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Self-assembled monolayers as a tunable platform for biosensor applications.

Considerable attention has been drawn during the last two decades to functionalize noble metal surfaces by forming ordered organic films of few nm to several hundred-nm thickness. Self-assembled monolayer (SAM) provides one simple route to functionalize electrode surfaces by organic molecules (both aliphatic and aromatic) containing free anchor groups such as thiols, disulphides, amines, silanes, or acids. The monolayer produced by self-assembly allows tremendous flexibility with respect to several applications depending upon their terminal functionality (hydrophilic or hydrophobic control) or by varying the chain length (distance control). For example, SAM of long chain alkane thiol produces a highly packed and ordered surface, which can provide a membrane like microenvironment, useful for immobilising biological molecules. The high selectivity of biological molecules integrated with an electrochemical, optical or piezoelectric transduction mode of analyte recognition offers great promise to exploit them as efficient and accurate biosensors. It is demonstrated with suitable examples that monolayer design plays a key role in controlling the performance of these SAM based biosensors, irrespective of the immobilisation strategy and sensing mechanism.

Biosensing Techniques↗

Bacteria-degraders as the base of an amperometric biosensor for detection of anionic surfactants.

Several strains belonging to genera Pseudomonas and Achromobacter and characterized by the ability to degrade anionic surfactants were tested as potential bases of microbial biosensors for surfactant detection. For each strain the substrate specificity and stability of sensor signals were studied. The total amount of the substrates tested (including carbohydrates, alcohols, aromatics, organic acids, etc.) was equal to 60; the maximal signals were observed towards the anionic surfactants. The lower limit of detection for sodium dodecyl sulfate used as a model surfactant was in the field of 1 microM for all the strains. The created microbial biosensor model can extend the practical possibilities for rapid evaluation of surfactants in water media.

Alcaligenes↗

Application of conducting polymers to biosensors.

Recently, conducting polymers have attracted much interest in the development of biosensors. The electrically conducting polymers are known to possess numerous features, which allow them to act as excellent materials for immobilization of biomolecules and rapid electron transfer for the fabrication of efficient biosensors. In the present review an attempt has been made to describe the salient features of conducting polymers and their wide applications in health care, food industries, environmental monitoring etc.

Biosensing Techniques↗

Mediated biosensors.

Direct electrode transfer between enzyme and the electrode in biosensors requires high efficiency therefore, synthetic replacement for oxygen led to the development of enzyme mediators and modified electrodes in biosensor fabrication. In this context, a number of electron acceptors and complexes have been used. Present paper gives an overview of various methodologies involved in the mediated systems, their merits and wide applications.

Biosensing Techniques↗

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents.

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.

Animals↗

Plant tissue-based chemiluminescence flow biosensor for determination of unbound dopamine in rabbit blood with on-line microdialysis sampling.

A novel plant tissue-based chemiluminescence (CL) biosensor for dopamine combined with flow injection analysis is presented in this paper. The potato roots act as molecular recognition elements. Dopamine is oxidized by oxygen under the catalysis of polyphenol oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of potato tissue to generate CL signal. The CL emission intensity was linear with dopamine concentration in the range of 1x10(-5)-1x10(-7) g/ml and the detection limit was 5.3x10(-8) g/ml (3sigma) with a relative standard deviation of 1.7%. Combined with microdialysis sampling, the biosensor was applied to monitor the variation of dopamine level in the blood of rabbit after the administration of dopamine to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.

Animals↗

Biosensors for rapid monitoring of primary-source drinking water using naturally occurring photosynthesis.

Working with primary-source freshwater drinking samples from the Clinch and Tennessee Rivers, we have developed a tissue-based biosensor detection system that uses naturally occurring aquatic photosynthetic tissue as the sensing material for detection of chemical antagonists in the water. Sensor readout is based on well-known principles of fluorescence induction by living photosynthetic tissue. The Clinch River is the main source of drinking water for Oak Ridge, Tennessee, while the Tennessee River is a major source for the city of Knoxville. We have successfully detected algae in every sample that we examined and readily monitored changes in the characteristic fluorescence induction curves when the samples were exposed to potassium cyanide (KCN), methyl parathion (MPt), N'(3,4-dichlorophenyl)-N,N-dimethylurea (DCMU), and paraquat. The percentage decreases in photochemical yields observed in Tennessee River samples after a 24-min exposure to KCN, MPt, and DCMU were, respectively, 21.89+/-0.76, 3.28+/-0.18, and 14.77+/-1.81. For a site at the Clinch River, the percentage decreases were 22.78+/-1.63, 8.32+/-0.21, and 17.71+/-1.32 (Table 1). The unique aspect of this approach to real-time water quality monitoring is that unlike conventional sensing devices, this sensor material is external to the detecting instrument and is continuously refreshed. These biosensors may be used as continuous rapid-warning sentinels for detection of chemical warfare agents in sunlight-exposed drinking water supplies.

