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PubMed2005

Implementation of laboratory order data in BioSense Early Event Detection and Situation Awareness System.

INTRODUCTION: Laboratory test orders constitute an early outbreak data source. CDC receives laboratory order data in HL7 format from the Laboratory Corporation of America (LabCorp) and plans to use the data in the BioSense Early Event Detection and Situation Awareness System. METHODS: These LabCorp data contain information on tests ordered and include the type of test ordered and the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)-coded reasons for the order. A consensus panel was formed to group test orders on the basis of expert opinion into eight standard syndrome categories to provide an additional data source for early outbreak detection. A laboratory order taxonomy was developed and used in the mapping consolidation phase. The five main classes of this taxonomy are miscellaneous functional tests, fluid screening tests, system-specific tests, tests for specific infections (by primary manifestation), and tests for specific noninfectious diseases. RESULTS: Summary of numbers of laboratory order codes in each syndrome category are fever (53), respiratory (53), gastrointestinal (27), neurological (35), rash (37), lymphadenitis (20), localized cutaneous lesion (11), and specific infection (63). CONCLUSION: With the daily use of laboratory order data in BioSense, the actual distribution of laboratory order codes in syndrome groups can be evaluated, allowing modification of the mapping.

Clinical Laboratory Information Systems↗
PubMed2006

Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection.

Among protein biosensors, those based on enzymatic responses to specific analytes offer convenient instruments for fast and ultra-fast molecular diagnosis, through the comparative analysis of the product formed in presence and in absence of the effector. We have explored here the performance of five beta-galactosidase substrates during the activation of a beta-galactosidase sensor by antibodies against the human immunodeficiency virus (HIV). Interestingly, the employed substrate determines the dynamic range of the allosteric signal and significantly influences the sensitivity of the senso-enzymatic reaction. While ortho-nitrophenyl beta-D-galactopyranoside allows the detection of a model anti-gp41 monoclonal antibody below 0.024 ng/microL, phenol red beta-D-galactopyranoside offers the most dynamic response with signal/background ratios higher than 12-fold and a detection limit around 0.071 ng/microL. The hydrolysis of both chromogenic substrates generates linear sensing responses to immune human sera and parallel time-course topologies of the allosteric reaction. Therefore, the obtained results stress the potential of chromogenic substrates versus those rendering quimioluminescent, amperometric, or fluorescent signals, for the further automatization, miniaturization, or adaptation of beta-galactosidase-based biosensing to high-throughput applications.

Allosteric Regulation↗
PubMed2006

Radical polymerization in biosensing.

This review briefly summarizes recently published work on radical polymerization in biosensor-related applications. Advancements in surface modification aimed at improving sensor biocompatibility and reducing nonspecific background noises are discussed. Direct applications of polymers as one of the key sensing elements in which they are used either as detection probes for the biomolecular binding events or as signal transducers to amplify sensing signals are detailed. Initial applications of radical polymerization reactions in biosensing are evident and appear promising.

Artifacts↗
PubMed2006

Anthozoa red fluorescent protein in biosensing.

The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.

Animals↗
PubMed1995

Biosensing with surface plasmon resonance--how it all started.

A subjective description is given of how the development of surface plasmon resonance for immunosensing began. The main differences between the initial experiments and a commercially available instrumentation are pointed out. For the practical use of surface oriented methods for biosensing it is noted that the arrangements around the optical system itself, such as the sensing chip or sample cell, are most important. It is concluded that the instrumentation developed can be used not only for immunosensing but also for "real time biospecific interaction analysis" in general. It is pointed out that the use of surface plasmon resonance for detection is only one possibility and that many new (optical) methods for real time biospecific interaction analysis have been and will be developed.

Biosensing Techniques↗
PubMed1996

Bispecific antibody modification of nicotinic acetylcholine receptors for biosensing.

Recent results show that bispecific antibodies can be used to tailor the selectivity of nicotinic acetylcholine receptors for biosensing purposes. The nicotinic acetylcholine receptors reconstituted in bilayer lipid membranes are inactivated when two bispecific antibodies, attached to the same receptor, bind to a single antigen molecule. Experiments with patch clamp recording equipment reveal that antigen levels of 10(-8) M completely and irreversibly inactivate small numbers of nicotinic acetylcholine receptors. This approach may lead to the construction of biosensors capable of detecting individual antibody-antigen (Ab-Ag) binding events.

Animals↗
PubMed2004

Properties of mixed alkanethiol-dendrimer layers and their applications in biosensing.

