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PubMed2005

Microelectrode biosensor for real-time measurement of ATP in biological tissue.

The purines ATP, ADP, and adenosine are important extracellular signaling agents. Analysis of purinergic signaling has been slowed by lack of direct methods for measurement of purine release in real-time during physiological activity. We have previously reported microelectrode biosensors for adenosine, but similar sensors for ATP have remained elusive. We now describe an ATP biosensor formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase. It responds rapidly (10-90% rise time <10 s) and exhibits a linear response to ATP over the physiologically relevant concentrations of 200 nM-50 microM and is very sensitive approximately 250 mA.M(-1).cm(-2). By including phosphocreatine kinase in the biolayer, we can optionally amplify the ATP signal and also make the sensor sensitive to external ADP. We have used our sensors to make the first demonstration that ATP is released from spinal networks in vivo during locomotor activity.

Adenosine Diphosphate↗

PubMed2005

Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites.

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl(2+/3+)] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to approximately 1 mA/cm(2) for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.

Biosensing Techniques↗

PubMed2004

DNA directed protein immobilization on mixed ssDNA/oligo(ethylene glycol) self-assembled monolayers for sensitive biosensors.

A stable and versatile biosensor surface is prepared by site-directed immobilization of protein-DNA conjugates onto a mixed self-assembled monolayer (SAM) composed of ssDNA thiols and oligo(ethylene glycol) (OEG) terminated thiols. The protein conjugates consist of an antibody chemically linked to a ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. Compared to standard antibody immobilization techniques, this approach offers many advantages. The exceptional specificity of DNA hybridization combined with the diversity of potential sequences makes this platform perfect for multichannel sensors. Once a surface is patterned with the appropriate probe sequences, sequence-specific hybridization will sort out the target conjugates and direct them to the appropriate spots on the surface. In addition, the DNA SAMs are very stable and well suited to recycling by dehybdridization of the conjugates from the surface-bound probes. In this work, we demonstrate the specificity, sensitivity, and convenience of using protein-DNA conjugates to convert a DNA/OEG SAM surface into a biosensor surface and apply this platform to the detection of human chorionic gonadotropin using surface plasmon resonance.

Antibodies↗

PubMed2005

Array biosensor for detection of ochratoxin A in cereals and beverages.

Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively.

Beverages↗

PubMed2004

Development of a whole-cell-based biosensor for detecting histamine as a model toxin.

A novel whole-cell potentiometric biosensor for screening of toxins has been developed. The constructed biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to an ion-selective cellulose triacetate (CTA) membrane modified with a covalently attached RGD (arginine-glycine-aspartic acid) peptide sequence. When the HUVECs form a confluent monolayer, ion transport is almost completely inhibited, thereby reducing the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase its permeability (e.g., toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was used in this study. When the cell-based membranes are exposed to varying concentrations of histamine, the overall response increases with increasing histamine concentration. Thus, the measured potential is an indirect measurement of the histamine concentration. Further experiments were performed for a similar molecule, l-histidine, to test for selectivity. The cell permeability was unaffected by l-histidine, and the sensor response remained unchanged. This type of sensor should find multiple applications in medical, food, and environmental fields and in homeland security.

Biosensing Techniques↗

PubMed2005

Selection of ligands for affinity chromatography using quartz crystal biosensor.

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.

Algorithms↗

PubMed2005

Complexing of heavy metals with DNA and new bioaffinity method of their determination based on amperometric DNA-based biosensor.

