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PubMed2004

Biosensors for determination of total antioxidant capacity of phytotherapeutic integrators: comparison with other spectrophotometric, fluorimetric and voltammetric methods.

Enzymatic electrodes based on superoxide dismutase (SOD) biosensors, working both in aqueous and non-aqueous solutions, recently developed by the present authors, were used to experimentally evaluate the antioxidant capacity of several phytotherapeutic diet integrators. The precision of this method of analysis was found to be reasonable (R. S. D. < or = 10%). The results were also compared with those obtained using a traditional spectrophotometric method as well as a spectrofluorimetric method described in literature. Lastly, the comparison was extended to another method based on cyclic voltammetry currently being trialled by the present authors.

Antioxidants↗

PubMed1999

Amperometric biosensors for clinical and therapeutic drug monitoring: a review.

The coupling of enzymes and electrode transducers permits the rapid and simple determination of endogeneous compounds and therapeutic drugs in clinical samples. New developments in the operation, miniaturization and microfabrication of electrochemical biosensors offer exciting possibilities for biomedical and pharmaceutical analysis. This review focuses on the current state of amperometric enzyme electrodes for biomedicine, with emphasis on recent advances, challenges and trends.

Animals↗

PubMed1998

Hydrogen peroxide determination in pharmaceutical formulations and cosmetics using a new catalase biosensor.

The possibility of evaluating the content of hydrogen peroxide in several authentic matrices, such as cosmetic and pharmaceutical formulations, was studied. A new catalase biosensor fabricated using an amperometric gas-diffusion oxygen sensor as electrochemical transducer and the catalase enzyme immobilized in kappa-carrageenan gel and capable of operating in both aqueous and non aqueous solvents was developed and tested for this purpose. Creams, emulsions and disinfectant solutions were analysed. To this end, a preliminary check was needed to establish the best conditions to analyse these matrices; the choice of solvent was one of the most important points studied. The solvents considered included dioxane, water-dioxane mixtures, water saturated chloroform and aqueous solutions. The different solubility properties of the matrices analysed were taken into account.

Biosensing Techniques↗

PubMed2000

Methods for reducing biosensor membrane biofouling.

The deleterious effect that biofouling has on sensor stability is a serious impediment to the development of long term implanted biosensors. This paper reviews the surface modification strategies currently employed to minimize membrane biofouling of in vivo sensors. Nine sensor modifications are discussed herein: hydrogels, phospholipid-based biomimicry, flow-based systems, Nafion, surfactants, naturally derived materials, covalent attachments, diamond-like carbons, and topology.

Journal Article↗

PubMed2001

P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays.

The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1' benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 microM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1-100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.

Biosensing Techniques↗

PubMed1998

Screening of xenobiotic compounds degrading microorganisms using biosensor techniques.

A screening device based on microorganisms immobilised onto a Clark-type oxygen electrode was used to monitor the potential of these microorganisms for the degradation and detection of xenobiotic compounds especially their chlorinated derivatives. The sensitivity and specificity of various species of Pseudomonas, Sphinomonas, Ralstonia, Rhodococcus were characterised in relation to xenobiotic compounds by using biosensor techniques. The following groups of xenobiotics were subjects of investigation: chlorophenols, chlorobenzoates, 2,4-D, PCB, dibenzofurane and their putative intermediates. Using this simple setup it proved possible to screen microbial strains for their potential to catabolize aromatic and chloroaromatic compounds under oxygen consumption. In a kinetic regime, a reproducible signal was obtained within minutes. Based on these results the sensor technique was a suitable method for the rapid characterization of microorganisms and allowed to gather information about the substrate spectrum.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Live-cell fluorescent biosensors for activated signaling proteins.

A new generation of live-cell fluorescent biosensors enables us to go beyond visualization of protein movements, to quantify the dynamics of many different protein activities. Alternate approaches can report post-translational modifications, ligand interactions and conformational changes, revealing how the location and subtle timing of protein activity controls cell behavior.

Animals↗

PubMed2000

Porous gold surfaces for biosensor applications.

The sensitivity of optical biosensors where the detection takes place on a planar gold surface can be improved by making the surface porous. The porosity allows a larger number of ligands per surface area resulting in larger optical shifts when interacting with specifically binding analyte molecules. The porous gold was deposited as a thin layer on a planar gold surface by electrochemical deposition in a solution of tetrachloroaurate and lead acetate. A protein, streptavidin, was adsorbed into the formed porous layer and the time course of the adsorption was monitored by in-situ ellipsometry. When the porous layer was 500 nm in thickness a six-fold increase of the ellipsometric response was obtained compared with a planar gold surface. The dependency of porosity and layer thickness was explained with a mathematical model of the gold/porous gold/protein/solution system.

