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PubMed1989

Stable-light-emitting Escherichia coli as a biosensor.

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 degrees C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.

Biosensing Techniques↗

PubMedDate not supplied

Fibre-optic biosensor based on luminescence and immobilized enzymes: microdetermination of sorbitol, ethanol and oxaloacetate.

We have investigated highly selective and ultrasensitive biosensors based on luminescent enzyme systems linked to optical transducers. A fibre-optic sensor with immobilized enzymes was designed; the solid-phase bioreagent was maintained in close contact contact with the tip of a glass fibre bundle connected to the photomultiplier tube of a luminometer. A bacterial luminescence fibre-optic sensor was used for the microdetermination of NADH. Various NAD(P)-dependent enzymes, sorbitol dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, were co-immobilized on preactivated polyamide membranes with the bacterial system and used for the microdetermination of sorbitol, ethanol and oxaloacetate at the nanomolar level with a good precision.

Biosensing Techniques↗

PubMed2006

Investigating oligonucleotide hybridization at subnanomolar level by surface plasmon resonance biosensor method.

We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.

Biosensing Techniques↗

PubMed2003

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors.

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.

Binding Sites↗

PubMed2003

Discrimination of peptides by using a molecularly imprinted piezoelectric biosensor.

Based on the direct formation of a molecularly imprinted polymer on gold electrodes, we have developed a peptide sensor for the detection of low-molecular-weight peptides. A new cross-linking monomer, (N-Acr-L-Cys-NHBn)(2), was employed to attach the surface of the chip and to copolymerize with other monomers. Interestingly, N-benzylacrylamide participates in the polymerization and recognition is carried out in an aqueous environment. By using quartz crystal microbalance detection, short peptides can be monitored by their interaction with plastic antibodies specific for the target peptides. The selectivity of molecularly imprinted polymers and the sensitivity of such artificial biosensors have been combined to differentiate between traces of oxytocin and vasopressin to the ng mL(-1) scale.

Amino Acid Sequence↗

PubMed2006

Controlled nanozeolite-assembled electrode: remarkable enzyme-immobilization ability and high sensitivity as biosensor.

An enzyme-immobilized nanozeolite-assembled electrode was prepared by controlled assembly of nanometer-sized Linder type-L zeolite (nano-LTL-zeolite) on an indium tin oxide (ITO) glass electrode surface, and subsequent immobilization of cytochrome c. Cyclic voltammetric (CV) and amperometric experiments showed that, relative to other reported electrodes, the enzyme-immobilized electrodes possess fast electron-transfer rates (2.2 s(-1)), a broad linear range (15-540 micromol L(-1)), a low detection limit (3.2 nmol L(-1)), a remarkably long lifetime (5 months), and high stability in the pH range 5-10. These characteristics could be due to the fact that nanozeolites assembled on ITO have high immobilization ability and facilitate interaction with enzymes. The function controllability of these enzyme electrodes, resulting from the facile manipulability of nanozeolite-assembled layers, may provide a possibility to rationally design biosensors.

Biosensing Techniques↗

PubMed2001

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.

Automation↗

PubMedDate not supplied

Photostructurized electrochemical biosensors for bioreactor control and measurement in body fluids.

This article details a new type of biosensor for the simultaneous analysis of glucose, glutamate and glutamine in complex biological fluids like fermentation broths and blood. Simultaneous analysis was made possible by the application of different enzyme layers onto different electrodes of one photostructurized sensor. Photostructuring was done by means of a new developed photopolymer. Preparation of the photopolymer and the enzyme layers as well as the characterization of the sensors thus constructed with respect to linearity, response time and sensitivity are described.

Biosensing Techniques↗

PubMedDate not supplied

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers.

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.

Biosensing Techniques↗

PubMedDate not supplied

Peptide biosensor for recognition of cross-species cell surface markers.

