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PubMed2004

Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique.

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 microA mmol(-1)lcm(-2) and a limit of detection of 0.20 mmoll(-1) being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H(2)O(2) produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.

Adsorption↗

PubMed2004

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element.

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.

Biosensing Techniques↗

PubMed2004

XPS and AFM analysis of antifouling PEG interfaces for microfabricated silicon biosensors.

In the past two decades, the biological and medical fields have seen great advances in the development of biosensors capable of quantifying biomolecules. Many of these biosensors have micro- and nano-scale features, are fabricated using biochip technology, and use silicon as a base material. The creation of antifouling sensor interfaces is critical to avoid serious consequences that arise due to their contact with biological fluids. To this end, we have created thin PEG interfaces of various grafting densities on silicon using a single-step PEG-silane coupling reaction scheme. Initial PEG concentration (5-50 mM) and coupling time (0.5-24 h) were varied to attain different grafting densities, and different PEG interfaces so created were analyzed using XPS and AFM. Furthermore, all the PEG interfaces were evaluated using XPS and AFM for their antifouling abilities using fibrinogen as the model protein. Results indicated that PEG interfaces created in this investigation are appropriate for biosensors with micro- and nano-scale features, and are efficient in controlling protein fouling.

Biosensing Techniques↗

PubMed2004

Single-wall carbon nanotube-based voltammetric sensor and biosensor.

The pH-sensitive property of the single-wall carbon nanotube modified electrode based on the electroactive group on the single-wall carbon nanotube was explored by differential pulse voltammetry technique. In pH range 1-13 investigated in Britton-Robinson (B-R) buffer, the anodic peak shifted negatively along with the increase of pH exhibiting a reversible Nernstian response. Experiments were carried out to investigate the response of the single-wall carbon nanotube (SWNT) modified electrode to analytes associated with pH change. The response behavior of the modified electrode to ammonia was studied as an example. The potential response could reach equilibrium within 5 min. The modified electrode had good operational stability. Voltammetric urease and acetylcholinesterase biosensors were constructed by immobilizing the enzymes with sol-gel hybrid material. The maximum potential shift could reach 0.130 and 0.220 V for urea and acetylthiocholine, respectively. The methods for preparing sensor and biosensor were simple and reproducible and the range of analytes could be extended to substrates of other hydrolyases and esterases. This broadened the biosensor application of carbon nanotube in electrochemical area.

Acetylcholine↗

PubMed2004

Immobilization of metallothionein as a sensitive biosensor chip for the detection of metal ions by surface plasmon resonance.

A biosensor based on mammalian metallothionein (MT) for the detection of metal ions was developed and characterized. MT was immobilized onto a carboxymethylated dextran matrix as a biosensor for the detection of metal ions by surface plasmon resonance (SPR). The optimal pH for the immobilization step was determined to be 4. The temperature for the analysis was also defined, and the highest interaction was observed at 30 degrees C. The MT sensor chip binds cadmium (Cd), zinc (Zn) or nickel (Ni), but not magnesium (Mg), manganese (Mn) and calcium (Ca). Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection is at the micromolar level. The interaction between the metal ions and the sensor chip is influenced significantly by the presence of NaCl, Tween 20 and the pH of the reaction buffer. By decreasing the NaCl in the reaction buffer to 1 mM, the MT chip effectively differentiates cadmium from zinc and nickel. Kinetic parameters of the metal-MT interactions were also determined by using this chip. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel, which is the same as that determined for MT in solution. Thus, the MT chip can be an effective biosensor for the detection and measurement of several metal ions.

Adsorption↗

PubMed2005

A novel L-lactate-selective biosensor based on flavocytochrome b2 from methylotrophic yeast Hansenula polymorpha.

A novel amperometric biosensor highly selective to L-lactate has been developed using L-lactate-cytochrome c oxidoreductase (flavocytochrome b2) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface. Moreover, the possibility of direct electron transfer from the reduced form of FCb2 to carbon electrodes has been evaluated. The bioanalytical properties of FCb2-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for L-lactate determination.

Biosensing Techniques↗

PubMed2004

The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors.