Adsorption↗

PubMed2000

Electrode potential-modulated cleavage of surface-confined DNA by hydroxyl radicals detected by an electrochemical biosensor.

Damage to DNA frequently involves interruption of DNA sugar-phosphate strands (strand breaks, sb). Under aerobic conditions, transition metal ions cause DNA damage through production of reactive oxygen species (frequently via Fenton-type reactions). Formation of sb in covalently closed supercoiled (sc) DNA can be detected using an electrochemical biosensor based on a scDNA-modified mercury electrode. By controlling the potential of the electrode, this technique can be employed in studies of redox reactions involved in formation of DNA strand breaks, and to detect species involved in these reactions. ScDNA anchored at HMDE was cleaved by catalytic amounts of iron/EDTA ions in the absence of chemical reductants when appropriate electrode potential (sufficiently negative to reduce [Fe(EDTA)]- to [Fe(EDTA)]2-) was applied. The process required oxygen or hydrogen peroxide. The extent of DNA damage increased with the shift of the electrode potential to negative values, displaying a sharp inflection point matching the potential of [Fe(EDTA)]2-/[Fe(EDTA)]- redox pair. In the absence of transition metal ions, significant DNA damage was observed at potentials sufficiently negative for reduction of dioxygen at the mercury electrode. This observation suggests cleavage of the surface-attached scDNA by radical intermediates of oxygen reduction at HMDE.

Biosensing Techniques↗

PubMed2000

A micro flow injection electrochemical biosensor for organophosphorus pesticides.

We describe a disposable, amperometric micro flow injection electrochemical biosensor that can be applied to the identification and quantification of highly toxic organophosphorus (OP) compounds in the environment, on the spot and in a short time. The system traces very small quantities of OP by monitoring the enzymatic reaction of acetylcholine esterase (AChE) and its inhibition. The sensor is sensitive, rapid, small, inexpensive, disposable and can be operated by non-professional technicians. The electrochemical cell consists of screen-printed electrodes covered with an enzymatic membrane and placed in a home-made flow cell. The electrodes are connected to a computer-controlled potentiostat. We quantitatively detected the OP compound, dimethyl 2,2-dichlorovinyl phosphate (DDVP), by monitoring the OP induced decrease in enzymatic degradation of the substrate, acetylthiocholine chloride (ATCh), to thiocholine and acetic acid. Thiocholine reacts with hexacyanoferrate ion in the working solution and the reduction of [Fe(CN)6](-3) to [Fe(CN)6](-4) and its subsequent reoxidization by the electrode generates very sharp, rapid and reproducible electric signals. The ability to detect low quantities is extremely important when dealing with hazardous environmental pollutants.

Acetylcholinesterase↗

PubMed2000

Enzyme biosensor for studying therapeutics of Alzheimer's disease.

An electrochemical method for the investigation and comparison of anti-Alzheimer medications that is based on the inhibition of the acetylcholinesterase is presented. The developed amperometric biosensor determines the in-vitro inhibition of the acetylcholinesterase that is co-immobilized with choline oxidase on the working electrode surface of a three-electrode system using gel entrapment. The sensor has been applied to determine the IC50 values of two known and one newly developed Alzheimer remedy. A simultaneous measurement with the photometric standard method shows the applicability of our method for fast drug screening.

Acetylcholinesterase↗

PubMed2000

Immobilization of pneumococcal polysaccharide vaccine on silicon oxide wafer for an acoustical biosensor.

One the most important aspects of a biosensor is related to immobilization and maintenance of specific reference compounds on sensing surfaces. A method for the immobilization of polysaccharides to a silicon oxide surface intended for Surface Acoustical Waves (SAW) sensors is described. Silicon oxide is a hydrophobic inorganic support used for the fabrication of many electronic devices. The pneumococcal polysaccharide (PPS) vaccine is immobilized via Protein A after pre-treatment of the surface with hydrochloric acid. The effects of non-specific binding are discussed. The results indicate that the immobilization of PPS via Protein A increases the sensitivity of detecting Streptococcus pneumoniae antibodies in human sera and offers greater reproducibility of response compared with ELISA methods. The principles of this technique are simple and are applicable to the immobilization of many capsular polysaccharides.

