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PubMed1999

Sol-gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium.

An acid-stable soybean-peroxidase biosensor was developed by immobilizing the enzyme in a sol-gel thin film. Methylene blue was used as a mediator because of its high electron-transfer efficiency. The sol-gel thin film and enzyme membrane were characterized by FT-IR, and the effects of pH, operating potential, and temperature were explored for optimum analytical performance by using the amperometric method. The H2O2 sensor exhibited a fast response (5 s), high sensitivity (27.5 microA/mM), as well as good thermostability and long-term stability. In addition, the performance of the biosensor was investigated using flow-injection analysis (FIA).

Enzymes, Immobilized↗

PubMed1999

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.

Antibodies↗

PubMed2000

Probing trace phenols based on mediator-free alumina sol--gel-derived tyrosinase biosensor.

A novel tyrosinase biosensor has been developed for the subnanomolar detection of phenols, based on the immobilization of tyrosinase in a positively charged Al2O3 sol-gel membrane on a glassy carbon electrode. It has been found that Al2O3 sol-gel is perfectly beneficial to the immobilization of tyrosinase, because it not only possesses the general advantages of sol-gel but it also is an effective promoter of the biosensor. The large microscopic surface area, porous morphology, and hydrophilic property of the sol-gel matrix result in high enzyme loading, and the enzyme entrapped in this matrix retains its activity to a large extent. The Al2O3 sol-gel-containing surface also displays an intrinsic electrocatalytic o-quinone response and, hence, offers a high-sensitivity (127 microA mM(-1)) monitoring of phenols. The detection limit is 0.2 nM at a signal-to-noise ratio of 3, the response time is less than 4 s reaching 95% of the steady-state value, and 70% of the activity is retained after 3 months.

Biosensing Techniques↗

PubMedDate not supplied

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids.

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.

Base Sequence↗

PubMedDate not supplied

Immunoassay reagents for thyroid testing. 2. Binding properties and energetic parameters of a T4 monoclonal antibody and its Fab fragment with a library of thyroxine analog biosensors using surface plasmon resonance.

A library of 16 thyroxine analog biosensors were prepared for use on the BIAcore surface plasmon resonance instrument. An anti-thyroxine monoclonal antibody and its Fab fragment were tested with each biosensor to generate the association and dissociation rate constants, equilibrium association constants, and Gibbs free energy values for characterizing the binding event.

Animals↗

PubMedDate not supplied

Fractal analysis of antigen-antibody binding kinetics: biosensor applications.

The diffusion-limited binding kinetics of antigen (or antibody) in solution to antibody (or antigen) immobilized on a biosensor surface is analyzed within a fractal framework. The estimated fractal dimension increases linearly from 2.28 to 2.75 for the antirabbit IgG (antibody) (Nygren, H.; Stenberg, M. J. Colloid Interface Sci. 1985, 107, 560-566) as the R1S1 antibody concentration in solution increases 28-fold from 6 to 170 micrograms/mL. Similarly, for the binding of ferritin (antigen) in solution by a "sandwich" immunoassay to antiferritin (antibody) immobilized on a surface (Bluestein, B. I.; Craig, M.; Slovacek, R., Stundter, L.; Urciuoli, C.; Walizak, I.; Luderer, A. In Biosensors with Fiberoptics; Wise, D., Wingard, L. B., Jr., Eds.; The Humana Press, Inc.: Clifton, NJ, 1991; pp 181-223), the estimated fractal dimension decreases from 1.33 to 1.02 as the initial concentration of ferritin increases from 20 to 1000 micrograms/mL. The analysis attempts to provide novel physical insights into the reactions occurring on the surface and the structure or nature of the surface. Diffusional limitations inherent in such systems need to be uncoupled from reaction kinetics and complexities, such as heterogeneity, that exist on the surface.

Antigen-Antibody Reactions↗

PubMedDate not supplied

Purification of recombinant human B-domain-deleted factor VIII using anti-factor VIII monoclonal antibody selected by the surface plasmon resonance biosensor.

The surface plasmon resonance (SPR) biosensor measures the real-time kinetics of noncovalent interaction between a receptor and its ligand. Monoclonal antibodies (mAbs) against recombinant factor VIII (rFVIII) were screened from 127 mAb candidates using the SPR biosensor for the purpose of affinity purification of rFVIII. Each mAb showed a different association and dissociation capacity for rFVIII at each buffer condition. One mAb, F8-38, was selected for immunopurification of rFVIII. To characterize the selected mAb F8-38, the immunopurification results on the anti-FVIII mAb F8-38 affinity gel and the anti-von Willebrand factor (vWF) mAb affinity gel were studied. Immunopurification by the anti-vWF affinity gel showed a lower binding capacity of rFVIII and resulted in low purification efficiency. On the other hand, immunopurification by the anti-FVIII affinity gel exhibited a 3.5-fold binding capacity and a 2-fold purification efficiency compared to those of the anti-vWF affinity gel. The amounts of proteins and DNAs derived from host cells and mouse IgGs derived from the affinity matrix in the affinity eluate were similar to those of the anti-vWF affinity gel. In conclusion, the SPR method of immunopurification is a useful technology in the screening of mAbs aimed at the development of an affinity purification procedure, and the mAb F8-38 was selected using this technology on the basis of the purification procedure of rFVIII.

Animals↗

PubMedDate not supplied

A thermostable sugar-binding protein from the Archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients.

