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.     

An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol-gel/chitosan hybrid composite film

A new type of amperometric cholesterol biosensor based on sol-gel chitosan/silica and multiwalled carbon nanotubes (MWCNTs) organic-inorganic hybrid composite material was developed. The hybrid composite film was used to immobilize cholesterol oxidase on the surface of Prussian blue-modified glass carbon electrode. Effects of some experimental variables such as enzyme loading, concentration of Triton X-100, pH, temperature, and applied potential on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results show that analytical performance of the biosensor can be improved greatly after introduction of the MWCNTs. Response time, sensitivity, linear range, limit of detection (S/N=3), and apparent Michaelis-Menten constant Km are 25s, 0.54 microA mM(-1), 8.0 x 10(-6) to 4.5 x 10(-4) M, 4.0 x 10(-6) M, and 0.41 mM for the biosensor without MWCNTs and 13 s, 1.55 microA mM(-1), 4.0 x 10(-6) to 7.0 x 10(-4) M, 1.0 x 10(-6) M, and 0.24 mM for the biosensor with MWCNTs, respectively. The activation energy of the enzyme-catalyzed reaction was measured to be 42.6 kJ mol(-1). This method has been used to determine the free cholesterol concentration in real human blood samples.     

Direct electrochemistry of hemoglobin in egg-phosphatidylcholine films and its catalysis to H(2)O(2)

Direct electrochemistry of hemoglobin was observed in stable thin film composed of a natural lipid (egg-phosphatidylcholine) and hemoglobin on pyrolytic graphite (PG) electrode. Hemoglobin in lipid films shows thin layer electrochemistry behavior. The formal potential E degrees ' of hemoglobin in the lipid film was linearly varied with pH in the range from 3.5 to 7.0 with a slope of -46.4 mV pH(-1). Hemoglobin in the lipid film exhibited elegant catalytic activity for electrochemical reduction of H(2)O(2), based which a unmediated biosensor for H(2)O(2) was developed.     

Glutamate optical biosensor based on the immobilization of glutamate dehydrogenase in titanium dioxide sol-gel matrix

A simple and novel titania sol-gel derived optical biosensor coupled with carboxy seminaphthorhodamine-1-dextran (SNARF-1-dextran) as the fluorescent dye was fabricated for the determination of glutamate in water and biological samples. The NADH-dependent glutamate dehydrogenase (GLDH) was trapped in titania sol-gel derived matrix prepared by vapor deposition method. In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the surface morphology of the spots. SEM and AFM images showed that the deposition of titania precursor at 27 degrees C for 6.5h was found to be suitable to form transparent titania sol-gel matrix to encapsulate GLDH and fluorescent probe. AFM images showed that the roughness of TiO(2) surface increased from 2.16 nm in the absence of GLDH and SNARF to 37.8 nm after the immobilization. The developed titania biosensor has good analytical performance with water samples. A dynamic range between 0.04 and 10mM with the detection limit of 5.5 microM were observed. The responses to glutamate in biological samples also showed good performances, and the dynamic range and detection limit were 0.02-10mM and 6.7 microM, respectively. High precision with relative standard deviations of 4.2 and 10.7% in water and biological samples, respectively, were also demonstrated. In addition, the biosensor showed a relatively high storage stability over more than 1 month. Results obtained in this study clearly demonstrate that this simple vapor deposition method can be successfully used to form transparent titania sol-gel film for the fabrication of glutamate biosensors that are suitable for optical detection of glutamate in water and biological samples.     

A high-sensitive and fast-fabricated glucose biosensor based on Prussian blue/topological insulator Bi(2)Se(3) hybrid film

A novel and fast-fabricated Prussian blue (PB)/topological insulator Bi(2)Se(3) hybrid film has been prepared by coelectrodeposition technique. Taking advantages of topological insulator in possessing exotic metallic surface states with bulk insulating gap, Prussian blue nanoparticles in the hybrid film have smaller size as well as more compact structure, showing excellent pH stability even in the alkalescent solution of pH 8.0. Based on the Laviron theory, the electron transfer rate constant of PB/Bi(2)Se(3) hybrid film modified electrode was calculated to be 4.05±0.49s(-1), a relatively big value which may be in favor of establishing a high-sensitive biosensor. An amperometric glucose biosensor was then fabricated by immobilizing glucose oxidase (GOD) on the hybrid film. Under the optimal conditions, a wide linear range extending over 3 orders of magnitude of glucose concentrations (1.0×10(-5)-1.1×10(-2)M) was obtained with a high sensitivity of 24.55μAmM(-1) cm(-2). The detection limit was estimated for 3.8μM defined from a signal/noise of 3. Furthermore, the resulting biosensor was applied to detect the blood sugar in human serum samples without any pretreatment, and the results were comparatively in agreement with the clinical assay.     

