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Construction of biosensors using a gold-binding polypeptide and a miniature integrated surface plasmon resonance sensor.

Surface plasmon resonance (SPR) biosensors were constructed on miniature integrated sensors. Recognition elements were attached to the sensor surface using a gold-binding repeating polypeptide. Biosensors with fluorescyl groups attached to their surfaces were functional for at least 1 month of daily use with little decrease in response to the binding of an anti-fluorescyl monoclonal antibody. The coupling of protein A to the gold-binding polypeptide on the sensor surface enabled the biosensor to detect the binding of antibodies to the protein A and provided a sensor with convertible specificity. The system described herein provides a simple and rapid approach for the fabrication of highly specific, durable, portable and low cost SPR-based biosensors.

Amino Acid Sequence↗

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites.

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

Biosensing Techniques↗

Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe.

The electropolymerized toluidine blue film deposited on the glassy carbon electrode show amperometrically detectable pH sensitivity. This feature of polytoluidine blue (PTOB) film was used for a construction of an amperometric urea biosensor. We have observed a linear shift of the formal redox potential with increasing pH value between 4 and 8 giving the slope of 81 mV(Delta) pH(-1). Polytoluidine blue film has had a significantly increased stability and higher electrochemical activity compared to the adsorbed monomeric dye. The polytoluidine blue urea biosensor has been operating at a working potential of -200 mV vs. SCE. The sensitivity of the biosensor was 980 nA mM(-1) cm(-2). The biosensor showed linearity in concentration range up to 0.8 mM with the detection limit of 0.02 mM (S/N=3).

Biosensing Techniques↗

Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity.

The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 microA mM(-1). The microbial biosensor exhibits a very low detection limit of 0.85 microM and a wide linear range from 2 to 270 microM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R(2)=0.998).

Biosensing Techniques↗

Biosensor for dopamine based on stabilized lipid films with incorporated resorcin[4]arene receptor.

This work reports a technique for the stabilization after storage in air of a lipid film with incorporated resorcin[4]arene receptor based biosensor for dopamine. 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 and the lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The stability of the lipid films by incorporation of a receptor for the preparation of stabilized lipid film biosensor is studied throughout this work. The response towards dopamine of the present stabilized for repetitive uses lipid membrane biosensor composed of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid 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. However, the response of the stabilized lipid films was slower than that of the freely suspended BLMs. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices, because it is now possible to prepare stabilized lipid film based biosensors and store them in the air.

1,2-Dipalmitoylphosphatidylcholine↗

An aptamer-based quartz crystal protein biosensor.

We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.

Antibodies, Anti-Idiotypic↗

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors.

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.

Biosensing Techniques↗

Integrating waveguide biosensor.

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.

Animals↗

Multianalyte pin-printed biosensor arrays based on protein-doped xerogels.

We report the first biosensor arrays based on pin printing protein-doped xerogels. The individual biosensor elements are on the order of 100 microm in diameter. Arrays are formed (1) onto a planar substrate that is excited by an external source (laser) or (2) directly on the face of a light-emitting diode. We illustrate the potential of our approach by fabricating, testing, and characterizing four types of pin-printed biosensor arrays (PPBSA) for the simultaneous detection of glucose and O2. The analytically reliable operating ranges for the PPBSAs are 0.1-10 mM for glucose and 0.1-100% for O2. The PPBSAs exhibit short- and long-term reproducibilities of no worse than 4 and 8%, respectively. The overall array-to-array response reproducibilities are < or = 12%. These results demonstrate for the first time the combination sol-gel processing and pin printing methods as a way to rapidly form ensembles of integrated, reusable, and stable biosensor arrays for simultaneous multianalyte detection.

Biosensing Techniques↗

Urea biosensors based on immobilization of urease into two oppositely charged clays (laponite and Zn-Al layered double hydroxides).

Enzyme-based field effect transistors (ENFETs) for urea determination were developed based on the immobilization of urease within two different clay matrixes, one cationic (Laponite) and the other anionic (layered double hydroxide (LDH)), cross-linked with glutaraldehyde. The biosensor based on the enzyme immobilized in Laponite shows a greater sensitivity and smaller dynamic linear range, because the enzymatic reaction is protected from the effect of the buffer capacity of the outer medium. The apparent Michaelis-Menten constant, Km(app), is quite similar for both biosensors. Inhibition of the enzyme by sodium tetraborate was investigated. Tetraborate acts as a competitive inhibitor for urease in the two different types of clay, the inhibitor effect being stronger for the LDH/urease biosensor. In particular, the maximum limit of the dynamic linear range extends from 1.4 mM in the absence of the inhibitor to 12 mM in the presence of 0.5 mM tetraborate. The Km(app) values in the presence of 0.5 mM tetraborate for Laponite and LDH biomembranes were 10 and 62 mM, respectively. Comparison of the inhibition constant values, Ki 0.16 and 0.05 mM for Laponite and LDH biosensors, respectively, clearly indicates a stronger enzyme-inhibitor interaction in the LDH/urease biomembrane.

Aluminum Silicates↗

Correlation of analyte structures with biosensor responses using the detection of phenolic estrogens as a model.

