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At least 109 records · Page 6Linked to original sources
PubMedDate not supplied

Discarding multidrug resistance inducers, the possible role of a biosensing reporter in antimicrobial discovery.

The aim of the present work was to determine whether the luminescence-based reporter plasmid pQacLux could be applied to drug discovery in order to discard compounds with defined properties. Non-pathogenic Staphylococcus aureus RN4220 cells bearing pQacLux were incubated with different concentrations of a disinfectant of common use in hospitals. The in vivo light emission response of the plasmid to the given stimuli was then quantified and compared to a negative control for the construction of dose-response curves. The selected disinfectant provided a convenient model for the activity of quaternary ammonium compounds. In spite of the use of a raw model solution, the system revealed high levels of sensitivity. According to the results obtained, pQacLux could be conveniently used in the first steps of drug development in order to discard all possible multidrug resistance inducers.

Biosensing Techniques↗
PubMed2006

Lung cell fiber evanescent wave spectroscopic biosensing of inhalation health hazards.

Health risks associated with the inhalation of biological materials have been a topic of great concern; however, there are no rapid and automatable methods available to evaluate the potential health impact of inhaled materials. Here we describe a novel approach to evaluate the potential toxic effects of materials evaluated through cell-based spectroscopic analysis. Anchorage-dependent cells are grown on the surface of optical fibers transparent to infrared light. The probe system is composed of a single chalcogenide fiber (composed of Te, As, and Se) acting as both the sensor and transmission line for infrared optical signals. The cells are exposed to potential toxins and alterations of cellular composition are monitored through their impact on cellular spectral features. The signal is collected via evanescent wave absorption along the tapered sensing zone of the fiber through spectral changes between 3,000 and 600 cm(-1) (3,333-16,666 nm). Cell physiology, composition, and function are non-invasively tracked through monitoring infrared light absorption by the cell layer. This approach is demonstrated with an immortalized lung cell culture (A549, human lung carcinoma epithelia) in response to a variety of inhalation hazards including gliotoxin (a fungal metabolite), etoposide (a genotoxin), and methyl methansesulfonate (MMS, an alkylating agent). Gliotoxin impacts cell metabolism, etoposide impacts nucleic acids and the cell cycle, and MMS impacts nucleic acids and induces an immune response. This spectroscopic method is sensitive, non-invasive, and provides information on a wide range of cellular damage and response mechanisms and could prove useful for cell response screening of pharmaceuticals or for toxicological evaluations.

Air Pollutants↗
PubMed2005

Gold nanoparticles for microfluidics-based biosensing of PCR products by hybridization-induced fluorescence quenching.

Colloidal gold nanoparticles were used to develop a simple microfluidics-based bioassay that is able to recognize and detect specific DNA sequences via conformational change-induced fluorescence quenching. In this method, a self-assembled monolayer of gold nanoparticles was fabricated on the channel wall of a microfluidic chip, and DNA probes were bonded to the monolayer via thiol groups at one end and a fluorophore dye was attached to the other end of the probe. The created construct is spontaneously assembled into a constrained arch-like conformation on the particle surface and, under which, the fluorescence of fluorophores is quenched by gold nanoparticles. Hybridization of target DNAs results in a conformational change of the construct and then restores the fluorescence, which serves as a sensing method for the target genes. The nanocomposite constructed on the glass surface was characterized by UV absorbance measurement and the quenching efficiency for different fluorophores was evaluated by Stern-Volmer studies. The applicability of proposed assay was first demonstrated by the use of a pair of synthesized complementary and noncomplementary DNA sequences. The method was further applied for the detection of the PCR product of dengue virus with the use of enterovirus as the negative control, and results indicate that the assay is specific for the target gene. Moreover, using this approach, dehybridization, hybridization, and detection of the target genes can be performed in situ on the same microfluidic channel. Thus, this method could be regarded as one-pot reaction and it holds great promises for clinical diagnostics.

Biosensing Techniques↗
PubMedDate not supplied

Proteins, recognition networks and developing interfaces for macromolecular biosensing.

Genomics and proteomics discovery is leading to the identification of all proteins and to the opportunity, and challenge, to reveal the protein recognition networks that drive virtually all biological processes. Over the past decade, biosensors have emerged as a key technology for detection and analysis of biomolecular interactions. An important limitation in developing such biosensors is that the focus has been mainly on sensor platforms, the transducing hardware that converts interaction signals into recorded data, without adequately considering the role of molecular interfaces, the elements of sensors that interact with analytes to produce signals. We have investigated this alternative focus by identifying and, where necessary, designing molecular interfaces that will more effectively drive new biosensor development and utilization in biomedical and biotechnological investigations. Here we describe our recent studies of coiled coil and lipid bilayer interfaces and the potential to use these to expand sensing technologies for multiplexed target detection and analysis in increasingly biologically relevant membrane like environments.

Amino Acid Motifs↗
PubMed2006

Biocatalytically induced formation of cupric ferrocyanide nanoparticles and their application for electrochemical and optical biosensing of glucose.

