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PubMed2005

Developing implantable optical biosensors.

Nanobiotechnologists are developing devices that can measure specific enzymes and proteins. These devices are expected to detect single enzyme or protein molecules accurately, providing highly sensitive biosensing applications. A recent study by Strano and co-workers shows that single-walled carbon nanotubes (SWNTs) hold great promise as implantable biosensors. Although most researchers have focused on substrate-oriented biosensors, Strano and colleagues have shown that the inherent fluorescent properties of suspended individual SWNTs can be used for solution-phase beta-D-glucose sensing.

Animals↗
PubMed2005

Electrochemical molecular analysis without nucleic acid amplification.

Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electrochemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.

Biosensing Techniques↗
PubMed2005

Microcantilever biosensors.

Biosensors are sensors in which biomolecular interactions are used as sensing reactions. Biomolecular interactions, when combined with a microcantilever platform, can produce an extremely powerful biosensing design. The resonance frequency of a microcantilever shifts sensitively due to mass loading from molecular interaction as in the case of any acoustic sensors. In addition, the microcantilevers also undergo bending if the molecular adsorption is confined to a single surface of a microcantilever. This cantilever bending is due to a differential surface stress caused by the forces involved in the adsorption process and is amplified by making the cantilever surfaces chemically different. Lack of specificity, the main disadvantage of the cantilevers, can be overcome by using the extremely selective biochemical reactions such as receptor-ligand, antibody-antigen, or enzyme-substrate reactions. Here we review the microcantilever technology and discuss a number of highly sensitive biochemical sensor applications based on microcantilevers.

Adsorption↗
PubMed2003

A real-time analysis of QacR-regulated multidrug resistance in Staphylococcus aureus.

Here we describe the construction and characterization of a biosensing reporter where luxCDABE genes from Photorhabdus luminescens, engineered for expression in Gram-positive organisms, are under the transcriptional control of QacR repressor from Staphylococcus aureus. In non-pathogenic S. aureus model system we analyzed the activity of the regulatory region acting as multidrug-resistance mediator in wild type strains. The use of full-length bacterial luciferase and the measurement of real-time light emission from intact, living cells make the present system suitable to follow the short term activity of different inducers. Among the tested molecules, tetracyclines showed a peculiar behavior by giving very high induction in a fashion seemingly more related to a general stress condition of the culture than to the direct binding and displacement of QacR from its operator. Temperature shocks confirmed that active transcription from qacA promoter is started in response to unspecific conditions where cell growth is strongly inhibited.

Anti-Infective Agents, Local↗
PubMed2003

Allosteric enzymes as biosensors for molecular diagnosis.

Biosensors are hybrid analytical devices that amplify signals generated from the specific interaction between a receptor and the analyte, through a biochemical mechanism. Biosensors use tissues, whole cells, artificial membranes or cell components like proteins or nucleic acids as receptors, coupled to a physicochemical signal transducer. Allosteric enzymes exhibit a catalytic activity that is modulated by specific effectors, through binding to receptor sites that are distinct from the active site. Several enzymes, catalyzing easily measurable reactions, have been engineered to allosterically respond to specific ligands, being themselves the main constituent of new-generation biosensors. The molecular basis, robustness and application of allosteric enzymatic biosensing are revised here.

Allosteric Regulation↗
PubMed1997

Detection of blood group antigens utilising immobilised antibodies and surface plasmon resonance.

Surface plasmon resonance (SPR) detection using the BIAcore biosensing system was employed for the detection of blood group-associated antigens (BGAA) on whole erythrocytes. The quantitative detection of erythrocytes was accomplished by covalently immobilising blood group-specific antibodies (IgM) to a dextran matrix and monitoring the cell binding response. Non-specific binding of erythrocytes to the IgM coated surface was not detected. Relatively mild regeneration conditions (20 mM NaOH) were employed to elute bound erythrocytes in order to preserve the activity of the immobilised antibody and allow the surface to be used repeatedly. Regeneration of the surface was particularly difficult when a high IgM immobilisation level was used and when the number of bound cells was high. Despite these considerations, a quantitative relationship between the cell binding response and erythrocyte concentration was confirmed. Erythrocyte preparations, diluted by a factor of ten as compared to physiological concentrations, were detectable. The occurrence of non-specific false positives appears to be minimal and allows the system to be used for blood typing. As a model study, the lectin concanavalin A (ConA) was covalently immobilised onto a hydrophilic dextran matrix and successfully used to support the capture of erythrocytes from suspension.