The apparent increase in hormone-induced cancers and disorders of the reproductive tract in wildlife and humans has led to a search for an accurate and reliable method for monitoring endocrine-disrupting chemicals (EDCs). This study presents a generic approach that may allow researchers to establish screening procedures for potential EDCs by correlating the analyte structures with biosensor responses and explain possible reaction mechanisms. A simple amperometric tyrosinase-based biosensor (Tyr-CPE) has been developed for the detection of phenolic EDCs. The investigation of the enzymatic oxidation of selected phenolic estrogens was first carried out using UV-vis spectroscopy. The result was used to correlate sensor responses to enzymatic activity. Natural phytoestrogen polyphenols, including resveratrol (RES), genistein (GEN), and quercetin (QRC), were compared with synthetic estrogens, for example, bisphenol A (BPhA), nonylphenol (NPh), and diethylstilbestrol (DES). The Tyr-CPE biosensor resulted in rapid, simple, and accurate measurement of phenolic estrogens with varying degrees of sensitivity, selectivity, and response times. The sensor responses have been evaluated for the detection of binary and tertiary mixtures of EDCs and natural estrogens. The results showed that BPhA could be successfully discriminated in a composite mixture containing NPh and DES at various ratios. In the case of natural phenolic estrogens GEN, RES, and QRC, the sensor allows the determination of a total phenolic content. The sensor was also validated for the detection of BPhA in a real environmental water sample, and the results was compared with standard ASTM method 9065. Mechanistically, our results indicated that the number of OH groups, the nature and the position of aryl ring substituents, or both could affect the detection limit and the biosensor sensitivity.

Animals↗

A wireless, remote query glucose biosensor based on a pH-sensitive polymer.

This paper describes a wireless, remote query glucose biosensor using a ribbonlike, mass-sensitive magnetoelastic sensor as the transducer. The glucose biosensor is fabricated by first coating the magnetoelastic sensor with a pH-sensitive polymer and upon it a layer of glucose oxidase (GOx). The pH-responsive polymer swells or shrinks, thereby changing mass, respectively, in response to increasing or decreasing pH values. The GOx-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the polymer mass. In response to a time-varying magnetic field, a magnetoelastic sensor mechanically vibrates at a characteristic resonance frequency, the value of which inversely depends on sensor mass loading. As the magnetoelastic films are magnetostrictive, the vibrations launch magnetic flux that can be remotely detected using a pickup coil. Hence, changes in the resonance frequency of a passive magnetoelastic transducer are detected on a remote query basis, without the use of physical connections to the sensors.The sensitivity of the glucose biosensors decreases with increasing ionic strength; at physiological salt concentrations, 0.6 mmol/L of glucose can be measured. At glucose concentrations of 1-10 mmol/L, the biosensor response is reversible and linear, with the detection limit of 0.6 mmol/L corresponding to an error in resonance frequency determination of 20 Hz. Since no physical connections between the sensor and the monitoring instrument are required, this sensor can potentially be applied to in vivo and in situ measurement of glucose concentrations.

Acrylates↗

Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor.

The development of label-free optical biosensors for DNA and other biomolecules has the potential to impact life sciences as well as screening in medical and environmental applications. In this report, we developed a localized surface plasmon resonance (LSPR) based label-free optical biosensor based on a gold-capped nanoparticle layer substrate immobilized with peptide nucleic acids (PNAs). PNA probe was designed to recognize the target DNA related to tumor necrosis factor. The nanoparticle layer was formed on a gold-deposited glass substrate by the surface modified silica nanoparticles using silane-coupling reagent. The optical properties of gold-capped nanoparticle layer substrate were characterized through monitoring the changes in the absorbance strength, as the thickness of the biomolecular layer increased with hybridization. The detection of PNA-DNA hybridization with target oligonucleotides and PCR-amplified real samples were performed with a limit of detection value of 0.677 pM target DNA. Selective discrimination against a single-base mismatch was also achieved. Our LSPR-based biosensor with the gold-capped nanoparticle layer substrate is applicable to the design of biosensors for monitoring of the interaction of other biomolecules, such as proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.

Biosensing Techniques↗

Control of the oxygen dependence of an implantable polymer/enzyme composite biosensor for glutamate.

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K(M)(Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K(M)(O2). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K(M)(O2) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.

Amino Acid Oxidoreductases↗

Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles.

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been self-assembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of approximately 915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted "V" shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 +/- 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADH-dependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors.

Biosensing Techniques↗

Dynamic delay and maximal dynamic error in continuous biosensors.

When biosensors are operated continuously, a dynamic delay and a dynamic error relate the sensor signal to the changing analyte concentration. The dynamic delay is the temporal displacement of the signal, or the lag, and is specified solely by properties of the biosensor and external mass transfer. The dynamic error is the difference between the actual concentration and the simultaneous reported concentration and is the product of the dynamic delay and the instantaneous rate of concentration change. In real-time operation of sensors, a maximal dynamic error based on the maximal expected rate of concentration change must be employed to estimate the worst-case error because the actual instantaneous rate is not independently known. Values of dynamic delay and maximal dynamic error that are acceptable in particular monitoring situations can be used in the design of acceptable continuous biosensors. This analysis suggests experimental alternatives to the standard response time approach for sensor characterization that are particularly advantageous for continuously operated biosensors. The concepts are applied here to in vitro operation of a continuous glucose sensor.

Biosensing Techniques↗

Amperometric glucose biosensor based on sol-gel organic-inorganic hybrid material.

A new type of sol-gel organic-inorganic hybrid material was developed and used for the production of biosensors. This material is composed of silica sol and a grafting copolymer of poly(vinyl alcohol) with 4-vinylpyridine. It prevents the cracking of conventional sol-gel-derived glasses and eliminates the swelling of the hydrogel. The optimum composition of the hybrid material was first examined, and then glucose oxidase was immobilized in this matrix to demonstrate its application. The characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The biosensor exhibited a series of good properties: high sensitivity (600 nA mmol-1 L-1), short response time (11 s) and remarkable long-term stability in storage (at least 5 months). In addition, the characteristics of the second-generation biosensor with the use of tetrathiafulvalene as a mediator were discussed.

Biosensing Techniques↗

Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode.

A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.

Biosensing Techniques↗