The enzymatically controlled growth of cupric ferrocyanide nanoparticles in the presence of glucose oxidase, its ferricyanide electron acceptor, and copper ions is described. The biocatalytically stimulated growth of these nanoparticles on the surface of carbon-paste electrodes results in an amplified electrochemical detection of the glucose substrate. This concept can readily be expanded for monitoring a wide range of biocatalytic processes involving the ferricyanide electron acceptor.

Biopolymers↗
PubMed1995

Lifetime-based fluorescence energy transfer biosensing of zinc.

A new type of fluorescence transduction method for determining zinc in solution is described. The approach is based upon energy transfer from a fluorescent label on an enzyme, human carbonic anhydrase II, to a colored inhibitor which binds to zinc in the enzyme active site. If zinc is present in solution, it binds to the apoenzyme, which in turn permits the inhibitor to bind to the enzyme; the inhibitor is thus in close proximity to the label on the enzyme and thereby quenches the label's fluorescence by Forster energy transfer with a concomitant reduction of its lifetime, which is quantitated by phase fluorometry.

Binding Sites↗
PubMed1999

Selectivity and sensitivity of fluorescence lifetime-based metal ion biosensing using a carbonic anhydrase transducer.

A key performance criterion for metal ion determinations in complex media like serum, cytoplasm of the cell, and sea water is selectivity: the ability to determine the analyte(s) of interest, in the presence of relatively high concentrations of interferents. Cu(II), Zn(II), Cd(II), Co(II), and Ni(II) may be determined by changes they induce in the fluorescence lifetime and intensity of site-specifically labeled fluorescent variants of apocarbonic anhydrase II. Free metal ion concentrations in the picomolar range (for Cu(II) and Zn(II)) and the nanomolar range (for Cd(II), Co(II), and Ni(II)) were determined, based on the affinity of the apoenzyme for these ions. Mg(II) at 50 mM and Ca(II) at 10 mM produced no effect. By the use of different fluorescent labels, transducers were made which responded well to Cu(II), Co(II), and Ni(II), but not to Zn(II) and Cd(II), and vice versa.

Biosensing Techniques↗
PubMed2000

Application of high-density optical microwell arrays in a live-cell biosensing system.

In this paper, we use optical imaging fibers to fabricate a chemical and biochemical sensor that utilizes the ability of living cells to respond to biologically significant compounds. The sensor is created by randomly dispersing single NIH 3T3 mouse fibroblast cells into an optically addressable fiber-optic microwell array such that each microwell accommodates a single cell. The cells are encoded to identify their location within the array and to correlate changes or manipulations in the local environment to responses of specific cell types. The entire array can be simultaneously measured, yielding a rapid, repetitive, and high-density analysis method.

3T3 Cells↗
PubMed1997

Screening and characterization of new enzymes for biosensing and analytics.

The development of new or improved analytical methods requires new enzymes. Screening techniques utilizing enrichment cultures and rapid assay methods supported by automated or miniaturized methods are useful tools to detect new enzyme producers. Notably, oxidoreductases are well suited for analytical purposes. The NAD(P)- and oxygen-independent quinoprotein dehydrogenases with a covalently bound redox cofactor can be used advantageously for the development of biosensors. Examples are given of selective enrichment methods used in screening for useful enzyme-producing microorganisms. Enrichment under chemostatic conditions proved to be successful because enzymes with a remarkably high affinity against the analyte could be obtained. This is demonstrated by the screening of a trimethylamine-converting enzyme. The frequently observed high selectivity of these enzymes against the substrate is demonstrated in a few examples. In exploitation of these new oxidoreductases, new analytical methods were developed which are useful for the detection and during monitoring of phenylketonuria (PKU) or maple syrup urine disease (MSUD).

Alcohols↗
PubMed1997

Thermistor-based biosensing.

In this review a universal thermistor-based biosensor system is described with examples from clinical chemistry, bioprocess monitoring and environmental control. The technique is based on the measurement of the small temperature changes associated with enzymatic reactions occurring in a microreactor with immobilized enzyme. The system has good operational stability and a sensitivity that permits measurements down to 1 microM concentrations. Current developments include devices constructed by micromachining for multisensing purposes and miniaturised instrumentation intended for use in portable monitoring. With use of special supports for enzyme immobilisation even untreated whole blood samples can be applied. Another current line of investigation involves hybrid biosensors, such as combinations of electrochemistry and calorimetry into bioelectrocalorimetric devices with interesting new properties.

Alcohols↗
PubMed2002

Quartz-crystal sensors for biosensing and chemical analysis.

The principle and applications of quartz-crystal sensors based on the three basic concepts for mass, viscosity, and viscoelastic changes are reported. In the general discussion the realization of a resonant frequency-resonant resistance diagram is described in detail. As an example of application to mass sensing, gas sensing with a carbon-coated quartz crystal is reported. Determination of the blood coagulation factor is used as an example of the application to viscosity sensing. As an example of viscoelastic measurement, an ion-exchange polymer-coated quartz crystal is investigated to show that viscoelasticity changes more than mass in the transport process. The possibility of developing new biosensors and chemical sensors is discussed on the basis of these results.

