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PubMed1995

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Animals↗

PubMed1997

The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted. Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently. Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.

ATP-Binding Cassette Transporters↗

PubMed2005

Laccase biosensors based on mercury thin film electrode.

A biosensor was developed by immobilizing laccase onto mercury thin film electrode (MTFE) by means of gelatin that is then crosslinked with glutaraldehyde. Mercury thin film (MTF) was deposited onto glassy carbon electrode (GCE) and the obtained biosensor was utilized for the determination of phenolic compounds. The measurement was based on the amperometric detection of oxygen consumption in relation to analyte oxidation. The optimum experimental conditions for the biosensor were investigated and the system was calibrated for both catechol and phenol. A linear relationship between sensor responses and analyte concentrations was obtained in concentration range between 0.5 x 10(-6)-5.0 x 10(-6)M for catechol and 2.5 x 10(-6)-2.0 x 10(-6)M for phenol, respectively. Mercury thin film was also formed onto the surface of screen printed graphite electrodes and applied for the catechol detection. The linearity was observed in concentration range between 2.5 x 10(-6)-3.0 x 10(-5)M.

Biosensing Techniques↗

PubMed2006

An amperometric biosensor for xanthine determination prepared from xanthine oxidase immobilized in polypyrrole film.

In order to prepare a biosensor for the determination of xanthine, electropolymerization of pyrrole on Pt surface was carried out with an electrochemical cell containing pyrrole, ferrocene (as a electron mediator) and tetrabutylamonium tetrafluoroborat in acetonitrile by cyclic voltammetry between 0.0 and 0.9V (vs SCE) at a scan rate of 50mV/s upon Pt electrode. Xanthine oxidase was immobilized by a glutaraldehyde/bovine serum albumin (BSA) crosslinking procedure on to polypyrrole film after the electropolymerization processes. The response of the biosensor against xanthine was measured after 3-4 min following the application of a constant potential of + 0.7 V (vs SCE). The resulting biosensor exhibits excellent electrocatalysis for the xanthine. The amperometric determination is based on the electrochemical detection of H202, which is generated in enzymatic reaction of xanthine. The effect of various experimental conditions was examined for the determination of the analytical performance. The sensor responds to xanthine with a detection limit of 1.0 x 10(-6)M. The response current increases linearly with xanthine concentration up to 4.0 x 10(-4) M. The sensor remains relatively stable for 45 days.

Animals↗

PubMed2006

A simple fluorescent biosensor for theophylline based on its RNA aptamer.

Theophylline is a potent bronchodilator with a narrow therapeutic index. A simple fluorescent biosensor that detects clinically relevant theophylline concentrations has been developed using the well-characterized theophylline binding RNA aptamer. Hybridization of the RNA aptamer to a fluorescently labeled DNA strand (FL-DNA) yields a fluorescent RNA:DNA hybrid that is sensitive to theophylline. The biosensor retains the remarkable selectivity of the RNA aptamer for theophylline over caffeine and is sensitive to 0-2 muM theophylline, well below the clinically relevant concentration (5-20 mg/L or approximately 10-50 muM). Adding a dabcyl quenching dye to the 3'-terminus of the fluorescently labeled DNA strand yielded a dual-labeled DNA strand (FL-DNA-Q) and increased the dynamic range of this simple biosensor from 1.5-fold to 4-fold.

Aptamers, Nucleotide↗

PubMed2003

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 +/- 8 nM amperometrically and 42 +/- 2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 +/- 3 and 70 +/- 14 nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highly correlated between brain and lymphocyte (r = .994) and between brain and blood (r = .997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Animals↗

PubMed2002

Biosensor for the enantioselective analysis of the thyroid hormones (+)-3,3',5-triiodo-L-thyronine (T3) and (+)-3,3',5,5'-tetraiodo-L-thyronine (T4).

An amperometric biosensor based on L-aminoacid oxidase is proposed for enantioselective assay of (+)-3,3',5-triiodo-L-thyronine (L-T3) and (+)-3,3',5,5'-tetraiodo-L-thyronine (L-T4), due to the fact that only the L enantiomer has the hormonal activity. The construction of the amperometric biosensor is simple and reproducible. The analytical information obtained from enantioselective analysis are reliable. The RSD <1% assured by using the amperometric biosensors for L enantiomers assay as raw materials, and from tablets, demonstrated their suitability for the analysis of T3 and T4 at ppb concentration levels.