Acoustics↗

PubMed2000

Coupling of a DNA piezoelectric biosensor and polymerase chain reaction to detect apolipoprotein E polymorphisms.

In this paper we report the coupling of the Polymerase Chain Reaction (PCR) with a piezoelectric biosensor to detect a point mutation in a human gene. Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridisation of the immobilised probes with a short sequence (23 mer) complementary, non-complementary and mismatched DNA was investigated: the device was able to distinguish the different synthetic oligonucleotides. Many cycles of measurements can be performed on the same crystal surface regenerating the single strand of DNA with 1 mM of HCl. The same hybridisation reaction was then performed using real samples of human DNA extracted from blood and amplified by PCR, following a standard procedure for genetic detection of the polymorphism of the apolipoprotein E (apoE) gene. The procedure was able to distinguish the sequences present in the different samples, which differ only in one base: in this way it was possible distinguish between different groups of genotypes with apoE typing. Experiments with 'blank' samples confirmed the absence of adsorption or non-specific effects on the quartz crystal treated with the reported procedure.

Apolipoproteins E↗

PubMed2000

A liquid crystal pixel array for signal discrimination in array biosensors.

A new optical design uses a liquid crystal pixel array (LCPA) to discriminate multiple fluorescence signals on a two-dimensional biosensor array. The LCPA can selectively control the transmission of fluorescence generated from multiple biosensing elements on a planar waveguide. This device sequentially acquires the fluorescence data from the substrate by making multiple individual measurements of the sensing elements on the waveguide. The biosensing elements are patterned according to the pixel layout of the LCPA and optically aligned so that each electronically driven pixel can either transmit or filter out the fluorescence signal as specified by the user. The primary advantage of this system is that a single detection channel (i.e. photomultiplier tube (PMT)) can be used to measure multiple fluorescence signals from a two-dimensional substrate while the LCPA provides for spatial resolution. We evaluate the performance of the LCPA by testing the optical homogeneity of the liquid crystal pixels and linear dynamic range for transmitting light. The LCPA is also used with well-developed biosensing chemistry modified for this optical format.

Biosensing Techniques↗

PubMed2000

Structure and activity of lipid membrane biosensor surfaces studied with atomic force microscopy and a resonant mirror.

Three variants of the liposome fusion (coalescence) method to produce supported lipid bilayers, containing the ganglioside GM1 on silicon nitride surfaces, were studied. The first procedure involved attachment and fusion of liposomes containing DMPC, GM1 and a small amount of biotinylated lipid (Biotin-LC-DPPE) to a streptavidin coated surface. Direct fusion of liposomes composed of a mixture of DPPC, DPPG, DPPE, GM1 and cholesterol to the surface were the second variant. The final method utilised the second type of liposomes, fused onto a streptavidin layer with a small amount of exposed hydrophobic tails. The methods produced similar lipid layers, but with different ways of attachment to the surface. The binding of cholera toxin B-subunit (CTB) towards these sensor surfaces was measured in a resonant mirror biosensor instrument and the activity and longer-term stability of the layers were examined. The prepared surfaces were also imaged by atomic force microscopy (AFM) in liquid to characterise the topography of the lipid layers. The binding efficiency of CTB towards these surfaces was discussed in terms of lipid fluidity and surface roughness.

Biosensing Techniques↗

PubMed2000

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer.

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Arachis↗

PubMed2000

Evaluation of antibody immobilization methods for piezoelectric biosensor application.

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.

Antibodies, Bacterial↗

PubMed2000

A comparison of protocols for the optimisation of detection of bacteria using a surface acoustic wave (SAW) biosensor.

A dual channel surface acoustic wave (SAW) device has been used as a biosensor to detect two different microorganisms, Legionella and Escherichia coli, simultaneously. A series of experiments was conducted to optimise the use of the SAW for bacterial detection using a novel protocol of coating bacteria on the sensor surface prior to addition of the antibody. Results were compared with an experiment in which a conventional protocol was utilised, where antibody was coated on the sensor surface prior to exposure to bacteria. The concentration of bacteria that attached to the surface of the SAW device was related to the antibody that specifically bound to it and therefore to frequency in a dose dependent fashion. Unlike conventional microbiological techniques quantitative results can be obtained for Legionella and E. coli down to 10(6) cells per ml within 3 h. In addition E. coli was detected down to 10(5) cells per ml in a modified protocol using sheep IgG as a blocking agent.

Bacteria↗