A novel sensor based on electrochemical polymerization of diglycolic acid for determination of acetaminophen

Diglycolic acid (DA) polymer was coated on glassy carbon (GC) electrode by cyclic voltammetry (CV) technique for the first time. The electrochemical performances of the modified electrode were investigated by CV and electrochemical impedance (EIS). The obtained electrode showed an excellent electrocatalytic activity for the oxidation of acetaminophen (ACOP). A couple of well-defined reversible electrochemical redox peaks were observed on the ploy(DA)/GC electrode in ACOP solution. Compared with bare GC electrode, the oxidation peak potential of ACOP on ploy(DA)/GC electrode moved from 0.289 V to 0.220 V. Meanwhile, the oxidation peak current was much higher on the modified electrode than that on the bare GC electrode, indicating DA polymer modified electrode possessed excellent performance for the oxidation of ACOP. This kind of capability of the modified electrode can be enlisted for the highly sensitive and selective determination of ACOP. Under the optimized conditions, a wide linear range from 2 × 10(-8) to 5.0 × 10(-4)M with a correlation coefficient 0.9995 was obtained. The detection limit was 6.7 × 10(-9)M (at the ratio of signal to noise, S/N=3:1). The modified electrode also exhibited very good stability and reproducibility for the detection of ACOP. The established method was applied to the determination of ACOP in samples. An average recovery of 100.1% was achieved. These results indicated that this method was reliable for determining ACOP.     

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.     

[AuCl4]− and Fe3+/[Fe(CN)6]3− ions-derivated immunosensing interface for electrochemical immunoassay of carcinoembryonic antigen in human serum

[AuCl₄]⁻ was initially deposited by electrochemical reduction on a glassy carbon electrode (GCE) to form porous nanogold layer, then prussian blue (PB) was electrodeposited onto the as-prepared nanogold layer, and then secondary nanogold particles were fabricated again on the PB surface by electrochemical reduction for the immobilization of anti-CEA antibodies. The presence of double-layer porous gold nanoparticles enhanced the immobilized amount of biomolecules, and improved the sensitivity of the immunoassay. PB, as a good redox probe, was facile to electrochemical analysis and measurement. Under optimal conditions, the developed immunoassay exhibited dynamic range from 3.0 to 80.0 ng/mL with a detection limit of 0.9 ng/mL CEA (S/N = 3). Moreover, the selectivity, reproducibility and stability of the immunosensor were acceptable.     

Nonenzymatic electrochemical detection of glucose using well-distributed nickel nanoparticles on straight multi-walled carbon nanotubes

A nonenzymatic electrochemical sensor device was fabricated for glucose detection based on nickel nanoparticles (NiNPs)/straight multi-walled carbon nanotubes (SMWNTs) nanohybrids, which were synthesized through in situ precipitation procedure. SMWNTs can be easily dispersed in solution after mild sonication pretreatment, which facilitates the precursor of NiNPs binding to their surface and results in the homogeneous distribution of NiNPs on the surface of SMWNTs. The morphology and component of the nanohybrids were characterized by scanning electron microscopy (SEM) and X-ray powder diffraction (XRD), respectively. Cyclic voltammetry (CV) and amperometry were used to evaluate the catalytic activity of the NiNPs/SMWNTs nanohybrids modified electrode towards glucose. It was found that the nanohybrids modified electrode showed remarkably enhanced electrocatalytic activity towards the oxidation of glucose in alkaline solution compared to that of the bare glass carbon electrode (GCE), the NiNPs and the SMWNTs modified electrode, attributing to the synergistic effect of SMWNTs and Ni²⁺/Ni³⁺ redox couple. Under the optimal detection conditions, the as-prepared sensors exhibited linear behavior in the concentration range from 1 μM to 1 mM for the quantification of glucose with a limit of detection of 500 nM (3σ). Moreover, the NiNPs/SMWNTs modified electrode was also relatively insensitive to commonly interfering species such as ascorbic acid (AA), uric acid (UA), dopamine (DA), galactose (GA), and xylose (XY). The robust selectivities, sensitivities, and stabilities determined experimentally indicated the great potential of NiNPs/SMWNTs nanohybrids for construction of a variety of electrochemical sensors.     

Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolase

A label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancer marker, was developed in this work via novel electrochemical catalysis for signal amplification. The new amplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles (NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous gold nanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (II/III). Subsequently, gold nanoparticles functionalized graphene nanosheets (Au-Gra) were coated on the surface of NiHCFNPs/AuNCs film. Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sensitive immunosensor of anti-NSE/Au-Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity and large specific surface area of Au-Gra. More importantly, the oxidation peak current can be enormously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in the further improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemical immunosensor exhibited a linear range of 0.001-100 ng/mL with a detection limit of 0.3 pg/mL (S/N=3). Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSE detection, which may hold a promise for clinical diagnosis.     

Zinc oxide/redox mediator composite films-based sensor for electrochemical detection of important biomolecules

Electrochemical oxidation of serotonin (SN) onto zinc oxide (ZnO)-coated glassy carbon electrode (GCE) results in the generation of redox mediators (RMs) that are strongly adsorbed on electrode surface. The electrochemical properties of zinc oxide-electrogenerated redox mediator (ZnO/RM) (inorganic/organic) hybrid film-coated electrode has been studied using cyclic voltammetry (CV). The scanning electron microscope (SEM), atomic force microscope (AFM), and electrochemical techniques proved the immobilization of ZnO/RM core/shell microparticles on the electrode surface. The GCE modified with ZnO/RM hybrid film showed two reversible redox peaks in acidic solution, and the redox peaks were found to be pH dependent with slopes of −62 and −60 mV/pH, which are very close to the Nernst behavior. The GCE/ZnO/RM-modified electrode exhibited excellent electrocatalytic activity toward the oxidations of ascorbic acid (AA), dopamine (DA), and uric acid (UA) in 0.1 M phosphate buffer solution (PBS, pH 7.0). Indeed, ZnO/RM-coated GCE separated the anodic oxidation waves of DA, AA, and UA with well-defined peak separations in their mixture solution. Consequently, the GCE/ZnO/RMs were used for simultaneous detection of DA, AA, and UA in their mixture solution. Using CV, calibration curves for DA, AA, and UA were obtained over the range of 6.0 × 10⁻⁶ to 9.6 × 10⁻⁴ M, 1.5 × 10⁻⁵ to 2.4 × 10⁻⁴ M, and 5.0 × 10⁻⁵ to 8 × 10⁻⁴ M with correlation coefficients of 0.992, 0.991, and 0.989, respectively. Moreover, ZnO/RM-modified GCE had good stability and antifouling properties.     

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA(21)) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADH measured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50 nM of AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.     

A sensitive amperometric immunosensor for carcinoembryonic antigen detection with porous nanogold film and nano-Au/chitosan composite as immobilization matrix

A sensitive amperometric immunosensor for carcinoembryonic antigen (CEA) was prepared. Firstly, a porous nano-structure gold (NG) film was formed on glassy carbon electrode (GCE) by electrochemical reduction of HAuCl₄ solution, then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, and finally the anti-CEA was adsorbed onto the surface of the bilayer gold nanoparticles to construct an anti-CEA/nano-Au/Chit/NG/GCE immunosensor. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry (CV). The gold colloid, chitosan and nano-Au/Chit were characterized by transmission electron microscopy and UV–vis spectroscopy. In addition, the performances of the immunosensor were studied in detail. The resulting immunosensor offers a high-sensitivity (1310 nA/ng/ml) for the detection of CEA and has good correlation for detection of CEA in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.06 ng/ml estimated at a signal-to-noise ratio of 3. The proposed method can detect the CEA through one-step immunoassay and would be valuable for clinical immunoassay.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.     