The immobilized single-stranded DNA (ssIDNA) has been found to be a very effective biospecific analytical reagent when used in a newly developed bioaffinity method of the determination of heavy metals based on the amperometric DNA-based biosensor. This has been concluded from the comparative study of the complexing of heavy metals with double-stranded DNA, single-stranded DNA, and ssIDNA, using Fe(III) and Cu(II) as a model (metal/nucleotide ratio and stability constants are maximum for ssIDNA), from the study of adsorption of Fe(III), Cu(II), Pb(II), and Cd(II) on nitrocellulose membranes, containing single-stranded DNA, and from the determination of their binding constants with ssIDNA. According to these data, the chosen heavy metals can be lined up in a series of binding strengths with ssIDNA: Cu(II) > Pb(II) > Fe(III) > Cd(II). The method of the determination of heavy metals is based on biospecific preconcentration of metal ions on the biosensor followed by the destruction of DNA-metal complexes with ethylenediaminetetraacetate and voltammogram recording has been proposed. The lower detection limits are 4.0 x 10(-11), 1.0 x 10(-10), 1.0 x 10(-9), and 5.0 x 10(-9) M for Cu(II), Pb(II), Cd(II), and Fe(III), respectively. The heavy metals have been assayed in multicomponent environmental and biological systems such as natural and drinking water, milk, and blood serum samples.

Biosensing Techniques↗

PubMed2006

Electrochemical nanoneedle biosensor based on multiwall carbon nanotube.

We report the fabrication and analytical functions of a biosensor based on a nanoneedle consisting of a multiwall carbon nanotube attached to the end of an etched tungsten tip. The devised electrode is the smallest needle-type biosensor reported to date. The nanoneedles prepared in this work are 30 nm in diameter and 2-3 microm in length. Dopamine and glutamate, which are physiologically important neurotransmitters, were successfully detected using these nanoneedles. Bare nanoneedles detected dopamine in the range from 100 to 1000 microM by differential pulse voltammetry, and enzyme-modified nanoneedles were able to respond to glutamate in the 100-500 microM range by potentiostatic amperometry.

Biosensing Techniques↗

PubMed2006

Au nanoparticle conjugation for impedance and capacitance signal amplification in biosensors.

Amplification of the electrochemical impedance and capacitance signals in a biosensor is demonstrated for the model fluorescein/anti-fluorescein system. Following immobilization of fluorescein onto Au through formation of a self-assembled monolayer, goat anti-fluorescein conjugated with 10-nm Au nanoparticles is introduced into the system. This results in an increase in the capacitance of approximately 400 nF/cm(2), whereas no change can be observed for goat anti-fluorescein without the Au nanoparticle conjugate. An even greater sensitivity is obtained by introduction of a redox probe, [Fe(CN)6]3-/4-, whereby the charge-transfer resistance (R(ct)) is reduced to approximately 25% of its original value. This allows construction of high-sensitivity electrochemical impedance biosensors at a single low frequency, where the signal is sensitive to the interfacial R(ct). This change in the electrochemical impedance signal upon binding to goat anti-fluorescein conjugated with Au nanoparticles can be attributed to the much higher electrochemical activity of Au surfaces relative to the underlying organic layer.

Amines↗

PubMed2006

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors.

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.

Animals↗

PubMed2006

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.

Acid Phosphatase↗

PubMed2006

Ozone monitoring based on a biosensor concept utilizing a reagentless alcohol oxidase electrode.

An electrochemical method based on the concept of a biosensor for the monitoring of ozone is described for first time. The proposed method includes two parts: a selective sorbent for ozone, that is, eugenol, and a formaldehyde amperometric biosensor mounted into a flow-through cell. Ozone adds rapidly to the double bond of the allyl group of eugenol, which has been immobilized onto a hydrophobic C-18 reactor and the so produced formaldehyde is collected into the working buffer solution (sampler) and pumped to the detector. A multimembrane assembly consisting of an alcohol oxidase-modified nylon membrane sandwiched between an outer polycarbonate and an inner cellulose acetate membrane was fitted onto a Pt electrode and the enzymatically produced H2O2 was monitored at +0.65 V (vs Ag/AgCl/KCl 3 M). Under optimum conditions, a linear calibration curve over the concentration range 3-200 microg x mL(-1) ozone was constructed. The detection limit (S/N = 3) was calculated at 1.1 microg x mL(-1) ozone. The proposed method is interference-free from other gases such as O2, Ar, N2, N2O, NOCl, SO2, NH3, and CO2, which were tested at concentrations >200-fold higher than that of 100 microg x mL(-1) ozone used for comparison. Besides selectivity, the method is easy to perform and reproducible; its applicability in synthetic gaseous samples is also demonstrated.