We studied the properties of mixed alkanethiol-dendrimer layers on a gold support and their application in biosensing. We showed that properties of glucose sensor can be modified using a different ratio of 1-hexadecanethiol (HDT) and poly(amidoamine) dendrimer of first generation (G1). The cyclic voltammetry in the presence of the redox couple, Fe(CN)(6)(3-)/Fe(CN)(6)(4-), was used for estimating how effectively the layer blocks the redox probe's access to the electrode surface. A scanning electrochemical microscope (SECM) was used to image the resulting distribution of the organic compounds. We found that with increasing content of dendrimers, the integrity of the layers was improved.

Biosensing Techniques↗
PubMed2026

From host response to genomic targets: electrochemical biosensing of tuberculosis biomarkers.

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, with timely diagnosis continuing to be a major challenge, particularly in resource-limited settings. Conventional TB diagnostic methods are limited by low sensitivity, long turnaround times, and an inability to reliably differentiate latent from active disease. Biomarker-based diagnostic strategies have therefore gained increasing attention as they offer the potential to improve early detection, disease differentiation, and treatment monitoring. Herein, we examine electrochemical biosensing strategies for TB diagnostics using a biomarker-class-driven framework, covering host-response biomarkers (IFN-γ and TNF-α), pathogen-derived antigens (ESAT6, CFP10, CFP10-ESAT6, MPT64, Ag85, HspX and LpqH), cell-wall signatures and whole-cell markers (LAM and whole cell Mtb), and genomic markers (Mtb DNA and IS6110). Through structured comparison of recognition elements, biointerface designs, signal amplification strategies, electrochemical techniques, matrices, and validation levels, this review identifies the most promising technical approaches for different TB biomarker classes. It further highlights key translational bottlenecks, including limited clinical validation, buffer-based testing, complex multistep amplification, redox-probe dependence, matrix fouling, and insufficient evidence of manufacturability. This review therefore provides practical guidance for developing electrochemical TB biosensors that are analytically sensitive, clinically relevant, and suitable for decentralized diagnostic applications.

Biosensing Techniques↗
PubMed2004

On-chip biosensing of estrogen receptor-alpha at single molecular level.

A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5'-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.

Binding Sites↗
PubMed2004

Development of an amperometric biosensing method for the determination of L-fucose in pretreated urine.

The first amperometric biosensing method for the determination of L-fucose is described. L-Fucose is the objective of much current research, as it is considered as a potential marker for various pathologic disorders. Recombinant L-fucose dehydrogenase, having as cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD+P), was cross-linked in a water-soluble photosensitive polymer matrix, that is, polyvinyl alcohol (PVA) modified with styrylpyridinium (SbQ), in the presence of BSA and glutaraldehyde. The resulting membrane was sandwiched between two polycarbonate membranes and was mounted in an amperometric cell. The oxidation of the enzymatically produced NADPH was monitored at a platinum anode at +0.25 V versus a silver pseudoreference electrode in the presence of ferricyanide. The system was fully optimized with respect to various analytical parameters. Regarding to the mechanical properties of the membrane and the storage stability of the immobilized enzyme, various parameters were also optimized. Several methods for the pretreatment of urine samples were investigated. Treatment of the samples with PbO2 found to eliminate the interference effect of various electroactive species exist in urine; optimum incubation time was determined since at prolonged incubation times L-fucose is also affected. Calibration curves for the direct and the mediated monitoring of NADPH were liner over the concentration ranges 0.04-1.0 mM (r2=0.9995) and 0.03-3.0 mM (r2=0.9997) fucose, respectively. The detection limits (S/N 3) were 2 and 1.5 microM fucose, respectively. The R.S.D. of the mediated biosensor is better than 1.5% (n=10, 0.5 mM fucose). The proposed biosensor correlates well with a reference enzymatic method and exhibits very good working and storage stability.

Biosensing Techniques↗
PubMed2005

Metal clad leaky waveguides for chemical and biosensing applications.

Novel metal clad leaky waveguide (MCLW) sensor devices have been developed for sensing applications. These chips are designed to confine the light in a low refractive index waveguide that encompasses the chemically-selective layer, maximising the overlap between the optical mode and the chemistry, thus improving the sensitivity. In this work, a thin metal layer was inserted between the substrate and the thick waveguide layer, increasing the reflectivity of the waveguide/metal interface and decreasing the light lost at each of reflection in the leaky mode, which in turn increases the propagation distance. The device has been used for a range of biosensing applications, including the detection of organophosphoros pesticides. The limit of detection for paraoxon, based on absorbance detection, was calculated to be 6 nM. Refractive index detection was demonstrated by monitoring the change in the out-coupled angle resulting from the binding of protein A to anti-protein A immobilized on agarose. The sensor was also used for detecting the quenching of the fluorescence of an acid-base sensitive ruthenium complex immobilized within the sol-gel and with glucose oxidase enzyme. The limit of detection for glucose was 3 microM. The advantage of using the metal layer in the MCLW was that an electrical potential could be applied to accelerate the diffusion of the analyte to the immobilised antibody, which resulted in a shortened analysis time and a reduction in non-specific binding.

