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At least 73 records · Page 4Linked to original sources
PubMed2003

Enzyme inhibition-based biosensors and biosensing systems: questionable analytical devices.

An overview of methods based on enzyme inhibition is presented. Both biosensors and biosensing systems (implemented in continuous and discontinuous approaches) are considered. The aim of the overview is to alert users on the use of these devices which, despite they can involve selective biocatalysts, the inhibition effect is not selective. Tables and examples illustrate this assertion.

Biosensing Techniques↗
PubMed2003

Biosensing under an applied voltage using optical waveguide lightmode spectroscopy.

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.

Biosensing Techniques↗
PubMed1997

Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers.

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected for different concentrations of antibody solutions. Oligolayers were studied in a second set of experiments. Dextran hydrogels were modified by thrombin inhibitors. The QCM response was used in a competitive binding assay to identify inhibitors for thrombin at different concentrations. Multilayers of proteins formed by self-assembly of a biotin-conjugate and streptavidin were investigated next. The QCM frequency response was monitored as a function of layer thickness up to 20 protein layers. A linear frequency decay was observed with increasing thickness. The decay per layer remained constant, thus indicating perfect mass coupling to the substrate. Frequency changes a factor of four higher were obtained in buffer solution as compared to measurements in dry air. This indicates a significant incorporation of water (75% weight) in the protein layers. This water behaves like a solid concerning the shear mode coupling to the substrate. The outlook discusses briefly the need for controlled molecular engineering of overlayers for subsequent QCM analysis, and the importance of an additional multiparameter analysis with other transducer principles and with additional techniques of interface analysis to characterize the mechanical coupling of overlayers as biosensor coatings. A promising trend concerns the use of QCM-arrays for screening experiments.

Biosensing Techniques↗
PubMed1998

Biosensing with G-protein coupled receptor systems.

Many cell membrane bound receptors communicate with the inside of the cell through guanine nucleotide binding proteins (G-proteins). This holds also for olfactory receptor neurons, which respond to odorants with G-protein mediated increases in the concentration of cyclic adenosine 3', 5'-monophosphate (cAMP) and/or inositol 1,4,5-triphosphate (InsP3). These substances regulate the ionic conductivity of the wall of the cilia. We have studied a similar system, namely G-protein coupled alpha 2-adrenoceptors, present for example in the cells of certain fish scales. These receptors react on, catecholamines and the G-protein mediates a decrease in cAMP, which causes an aggregation of pigment containing granulas to the middle of the cells. The light transmission of the cell increases due to this aggregation. This simple physiological response has been used in a sensitive biosensor for noradrenaline and for pertussis toxin that is based on isolated fish scales from cuckoo wrasse (Labrus ossifagus). The results were obtained with a simple photometer. Measurements can be performed also on single isolated melanophores. The main purpose of this contribution is, however, to point out that G-protein coupled receptors together with a simple physiological response form a principle for biosensing, which could also be an interesting alternative for odour sensing.

Animals↗
PubMed1998

Covalent photolinker-mediated immobilization of an intermediate dextran layer to polymer-coated surfaces for biosensing applications.

A new method is presented for the covalent binding of dextran as an intermediate layer on surface acoustic wave (SAW) devices. For biosensing applications in aqueous media commercially available SAW devices require surface passivation to prevent corrosion of the aluminum device structures in electrolytes. Thin films of polyimide and parylene revealed exceptional passivation properties. They were used as a base for dextran immobilization. Covalent binding of dextran to polymer-coated surfaces was achieved by photoimmobilization. Aryldiazirine-functionalized bovine serum albumin served as the multifunctional light-activable linking agent (photolinker polymer). Dextran and photolinker polymer were mixed and photobonded to sensor surfaces. Essential photoimmobilization parameters were optimized. The binding of proteins to dextran applying carbodiimide chemistries was exemplified with antiurease antibodies and the feasibility of specific immunosensing was investigated on SAW sensors connected to a fluid handling system.

Acoustics↗
PubMed1999

Composite films of Prussian blue and N-substituted polypyrroles: covalent immobilization of enzymes and application to near infrared optical biosensing.

We demonstrate the feasibility of optical biosensing using a material which, in essence, is a modified inorganic film to which various enzymes were covalently attached. Thin and transparent blue films composed of Prussian blue and incorporated into a network of N-substituted polypyrroles are sensitive to pH in the 5-9 range at 720 nm wavelength and can be modified with enzymes to result in the respective biosensors. Several methods of enzyme immobilization, using bifunctional crosslinking reagents, and various enzymes were tested. The best results were obtained using the one-step carbodiimide method which resulted in highly active, stable and transparent biosensor films for optical determination of urea and acetylcholine. The operational stability exceeded 1 month and even after 2 months of dry storage at room temperature the activity did not drop. The biosensors allow optical determination of the respective substrates in the millimolar concentration range.

