Biosensor based on cellobiose dehydrogenase for detection of catecholamines

A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs Ag|AgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A.M(-1) x cm(-2) (approximately 1150 nA/microM) for noradrenaline, with a coverage of less than 2.5 microg of CDH adsorbed on the electrode surface (0.073 cm(2)). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.     

V-type nerve agent detection using a carbon nanotube-based amperometric enzyme electrode

An enzyme electrode for the detection of V-type nerve agents, VX (O-ethyl-S-2-diisopropylaminoethyl methylphosphonothioate) and R-VX (O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate), is proposed. The principle of the new biosensor is based on the enzyme-catalyzed hydrolysis of the nerve agents and amperometric detection of the thiol-containing hydrolysis products at carbon nanotube-modified screen-printed electrodes. Demeton-S was used as a nerve agent mimic. 2-(Diethylamino)ethanethiol (DEAET) and 2-(dimethylamino)ethanethiol (DMAET), the thiol-containing hydrolysis product and hydrolysis product mimic of R-VX and VX, respectively, were monitored by exploiting the electrocatalytic activity of carbon nanotubes (CNT). As low as 2 microM DMAET and 0.8 microM DEAET were detected selectively at a low applied potential of 0.5 V vs Ag/AgCl at a CNT-modified mediator-free amperometric electrode. Further, the large surface area and the hydrophobicity of CNT was used to immobilize organophosphorus hydrolase mutant with improved catalytic activity for the hydrolysis of the P-S bond of phosphothiolester neurotoxins including VX and R-VX nerve gases to develop a novel, mediator-free, membrane-free biosensor for V-type nerve agents. The applicability of the biosensor was demonstrated for direct, rapid, and selective detection of V-type nerve agents' mimic demeton-S. The selectivity of the sensor against interferences and application to spiked lake water samples was demonstrated.     

Protease amperometric sensor

An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H2O2 and hence an amperometric signal due to H2O2 electrooxidation was generated by potentiostating the electrode at 0.6 V. The biosensor detects the change in the increase in the maximum current caused by the proteolytic digestion of gelatin, which covers the platinum electrodes, thereby facilitating a speedier access for the glucose substrate to the electrode modified with both poly(pyrrole-alkylammonium) and glucose oxidase molecules. Our biosensor detected low trypsin concentrations down to 42 pM with a response time of approximately 10 min, making it a very sensitive device in the detection of lower trypsin levels with such future putative applications as the diagnosis of pancreatic diseases.     

Functionalized conducting polymer as an enzyme-immobilizing substrate: an amperometric glutamate microbiosensor for in vivo measurements

The functionalized conducting polymer (CP) of 5, 2':5', 2' '-terthiophene-3'-carboxylic acid on a platinum microelectrode was prepared through the electropolymerization process using cyclic voltammetry and was used as a substrate for the immobilization of enzymes. The nanoparticles of the CP were obtained at a high scan rate in the cyclic voltammetric experiment. A needle-type amperometric glutamate microbiosensor based on the covalent immobilization of glutamate oxidase (GlOx) onto the CP layer was fabricated for in vivo measurements. The surfaces of the CP/Pt and GlOx/CP/Pt were characterized by QCM, ESCA, and AFM. The biosensor efficiently detected glutamate through the oxidation of enzymatically generated H2O2 at approximately +0.45 V versus Ag/AgCl. Various experimental parameters, such as pH, temperature, and the applied potential in the detection step were optimized. The interference effects from other biological compounds were examined, and ascorbate and dopamine interferences were observed, which were completely minimized by coimmobilizing ascorbate oxidase and by coating the sensor surface with a cationic polymer, polyethyleneimine. A linear calibration plot for glutamate was obtained between 0.2 and 100 microM with a detection limit of 0.1 +/- 0.03 microM. The proposed glutamate microbiosensor was successfully used for in vivo monitoring of the extracellular glutamate released by cocaine stimulation.     