Alcohol Oxidoreductases↗

PubMed2007

Peptide-nucleic acid-modified ion-sensitive field-effect transistor-based biosensor for direct detection of DNA hybridization.

Here, we report the development of a peptide-nucleic acid (PNA)-modified ion-sensitive field-effect transistor (IS-FET)-based biosensor that takes advantage of the change in the surface potential upon hybridization of a negatively charged DNA. PNA was immobilized on a silicon nitride gate insulator by an addition reaction between a maleimide group introduced on the gate surface, the succinimide group of N-(6-maleimidocaproyloxy) succinimide, and the thiol group of the terminal cysteine in PNA. The surface was characterized after each step of the reaction by X-ray photoelectron spectroscopy analysis, and the kinetic analysis of the hybridization events was assessed by surface plasmon resonance. In addition, we measured the -potential before and after PNA-DNA hybridization in the presence of counterions to investigate the change in surface charge density at the surface-solution interface within the order of the Debye length. On the basis of the zeta-potential, the surface charge density, DeltaQ, calculated using the Grahame equation was approximately 4.0 x 10(-3) C/m2 and the estimated number of hybridized molecules was at least 1.7 x 10(11)/cm2. The I-V characteristics revealed that the PNA-DNA duplexes induce a positive shift in the threshold voltage, VT, and a decrease in the saturated drain current, ID. These results demonstrate that direct detection of DNA hybridization should be possible using a PNA-modified IS-FET-based biosensor. PNA is particularly advantageous for this system because it enables highly specific and selective binding at low ionic strength.

Base Sequence↗

PubMed1996

DNA electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus.

An electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus type 1 (HIV-1) is described. The sensor relies on the immobilization and hybridization of the 21- or 42-mer single-stranded oligonucleotide from the HIV-1 U5 long terminal repeat (LTR) sequence at carbon paste or strip electrodes. The extent of hybridization between the complementary sequences is determined by the enhancement of the chronopotentiometric peak of the Co(phen)3(3+) indicator. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions are optimized to maximize the sensitivity and speed the assay time. A detection limit of 4 x 10(-9) M HIV-1 U5 LTR segment is reported following a 30 min hybridization. The hybridization biosensor format obviates the use of radioisotopes common in radioactive methods for the detection of HIV-1 DNA. We also report on the direct adsorptive chronopotentiometric stripping measurements of trace levels of various HIV-1 DNAs.

Base Sequence↗

PubMed1997

A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole-derived bipyridinium.

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBP-Nir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 +/- 0.5) x 10(3) M-1 S-1 was obtained for the rate constant.

Biosensing Techniques↗

PubMed1998

Regenerable biosensor platform: a total internal reflection fluorescence cell with electrochemical control.

A new biosensor platform that provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed and investigated using antibody-antigen and streptavidin-biotin interactions. Specifically, biotin was covalently bound to a transparent indium-tin oxide (ITO) working electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions, while electrochemical polarization was employed to control interactions between biotin and streptavidin or between biotin and anti-biotin antibodies. Both streptavidin and polyclonal anti-biotin antibodies bound kinetically irreversibly to the biotinylated surface. In the absence of electrochemical control, the assay exhibited an extremely slow release of the bound analytes, causing poor regeneration ability of the biosensor surface. However, electrochemical polarization was found to stimulate dissociation of kinetically irreversibly bound biotin-streptavidin and antibody-antigen complexes. A "square wave" polarization function stimulated dissociation more effectively than a "saw tooth" function over the same voltage range. Application of the square wave polarization resulted in regeneration of an active biotinylated surface. Electrochemical polarization also modified affinity and kinetics of protein adsorption, which could likely be used to promote biospecific interactions and/or to suppress nonspecific adsorption.

Antigen-Antibody Reactions↗

PubMed1998

Polishable and renewable DNA hybridization biosensors.

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.

Animals↗

PubMed1998

Adapting selected nucleic acid ligands (aptamers) to biosensors.

A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.

Anisotropy↗