Contamination of food and water supplies by microorganisms such as Escherichia coli, the need for point-of-care bedside analysis of biological samples, and concerns about terrorist attacks using biological organisms, have made the development of fast, reliable, and sensitive analytical methodologies for use in monitoring of pathogens very important. With a variety of biosensors being developed for extremely sensitive and rapid nucleic acid diagnostics, it has become even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample preparation. The application of ultrasound has the potential to create DNA fragments from genomic material with lengths that are suitable for determination using biosensors and microarrays. For example, application of 85 W power at a frequency of 20 kHz can produce a preponderance of fragments of 100-400 base pairs (bp) within several seconds, and sample processing can lead to over 75% conversion from genomic material to fragments in times of 20-30 s. A proportion of these fragments are in a single-stranded state and are suitable for hydridization with immobilized single-stranded DNA probe oligonucleotides using a fiber optic biosensor. Control of factors such as salt concentration, exposure time, ultrasound power, and the initial temperature of the solution, can affect the length and form (single- or double-stranded) of DNA fragments that are generated by ultrasound, and average fragment length can be adjusted by selection of these operating parameters.

Biosensing Techniques↗

PubMed2005

Improved selectivity and stability of glucose biosensor based on in situ electropolymerized polyaniline-polyacrylonitrile composite film.

A new type of in situ electropolymerization method was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous polyacrylonitrile-coated platinum electrode in the presence of glucose oxidase. The novel glucose biosensor exhibited good selectivity, sensitivity and stability, which showed no apparent loss of activity after 100 consecutive measurements and intermittent usage for 100 days with storage in a phosphate buffer at 4 degrees C. Blood glucose determinations agreed well with standard hospital laboratory analysis. The construction and operational parameters of the biosensor were also optimized.

Acrylic Resins↗

PubMed2005

Stabilization of enzymes in nanoporous materials for biosensor applications.

In this study we present the results obtained from efforts to stabilize the inherently unstable m-AChE in nanoporous materials, for the development of biosensors with increased operational stability. Based on existing theoretical models, the entrapment of proteins into relatively small rigid cages drastically increases the stability of these proteins, as this is manifested by their decreased tendency to unfold. The use of two different meso/nanomaterials for the immobilization of the m-AChE shows that there is both a decrease in the leaching of the protein from the biosensor membrane to the test solution, as well as a drastic increase in the operational stability of the resulting biosensor.

Acetylcholinesterase↗

PubMed2005

An amperometric glucose biosensor prototype fabricated by thermal inkjet printing.

The prototype of an amperometric glucose biosensor was realized by thermal inkjet printing using biological and electronic water-based inks, containing a glucose oxidase (GOD) from Aspergillus niger and the conducting polymer blend poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS), respectively. The biosensor was fabricated microdepositing PEDOT/PSS and GOD, in sequence, on ITO-glass, by a commercial inkjet printer, with the help of a commercial software. High density microdots matrices were so-realized, with a calculated resolution of about 221 x 221 dpi (dot per inch). By means of a rapid and easy assay it was demonstrated that no activity loss occurred upon the printing of GOD, despite of the use of a thermal printhead. The device was encapsulated in a semipermeable membrane of cellulose acetate, applied by dip-coating, in order to prevent dissolution of the enzyme and/or PEDOT/PSS in water. The preliminary response of the electrode was measured in an aqueous glucose solution in the presence of ferrocenemethanol (FeMeOH) as a mediator, and resulted linear up to 60 mM in glucose. The best sensitivity value achieved was 6.43 microAM(-1) cm(-2) (447 nAM(-1) U(-1) cm(-2)). The characteristics of the device, and the possible performance improvements have been analyzed and discussed. The reported findings indicate that inkjet printing could be a viable instrument for the easy construction of a working biosensor via direct digital design using biological and conductive polymer based inks. Such an approach may be seen as an example of "biopolytronics".

Aspergillus niger↗

PubMed2004

The potential use of hydrazine as an alternative to peroxidase in a biosensor: comparison between hydrazine and HRP-based glucose sensors.