A convenient alcohol sensor using one-pot nanocomposite entrapping alcohol oxidase and magnetic nanoparticles as peroxidase mimetics

A colorimetric biosensor for convenient quantification of ethanol and methanol is described. The biosensor utilizes a 'one-pot' nanocomposite consisting of Fe3O4 magnetic nanoparticles (MNPs) and alcohol oxidase (Al Ox) simultaneously entrapped in large pore sized mesocellular silica. Al Ox immobilized in the silica generates H2O2 in the presence of alcohol in a sample, which subsequently activates MNPs in the mesopores of the silica to convert a colorimetric substrate into a colored product. Using this strategy, a target alcohol was specifically detected through a very convenient colorimetric signal resulting from the combined reactions. This strategy enabled successful sensing of ethanol and methanol in a linear concentration range from 100 to 500 microM with a detection limit as low as 25 microM by employing 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) as a peroxidase substrate. Along with excellent reusability via simple magnetic capturing, enhanced operational stability was achieved by the nanocomposite system. The present nanocomposite would serve as a novel platform for rapid and convenient analysis of alcohol.     

Subnanomolar cyanide detection at polyphenol oxidase/clay biosensors

A novel, inexpensive, and simple amperometric biosensor based on immobilization of polyphenol oxidase (PPO) into Zn-Al layered double hydroxides, also called anionic clays, is applied for determination of cyanide. The detection of cyanide was performed via its inhibiting action on the PPO electrode. Measurement was carried out with 3,4-dihydroxyphenylacetic acid as enzyme substrate, the enzymatically generated quinoid products being electroreduced at -0.2 V. An extremely sensitive detection limit (0.1 nM) was obtained for cyanide. Enzyme immobilization into an anionic exchanger clay seems to cause an increase in cyanide inhibition effects because of anion accumulation in the clay matrix.     

Extended-range glucose sensor employing engineered glucose dehydrogenases

An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy. This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range. We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold). We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range. The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error. The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system. Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp. The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes.     

Electrochemical multianalyte immunoassays using an array-based sensor

A novel amperometric biosensor for performing simultaneous electrochemical multianalyte immunoassays is described. The sensor consisted of eight iridium oxide sensing electrodes (0.78 mm(2) each), an iridium counter electrode, and a Ag/AgCl reference electrode patterned on a glass substrate. Four different capture antibodies were immobilized on the sensing electrodes via adsorption. Quantification of proteins was achieved using an ELISA in which the electrochemical oxidation of enzyme-generated hydroquinone was measured. The spatial separation of the electrodes enabled simultaneous electrochemical immunoassays for multiple proteins to be conducted in a single assay without amperometric cross-talk between the electrodes. The simultaneous detection of goat IgG, mouse IgG, human IgG, and chicken IgY was demonstrated. The detection limit was 3 ng/mL for all analytes. The sensor had excellent precision (1.9-8.2% interassay CV) and was comparable in performance to commercial single-analyte ELISAs. We anticipate that chip-based sensors, as described herein, will be suitable for the mass production of economical, miniaturized, multianalyte assay devices.     

Sensitive Label-Free Immunoassay by Colorimetric Plasmonic Biosensors Using Silver Nanodome Arrays

The sensitive direct detection of biomolecules is demonstrated by a colorimetric plasmonic biosensor utilizing the surface colors of plasmonic metasurfaces named Ag nanodome arrays. The Ag nanodome arrays consist of polystyrene bead monolayers coated with Ag thin films whose surface colors are optimized by changing the size of the polystyrene beads. The bulk refractive index sensitivity of colorimetric detection evaluated using the hue angle is 590° RIU⁻¹ (RIU: refractive index unit). For selected geometry, the refractive index resolution (5.0 × 10⁻⁵ RIU) obtained by colorimetric detection surpasses that of spectroscopic detection evaluated via the dip wavelength in the reflection spectrum. The numerical simulations predict an enhanced sensing performance when the hue angle of the surface colors of the Ag nanodome arrays changes from 300° to 200°, corresponding to changes in the dip wavelength from 570 to 600 nm in the reflection spectra. Furthermore, the detection sensitivity of the biomolecules is characterized using a direct IgG immunoassay format. The detection limit of the IgG concentration is as low as 134 pM using simple colorimetric detection. The feasibility of sensitive label-free immunoassays using a colorimetric plasmonic biosensor is expected to accelerate the development of highly sensitive and reliable smartphone-based plasmonic biosensors.     