Highly selective and sensitive determination of dopamine using a Nafion/carbon nanotubes coated poly(3-methylthiophene) modified electrode

A poly(3-methylthiophene) modified glassy carbon electrode coated with Nafion/single-walled carbon nanotubes film was fabricated and used for highly selective and sensitive determination of dopamine. The hybrid film surface of the modified electrode was characterized by scanning electrochemical microscopy (SECM) and the results indicated that the carbon nanotubes were dispersed uniformly on the conductive polymer. The experimental results suggest that the hybrid film modified electrode combining the advantages of poly(3-methylthiophene), carbon nanotubes with Nafion exhibits dramatic electrocatalytic effect on the oxidation of dopamine (DA) and results in a marked enhancement of the current response. In 0.1M phosphate buffer solution (PBS) of pH 7.0, the differential pulse voltammetric (DPV) peak heights are linear with DA concentration in three intervals, viz. 0.020-0.10 microM, 0.10-1.0 microM and 1.0-6.0 microM, with correlation coefficients of 0.9993, 0.9996 and 0.9993, respectively. The detection limit of 5.0 nM DA could be estimated (S/N=3). Moreover, the interferences of ascorbic acid (AA) and uric acid (UC) are effectively diminished. This hybrid film modified electrode can be applied to the determination of DA contents in dopamine hydrochloride injection and human serum. These attractive features provide a potential application for either in vitro measurement of DA in the presence of excess AA and UA or as detectors in flow injection analysis (FIA) and high performance liquid chromatography (HPLC).     

Carbon nanotubes-polymer-redox mediator hybrid thin film for electrocatalytic sensing

Electrocatalytic sensing of NADH using a hybrid thin film derived from multi-wall carbon nanotubes (CNTs), Nafion (Nf) polymer and electrogenerated redox mediator is described. The redox mediator was electrochemically generated by the oxidation of serotonin on the hybrid thin film modified glassy carbon electrode (GC/Nf-CNT). Controlled potential electrolysis of serotonin at 0.1 V in neutral solution results in the generation of the redox mediator 5,5'-dihydroxy-4,4'-bitryptamine (DHB) on the hybrid thin film. The electrogenerated DHB has redox active quinone-imine structure and was electrochemically characterized by studying the pH dependent redox response. DHB on the hybrid thin film exhibits reversible redox peak at -0.05 V and the formal potential shifts by -55 mV while increasing the solution pH by 1 unit. The quinone-imine structure of DHB efficiently catalyzes the oxidation of NADH with a decrease in the overpotential of about 500 mV compared to the unmodified electrode. The CNTs of the hybrid thin film facilitates the mediated electrocatalytic oxidation of NADH. The hybrid thin film modified electrode exhibits stable amperometric response and it linearly responds to NADH (0.5-400 microM). This hybrid thin film modified electrode could detect NADH as low as 0.1 microM at -0.05 V with a sensitivity of 11.1 nA/microM in physiological pH.     

A facile strategy for nonenzymatic glucose detection

Glassy carbon electrode modified with boron oxide nanoparticles supported on multiwall carbon nanotubes was obtained via a facile approach. The as-prepared modified electrode exhibits excellent electrocatalytic activity toward the redox of glucose in pH 7.0 phosphate buffer solution. The electrochemical response of the modified electrode to glucose shows a linear range of 1.5-260 μM with a correlation coefficient of 0.9986 and the calculated detection limit is 0.8 μM at a signal-to-noise ratio of 3, which makes it useful for developing the electrochemical determination of glucose concentrations without using glucose oxidase at physiological pH.     