Enhanced electron transfer for hemoglobin entrapped in a cationic gemini surfactant films on electrode and the fabrication of nitric oxide biosensor

The direct electrical communication between hemoglobin (Hb) and GCE surface was achieved based on the immobilization of Hb in a cationic gemini surfactant film and characterized by electrochemical techniques. The cyclic voltammograms showed that direct electron transfer between Hb and electrode surface was obviously promoted and then a novel unmediated nitric oxide (NO) biosensor was constructed in view of this protein-based electrode. This modified electrode showed an enzyme-like activity towards the reduction of NO and its amperometric response to NO was well-behaved with a rapid response time and displaying Michaelis-Menten kinetics with a calculated Km(app) value of 84.37 micromol L(-1). The detection limit was estimated to be 2.00 x 10(-8)mol L(-1). This biosensor was behaving as expected that it had a good stability and reproducibility, a higher sensitivity and selectivity and should has a potential application in monitoring NO released from biologic samples.     

An ultrasensitive chemiluminescence biosensor for cholera toxin based on ganglioside-functionalized supported lipid membrane and liposome

A novel chemiluminescence biosensor based on a supported lipid layer incorporated with ganglioside GM1 was developed for the detection of cholera toxin. The planar supported lipid membrane was prepared as biosensing interface via spontaneous spread of ganglioside-incorporated phospholipid vesicles on the octadecanethiol-coated gold surface. The specific interaction of multivalent CT by ganglioside GM1 molecules enables the biosensor to be implemented via a sandwiched format using a liposome probe functionalized with GM1 and horseradish peroxidase (HRP). Then, the presence of the target CT could be determined via the HRP-catalyzed enhanced chemiluminescence reaction. The developed strategy offers several unique advantages over conventional biosensors in that it allows for an easy construction and renewal of the sensing interface, a small background signal due to low non-specific adsorption of serum constituents on the lipid membrane, and effective immobilization of multiple biocatalytic amplifiers and recognition components via common phospholipid reagents. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1pgmL(-1) to 1ngmL(-1) with readily achievable detection limit of 0.8pgmL(-1).     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.     

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.     

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 facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode

A kind of solid substrate, glassy carbon (GC) electrode, was selected to support self-assembled lipid layer membranes. On the surface of GC electrode, we made layers of dimyristoylphosphatidylcholine (DMPG, a kind of lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We immobilized horseradish peroxidase (HRP) into the supported bilayer lipid membranes (s-BLM) to develop a kind of mediator-free biosensor for H2O2. The biosensor exhibited fine electrochemical response, stability and reproducibility due to the presence of the s-BLM. As a model of biological membrane, s-BLM could supply a biological environment for enzyme and maintain its activity. So s-BLM is an ideal choice to immobilize enzyme for constructing the mediator-free biosensor based on GC electrode.     

Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Na?on film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062-14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM(-1)cm(-2)). The Michaelis-Menten constant (K(m)) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.     

Amperometric phenol biosensor based on laponite clay-chitosan nanocomposite matrix

A novel strategy to fabricate an amperometric biosensor for phenol determination based on chitosan/laponite nanocomposite matrix was described. The composite film was used to immobilize PPO on the surface of a glassy carbon electrode. Chitosan was utilized to improve the analytical performance of the pure clay-modified bioelectrode. The biosensor exhibited a series of properties: good affinity to its substrate (the apparent Michaelis-Menten constant for the sensor was found to be 0.16 mM), high sensitivity (674 mA M(-1)cm(-2) for catechol) and remarkable long-term stability in storage (it retains 88% of the original activity after 60 days). In addition, optimization of the biosensor construction as well as effects of experimental variables such as pH, operating potential and temperature on the amperometric response of the sensor were discussed.     

A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles

A mediator-free phenol biosensor was developed. The low-isoelectric point tyrosinase was adsorbed on the surface of high-isoelectric point ZnO nanoparticles (nano-ZnO) facilitated by the electrostatic interactions and then immobilized on the glassy carbon electrode via the film forming by chitosan. It was found that the nano-ZnO matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retaining its activity to a large extent. Moreover, there is no need to use any other electron mediators. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at -200mV (vs. saturated calomel electrode). The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within 10s, and the sensitivity was as high as 182microAmmol(-1)L. The linear range for phenol determination was from 1.5x10(-7) to 6.5x10(-5)molL(-1) with a detection limit of 5.0x 10(-8)molL(-1) obtained at a signal/noise ratio of 3. In addition, the apparent Michaelis-Menten constant (K(m)(app)) and the stability of the enzyme electrode were estimated. The performance of the developed biosensor was compared with that of biosensors based on other immobilization matrices.     

