Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Disposable superoxide anion biosensor based on superoxide dismutase entrapped in silica sol–gel matrix at gold nanoparticles modified ITO electrode

A novel disposable biosensor based on direct electron transfer of superoxide dismutase (SOD) was fabricated for the determination of superoxide anion. The biosensor was constructed by electrodeposition of gold nanoparticles (GNPs) on the indium tin oxide (ITO) electrode and then immobilization of SOD in silica sol–gel (SG) network in the presence of cysteine on GNPs/ITO modified electrode surface. The distribution of GNPs on ITO electrode surface was examined by scanning electron microscopy (SEM). The immobilized SOD exhibited high catalytical activity towards superoxide anion. Parameters affecting the performance of the biosensor were also investigated. A linear calibration curve was obtained over the range from 0.08 to 0.64 μM with a correlation coefficient of 0.9937. The resulted biosensors were demonstrated to possess striking analytical properties for superoxide anion determination, such as high sensitivity, good accuracy, and long-term stability. It provides a promising platform for the fabrication of disposable biosensors.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Reductive determination of hydrogen peroxide with MWCNTs-Pd nanoparticles on a modified glassy carbon electrode

This paper introduces the use of multi walled carbon nanotubes (MWCNTs) with palladium (Pd) nanoparticles in the electrocatalytic reduction of hydrogen peroxide (H(2)O(2)). We have developed and characterized a biosensor for H(2)O(2) based on Nafion(?) coated MWCNTs-Pd nanoparticles on a glassy carbon electrode (GCE). The Nafion(?)/MWCNTs-Pd/GCE electrode was easily prepared in a rapid and simple procedure, and its application improves sensitive determination of H(2)O(2). Characterization of the MWCNTs-Pd nanoparticle film was performed with transmission electron microscopy (TEM), Raman, and X-ray photoelectron spectroscopy (XPS). Cyclic voltammetry (CV) and amperometry (at an applied potential of -0.2V) measurements were used to study and optimize performance of the resulting peroxide biosensor. The proposed H(2)O(2) biosensor exhibited a wide linear range from 1.0 μM to 10 mM and a low detection limit of 0.3 μM (S/N=3), with a fast response time within 10s. Therefore, this biosensor could be a good candidate for H(2)O(2) analysis.     

Design and development of a highly stable hydrogen peroxide biosensor on screen printed carbon electrode based on horseradish peroxidase bound with gold nanoparticles in the matrix of chitosan

The design and development of a screen printed carbon electrode (SPCE) on a polyvinyl chloride substrate as a disposable sensor is described. Six configurations were designed on silk screen frames. The SPCEs were printed with four inks: silver ink as the conducting track, carbon ink as the working and counter electrodes, silver/silver chloride ink as the reference electrode and insulating ink as the insulator layer. Selection of the best configuration was done by comparing slopes from the calibration plots generated by the cyclic voltammograms at 10, 20 and 30 mM K(3)Fe(CN)(6) for each configuration. The electrodes with similar configurations gave similar slopes. The 5th configuration was the best electrode that gave the highest slope. Modifying the best SPCE configuration for use as a biosensor, horseradish peroxidase (HRP) was selected as a biomaterial bound with gold nanoparticles (AuNP) in the matrix of chitosan (HRP/AuNP/CHIT). Biosensors of HRP/SPCE, HRP/CHIT/SPCE and HRP/AuNP/CHIT/SPCE were used in the amperometric detection of H(2)O(2) in a solution of 0.1M citrate buffer, pH 6.5, by applying a potential of -0.4V at the working electrode. All the biosensors showed an immediate response to H(2)O(2). The effect of HRP/AuNP incorporated with CHIT (HRP/AuNP/CHIT/SPCE) yielded the highest performance. The amperometric response of HRP/AuNP/CHIT/SPCE retained over 95% of the initial current of the 1st day up to 30 days of storage at 4 degrees C. The biosensor showed a linear range of 0.01-11.3mM H(2)O(2), with a detection limit of 0.65 microM H(2)O(2) (S/N=3). The low detection limit, long storage life and wide linear range of this biosensor make it advantageous in many applications, including bioreactors and biosensors.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

An electrochemical biosensor for the detection of tyrosine oxidation induced by Fenton reaction

A simple and sensitive electrochemical biosensor was used to detect tyrosine oxidation induced by hydroxyl radicals generated by Fenton reaction (Fe(2+)/H(2)O(2)). Poly(glu, tyr) (4:1) peptides were immobilized on indium tin oxide (ITO) electrode surface via layer-by-layer assembly technique, and Os(bpy)(3)(2+)-mediated tyrosine oxidation current was employed as the signal reporter of the biosensor. It was found that the electrochemical signal of the peptide decreased markedly after incubation with Fenton reagents. Interestingly, L-dopa, the oxidation product of tyrosine, was likely to form complexes with Fe(III), which could suppress the electro-oxidation of L-dopa and resulted in decrease of current response. Our results indicate that the peptide damage involved two steps and was a second-order reaction. X-ray photoelectron spectroscopy was used to quantitatively determine nitrogen elemental percentage on peptide-coated electrode surface, which eliminated the possibility that signal decrease was caused by peptide backbone cleavage. Moreover, the lowest concentration of Fenton reagents that could be detected was 10 μM Fe(2+) or H(2)O(2), similar to the level in vivo. We suggest that the biosensor can be used to detect protein damage induced by Fenton reaction.     