Synthesis of silver nanoparticle-hollow titanium phosphate sphere hybrid as a label for ultrasensitive electrochemical detection of human interleukin-6

A silver nanoparticle-hollow titanium phosphate sphere (AgNP-TiP) hybrid is successfully synthesized and used as a label for electrochemical detection of human interleukin-6 (IL-6). Hollow TiP spheres with a diameter of 430 nm and an average thickness of 40 nm are synthesized by a template approach. The AgNPs are incorporated in situ into the TiP shell via an exchange process. The as-prepared AgNP-TiP hybrid shows outstanding biocompatibility, good dispersity and solubility in water, and high silver loading properties (289.2 mg of silver in 1.0 g of TiP). These advantages make the AgNP-TiP hybrid an effective candidate as an amplification label in immunoassay systems. Herein, the as-prepared AgNP-TiP hybrid is attached to a signal antibody (Ab(2) ) to produce Ab(2) -AgNP-TiP labels in the fabrication of an electrochemical immunosensor. The nanoparticle-based amplification labels, upon coupling with a magnetic sensing array, give rise to an extremely sensitive response to IL-6 in a linear range of 0.0005-10 ng mL(-1) with a detection limit of 0.1 pg mL(-1) . The proposed sensor exhibits high specificity, good reproducibility, and long-term stability, and may be a promising technique for protein and DNA detection.     

A lactate electrochemical biosensor with a titanate nanotube as direct electron transfer promoter

Hydrogen titanate (H(2)Ti(3)O(7)) nanotubes (TNTs) have been synthesized by a one-step hydrothermal processing. Lactate oxidase (LOx) enzyme has been immobilized on the three-dimensional porous TNT network to make an electrochemical biosensor for lactate detection. Cyclic voltammetry and amperometry tests reveal that the LOx enzyme, which is supported on TNTs, maintains their substrate-specific catalytic activity. The nanotubes offer the pathway for direct electron transfer between the electrode surface and the active redox centers of LOx, which enables the biosensor to operate at a low working potential and to avoid the influence of the presence of O(2) on the amperometric current response. The biosensor exhibits a sensitivity of 0.24?μA?cm(-2)?mM(-1), a 90% response time of 5?s, and a linear response in the range from 0.5 to 14?mM and the redox center of enzyme obviates the need of redox mediators for electrochemical enzymatic sensors, which is attractive for the development of reagentless biosensors.     

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.     

Mixed ceria-based metal oxides biosensor for operation in oxygen restrictive environments

The unique catalytic, electrochemical, and oxygen storage properties of ceria and mixed ceria/titania hybrid composites were used to fabricate a new type of electrochemical enzyme biosensor. These materials provided increased analytical performance and possibilities for operation in oxygen-free conditions of an oxidase enzyme biosensor using tyrosinase as a model example. The investigation of the enzymatic reaction in the presence and absence of oxygen was first carried out using cyclic voltammetry. The results were used to identify the role of each metal oxide in the immobilization matrix and fabricate a simple amperometric tyrosinase biosensor for the detection of phenol and dopamine. The biosensor was optimized and characterized with respect to response time, detection limit, linear concentration range, sensitivity, and kinetic parameters. The detection limit for phenol was in the nanomolar range, with a detection limit of 9.0 x 10(-9) M and a sensitivity of 86 mA M(-1) in the presence of oxygen and of 5.6 x 10(-9) M and a sensitivity of 65 mA M(-1) in the absence of oxygen. The optimized biosensor also showed selective determination of the neurotransmitter dopamine with a detection limit of 3.4 x 10(-8) M and a sensitivity of 14.9 mA M(-1) in the presence of oxygen and of 4.2 x 10(-8) M and 14.8 mA M(-1) in the absence of oxygen. This strategy shows promise for increasing the sensitivity of oxidase enzyme sensors and provides opportunities for operation in oxygen limited conditions. It can also be extended for the development of other enzyme biosensors.     

Amperometric thick-film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophosphorus hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.     