Electrochemical sensor based on nitrogen doped graphene: simultaneous determination of ascorbic acid, dopamine and uric acid

Nitrogen doped graphene (NG) was prepared by thermally annealing graphite oxide and melamine mixture. After characterization by atomic force microscopy and X-ray photoelectron spectroscopy etc., the electrochemical sensor based on NG was constructed to simultaneously determine small biomolecules such as ascorbic acid (AA), dopamine (DA) and uric acid (UA). Due to its unique structure and properties originating from nitrogen doping, NG shows highly electrocatalytic activity towards the oxidation of AA, DA and UA. The electrochemical sensor shows a wide linear response for AA, DA and UA in the concentration range of 5.0×10(-6) to 1.3×10(-3)M, 5.0×10(-7) to 1.7×10(-4)M and 1.0×10(-7) to 2.0×10(-5)M with detection limit of 2.2×10(-6)M, 2.5×10(-7)M and 4.5×10(-8)M at S/N=3, respectively. These results demonstrate that NG is a promising candidate of advanced electrode material in electrochemical sensing and other electrocatalytic applications.     

Simultaneous determination of catecholamines, uric acid and ascorbic acid at physiological levels using poly(N-methylpyrrole)/Pd-nanoclusters sensor

An interesting electrochemical sensor has been constructed by the electrodeposition of palladium nanoclusters (Pd_nano) on poly(N-methylpyrrole) (PMPy) film-coated platinum (Pt) electrode. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and scanning electron microscopy were used to characterize the properties of the modified electrode. It was demonstrated that the electroactivity of the modified electrode depends strongly on the electrosynthesis conditions of the PMPy film and Pd_nano. Moreover, the modified electrode exhibits strong electrocatalytic activity toward the oxidation of a mixture of dopamine (DA), ascorbic acid (AA), and uric acid (UA) with obvious reduction of overpotentials. The simultaneous analysis of this mixture at conventional (Pt, gold [Au], and glassy carbon) electrodes usually struggles. However, three well-resolved oxidation peaks for AA, DA, and UA with large peak separations allow this modified electrode to individually or simultaneously analyze AA, DA, and UA by using differential pulse voltammetry (DPV) with good stability, sensitivity, and selectivity. This sensor is also ideal for the simultaneous analysis of AA, UA and either of epinephrine (E), norepinephrine (NE) or l-DOPA. Additionally, the sensor shows strong electrocatalytic activity towards acetaminophen (ACOP) and other organic compounds. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.05 to 1 mM, 0.1 to 10 μM, and 0.5 to 20 μM, respectively. The detection limits (signal/noise [S/N] = 3) were 7 μM, 12 nM, and 27 nM for AA, DA, and UA, respectively. The practical application of the modified electrode was demonstrated by measuring the concentrations of AA, DA, and UA in injection sample, human serum, and human urine samples, respectively, with satisfactory results. The reliability and stability of the modified electrode gave a good possibility for applying the technique to routine analysis of AA, DA, and UA in clinical tests.     

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.     

A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.     

Voltammetric detection of trimethylamine using immobilized trimethylamine dehydrogenase on an electrodeposited goldnanoparticle electrode

A voltammetric enzyme electrode was developed based on nicotinamide-independent trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), which catalyses the oxidation of trimethylamine (TMA) to dimethylamine and formaldehyde. A quaternized osmium hydrogel polymer, poly(vinylimidazole-[Os(4,4′-dimethyl-2,2′-bipyridine)₂Cl]^+/2+) with ethylamine (PVI-Os-EA), was prepared as a potential redox mediator in an electrochemical biosensor. TMA was detected using TMADH that was co-immobilized with an osmium hydrogel polymer on electrodeposited gold nanoparticles (Au-NPs) on screen-printed carbon electrodes (SPCEs). The Au-NPs deposited onto SPCEs provided about a three times higher electrochemical response compared to that of a planar gold electrode. As TMA was catalyzed by wired TMADH, the electrical signal was monitored at 0.3 V versus Ag/AgCl by cyclic voltammetry and chronoamperometry. The anode currents increased linearly in proportion to the TMA concentration over the 0 ∼ 2.5 mM range with a detection limit of 1 μM (R = 0.9972).     