Antibodies, Monoclonal↗
PubMed2000

Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.

We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.

Animals↗
PubMed2001

Manipulating redox systems: application to nanotechnology.

Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure-function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are "wired up" in efficient electron-transfer chains, are "assembled" in artificial multidomain structures (molecular Lego), are "linked" to surfaces in nanodevices for biosensing and nanobiotechnological applications.

Biosensing Techniques↗
PubMed2003

What can spores do for us?

Many organisms have the ability to form spores, a remarkable phase in their life cycles. Compared with vegetative cells, spores have several advantages (e.g. resistance to toxic compounds, temperature, desiccation and radiation) making them well suited to various applications. The applications of spores that first spring to mind are bio-warfare and the related, but more positive, field of biological control. Although they are often considered metabolically inert, spores can also be used as biocatalysts. Other uses for spores are found in the fields of probiotics, tumour detection and treatment, biosensing and in the "war against drugs".

Animals↗
PubMed2001

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring.

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.

Biosensing Techniques↗
PubMed1998

Cytotoxicity and DNA binding characteristics of dextran-conjugated doxorubicins.

The antitumor antibiotic doxorubicin was conjugated with polymeric dextrans of various molecular weights and the cytotoxicity of the conjugates against human carcinoma KB-3-1 cells and its multidrug-resistant subclone KB-V-1 cells was measured by tetrazolium salt MTT assay. The conjugates were much less toxic to the KB-3-1 cells than the free doxorubicin but exhibited similar toxicity to the KB-V-1 cells. The conjugate-DNA interactions were monitored in real-time using an optical biosensor based on evanescent wave detection to obtain the association (ka) and dissociation (kd) rate constants as well as the equilibrium binding constants (KA) of the bindings. Both ka and kd values for the conjugates are more than three magnitudes smaller than those for free doxorubicin, while the KA values of the conjugate-DNA complexes are only about 10 times smaller than that of the free doxorubicin-DNA complex. The results indicate that the cytotoxicity and the DNA-binding kinetics of doxorubicin may be modified with dextran conjugation. The KA values obtained from the biosensor measurements were in close agreement with those determined in solution by fluorescent titration method, verifying the utility of the label-free biosensing measurements as an efficient method for studying ligand-DNA interactions.

Binding Sites↗
PubMed2001

An integrated CMOS microluminometer for low-level luminescence sensing in the bioluminescent bioreporter integrated circuit.

We report an integrated CMOS microluminometer for the detection of low-level bioluminescence in whole cell biosensing applications. This microluminometer is the microelectronic portion of the bioluminescent bioreporter integrated circuit (BBIC). This device uses the n-well/p-substrate junction of a standard bulk CMOS IC process to form the integrated photodetector. This photodetector uses a distributed electrode configuration that minimizes detector noise. Signal processing is accomplished with a current-to-frequency converter circuit that forms the causal portion of the matched filter for dc luminescence in wide-band white noise. Measurements show that luminescence can be detected from as few as 4 x 10(5) cells/ml.

Biosensing Techniques↗
PubMed2000

Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods.

The present report reviews immobilisation techniques of purified oligonucleotides on electrochemical transducers and their corresponding detection techniques. Most of the literature reviewed was published in the 1990s. The immobilisation techniques of a DNA probe to the surface of an electrochemical transducer made from carbon, gold, platinum or polypyrrole, ranged from simple adsorption to covalent bonding. Recent efforts to couple the recognition layer containing the immobilised nucleic acid recognition layer with the electrochemical signal transducer are discussed. Special attention is given to hybridisation biosensing based on electroactive indicators.

Adsorption↗
PubMed2000

Nanoporous anti-fouling silicon membranes for biosensor applications.

