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.     

A new film for the fabrication of an unmediated H2O2 biosensor

A novel and stable film made from polyethylene glycol (PEG) on pyrolytic graphite (PG) electrode was presented in this paper for incorporating horseradish peroxidase (HRP) to study the direct electrochemistry of the enzyme. In PEG film, HRP showed a thin-layer electrochemistry behavior. The apparent standard potential (E degrees ') was -0.379 V versus SCE at pH 7.2. Moreover, the PEG-HRP modified electrode exhibited excellent electrocatalytical response to the reduction of H2O2 with a calibration range between 2.0 x 10(-6) and 6.0 x 10(-4) M and a good linear relation from 2.0 x 10(-6) to 1.0 x 10(-4) M, on which an unmediated H2O2 biosensor was based. The detection limit of 6.7 x 10(-7) M was estimated when the signal-to-noise ratio was 3. The relative standard deviation (R.S.D.) was 4.7% for six successive determinations at a concentration of 4.0 x 10(-5) M. The apparent Michaelis-Menten constant (Km app) of the sensor was found to be 1.38 mM. Epinephrine, dopamine, and ascorbic acid did not interfere with the sensitive determination of H2O2.     

Glucose biosensor based on Au nanoparticles-conductive polyaniline nanocomposite

In this paper, we report a novel glucose biosensor based on composite of Au nanoparticles (NPs)-conductive polyaniline (PANI) nanofibers. Immobilized with glucose oxidase (GOx) and Nafion on the surface of nanocomposite, a sensitive and selective biosensor for glucose was successfully developed by electrochemical oxidation of H2O2. The glucose biosensor shows a linear calibration curve over the range from 1.0x10(-6) to 8.0x10(-4) mol/L, with a slope and detection limit (S/N=3) of 2.3 mA/M and 5.0x10(-7) M, respectively. In addition, the glucose biosensor system indicates excellent reproducibility (less than 5% R.S.D.) and good operational stability (over 2 weeks).     

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.     

Direct electrochemistry of horseradish peroxidase based on biocompatible carboxymethyl chitosan-gold nanoparticle nanocomposite

The nanocomposite composed of carboxymethyl chitosan (CMCS) and gold nanoparticles was successfully prepared by a novel and in situ process. It was characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectrophotometer (FTIR). The nanocomposite was hydrophilic even in neutral solutions, stable and inherited the properties of the AuNPs and CMCS, which make it biocompatible for enzymes immobilization. HRP, as a model enzyme, was immobilized on the silica sol-gel matrix containing the nanocomposite to construct a novel H(2)O(2) biosensor. The direct electron transfer of HRP was achieved and investigated. The biosensor exhibited a fast amperometric response (5s), a good linear response over a wide range of concentrations from 5.0 x 10(-6) to 1.4 x 10(-3)M, and a low detection limit of 4.01 x 10(-7)M. The apparent Michaelis-Menten constant (K(M)(app)) for the biosensor was 5.7 x 10(-4)M. Good stability and sensitivity were assessed for the biosensor.     

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H(2)O(2) was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H(2)O(2) without electron mediator or promoter, at a potential of +0.10 V versus Agmid R:AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N=3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.     

HRP/[Zn-Cr-ABTS] redox clay-based biosensor: design and optimization for cyanide detection

A novel inexpensive and simple amperometric biosensor, based on the immobilization of HRP into redox active [Zn-Cr-ABTS] layered double hydroxide, is applied to the determination of cyanide. The electrochemical transduction step corresponds to the reduction at 0.0 V of ABTS+* enzymatically formed in the presence of H2O2. The biosensor has a fast response to H2O2 (8s) with a linear range of 1.7 x 10(-9) to 2.1 x 10(-6) M and a sensitivity of 875 mA M(-1) cm(-2). The apparent Michaelis-Menten constant (KMapp) is 12 microM. The detection of cyanide is performed via its non competitive inhibiting action on the HRP/[Zn-Cr-ABTS] electrode. The concentration range of the linear response and the apparent inhibition constant (ki) are 5 x 10(-9) to 4 x 10(-8) and 1.4 x 10 (-7) M, respectively.     

Nanosized flower-like ZnO synthesized by a simple hydrothermal method and applied as matrix for horseradish peroxidase immobilization for electro-biosensing

Nanosized flower-like ZnO was synthesized by a simple hydrothermal method which is a convenient, environment friendly, inexpensive and efficient process. Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscope (SEM) were used to confirm the material structure and the crystallite microstructure. Then ZnO was dispersed in the chitosan solution to form a ZnO/chitosan composite matrix for the fabrication of H2O2 biosensor. This composite combined the advantages of inorganic species (ZnO) and organic polymer (chitosan). The parameters affecting the fabrication and experimental conditions of biosensors were optimized. Using hydroquinone as the mediator, the biosensor showed a fast response of less than 5s with the linear range of 1.0x10(-5) to 1.8x10(-3) M H2O2 with a correlation coefficient of 0.995 (n=20). The detection limit of the sensor was found to be 2.0 microM, based on a signal-to-noise ratio of 3. The biosensor exhibited satisfactory reproducibility and stability and retained about 78% of its original response after 40 days storage in a phosphate buffer at 4 degrees C.     

Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)M(-1)s(-1), for azurin coupled to gold by 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.     