Biosensors based on phage display-derived peptides as biorecognition molecules were used for the detection of cell surface cross-species markers in tissue homogenates. The peptide selected for murine myofibers was immobilized onto the surface of an acoustic wave sensor by biotin-streptavidin coupling. To detect peptide-receptor interaction, the sensors were exposed to muscle and control (kidney, liver, brain) tissue homogenates. The sensor showed a strong response to murine muscle. The amplitudes of the responses to the feline muscle homogenates were lower compared to those of the murine muscle, while the same K(d) indicated that the peptide has cross-species affinity. In contrast, murine kidney, liver and brain homogenates produced insignificant responses. Specificity of the sensor was shown in a blocking experiment, as reduced signal was detected when muscle preparations were preincubated with free peptide. Additionally, when muscle-specific peptide was replaced with two different random control peptides, the sensors produced no response to murine muscle. Suitability of peptide ligands for a variety of species can be evaluated using this technology.

Acoustics↗

PubMedDate not supplied

The use of the resonant mirror biosensor to detect point mutations, as demonstrated with synthetic oligonucleotides.

The possibility of using the resonant mirror biosensor to detect point substitutions in oligonucleotides was demonstrated with a fragment of the Helicobacter pylori 23S rRNA gene, point mutations in which are responsible for clarythromycin resistance. Conditions were optimized for the interaction of a probe immobilized on the sensing surface with targets containing various nucleotide substitutions. A probe allowing reliable discrimination of mutant targets was selected. The mismatch position in the probe was shown to affect the kinetic parameters (response) of hybridization with mutant targets, reporting not only the position, but also the character (G or C) of a substitution.

Base Pair Mismatch↗

PubMedDate not supplied

Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor.

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.

Cystic Fibrosis↗

PubMed2001

Monitoring arterio-venous differences of glucose and lactate in the anesthetized rat with or without brain damage with ultrafiltration and biosensor technology.

Continuous monitoring of arterio-venous glucose and lactate differences may serve as a diagnostic tool to assess normal brain function and brain pathology. We describe a method and some results obtained with arterio-venous measurements of glucose and lactate in the blood of the halothane-anesthetized rat and after brain injury. The method is based on low flow rate ultrafiltration for continuous collection of blood filtrate combined with flow injection analysis and biosensors for the detection of glucose and lactate. We measured the glucose and lactate concentration every minute in the jugular vein and the aorta at control conditions and during and after inflation of an embolectomy-balloon for 2 min. Net cerebral lactate efflux and glucose uptake was seen under control conditions and at low blood lactate levels. During brain injury both lactate release and glucose uptake were reduced and there was a net lactate influx at high arterial lactate levels. These results indicate that the flux of lactate in and out of the brain is not only dependent on the lactate concentration in the brain, but on blood levels as well, possibly because of bi-directional flux through the monocarboxylate transporter type 1.

Anesthetics↗

PubMed2004

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor.

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.

Antibodies↗

PubMed2004

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase.

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.

Adrenodoxin↗

PubMed1999

The optical biosensor studies on the role of hydrophobic tails of NADPH-cytochrome P450 reductase and cytochromes P450 2B4 and b5 upon productive complex formation within a monomeric reconstituted system.

The optical biosensor study of interaction between microsomal proteins-NADPH-cytochrome P450 reductase, cytochrome P450 2B4, and cytochrome b5-was carried out in the monomeric reconstituted system in the absence of phospholipids. The formation of individual complexes was kinetically characterized and their association and dissociation rate constants were determined. The association rate constants for the complexes formed were found to be close to the diffusiion limit-(0.5-4) x 10(6) M-1 s-1-while their dissociation rate constants did not exceed 0.5 s-1. It was shown that the interprotein electron transfer can occur both through complex formation and due to random collision. The dominant role of hydrophobic membraneous protein fragments in formation of productive electron transfer complexes was demonstrated.

Animals↗

PubMed2001

Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.

Binding Sites↗

PubMed1993

A flow-injection biosensor system for the amperometric determination of creatinine: simultaneous compensation of endogenous interferents.

A flow-injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100-microliters injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (< 3%) and long operational stability (> 1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.

Amino Acid Oxidoreductases↗