Enzyme biosensor based on plasma-polymerized film-covered carbon nanotube layer grown directly on a flat substrate

We report a novel approach to fabrication of an amperometric biosensor with an enzyme, a plasma-polymerized film (PPF), and carbon nanotubes (CNTs). The CNTs were grown directly on an island-patterned Co/Ti/Cr layer on a glass substrate by microwave plasma enhanced chemical vapor deposition. The as-grown CNTs were subsequently treated by nitrogen plasma, which changed the surface from hydrophobic to hydrophilic in order to obtain an electrochemical contact between the CNTs and enzymes. A glucose oxidase (GOx) enzyme was then adsorbed onto the CNT surface and directly treated with acetonitrile plasma to overcoat the GOx layer with a PPF. This fabrication process provides a robust design of CNT-based enzyme biosensor, because of all processes are dry except the procedure for enzyme immobilization. The main novelty of the present methodology lies in the PPF and/or plasma processes. The optimized glucose biosensor revealed a high sensitivity of 38 μA mM(-1) cm(-2), a broad linear dynamic range of 0.25-19 mM (correlation coefficient of 0.994), selectivity toward an interferent (ascorbic acid), and a fast response time of 7 s. The background current was much smaller in magnitude than the current due to 10 mM glucose response. The low limit of detection was 34 μM (S/N = 3). All results strongly suggest that a plasma-polymerized process can provide a new platform for CNT-based biosensor design.     

Electrochemical enzyme immunoassays on microchip platforms.

A microfluidic device for conducting electrochemical enzyme immunoassays is described. The new "lab-on-a-chip" protocol integrates precolumn reactions of alkaline phosphatase-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibody-antigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the 4-aminophenyl phosphate substrate and a downstream amperometric detection of the liberated 4-aminophenol product Factors influencing the reaction, separation, and detection processes were optimized, and the analytical performance was characterized. An applied field strength of 256 V/cm results in free antibody and antibody-antigen complex migration times of 125 and 340 s, respectively. A remarkably low detection limit of 2.5 x 10(-16) g/mL (1.7 x 10(-18) M) is obtained for the mouse IgG model analyte. Such combination of a complete integrated immunoassay, an attractive analytical performance, and the distinct miniaturization/portability advantages of electrochemical microsystems offers considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics or on-site environmental testing.     

Specific determination of As(V) by an acid phosphatase-polyphenol oxidase biosensor

An original amperometric biosensor based on the simultaneous entrapment of acid phosphatase (AcP) and polyphenol oxidase (PPO) into anionic clays (layered double hydroxides) was developed for the specific detection of As(V). The functioning principle of the bienzyme electrode consisted of the successive hydrolysis of phenyl phosphate into phenol by AcP, followed by the oxidation of phenol into o-quinone by PPO. The phenyl phosphate concentration was, thus, monitored by potentiostating the biosensor at -0.2 V vs Ag/AgCl to detect amperometrically the generated quinone. The detection of As(V) was based on its inhibitory effect on AcP activity toward the hydrolysis of phenyl phosphate into phenol. The As(V) can be specifically determined in pH 6.0 acetate buffer without any interferences of As(III) or phosphate, the detection limit being 2 nM or 0.15 ppb after an incubation step for 20 min.     

Preparation of porous titania sol-gel matrix for immobilization of horseradish peroxidase by a vapor deposition method

A new and facile vapor deposition method has been developed for the preparation of sol-gel matrix. This method was used to form a titania sol-gel thin film and to immobilize horseradish peroxidase (HRP) on a glassy carbon electrode surface for the production of an amperometric hydrogen peroxide biosensor. This process prevented the cracking of conventional sol-gel-derived glasses. The morphologies of both titania sol-gel and the enzyme membranes were characterized using scanning electron microscopy and proved to be chemically clean, porous, and homogeneous and to have a very narrow particle size distribution. The sol-gel-derived titania-modified electrode retained the enzyme bioactivity and provided for long-term stability of the enzyme in storage. In the presence of catechol as a mediator, the sensor exhibited a rapid electrocatalytic response (less than 5 s), a linear calibration range from 0.08 to 0.56 mM with a detection limit of 1.5 microM and a high sensitivity (61.5 microA mM(-1)) for monitoring of H2O2. Effects of pH and operating potential were also explored for optimum analytical performance by using the amperometric method. The apparent Michaelis-Menten constant of the encapsulated HRP was 1.89 +/- 0.21 mM.