The potential use of hydrazine sulfate was examined for the catalytic reduction of enzymatically generated H2O2 in a biosensor system. The performance of the hydrazine-based sensor was compared with an HRP-based glucose sensor as a model of a biosensor. Hydrazine and HRP were covalently immobilized onto a conducting polymer layer with glucose oxidase. The direct electron transfer reactions of the immobilized hydrazine and HRP onto the poly-5,2':5,2''-terthiophene-3'-carboxylic acid (poly-TTCA) layer were investigated by using cyclic voltammetric method and the electron transfer rate constants were determined. The glucose oxidase- and hydrazine-immobilized sensor efficiently reduced the enzymatically generated H2O2 at -0.15 V versus Ag/AgCl. The surface of this GOx/hydrazine/poly-TTCA-based glucose sensor was characterized by QCM, SEM, and ESCA. Glucose-sensing properties were studied using cyclic voltammetric and chronoamperometric techniques. Various experimental parameters were optimized according to the amount of hydrazine, pH, the temperature, and the applied potential. A linear calibration plot was obtained in the concentration range between 0.1 and 15.0 mM, and the detection limit was determined to be 40.0+/-7.0 microM. Interferences from other biological compounds were studied. The long-term stability of the GOx/hydrazine sensor was better than that of the one based on a GOx/HRP biosensor. The proposed glucose sensor was successfully applied to human whole blood and urine samples for the detection of glucose.

Biosensing Techniques↗

PubMed2005

Biosensors-a perspective.

Biosensors have been under development for over 35 years and research in this field has become very popular for 15 years. Electrochemical biosensors are the oldest of the breed, yet sensors for only one analyte (glucose) have achieved widespread commercial success at the retail level. This perspective provides some cautions related to expectations for biosensors, the funding of science, and the wide gap between academic and commercial achievements for sensor research. The goal of this commentary is not to arrive at any particular truth, but rather to stimulate lively discussion.

Biosensing Techniques↗

PubMed2005

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand.

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.

Biofilms↗

PubMed2005

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase.

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.

Acrylic Resins↗

PubMed2004

Evanescent wave fluorescence biosensors.

Since discovery and first use in the mid-1970s, evanescent wave fluorescence biosensors have developed into a diverse range of instruments, each designed to meet a particular detection need. In this review, we provide a brief synopsis of what evanescent wave fluorescence biosensors are, how they work, and how they are used. In addition, we have summarized the important patents that have impacted the evolution from laboratory curiosities to fully automated commercial products. Finally, we address the critical issues that evanescent wave fluorescence biosensors will face in the coming years.

Biosensing Techniques↗

PubMed2004

Glucose biosensor based on multi-wall carbon nanotubes and screen printed carbon electrodes.

This paper describes a disposable electrochemical biosensor for glucose monitoring. The sensor was based on multi-wall carbon nanotubes (MWCNTs) immobilized with glucose oxidase and upon screen printed carbon electrode. The effect of MWCNTs on the response of amperometric glucose oxidase electrode for glucose was examined. Results obtained, of interest for basic and applied biochemistry, represent a first step in construction of a MWCNT-enzyme electrode biosensor with potentialities for a successful application in the biosensor area.

Adsorption↗

PubMed2005

An integrated approach for the design and synthesis of oligonucleotide probes and their interfacing to a QCM-based RNA biosensor.

The quantitative determination of specific cellular messenger-RNA is extremely important both in basic and applied research, especially in diagnostic and pharmacological fields. In order to perform a direct and easy quantification of transcripts on cell extracts, the feasibility of an analytical device able to selectively detect a defined target RNA in a complex mixture while avoiding labelling, retrotranscription and amplification steps, has been explored. In particular, several aspects necessary to obtain good selectivity in target recognition, stability, reusability and sensitivity of a gene specific biosensor were considered. For the development of suitable probe-receptors, analysis of the nucleotide sequence of the target mRNA was carried out to localise the preferred binding regions. As criteria for optimisation, we selected accessibility and uniqueness. Oligonucleotide probes, designed to specifically bind these sequences, were synthesised by using particular monomers producing nuclease-resistant RNA strands with high affinity towards the target. Quartz crystal microbalance (QCM) technology was selected to realise a microgravimetric sensor able to bind the RNA under investigation through a complementary oligonucleotide probe. Covalent immobilisation of bioreceptor molecules to the transducer sensitive surface ensured a stable integration between the two. The binding ability of immobilised probes was tested evaluating their annealing behaviour with both complementary oligonucleotides and full-length target mRNA. The conditions necessary for the regeneration of biosensor were also assessed. Measurements of shift in QCM resonant frequency, performed by hybridisation experiments in liquido, demonstrate that a label-free RNA-biosensor with high specificity, reusability and the ability to give quantitative information, can be realised.

Biosensing Techniques↗

PubMed2005

Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element.

A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.

Alcohol Dehydrogenase↗