Animals↗
PubMed2005

Synthesis and analytical properties of micrometric biosensing lipobeads.

This paper describes the preparation for the first time of lipobead-based micrometric fluorescence biosensors and the optimization of their analytical properties. The study focused on the well-established urea biosensors as a model system. Fluorescence-sensing lipobeads were prepared by coating carboxyl-functionalized silica microspheres with phospholipids. The enzyme urease and the pH indicator fluorescein-5-thiosemicarbazide were then attached covalently to the phospholipid membrane of the lipobeads. Urease converts urea to ammonia, which results in a pH increase in the analyte solution and to a urea concentration-dependent increase in the fluorescence intensity of the sensing lipobeads. Previous fluorescence-sensing lipobeads were synthesized by coating polystyrene particles with a phospholipid membrane. The membrane was physically attached to the particles and the fluorophores were entrapped in the membrane. In this study, we prepared improved fluorescence-sensing lipobeads by utilizing covalent chemistry to bind the phospholipid membrane to the silica particles and the fluorophores to the membrane. This led to improvement in the stability of the newly developed urea-sensing lipobeads compared to previously developed miniaturized fluorescence biosensors.

Biosensing Techniques↗
PubMed2006

ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls.

Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Among the metabolites of PCBs, dihydroxylated PCBs are also considered as toxic compounds. Various studies have shown that dihydroxylated PCBs affect the reproductive, immune, nervous, and endocrine systems. Detection of these chemicals in environmental and biological samples could provide first-hand information about their levels and lead to a better understanding of their role in toxicity. To that end, we developed a sensing system for the detection of dihydroxylated PCBs based on the clc operon. The Pseudomonas putida clc operon encodes a catabolic pathway for degradation of chlorocatechols, which are major metabolites of a large number of chlorinated compounds. In P. putida, the expression of these genes is regulated by a protein encoded by the gene clcR located upstream from the clcABD genes. We demonstrate here for the first time that dihydroxy PCBs can also induce the clc operon. Our sensing system employs P. putida bacteria harboring a plasmid in which the reporter gene, lacZ, is under the control of the regulatory protein ClcR. Consequently, when exposed to dihydroxy PCBs, the bacteria express beta-galactosidase in an amount related to the concentration of the corresponding dihydroxy PCB. Various dihydroxylated PCBs, differing in the number and position of chlorines and in the position of hydroxyls, were tested for their ability to induce expression of beta-galactosidase. Detection limits as low as 1 x 10(-6) mol L(-1) were obtained for various dihydroxylated PCBs.

Biosensing Techniques↗
PubMed1996

Potentiometric alternating biosensing system for in continuum toxicity tests on cell population.

In this paper, we investigate the possibility to monitor, in continuum, changes of cellular metabolism. Examples of this feature are shown: metabolic changes due to drugs administration are evaluated as a decrease of extracellular acidification rate on CHO-K1 and HeLa cells. Parallel experiments with a standard test were performed to validate the results obtained with the biosensor.

Animals↗
PubMed1992

A lysine dehydrogenase-based electrode for biosensing of L-lysine.

An amperometric biosensor for L-lysine based on the recently isolated enzyme lysine dehydrogenase is described. Immobilization of the enzyme onto a platinum electrode is achieved via entrapment within a gelatin support on a cellulose membrane. Anodic detection (at 0.4 V vs. Ag/AgCl) is facilitated by the presence of a redox-mediating ferricyanide ion. The effect of experimental variables such as pH, enzyme loading, applied potential, cofactor and mediator concentrations were evaluated in order to optimize the analytical performance. A detection limit of 7 x 10(-8) M, and linearity up to 7 x 10(-4) M are reported. The fast response permits adaptation for flow injection operation with good precision (RSD = 1.9%) and high sample throughout (40 samples per hour). The high specificity offered by this new enzyme is indicated by the lack of interference by other L-amino acids, alcohols or carbohydrates.

Amino Acid Oxidoreductases↗
PubMed1994

Organic-phase biosensing of secondary alcohols with a Ta. brockii alcohol dehydrogenase electrode.

An organic-phase enzyme electrode for monitoring secondary alcohols is described. The new biosensor relies on the biocatalytic activity of Ta. brockii alcohol dehydrogenase in non-aqueous media. The Eastman-AQ 55D polymeric coating, known for its stability in organic solvents, is used to entrap the enzyme and its NADP+ cofactor onto the glassy carbon surface. Various experimental variables, influencing the response of the reagentless sensor, are explored for optimum performance. The solvent-induced changes in the enzymatic activity result in an extended linear range. The work expands the scope of organic-phase biosensors towards additional substrates of analytical significance.

Alcohol Dehydrogenase↗