Fabrication of a glucose sensor based on a novel nanocomposite electrode

The direct electrocatalytic oxidation of glucose in alkaline medium at nanoscale nickel hydroxide modified carbon ionic liquid electrode (CILE) has been investigated. Enzyme free electro-oxidation of glucose have greatly been enhanced at nanoscale Ni(OH)(2) as a result of electrocatalytic effect of Ni(+2)/Ni(+3) redox couple. The sensitivity to glucose was evaluated as 202 microA mM(-1)cm(-2). From 50 microM to 23 mM of glucose can be selectively measured using platelet-like Ni(OH)(2) nanoscale modified CILE with a detection limit of 6 microM (S/N=3). The nanoscale nickel hydroxide modified electrode is relatively insensitive to electroactive interfering species such as ascorbic acid (AA), and uric acid (UA) which are commonly found in blood samples. Long-term stability, high sensitivity and selectivity as well as good reproducibility and high resistivity towards electrode fouling resulted in an ideal inexpensive amperometric glucose biosensor applicable for complex matrices.     

Catechol sensor using poly(aniline-co-o-aminophenol) as an electron transfer mediator

In this work, poly(aniline-co-o-aminophenol) (copolymer) was used as an electron transfer mediator in the electrochemical oxidation of catechol due to its reversible redox over a wide range of pH. The experimental results indicate that the anodic peak potential of catechol at the copolymer electrode is lower than that at the platinum electrode in a solution consisting of catechol and sodium sulfate with pH 5.0, and the activation energy for the electrochemical oxidation of catechol at the copolymer electrode is low (23.6 kJ mol(-1)). These are strong evidence for the electrocatalytic oxidation of catechol at the copolymer electrode. The -OH group on the copolymer chain plays an important role in the electron transfer between the copolymer electrode and catechol in the solution. Based on the catalytic oxidation, the copolymer is used as a sensor to determine the concentration of catechol. The response current of the sensor depends on the concentration of catechol, pH, applied potential and temperature. At 0.55 V (versus saturated calomel reference electrode (SCE)) and pH 5.0, the sensor has a fast response (about 10s) to catechol and good operational stability. The sensor shows a linear response range between 5 and 80 microM catechol with a correlation coefficient of 0.997. It was found that phenol and resorcinol cannot be oxidized at the copolymer electrode at potentials < or =0.55 V, so controlling the sensor potential affords a good way of avoiding the effect of phenol and resorcinol on the determination of catechol.     

Sensitive immunoassay of human chorionic gonadotrophin based on multi-walled carbon nanotube–chitosan matrix

A novel amperometric immunosensor for human chorionic gonadotropin (HCG) assay has been fabricated through incorporating toluidine blue (TB) and hemoglobin (Hb) on the multiwall carbon nanotube (MWNT)-chitosan (CS) modified glassy carbon electrode, followed by electrostatic adsorption of a conducting gold nanoparticles (nanogold) film as sensing interface. The MWNT-CS matrix provided a congenial microenvironment for the immobilization of biomolecules and promoted the electron transfer to enhance the sensitivity of the immunosensor. Due to the strong electrocatalytic properties of Hb and MWNT toward H(2)O(2), the Hb and MWNT significantly amplified the current signal of the antigen-antibody reaction. The immobilized toluidine blue as an electron transfer mediator exhibited excellent electrochemical redox property. After the immunosensor was incubated with HCG solution, the access of activity center of the Hb to toluidine blue was partly inhibited, which leaded to a linear decrease in the catalytic efficiency of the Hb to the oxidation of immobilized toluidine blue by H(2)O(2) over HCG concentration ranges from 0.8 to 500 mIU/mL. Under optimal condition, the detection limit for the HCG immunoassay was 0.3 mIU/mL estimated at a signal-to-noise ratio of 3. Moreover, the proposed immunosensor displayed a satisfactory stability and reproducibility.