In this work is presented the first attempt to develop an innovative ultrastable protein-based biosensor for blood glucose detections. The gene of a putative thermostable sugar-binding protein has been cloned and expressed in E. coli. The recombinant protein has been purified to homogeneity by thermoprecipitation and affinity chromatography steps. The recombinant protein is a monomer with an apparent molecular weight of 55,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel eletrophoresis. Circular dichroism experiments showed that the protein possesses a secondary structure content rich in alpha-helices and beta-structures and that the protein is highly stable as investigated in the range of temperature between 20 and 95 degrees C. Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar very close to the concentration of glucose present in the human blood. A docking simulation on the modeled structure of the protein confirms its ability to bind glucose and proposes possible modifications to improve the affinity for glucose and/or its detection. The obtained results suggest the use of the protein as a probe for a stable glucose biosensor.

Archaea↗

PubMed2006

Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor.

Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA II has a K d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5-10 pM in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-Zn complex and TPEN are both apoptogenic for this cell line.

Animals↗

PubMed2003

A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles.

A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5'-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The detection level can be tuned to several orders of magnitude, from 100 nM to over 200 muM, through addition of an inactive variant of the DNAzyme. The concept developed here can be applied to the design of nucleic acid enzyme/nanoparticle sensors for analytes that are subject to in vitro selection, and thus can significantly expand the scope of nanomaterial applications and provide a novel approach to designing simple colorimetric biosensors.

Biosensing Techniques↗

PubMed2004

Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase.

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.

Animals↗

PubMed2005

Protein biosensors based on biofunctionalized conical gold nanotubes.

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.

Animals↗

PubMed2004

Development of a functionalized xenon biosensor.

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

Avidin↗

PubMed2005

Combined affinity and catalytic biosensor: in situ enzymatic activity monitoring of surface-bound enzymes.

Surface Plasmon Resonance Spectroscopy (SPR) and miniature Fiber Optic Absorbance Spectroscopy (FOAS) were combined to monitor in situ and quantitatively an enzymatic model reaction catalyzed by beta-lactamase. The enzyme was covalently immobilized to the gold surface of a SPR chip, which was functionalized with NeutrAvidin through a biotinylated alkanethiol self-assembled monolayer, thus serving as a highly sensitive affinity biosensor. SPR was used to control the density of the surface-bound enzyme. Nitrocefin as the enzymatic substrate was allowed to react with the immobilized enzyme in the SPR flow cell, and its turnover was detected with the FOAS system acting as the catalytic biosensor. The coupling of the two techniques has a substantial potential for highly controlled on-line monitoring of surface-bound enzyme activity. The FOAS technique may also be easily employed as an add-on device to other types of affinity sensing instruments.

Biosensing Techniques↗

PubMed2006

A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes.

Fluorescent biosensors that facilitate reagentless sensitive detection of small molecules are crucial tools in the areas of therapeutics and diagnostics. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. An ATP-binding ribonucleopeptide (RNP) receptor was converted to a fluorescent ATP sensor without chemically modifying the nucleotide in the ATP-binding RNA. The RNA subunit of the ATP-binding RNP and a peptide modified with a pyrenyl group formed a stable fluorescent RNP complex that showed an increase in the fluorescence intensity upon binding to ATP. The strategy to convert the ATP-binding RNP receptor to a fluorescent ATP sensor was applied to generate fluorescent ATP-binding RNP libraries by using a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and a series of fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded fluorescent ATP or GTP sensors with emission wavelengths varying from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which provided ATP sensors responding at wide concentration ranges of ATP. The combinatorial strategy using the modular RNP receptor reported here enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor.

Adenosine Triphosphate↗

PubMed2004

Development of a new method, based on a bioreactor coupled with an L-lactate biosensor, toward the determination of a nonspecific inhibition of L-lactic acid production during milk fermentation.

The development and characteristics of a bioreactor employing bacteria (Streptococcus thermophilus) encapsulated in Ca-alginate beads coupled with an L-lactate biosensor are reported. The biosensor comprises a carbon paste electrode modified with enzymes HRP (horseradish peroxidase), LOD (lactate oxidase), and FcH (ferrocene) as redox mediator. The measurement of L-lactate is based on the signal produced by H(2)O(2), the product of the enzymatic oxidation of L-lactate by LOD. The detection of H(2)O(2) is performed at the electrode surface via HRP/FcH at low operating potential (-100mV vs Ag/AgCl). Optimization studies were performed using the bioreactor in conjunction with an L-lactate electrode operating in a flow injection system to assess the ability of encapsulated bacteria to ferment carbohydrate solutions. The possibility of using the developed method to assess the fermentation capability of milk samples was evaluated. Bronopol (2-bromo-2-nitro propane-1,3-diol) was chosen to simulate the effect of an inhibitory agent of milk fermentation. The obtained results indicated that the evaluation of the amount of L-lactate amount produced through the bioreactor could be used as a measure of inhibition of lactic acid production in milk samples.

Alginates↗

PubMed2004

Determination of beta-lactams in milk using a surface plasmon resonance-based biosensor.

Two surface plasmon resonance (SPR)-based biosensor assays for detection of beta-lactam antibiotics in milk are reported. The assays are based on the enzymatic activity of a carboxypeptidase converting a 3-peptide into a 2-peptide, a reaction that is inhibited in the presence of beta-lactams. Antibodies were used to measure either the amount of formed enzymatic product or the amount of remaining enzymatic substrate. Both assays detected different beta-lactams at or below European maximum residue limits (MRLs), and the detection limit for penicillin G was 1.2 microg/kg and 1.5 microg/kg for the 2- and 3-peptide assays, respectively. The precision (CV) was < 5%, both within and between assays at the penicillin G MRL (4 microg/kg). The biosensor results obtained upon analysis of incurred milk samples were compared with results obtained by liquid chromatography (HPLC), and the method agreements were, in general, good.

Animals↗

PubMed2004

Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor.

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.

Alcohol Oxidoreductases↗