Electrospun polystyrene-poly(styrene-co-maleic anhydride) nanofiber as a new aptasensor platform

Here we report on a new approach for the electrochemical detection of hydrogen peroxide (H(2)O(2)) based on the co-immobilization of horseradish peroxidase and methylene blue on the functionalized carbon buckypaper supported by a titanium substrate. Cyclic voltammetry was used to study and optimize the performance of the resulting electrochemical biosensor. The proposed biosensor exhibited high analytical performance towards the quantification of H(2)O(2) at the physiological pH 7.4. Under optimized conditions, the biosensor shows a wide linear response range from 0.1 × 10(-6) to 5 × 10(-4)M concentrations of H(2)O(2). The detection limit was determined to be 7.5 × 10(-8)M (based on S/N=3). Reproducibility and stability of the fabricated biosensor were examined with satisfactory results. The biological relevance of the developed electrochemical biosensor has been further studied by the determination of H(2)O(2) in human urine samples of normal volunteers prior to and following the ingestion of coffee. Increased levels of urinary H(2)O(2) concentration suggest that oxidative stress is induced by coffee drinking in humans. There is considerable interest in oxidative stress as relates to human physiology. The sensitive determination of H(2)O(2) in human urine may serve as a valuable biomarker to effectively elucidate specific levels of oxidative stress in vivo.     

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.     

Enhanced resonance light scattering based on biocatalytic growth of gold nanoparticles for biosensors design

The biocatalytic growth of gold nanoparticles (Au-NPs) has been employed in the design of new optical biosensors based on the enhanced resonance light scattering (RLS) signals. Both absorption spectroscopy and transmission electron microscopy (TEM) analysis revealed Au-NP seeds could be effectively enlarged upon the reaction with H(2)O(2), an important metabolite that could be generated by many biocatalytic reactions. Upon the stepwise enlargement of Au-NPs, the light scattering intensity could be greatly enhanced, which then allowed the quantitative detection of the analyte, H(2)O(2). Further combination of the biocatalytic reaction that can yield H(2)O(2) by using the enzyme, glucose oxidase, with the enlargement of Au-NPs enabled the design of a sensitive glucose biosensor using the RLS technique. In the present study, we could achieve the detection of glucose in a linear range of 1.0 x 10(-6) M to 1.1 x 10(-4) M, with detection limit of 6.8 x 10(-7) M.     

A novel hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto Au-modified titanium dioxide nanotube arrays

In this study, we report on a promising H(2)O(2) biosensor based on the co-immobilization of horseradish peroxidase (HRP) and chitosan onto Au-modified TiO(2) nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then uniformly coated onto the TiO(2) nanotube arrays by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO(2) nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and chronoamperometry were used to study and to optimize the performance of the resulting electrochemical biosensor. The effect of pH, applied electrode potential, the presence of the electron-mediator methylene blue, and the anodic oxidation time of the Ti substrate on the electrochemical biosensor has been systemically studied. Our electrochemical measurements show that the Au-modified TiO(2) nanotube arrays provide excellent matrices for the immobilization of HRP and that the optimized electrochemical biosensor exhibits long linearity, a low detection limit, high stability and very good reproducibility for the detection of H(2)O(2). Under the optimized conditions the linearity of the developed biosensor for the detection of H(2)O(2) is observed from 5 x 10(-6) to 4 x 10(-4) moll(-1) with a detection limit of 2 x 10(-6) moll(-1) (based on the S/N=3).     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

A reagentless optical biosensor based on the intrinsic absorption properties of peroxidase

During the reversible reaction between peroxidase (HRP) and H(2)O(2), several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for H(2)O(2) determination; when H(2)O(2) is generated in a previous enzymatic reaction, the substrate involved in this reaction can also be determined. On this basis, a new family of fully reversible reagentless optical biosensors containing HRP is presented; glucose determination is used as a model. The biosensor (which can be used for at least 6 months and/or more than 750 measurements) is prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the substrate concentration is also presented together with a simple methodology for characterization of this kind of biosensor. Regarding the optical model, the Kubelka-Mulk theory of reflectance does not give good results and the biosensors are better described by the Rayleigh theory of polymer solutions. Under working conditions, linear response ranges from 1.5x10(-6) to 3.0x10(-4)M glucose and CV was about 4%. This biosensor has been applied for glucose determination in fruit juices and synthetic serum samples without sample pretreatment.     

Direct electron transfer and enzymatic activity of hemoglobin in a hexagonal mesoporous silica matrix

The direct electrochemistry of hemoglobin (Hb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Hb and the HMS was investigated using UV-Vis spectroscopy, FT-IR, and electrochemical methods. The direct electron transfer of the immobilized Hb exhibited two couples of redox peaks with the formal potentials of -0.037 and -0.232 V in 0.1 M (pH 7.0) PBS, respectively, which corresponded to its two immobilized states. The electrode reactions showed a surface-controlled process with a single proton transfer at the scan rate range from 20 to 200 mV/s. The immobilized Hb retained its biological activity well and displayed an excellent response to the reduction of both hydrogen peroxide (H2O2) and nitrate (NO2-). Its apparent Michaelis-Menten constants for H2O2 and NO2- were 12.3 and 49.3 microM, respectively, showing a good affinity. Based on the immobilization of Hb on the HMS and its direct electrochemistry, two novel biosensors for H2O2 and NO2- were presented. Under optimal conditions, the sensors could be used for the determination of H2O2 ranging from 0.4 to 6.0 microM and NO2- ranging from 0.2 to 3.8 microM. The detection limits were 1.86 x 10(-9) M and 6.11 x 10(-7) M at 3sigma, respectively. HMS provided a good matrix for protein immobilization and biosensor preparation.     

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).     

Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples

An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min.     

Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.