Amino Acid Oxidoreductases↗

PubMed1995

Recent advances in amperometric glucose biosensors for in vivo monitoring.

Electrochemical biosensors for glucose, based on the specific glucose oxidizing enzyme glucose oxidase, have generated considerable interest. Several commercial devices based on this principle have been developed and are widely used for in vitro monitoring of glucose e.g. in hospitals, doctors surgeries and for home monitoring by patients themselves. A significant advance in the application of biosensor technology would be the development of portable, implantable sensors which could continuously indicate the blood glucose concentration, enabling swift corrective action to be taken by the patient. This review highlights recent developments in amperometric glucose biosensors for in vivo monitoring and also considers the remaining barrier which need to be overcome to enable successful introduction of an implantable sensor.

Biosensing Techniques↗

PubMed2006

A long-wavelength fluorescent glucose biosensor based on bioconjugates of galactose/glucose binding protein and Nile Red derivatives.

BACKGROUND: Fluorescent biosensors based on galactose/glucose binding protein (GGBP) and environmentally sensitive derivatives of the phenoxazine dye Nile Red are described. These biosensors are proposed as the sensing platform for a minimally invasive, continuous glucose monitoring system that can be implanted under the skin and read transdermally using an external fluorometer. METHODS: To construct the biosensors, the thiol-reactive Nile Red derivatives INR and IANR were prepared and conjugated to GGBP proteins possessing cysteine mutations that were designed for optimal site-specific fluorophore attachment. The attachment sites were selected to maximize the local environment change for attached dyes between the bound and unbound conformations of GGBP. RESULTS: Fluorescence responses at the selected cysteine sites of GGBP upon binding to glucose showed that the conjugates typically yielded fluorescence emission around 640-650 nm with up to 50% changes in fluorescence intensity. Conjugate E149C/A213C/L238S INR GGBP also displayed glucose binding in the human physiological range (K (D) = 7.4 mM). CONCLUSIONS: The phenoxazine derivatives fluoresced at longer wavelengths (>600 nm) approaching the near-infrared spectral window, where interference from scattering and tissue absorbance are minimal. Ultimately, we expect that monitoring systems based on GGBP and longwavelength dyes will be implanted for up to 6 months and can be used to transmit information through the skin to an external monitor.

Biosensing Techniques↗

PubMed1996

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Antibodies, Monoclonal↗

PubMed2003

Biosensor reporting of root exudation from Hordeum vulgare in relation to shoot nitrate concentration.

The aim of this study was to determine the relationship between shoot nitrate concentration, mediated by nitrate supply to roots, and root exudation from Hordeum vulgare. Plants were grown for 14 d in C-free sand microcosms, supplied with nutrient solution containing 2 mM nitrate. After this period, three treatments were applied for a further 14 d: (A) continued supply with 2 mM nitrate (zero boost), (B) supply with 10 mM nitrate (low boost), and (C) supply with 20 mM nitrate (high boost). At the end of the treatment period, a bacterial biosensor (Pseudomonas fluorescens 10586 pUCD607, marked with the lux CDABE genes for bioluminescence) was applied to the microcosms to report on C-substrate availability, as a consequence of root exudation. The nitrate boost treatments significantly affected shoot nitrate concentrations, in the order C>B>A. In treatments receiving a nitrate boost (B, C), increased shoot nitrate concentration was correlated with increased plant biomass, reduced root length, reduced number of root tips, and increased mean root diameter, relative to the no boost treatment (A). Imaging of biosensor bioluminescence (proportional to metabolic activity in response to availability of root exudates) indicated that root exudation increased with decreasing shoot nitrate concentration. Biosensor reporting of root C-flow indicated that exudation was greater from root tip regions than from the whole root, but that specific exudation rates for all sites were unaffected by treatments. Total root exudation across treatments was found to be closely correlated with total root length, indicating that increased root exudation, per unit root biomass, with decreasing nitrate supply was associated with altered root morphology, as a consequence of systemic plant responses to internal N-status.

Biomass↗

PubMed2000

From DNA biosensors to gene chips.