A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film

A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05mM and a detection limit of 0.5muM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode.     

A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode

A screen-printed three-electrode system is fabricated to prepare a novel disposable screen-printed immunosensor for rapid determination of alpha-1-fetoprotein (AFP) in human serum. The immunosensor is prepared by entrapping horseradish peroxidase (HRP)-labeled AFP antibody in chitosan membrane to modify the screen-printed carbon electrode. The membrane is characterized with scanning electron microscope and electrochemical methods. After the immunosensor is incubated with AFP at 30 degrees C for 35 min, the access of the active center of HRP catalyzing the oxidation reaction of thionine by H(2)O(2) is partly inhibited. In presence of 1.2 mM thionine and 6 mM H(2)O(2), the electrocatalytic current decreases linearly in two concentration ranges of AFP from 0 to 20 and from 20 to 150 ng/mL with a detection limit of 0.74 ng/mL. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6 and 4.2% at 10 and 100 ng/mL, respectively. The storage stability is acceptable in pH 7.0 phosphate buffer solution at 4 degrees C for more than 10 days. The proposed method can detect the AFP through one-step immunoassay and would be valuable for clinical immunoassay.     

Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer

A disposable amperometric biosensor for the measurement of ethanol has been developed. It comprises a screen-printed carbon electrode doped with 5% cobalt phthalocyanine (CoPC-SPCE), and coated with alcohol oxidase; a permselective membrane on the surface acts as a barrier to interferents. The measurement of ethanol is based on the signal produced by H2O2, the product of the enzymatic reaction. Optimisation studies were performed using amperometry in stirred solution and the magnitude of the signal was found to be dependent on pH, enzyme loading, type of membrane and applied potential. The same technique was used to evaluate the biosensor for the determination of ethanol in samples. The results obtained compared well with the manufacturers specifications. In order to test the possibility of using the devices in the field, chronoamperometry was also used to determine ethanol in the same beer samples. The precision and recovery data again indicated that the biosensor should give reliable results under the conditions described.     

A novel hydrogen peroxide biosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) film

The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface. Hemoglobin (Hb) has been successfully immobilized on the 3DOM GTD film and the fabrication process was characterized by Raman and UV-vis spectra. The results indicated that the Hb immobilized on the film retained its biological activity and the secondary structure of Hb was not destroyed. The direct electrochemistry and electrocatalysis of Hb immobilized on this film have been investigated. The Hb/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the Hb intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.20 and -0.48 V in 0.1M PBS (pH7.0), respectively. The Hb/3DOM GTD/ITO electrode exhibits an excellent eletrocatalytic activity, a wide linear range for H(2)O(2) from 5.0 μM to 1.0mM with a limit of detection of 0.6μM, high sensitivity (144.5 μA mM(-1)), good stability and reproducibility. Compared with the TiO(2) nanoneedles modified electrode, the GTD modified electrode has higher sensitivity and response peak current. The 3DOM GTD provided a good matrix for bioactive molecules immobilization, suggesting it has the potential use in the fields of H(2)O(2) biosensors.     

TiO(2) nanocrystals electrochemiluminescence quenching by biological enlarged nanogold particles and its application for biosensing

Electrochemiluminescence (ECL) of TiO(2) nanocrystals with different crystal styles modified fluorine-doped tin oxide (FTO) electrode was investigated in H(2)O(2) solution. The amorphous TiO(2) nanospheres were facilely synthesized by the hydrothermal and condensation method. Crystal TiO(2), namely anatase and rutile, were prepared by calcination of the amorphous TiO(2) nanospheres at 450 and 800°C, respectively. The transmission electron microscope (TEM) and electron diffraction pattern were used to characterize the obtained TiO(2) nanoparticles morphology and the corresponding crystal styles. The electrochemical and ECL behaviors were investigated by cyclic voltammetry. The ECL quenching was observed by introduction of gold nanoparticles. Based on the quenching effect, a sensitive glucose ECL biosensor as a model was fabricated by in-situ growing-up gold seeds in AuCl(4)(-) solution induced by biologically generated H(2)O(2). The linear range to detect glucose is from 5.0×10(-7)M to 4.0×10(-3)M with the limit of detection of 2.5×10(-7)M.     