Biosensing Techniques↗
PubMed2005

Frog melanophores cultured on fluorescent microbeads: biomimic-based biosensing.

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.

Animals↗
PubMed2006

Maltose-binding protein: a versatile platform for prototyping biosensing.

The bacterial periplasmic-binding protein (PBP) superfamily members, in particular the maltose-binding protein, have been used extensively to prototype a variety of biosensing platforms. Although quite diverse at the primary sequence level, this protein superfamily retains the same basic two-domain structure, and upon binding a recognized ligand almost all PBPs undergo a conformational change to a closed structure. This process forms the basis for most, but not all, PBP-based biosensor signal transduction. Many direct detection or reagentless sensing modalities have been utilized with maltose-binding protein for both in vitro and in vivo detection of target compounds. Signal transduction modalities developed to date include direct fluorescence, electrochemical detection, fluorescence resonance energy transfer (FRET)-based detection, surface-tethered FRET sensing, hybrid quantum dot FRET sensing, and enzymatic detection, each of which have different benefits, potential applications and limitations.

Biosensing Techniques↗
PubMed2005

Electrothermally activated microchips for implantable drug delivery and biosensing.

Novel drug delivery and biosensing devices have the potential to increase the efficacy of drug therapy by providing physicians and patients the ability to precisely control key therapy parameters. Such "intelligent" systems can enable control of dose amount and the time, rate, and location of drug delivery. We have developed and demonstrated the operation of an electrothermal mechanism to precisely control the delivery of drugs and exposure of biosensors. These microchip devices contain an array of individually sealed and actuated reservoirs, each capped by a thin metal membrane comprised of either gold or multiple layers of titanium and platinum. The passage of a threshold level of electric current through the membrane causes it to disintegrate, thereby exposing the protected contents (drugs or biosensors) of the reservoir to the surrounding environment. This paper describes the theory and experimental characterization of the electrothermal method and includes in vitro release results for a model compound.

Algorithms↗
PubMed2006

Kinetic analysis of beta-amyloid peptide aggregation induced by metal ions based on surface plasmon resonance biosensing.

Recent studies suggest that beta-amyloid (Abeta) aggregation and toxicity are facilitated by metal ions. This study aims to evaluate the kinetics of Abeta aggregation/dissociation in the presence of metal ions and to investigate the efficacy of a metal chelator to disrupt the metal ion-induced Abeta aggregates. Soluble Abeta(1-40) peptide was immobilized on a surface plasmon resonance biosensing surface and aggregation induced by contact with soluble Abeta with or without metal ions. Our study revealed that all the tested metal ions promoted Abeta aggregation but with different kinetics. Among them, Cu(II) ions had the highest association constant, and reached the maximum binding in 10 min. However, the Cu(II)-induced Abeta aggregates were unstable. Other ions attained the maximum Abeta binding at much longer times: 45 min for Ca(II), 60 min for Fe(II), Fe(III), and Zn(II) ions. The Abeta aggregates induced by Fe(III) ions had the greatest stability. The metal ion-induced Abeta(1-40) aggregates could be disrupted by the metal chelator, EDTA, suggesting a metal chelator may serve as a pharmacological agent to interfere with Abeta aggregation. Finally, this study demonstrates that the SPR biosensor can be an effective and efficient setup to investigate the mechanism of Abeta aggregation.

Algorithms↗
PubMed2001

Fabrication of ultra-thin polypyrrole-glucose oxidase film from supporting electrolyte-free monomer solution for potentiometric biosensing of glucose.

A simple electropolymerisation process is described for the fabrication of an ultra-thin ( approximately 55 nm) polypyrrole (PPy)-glucose oxidase (GOD) film in a supporting electrolyte-free monomer solution for potentiometric biosensing of glucose. The optimum conditions for growing the ultra-thin film include 0.1 M pyrrole, 55-110 U/ml GOD, an applied current density of 0.05 mA/cm(2) and an electrical charge of 25 mC/cm(2). Long-term storage of the biosensor in acetate buffer improved the sensitivity of the biosensor by a factor of approximately two. The biosensor can also be used repeatedly for over 2 months with little or no loss in sensitivity. The interference effect of ascorbic acid was successfully reduced by inclusion of an outer PPy-Cl layer.

Biosensing Techniques↗
PubMed2002

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

Biosensing Techniques↗
PubMed2002

Biosensing of opioids using frog melanophores.

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.

Analgesics, Opioid↗