Acetylcholinesterase↗
PubMed2005

Electrochemical detection of single a-g mismatch using biosensing surface based on gold nanoparticles.

The study of small drug molecules interacting with nucleic acids is an area of intense research that has particular relevance in our understanding of relative mechanism in chemotherapeutic applications and the association between genetics (including sequence variation) and drug response. In this contribution, we demonstrate how the sequence-specific binding of an anticancer drug Dacarbazine (DTIC) to single base (A-G) mismatch could be sensitively detected by combining electrochemical detection with biosensing surface based on gold nanoparticles.

Base Pair Mismatch↗
PubMed2005

NMR-based biosensing with optimized delivery of polarized 129Xe to solutions.

Laser-enhanced (LE) 129Xe nuclear magnetic resonance (NMR) is an exceptional tool for sensing extremely small physical and chemical changes; however, the difficult mechanics of bringing polarized xenon and samples of interest together have limited applications, particularly to biological molecules. Here we present a method for accomplishing solution 129Xe biosensing based on flow (bubbling) of LE 129Xe gas through a solution in situ in the NMR probe, with pauses for data acquisition. This overcomes fundamental limitations of conventional solution-state LE 129Xe NMR, e.g., the difficulty in transferring hydrophobic xenon into aqueous environments, and the need to handle the sample to refresh LE 129Xe after an observation pulse depletes polarization. With this new method, we gained a factor of >100 in sensitivity due to improved xenon transfer to the solution and the ability to signal average by renewing the polarized xenon. Polarized xenon in biosensors was detected at very low concentrations, </=250 nanomolar, while retaining all the usual information from NMR. This approach can be used to simultaneously detect multiple sensors with different chemical shifts and is also capable of detecting signals from opaque, heterogeneous samples, which is a unique advantage over optical methods. This general approach is adaptable for sensing minute quantities of xenon in heterogeneous in vitro samples, in miniaturized devices and should be applicable to certain in-vivo environments.

Biosensing Techniques↗
PubMed2006

Aptameric enzyme subunit for biosensing based on enzymatic activity measurement.

The aptameric enzyme subunit (AES), which is a DNA aptamer composed of an enzyme-inhibiting aptamer and a target molecule-binding aptamer, has been developed for the biosensing of target molecules. We used a thrombin-inhibiting aptamer as the aptamer that inhibits enzymatic activity. The thrombin-inhibiting aptamer folds into the G-quartet structure, which plays an important role in its inhibitory activity. As a target molecule-binding aptamer, an adenosine-binding aptamer was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to enable the change of the G-quartet structure upon the recognition of adenosine. In the present study, the change in the G-quartet structure led to a change in the thrombin inhibition activity, and adenosine was successfully detected by measuring the thrombin activity in a homogeneous solution without bound/free separation. We constructed two kinds of AESs; one of the structures is universal and can be used for designing any target molecule-binding aptamer. Since the enzyme activity is measured, AESs enable the simple and high-sensitivity detection of target molecules in a homogeneous assay.

Adenosine↗
PubMed1999

Myoglobin-containing carbon-paste enzyme microelectrodes for the biosensing of glucose under oxygen-deficit conditions.

The response of first-generation glucose oxidase (GOx) amperometric glucose biosensors is strongly dependent on the concentration of the oxygen cosubstrate. The incorporation of the natural oxygen binder myoglobin into a GOx-containing carbon-paste matrix is shown to satisfy the oxygen demand of the enzymatic reaction and to provide convenient biosensing of glucose in oxygen-free solutions. Such use of myoglobin-containing mineral oil thus offers an attractive alternative to the use of oxygen-rich fluorocarbon pasting liquids. Further improvements are observed upon doping the fluorocarbon oil with myoglobin. Factors affecting the oxygen independence of the new enzyme microelectrodes, including the myoglobin loading or length of the oxygen reservoir, have been optimized. The myoglobin-doped mineral oil or Kel-F-based carbon-paste enzyme microelectrodes display a highly stable glucose response over prolonged (6-7 h) operations in oxygen-free solutions, indicating no depletion of the internal oxygen supply.

Biosensing Techniques↗
PubMedDate not supplied

Synthesis and characterization of a ruthenium(II)-based redox conjugate for reagentless biosensing.

Synthesis of a novel sulfhydryl-specific, tetraammine Ru(II)polypyridyl complex, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2), which exhibits environment-sensitive electrochemical properties is described. When conjugated to an allosteric site in a genetically engineered mutant of maltose binding protein, the formal potential of the conjugated redox probe is shifted to higher potential upon maltose binding. The magnitude of this potential shift was used to measure maltose affinity of the protein-redox conjugate complex and to monitor maltose concentration in solution. These results are presented in context of reagentless biosensing.