The effect of ultra small doses of thyroliberin on the structural changes in endoplasmic reticulum membranes in vitro

Peptides are known to have the ability of modulating the activity of important regulatory cellular systems. One of them--thyroliberin, i.e. thyreotropin-releasing hormone (TRH), causes changes in the membrane structure and morphology of rat erythrocytes, as well as activates retractive activity of lymphatic vessels in ultra low concentrations (10(-10) to 10(-16) mol/l). In this study we used an electron spin resonance (ESR) method to explore the effect of TRH in a wide range of concentrations (10(-4) to 10(-18) mol/l) on thermo-induced structural transitions and microviscosity of lipid bilayer of the endoplasmic reticulum membrane of mice (C57 bI) liver cells. Two stable free radicals were used as paramagnetic probes: 2,2,6,6-tetramethil-4-capryolyl-1-oxyl and 16-doxyl-stearic acid, that are localized in superficial and deep layers of the membrane respectively. TRH caused a statistically significant change (p < 0.001) in microviscosity of the membrane surface layer. The largest effect (up to 30% decrease) was observed at TRH concentrations of 10(-10) and 10(-16) mol/l. It was also demonstrated that an addition of 10(-4), 10(-10) and 10(-16) mol/l of TRH decreases effective activation energy and temperature (by several degrees) of the thermo-induced structural transitions. The observed changes in the parameters of the membrane surface layer induced by TRH may be essential for its physiological activity, because of the obtained negative correlation (r = 0.99; p < 0.001) between the membrane microviscosity and frequency of lymphatic vessels' contraction. Complex changes in the structure of deep hydrophobic layer of the membrane caused by TRH were observed in this study as well. Higher concentrations of TRH (10(-4) and 10(-10) mol/l) produced results that were similar to the effect of TRH on the superficial lipid layer of the membrane, whereas the effect of ultra low TRH concentration (10(-16) mol/l) was reversed for microviscosity, number and activation energy of structural transitions in contrast with the case of surface layer. The results of this study suggest presence of a nonspecific factor in the effect of TRH on structural characteristics of the lipid component of biological membranes. It is possible, that the change of structural properties of biological membranes may be a part of the mechanism of TRH action at ultra low concentrations.     

Stabilized lipid film based biosensor for atenolol

This work reports a technique for the stabilization after storage in air of a lipid film based biosensor for atenolol. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices. The lipid film is formed on the filter by polymerization prior to its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The method for preparation of stabilized lipid film biosensor is studied throughout this work. The response towards atenolol of these stabilized lipid membrane biosensor, for repetitive use, composed of phosphatidylcholine was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices.     

Third-generation superoxide anion sensor based on superoxide dismutase directly immobilized by sol-gel thin film on gold electrode

A third-generation biosensor for superoxide anion (O(2)*-) was developed based on superoxide dismutase (SOD) immobilized by thin silica-PVA sol-gel film on gold electrode surface. A rapid and direct electron transfer of SOD in the thin sol-gel film at the gold electrode was realized without any mediators or promoters. The characterization of the SOD electrodes showed a quasi-reversible electrochemical redox behavior with a formal potential of 80 + 5 mV (versus SCE) in 50 mmol l(-1) phosphate buffer solution (PBS), pH 7.0. The heterogeneous electron transfer rate constant was evaluated to be about 2.1s(-1). The anodic and cathodic transfer coefficients are 0.6 and 0.4, respectively. Based on biomolecular recognition for specific reactivity of SOD toward O(2)*- the SOD electrode was applied to a sensitive and selective measurement of O(2)*- with the low operation potential (-0.15 V versus SCE) in phosphate buffer solution, pH 7.0. The amperometric response was proportional to O(2)*- concentration in the range of 0.2-1.6 micromol l(-1) and the detection limit was 0.1 micromol l(-1) at a signal-to-noise ration of 3. The preparation of SOD electrode is easy and simple. The uniform porous structure of the silica-PVA sol-gel matrix results in a fast response rate of immobilized SOD and is very efficient for stabilizing the enzyme activity.     

Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.