The ability to create biocompatible well-controlled membranes has been an area of great interest over the last few years, particularly for biosensor applications. The present study describes the fabrication and characterization of novel nanoporous micromachined membranes that exhibit selective permeability and low biofouling. Results indicate that such membranes can be fabricated with uniform pore sizes capable of the simultaneous exclusion of albumin and diffusion of glucose. Compared to polymeric membranes of similar pore size, micromachined silicon membranes allowed more than twice the amount of glucose diffusion after 240 min and complete albumin exclusion. Moreover, membranes exhibit no morphological change or degradability in the presence of biological proteins and fluids at 37 degrees C. The results point to the potential of using such membranes for implantable biosensor applications. With monodisperse pores sizes as small as 10 nm, these membranes offer advantages in their reproducibility, stability, and ability to be integrated in silicon-based biosensing technology.

Albumins↗
PubMed2000

An integrated NAD+-dependent enzyme-functionalized field-effect transistor (ENFET) system: development of a lactate biosensor.

An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD+/PQQ-assembly and the NAD+-cofactor-dependent lactate dehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET-device for the analysis of lactate. Biocatalyzed oxidation of lactate generates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH2, is oxidized by O2 in a process that constantly regenerates PQQ at the gate interface. The biocatalyzed formation of NADH and the O2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the solution near the gate interface and, consequently, the gate potential are controlled by the substrate (lactate) concentration in the solution. The device reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivity of 24+/-2 mV dec(-1). The response time of the device is as low as 15 s.

Biosensing Techniques↗
PubMed2001

The electric charge of pigment granules in pigment cells.

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be -1.5 x 10(-16) and -1.7 x 10(-16) C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.

Animals↗
PubMed2001

Voltammetric biosensors for the determination of formate and glucose-6-phosphate based on the measurement of dehydrogenase-generated NADH and NADPH.

This paper describes the development of a modified electrode for the electrocatalytic oxidation of beta-nicotinamide adenine dinucleotide (beta-NADH) and beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) using electropolymerised 3,4-dihydroxybenzaldehyde (3,4-DHB). Two voltammetric biosensors using enzyme-immobilised membranes were constructed for the determination of formic acid and glucose-6-phosphate (G6P), respectively. The formic acid biosensor based on the combination of formate dehydrogenase (FDH)-modified membrane with 3,4-DHB-coated glassy carbon electrode is one to two orders more sensitive (LOD, 5.0x10(-5) M) than previously reported electrochemical biosensors. Similarly, lower detection limit (4.0x10(-5) M) for the measurement of G6P was achieved using glucose-6-phosphate dehydrogenase (G6PDH) in the presence of beta-NADP(+). The interference of uric acid and ascorbate was minimised by incorporating an additional membrane modified with uricase and ascorbate oxidase, respectively. The biosensing scheme developed in this study can be adopted universally with a number of dehydrogenases for the detection of different substrates.

Benzaldehydes↗
PubMed2001

Ultrasensitive quartz crystal microbalance sensors for detection of M13-Phages in liquids.

Quartz crystal microbalance (QCM) sensors are widely used for determining liquid properties or probing interfacial processes. For some applications the sensitivity of the QCM sensors typically used (5-20 MHz) is limited compared with other biosensor methods. In this study ultrasensitive QCM sensors with resonant frequencies from 39 to 110 MHz for measurements in the liquid phase are presented. The fundamental sensor effect of a QCM is the decrease of the resonant frequency of an oscillating quartz crystal due to the binding of mass on a coated surface during the measurement. The sensitivity of QCM sensors increases strongly with an increasing resonant frequency and, therefore, with a decreasing thickness of the sensitive area. The new kind of ultrasensitive QCM sensors used in this study is based on chemically milled shear mode quartz crystals which are etched only in the center of the blank, forming a thin quartz membrane with a thick, mechanically stable outer ring. An immunoassay using a virus specific monoclonal antibody and a M13-Phage showed an increase in the signal to noise ratio by a factor of more than 6 for 56 MHz quartz crystals compared with standard 19 MHz quartz crystals, the detection limit was improved by a factor of 200. Probing of acoustic properties of glycerol/water mixtures resulted in an increase in sensitivity, which is in very good agreement with theory. Chemically milled QCM sensors strongly improve the sensitivity in biosensing and probing of acoustic properties and, therefore, offer interesting new application fields for QCM sensors.

Acoustics↗