Biosensing Techniques↗
PubMed2005

A modular nanoparticle-based system for reagentless small molecule biosensing.

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.

Biosensing Techniques↗
PubMed2004

Biosensing properties of diamond and carbon nanotubes.

The biochemical properties of boron-doped diamond (BDD), carbon nanofiber, fullerene, and multiwalled carbon nanotube (MWCNT) electrodes have been investigated comparatively. Physiochemical factors which affect the biosensing properties such as surface hydrophobicities, effective surface area, and intrinsic material properties are studied. Voltammetric responses of the as-grown thin film electrode and surface-modified electrode to biomolecules such as L-ascorbic acid (L-AA), dopamine (DA), and uric acid are examined. As-grown MWCNT electrodes exhibit selective voltammetric responses to the different biomolecules and faster electron-transfer kinetics compared to BDD. The selective response is due to the considerably lower anodic potential of L-AA on MWCNT (-48 mVvs Ag/AgCl compared to 575 mV on BDD). This electrocatalytic response can be replicated on a nonselective carbon nanofiber electrode by coating it with gold nanoparticles. BDD has no intrinsic selective response to L-AA, and surface modification by anodic polarization is necessary for resolving L-AA and DA.

Ascorbic Acid↗
PubMed2004

Heat-stabilized glycosphingolipid films for biosensing applications.

We have investigated a means of producing thin, oriented lipid monolayers which are stable under repeated washing and which may be useful in biosensing or surface-coating applications. Phosphatidylcholine and the glycosphingolipid GM1 were used as representative lipids for this work. Initially, a mixed self-assembled monolayer of octanethiol and hexadecanethiol was produced on a gold surface. This hydrophobic monolayer was then brought into contact with a thin lipid film that had been assembled at the liquid/air interface of a solution, allowing the lipid to deposit on the gold surface through hydrophobic interactions. The lipid layer was then heated to cause intermingling of the fatty acid and alkanethiol chains and cooled to form a highly stable film which withstood repeated rinsing and solution exposure. Presence and stability of the film were confirmed via ellipsometry, Fourier transform infrared spectroscopy, and quartz crystal microbalance (QCM), with an average overall film thickness of approximately 3.5 nm. This method was then utilized to produce GM1 layers on gold-coated QCM crystals for affinity sensing trials with cholera toxin. For these sensing elements, the lower detection limit of cholera toxin was found to be approximately 0.5 microg/mL, with a logarithmic relationship between toxin concentration and frequency response spanning over several orders of magnitude. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.

Animals↗
PubMed2006

Immobilization of antibodies on biosensing devices by nanoarrayed self-assembled monolayers.

This work presents an original and straightforward technique for antibody immobilization onto a surface, keeping the antibody in a biologically reactive configuration. Self-assembly of molecular monolayers and plasma-based colloidal lithography were combined to create chemical nanopatterns on the surface of a biosensing device. This technique was employed to create an array of 100 nm wide motifs having a hexagonal 2-D crystalline structure, characterized by COOH-terminated nanospots in a CH3-terminated matrix. The quality control of the chemical nanopattern was carried out by combining atomic force microscopy, ellipsometry, and contact angle measurements. Enzyme-linked immunosorbent assay experiments were set up showing that the COOH/CH3 nanopatterned surface constrains the immobilization of the antibodies in a biologically reactive configuration, thus significantly improving the device performances as compared to those of more conventional nonpatterned COOH-terminated or CH3-terminated surfaces.

Adsorption↗
PubMed2006

Shape-coded silica nanotubes for biosensing.

Shape-coded silica nanotubes (SNTs) were fabricated on the basis of template synthesis as a new dispersible microarray system. The template synthesis of shape-coded SNTs begins with the fabrication of a porous alumina film that has well-defined cylindrical pores with two or more different diameter segments by multistep anodization of an aluminum substrate. Then, SNTs were fabricated with a surface sol-gel method that can control the wall thickness of SNTs on the single-nanometer level. Attractively, the difference in optical reflectance between the segmented parts of individual silica nanotube makes it very convenient to identify each nanotube and enables these shape-coded SNTs to work as coding materials for biosensing.

Biosensing Techniques↗
PubMed2005

Novel electrochemical biosensing platform using self-assembled peptide nanotubes.

Here we describe a novel electrochemical biosensing platform based on biocompatible, well-ordered, self-assembled diphenylalanine peptide nanotubes. Voltammetric and time-based amperometric techniques were applied to demonstrate the ability of the peptide nanotubes to improve the electrochemical parameters of graphite electrodes. The findings clearly show that this novel class of peptide nanotubes provides an attractive component for future electroanalytical devices.

Biosensing Techniques↗
PubMed1999

Optical biosensing of nitric oxide using the metalloprotein cytochrome c'.

The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1.

Biosensing Techniques↗