A mimic peroxidase biosensor based on calcined layered double hydroxide for detection of H2O2

An enzymeless biosensor was explored from Cu-Mg-Al calcined layered double hydroxide (CLDH) modified electrode in this study. The Cu-Mg-Al CLDH greatly promotes the electron transfer between H(2)O(2) and GCE, and it is exemplified toward the non-enzymatic sensing of H(2)O(2). The results indicate that the Cu-Mg-Al CLDH exhibits excellent electrocatalytic property, high sensitivity, good reproducibility, long-term stability, and fast amperometric response toward reduction of H(2)O(2), thus is promising for the future development of man-made mimics of enzyme in H(2)O(2) sensors. This work opens a way to utilize simply Cu-Mg-Al CLDH as an electron mediator to fabricate an efficient H(2)O(2) biosensor, which exhibits great potential applications in varieties of simple, robust, and easy-to-make analytical approaches in the future.     

A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film

The direct electron transfer of immobilized horseradish peroxidase (HRP) on silica-hydroxyapatite (HAp) hybrid film-modified glassy carbon electrode (GCE) and its application as H(2)O(2) biosensors were investigated. On silica/HRP-HAp/GCE, HRP displayed a fast electron transfer process accompanied with one proton participate in. This sensor exhibited an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The proposed biosensor showed good reproducibility and high sensitivity to H(2)O(2) with the detection limit of 0.35 microM. In the range of 1.0-100 microM, the catalytic reduction current of H(2)O(2) was proportional to H(2)O(2) concentration. The apparent Michaelis-Menten constant (k(m)(app)) of the biosensor was calculated to be 21.8 microM, exhibiting a high enzymatic activity and affinity for H(2)O(2).     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Direct electrochemistry of horseradish peroxidase bonded on a conducting polymer modified glassy carbon electrode

Direct electron transfer process of immobilized horseradish peroxidase (HRP) on a conducting polymer film, and its application as a biosensor for H2O2, were investigated by using electrochemical methods. The HRP was immobilized by covalent bonding between amino group of the HRP and carboxylic acid group of 5,2':5',2"-terthiophene-3'-carboxylic acid polymer (TCAP) which is present on a glassy carbon (GC). A pair of redox peaks attributed to the direct redox process of HRP immobilized on the biosensor electrode were observed at the HRPmid R:TCAPmid R:GC electrode in a 10 mM phosphate buffer solution (pH 7.4). The surface coverage of the HRP immobilized on TCAPmid R:GC was about 1.2 x 10(-12) mol cm(-2) and the electron transfer rate (ks) was determined to be 1.03 s(-1). The HRPmid R:TCAPmid R:GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the reduction of H2O2 without the aid of an electron transfer mediator. The calibration range of H2O2 was determined from 0.3-1.5 mM with a good linear relation.     

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.     

One-step construction of biosensor based on chitosan-ionic liquid-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of hydrogen peroxide (H(2)O(2)) biosensors by in situ formation of chitosan-ionic liquid-horseradish peroxidase (CS-IL-HRP) biocomposite film on electrode surface. A highly porous surface with orderly three-dimensional network was revealed by scanning electron microscopy (SEM) investigation. The biocomposite provided improved conductivity and biocompatible microenvironment. The developed biosensor exhibited a fast amperometric response for the determination of H(2)O(2) and 95% of the steady-state current was obtained within 2s. The linear response of the developed biosensor for the determination of H(2)O(2) ranged from 6.0x10(-7) to 1.6x10(-4)M with a detection limit of 1.5x10(-7)M. Performance of the biosensor was evaluated with respect to possible interferences and a good selectivity was revealed. The fabricated biosensor exhibited high reproducibility and long-time storage stability. The ease of the one-step non-manual technique and the promising feature of biocomposite could serve as a versatile platform for the fabrication of electrochemical biosensors.     

Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite

A novel amperometric biosensor for hydrogen peroxide (H(2)O(2)) was developed by entrapping horseradish peroxidase (HRP) in a new ormosil composite doped with ferrocene monocarboxylic acid-bovine serum albumin conjugate and multiwall carbon nanotubes (MWNTs). The ormosil was prepared using 3-(aminopropyl)triethoxysilane and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers. The encapsulated conjugate showed excellent electrochemistry and acted as an electron transfer mediator. The presence of MWNTs improved the conductivity of the composite film. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H(2)O(2). The proposed H(2)O(2) biosensor exhibited a linear range of 0.02-4.0 mM with a detection limit of 5.0 microM (S/N = 3) and a K(M)(app) value of 2.0 mM. It could be used for flow injection analysis of hydrogen peroxide with a liner range from 0.02 to 4.5 mM, sensitivity of 0.042 microA/mM and analytical time of 20 s per sample. This biosensor possessed good analytical performance and storage stability.