Wide-scale DNA testing requires the development of small, fast and easy-to-use devices. This article describes the preparation, operation and applications of biosensors and gene chips, which provide fast, sensitive and selective detection of DNA hybridization. Various new strategies for DNA biosensors and gene chips are examined, along with recent trends and future directions. The integration of hybridization detection schemes with the sample preparation process in a 'Lab-on-a-Chip' format is also covered. While the use of DNA biosensors and gene chips is at an early stage, such devices are expected to have an enormous effect on future DNA diagnostics.

Biosensing Techniques↗

PubMed2005

Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies.

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic -794 CATT(5-8) repeat and -173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

Biosensing Techniques↗

PubMed1987

Introduction to the principles and applications of biosensors.

A biosensor is an analytical device that responds to an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and an electrochemical transducer. As a result of recent scientific and technological progress, such devices are likely to play an increasingly important role in generating analytical information in all sectors of human endeavour, from medicine to the military. In particular, biosensors will form the basis of cheap, simple devices for acquiring chemical information, bringing sophisticated analytical capabilities to the non-specialist and general public alike. The market opportunities for the rapid exploitation of novel developments in this sector are substantial. Biosensor research is also likely to have a significant impact on the development of modern electronics.

Biotechnology↗

PubMed2005

Communication scheduling to minimize thermal effects of implanted biosensor networks in homogeneous tissue.

A network of biosensors can be implanted in a human body for health monitoring, diagnostics, or as a prosthetic device. Biosensors can be organized into clusters where most of the communication takes place within the clusters, and long range transmissions to the base station are performed by the cluster leader to reduce the energy cost. In some applications, the tissues are sensitive to temperature increase and may be damaged by the heat resulting from normal operations and the recharging of sensor nodes. Our work is the first to consider rotating the cluster leadership to minimize the heating effects on human tissues. We explore the factors that lead to temperature increase, and the process for calculating the specific absorption rate (SAR) and temperature increase of implanted biosensors by using the finite-difference time-domain (FDTD) method. We improve performance by rotating the cluster leader based on the leadership history and the sensor locations. We propose a simplified scheme, temperature increase potential, to efficiently predict the temperature increase in tissues surrounding implanted sensors. Finally, a genetic algorithm is proposed to exploit the search for an optimal temperature increase sequence.

Biosensing Techniques↗

PubMed2004

Pure shear horizontal SAW biosensor on langasite.

The undetected introduction of pathogens into food or water supplies can produce grave consequences in terms of economic loss and human suffering. Sensitive and selective sensors capable of quickly detecting microbial pathogens are urgently needed to limit the effects of bioterrorist incidents, accidents, or pollution. Shear horizontal surface acoustic wave (SH SAW) devices provide an attractive platform for the design of microbial biosensors that function in liquid media, where Rayleigh-type modes are rapidly attenuated. This paper reports on an exploratory SH SAW delay line designed and fabricated on langasite, La3Ga5SiO14 (LGS), along the novel Euler propagation direction (0 degrees, 22 degrees, 90 degrees). A liquid chamber was fabricated and attached to the top surface, and the device was submitted to liquid and biochemical tests. Moderate (6 dB) additional attenuation of the transmission coefficient, /S21/, was consistently observed when the SH SAW delay line was assembled in the test fixture and submitted to the liquid tests, indicating that LGS is an attractive candidate for liquid sensing. Sensor selectivity can be achieved by integrating the LGS SH SAW delay line with a biochemical recognition layer. A test setup was implemented for the characterization of LGS SH SAW-based biosensors. The delay line response to biomolecule binding was shown by detection of sequential binding of proteins to the SH SAW device delay path. The biotinylated sensor was exposed sequentially to biotin-binding deglycosylated avidin, biotin-modified rabbit IgG, and goat anti-rabbit IgG antibody. As each protein was bound to the sensing surface, marked changes in the delay-line phase were recorded. The reported results demonstrate the capability of these devices to act as biochemical detectors in aqueous solutions, and this work represents the first effort using the novel material LGS in SAW-based biosensor technology.

Acoustics↗

PubMed2002

Construction of a fluorescent biosensor family.

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

Amino Acid Sequence↗

PubMed2000

Resonant mirror biosensor analysis of type Ialpha cAMP-dependent protein kinase B domain--cyclic nucleotide interactions.

A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ialpha regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (Ki' = Ki(cAMP)/Ki(analog)) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published Ki' values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The Ki' values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions.

Amino Acid Substitution↗