzyme reaction using the HRP electrode is carried out at −50 mV vs. Ag/AgCl where the noise and background are low and interferences are negligible. After optimization of the solution pH, the dual enzyme biosensor was found to have a limit of detection (LOD) of 24 μM (7.6 ppm), and a sensitivity of 0.095 ± 0.024 nA/μM for dichlorofenthion. By detecting the phenolic leaving groups from the OP targets using the HRP electrode, biosensors made using this new platform have the potential to detect a broad range of important OP compounds.     

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.     

An electrochemical sensor for trace uranium determination based on 6-O-palmitoyl-l-ascorbic acid-modified graphite electrodes

The development of a preconcentrating sensor based on 6-O-palmitoyl-l-ascorbic acid (PAA)-modified graphite (GRA) electrodes for the determination of uranium is described. PAA, a water insoluble compound of ascorbic acid, was immobilized onto the surface of the GRA electrodes through physical adsorption from acetone solutions. Uranium was accumulated by heterogeneous complexation (10 min, in 0.1 M H₃BO₃, pH 4.3) and then, it was reduced by means of a differential pulse voltammetric scan in 0.1 M H₃BO₃, pH 3.4. Alternatively, the performance of both preconcentration and voltammetric steps in a single run, at 0.1 M H₃BO₃, pH 3.65, was also examined; however, in this case the observed current signals were lower by 30%. The experimental variables were investigated and under the selected conditions, a linear calibration curve in the range 2.7-67.5 μg L⁻¹ U(VI) was constructed (r² = 0.9981). The 3σ limit of detection and the relative standard deviation of the method were 1.8 μg L⁻¹ U(VI) and 8% (n = 5, 20 μg L⁻¹ U(VI), preconcentration time 10 min), respectively. By increasing the preconcentration time to 30 min, a limit of detection as low as 0.26 μg L⁻¹ U(VI) can be achieved. The effect of potential interferences was also examined. The accuracy of the method was established by recovery studies in inoculated tap and lake water samples. A simple and fast procedure based on filtering of the sample through a C-18 microcolumn was successfully used to remove the organic matter from the lake water samples.     

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu₂(C₇H₅O₂)₄(C₂H₆O)₂] (abbreviated as CuR₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR₂ presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR₂ was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10⁻¹⁰ M for complementary target DNA with a good stability.     

Flow injection determination of sialic acid based on amperometric detection

In the present paper, we continue to utilize the sequential action of sialic acid aldolase and pyruvate oxidase to construct improved sialic acid (SA) amperometric biosensors and immobilized enzyme reactors (IERs). The enzymes were co-immobilized using glutaraldehyde crosslinking at the surface of a platinum disc electrode and by covalent attachment to the modified controlled pore glass beads packed in stainless steel columns to prepare the SA biosensors and IERs, respectively. Two different configurations were adapted in the present work to produce the first report on flow-injection amperometric determination of SA. In the first configuration, the SA biosensor was used as a detector in a flow injection setup. In the second configuration, an IER was used in conjunction with a flow-through amperometric cell equipped with a Pt disc electrode polarized at 0.7 V vs Ag/AgCl for the anodic detection of the produced hydrogen peroxide. We also report a simple way to control the temperature in the flow injection setups. The FI determination of SA based on IER was found to be more superior and showed linear dependence of the FI peak heights on SA concentration in the range of 0.002-5.0 mM (r² = 0.9997), RSD of 1.1% at 250 μM SA and a sample throughput of 35 sample h⁻¹.     

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.     

Biocompatible hybrid film of β-cyclodextrin and ionic liquids: A novel platform for electrochemical biosensing

A novel electrochemical biosensing platform was developed based on the modification of biocompatible hybrid film of β-cyclodextrin (β-CD) and ionic liquids (ILs), 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF₄) onto glass carbon electrode (GCE). Then an unmediated biosensor was successfully prepared by immobilizing the horseradish peroxidase (HRP) into the β-CD/ILs composite film. β-CD could provide a biocompatible microenvironment for HRP, and ILs could accelerate the electron transfer between HRP and the electrode. Results showed that the HRP entrapped in the β-CD/ILs film could maintain its native structure and the direct electrochemistry of HRP were facilely achieved. A couple of well-defined redox peaks of HRP were observed at about −0.32 V (vs. SCE), corresponding to the protein heme Fe(III)/Fe(II) redox couples. The electrocatalysis of this biosensor to both quercetin and hydrogen peroxide was characterized. The biosensor exhibited a low operating potential (−0.05 V vs. SCE), fast amperometric response, high sensitivity, good selectivity and sub-micromolar limit of detection.     

One-pot electrodeposition of 3-aminopropyltriethoxysilane-chitosan hybrid gel film to immobilize glucose oxidase for biosensing

We report on the electrodeposition of a 3-aminopropyltriethoxysilane-chitosan (APTES-CS) hybrid gel film for in situ immobilization of glucose oxidase (GOx) on an Au or platinized Au (Pt_nano/Au) electrode for biosensing of glucose. Controllable electroreduction of p-benzoquinone is used to lift the electrode-surface pH for the GOx-APTES-CS codeposition, which was monitored by an electrochemical quartz crystal microbalance. The fabrication procedures of the biosensor and the parameters influencing the biosensing performance were optimized. The prepared porous GOx-APTES-CS/Pt_nano/Au and GOx-APTES-CS/Au electrodes can be used to detect the enzymatically generated H₂O₂ at 0.5 and 0.7 V vs SCE, respectively. The enzyme electrodes exhibited linear responses to glucose concentration from 0.2 μM to 8.2 mM (R = 0.998, at Pt_nano/Au substrate) and from 0.2 μM to 5.5 mM (R = 0.998, at Au substrate), with current sensitivities of 69.5 (Pt_nano/Au) and 65 (Au) μA mM⁻¹ cm⁻², respectively, and a detection limit of 0.2 μM (S/N = 3) was achieved for each electrode. The response time was less than 5 (Pt_nano/Au) or 8 (Au) s. It is striking that the enzyme electrodes remained their initial response sensitivity after storage for 5 (Au) and >6 (Pt_nano/Au) months in 0.10 M PBS (pH 7.0) at 4 °C.