Biosensing Techniques↗

Hydrogen peroxide sensitive amperometric biosensor based on horseradish peroxidase entrapped in a polypyrrole electrode.

The enzyme horseradish peroxidase (HRP) has been entrapped in situ by electropolymerization of pyrrole onto a platinum electrode. The latter was previously coated by a polypyrrole layer for better adhesion of the biocatalyst film and in order to avoid the enzyme folding onto the Pt electrode. The biosensor allowed the determination of hydrogen peroxide in the concentration range comprised between 4.9 x 10(-7) and 6.3 x 10(-4) M. The biosensor retained more than 90% of its original activity after 35 days of use.

Biosensing Techniques↗

GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates.

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.

Biosensing Techniques↗

Detection of activity of telomerase in tumor cells using fiber optical biosensors.

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.

Biomarkers, Tumor↗

Use of surface plasmon resonance biosensor technology as a possible alternative to detect differences in binding of enantiomeric drug compounds to immobilized albumins.

The use of biosensors for monitoring real time interactions between biomolecules and drug compounds has a lot of advantages over presently existing detection methods, the major ones being the elimination of radio labels and rapid screening. We can also obtain information about the kinetic parameters and these values may serve as useful indicators towards subtle differences in the binding strength and characteristics of closely related drug compounds and enantiomers. The Biacore 3000 biosensor based on the Surface Plasmon Resonance (SPR) technology was used to assess the albumin protein binding differences between two enantiomers of a drug compound. Normalized responses (NRU) and affinity constants (K(D)) were readily calculated. Statistical parameters like mean normalized responses, %CV values were determined to make the technique robust. The %CV values obtained were within the preset limits of < or = 25% (FDA limits for drug development and method validation protocols) for the binding interactions for majority of the concentrations studied. For example, the %CV values for the normalized responses for the binding of the control drug warfarin to human albumin ranged from 7.9 to 24.3%. The method gave reproducible results, and the results indicated slight differences in binding patterns of the enantiomers to human and rat albumin.

Animals↗

Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides.

An optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level.

Atrazine↗

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability.

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.

Biosensing Techniques↗

Novel carbon materials in biosensor systems.

In this work, novel carbon materials are evaluated as transducers, stabilizers and mediators for the construction of amperometric biosensors. It is shown that materials such as fullerenes and carbon nanotubes are promising materials as electrochemical mediators and enzyme stabilizers. Additionally porous carbon and porous glassy carbon are excellent transducers for amperometric measurements, while they provide cavities adequate for enzyme immobilization. At the same time, the sensitivity to peroxide is shown to depend on the activation procedures. Treatment that introduces oxygen groups increases the sensitivity of the carbon-based sensor to hydrogen peroxide considerably. These materials are used for the construction, mediation and stabilization of glucose biosensor.

Adsorption↗

Design of acetylcholinesterases for biosensor applications.

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.

Acetylcholinesterase↗

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes.

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.

Biosensing Techniques↗

Immobilisation of glucose oxidase within metallic nanotubes arrays for application to enzyme biosensors.

Arrays of nanoscopic gold tubes were prepared by electroless deposition of the metal within the pores of polycarbonate particle track-etched membranes (PTM). Glucose oxidase (Gox) was immobilised onto preformed self-assembled monolayers (SAMs) (mercaptoethylamine or mercaptopropionic acid) on electroless gold via cross-linking with glutaraldehyde or covalent attachment by carbodiimide coupling. The effectiveness of the different steps in the Gox immobilisation procedure was assessed by contact angle measurements, cyclic voltammetry and X-ray photoelectron spectroscopy. The enzyme loading was estimated by radioactivity measurements. The sensitivity to beta-glucose of these different biosensors has been evaluated. Glucose responses as large as 400 nA mM(-1) cm(-2) have been obtained. To our knowledge, this sensitivity value is amongst the highest values reported in the literature for comparable biosensor systems.

Biosensing Techniques↗

Integrated capillary fluorescence DNA biosensor.

Covalent attachment of dsDNA molecules inside a glass capillary without the need for hybridization is described. It is shown that the glass capillary has a surface density of 2.5 x 10(13) molecules/cm(2) with specific binding capacity of 62.5%. The resulting substrate was used to develop a biosensor for determining fluorescent organic analytes and metal binding with DNA. The biosensor combines highly specific immobilization chemistry with a capillary-geometry flow cell arrangement. The results show that fluorescent dyes are retained in the dsDNA-modified surface and that exposure to concentrations of nickel and lead ions resulted in a recoverable, highly reproducible diminishment of the fluorescence intensity.

Adsorption↗