Application of cyanide hydrolase from Klebsiella sp. in a biosensor system for the detection of low-level cyanide

Abstract A partially purified preparation of cyanide hydrolase (cyanidase) from a bacterium, Klebsiella sp., was applied as a biocatalyst in a biosensor system for low-level cyanide detection. In the biosensor system cyanide hydrolase converts cyanide into formate and ammonia. The formate produced in the cyanide degradation was detected with a formate biosensor, in which formate dehydrogenase (FDH; E.C. 1.2.1.2) was co-immobilized with salicylate hydroxylase (SHL; E.C. 1.14.13.1) on a Clark electrode. The principle of the formate sensor is that FDH converts formate into carbon dioxide using -nicotinamide adenine dinucleotide hydrate (NAD⁺). The corresponding NADH produced is then oxidized to NAD⁺ by SHL using salicylate and oxygen. The oxygen consumption is monitored with the Clark electrode. The optimum buffer pH and temperature for the enzymatic hydrolysis of potassium cyanide were studied. The preliminary experiments including the pretreatment of cyanide with cyanide hydrolase and then detection by the formate sensor gave a detection limit at 7.3 mol l^–1 cyanide. The linear range of the calibration curve was between 30 mol l^–1 and 300 mol l^–1 cyanide.     

Microtubular conductometric biosensor for ethanol detection

A conductometric sensor using microtubules of polyaniline as transducer cum immobilization matrix is reported, capable of detecting ethanol in liquid phase. Enzyme ADH (alcohol dehydrogenase) and its coenzyme NAD+ have been used to improve the selectivity of the sensor. The sensor concept is based on the protonation of the polyaniline by the hydrogen ion produced in the enzyme-catalyzed reaction, leading to changes in the electrical conductance of the polyaniline. The sensor works well on the physiological pH, can detect ethanol as low as 0.02% (v/v) (0.092 M) and has a linear trend at par healthcare guidelines. The sensor responses were measured in various permutation and combination of enzyme and coenzyme concentrations and site of immobilization. The sensor shows minor interference with other functional groups and alcohols. The possible causes for such interference have been discussed.     

A new biosensor for osteoporosis detection

Abstract

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment.

In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45?min and 10–60?pg µL^?1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.     

Metal-enhanced biosensor for genetic mismatch detection

DNA biosensors are increasingly used in hybridization reactions, mutation detection, genomic sequencing, and identification of pathogens. However, the inability to monitor the recognition signals without resorting to the use of labels or electroactive mediators has led to DNA devices with inadequate sensitivity. Moreover, some electroactive species require high redox potentials that often destroy the DNA complementarity. This article presents the concept of metal-enhanced detection (MED) for the determination of DNA-DNA reactions and presents the application of this concept for mismatch detection. The MED concept relies on the idea that metallic films deposited as a continuous layer or monolayer onto a solid electrode, or even electrostatically held, could greatly enhance the rate of electron transfer by reducing the distance between the donor and acceptor species and could lead to label-free assays during DNA hybridization reactions. The MED concept has been tested for voltammetric detection of gene sequence of Microcystis spp. The resulting biosensor involved the immobilization of a 17-mer DNA probe that is complementary to a specific gene sequence of Microcystis spp. on a gold electrode via avidin-biotin chemistry. Electrochemical reduction and oxidation of DNA-captured Ag(+) ions provided the detection signals for the target gene sequence in solution. A linear response of silver cathodic peak current with concentration of the target oligonucleotide sequence was observed with a detection limit of 7 x 10(-9)M. This label-free approach was successfully applied to detecting two-base-pair mismatches in the gene sequence of Microcystis spp.     

Combinatorial augmentation for a multi-pathogen biosensor: signal analysis and design

Recent advances in combinatorial chemistries have revolutionized approaches to drug candidate synthesis and screening. Combinatorial approaches are also beginning to be used to increase the performance of diagnostic devices for both clinical and field uses. The use of combinatorial technologies is motivated by a general desire to detect as many different pathogens using the smallest, most inexpensive and fastest system possible. We examine the potential for rational design approaches to enhance the performance and miniaturization of biosensors. We describe novel combinatorial biosensor systems, in addition to mathematical frameworks for their optimization and performance prediction. The biosensors are assumed to be composed of multiple detection channels with the following characteristics. Each channel has a single output and can be dynamically set to respond to some or all of a set of pathogens. Regardless of the number of pathogens detected, however, there is a single numerical output from a channel. We evaluate the amount of ambiguity of positive signals produced as a result of increasing both the number of channels and the number of pathogens detected per channel and the effect this ambiguity has on system performance. We further discuss strategies for disambiguating positive signals. Finally we cite specific biosensor configurations that exploit the findings above and compare them to “brute force” approaches. Overall we suggest the approach we refer to as “n-squared” to simultaneously optimize device cost, speed and reagent usage.     

Materials and techniques for electrochemical biosensor design and construction

New developments in biosensor design are appearing at a high rate as these devices play increasingly important roles in daily life. This review aims to highlight recent developments in materials and techniques for electrochemical biosensor design and construction. Rapid growth in biomaterials, especially the availability and application of a vast range of polymers and copolymers associated with new sensing techniques have led to remarkable innovation in the design and construction of biosensors, significant improvements in sensor function and the emergence of new types of biosensor. Nevertheless, in vivo applications remain limited by functional deterioration due to surface fouling by biological components. However, new copolymers based upon biomembrane mimicry have been extensively investigated during the last two decades, raising hopes that the problems related to interactions between foreign surfaces and biological fluids and tissues may soon be solved.     

Porous silicon biosensor for detection of viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods.     

Novel cyanobacterial biosensor for detection of herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Chip-NMR biosensor for detection and molecular analysis of cells

Rapid and accurate measurement of biomarkers in tissue and fluid samples is a major challenge in medicine. Here we report the development of a new, miniaturized diagnostic magnetic resonance (DMR) system for multiplexed, quantitative and rapid analysis. By using magnetic particles as a proximity sensor to amplify molecular interactions, the handheld DMR system can perform measurements on unprocessed biological samples. We show the capability of the DMR system by using it to detect bacteria with high sensitivity, identify small numbers of cells and analyze them on a molecular level in real time, and measure a series of protein biomarkers in parallel. The DMR technology shows promise as a robust and portable diagnostic device.     

Versatile biosensor vectors for detection and quantification of mercury

Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp. In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively. Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments. The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria. The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.     

Metalloprotein and redox protein design

Metalloprotein and redox protein design are rapidly advancing toward the chemical synthesis of novel proteins that have predictable structures and functions. Current data demonstrate a breadth of successful approaches to metallopeptide and metalloprotein design based on de novo, rational and combinatorial strategies. These sophisticated synthetic analogs of natural proteins constructively test our comprehension of metalloprotein structure/function relationships. Additionally, designed redox proteins provide novel constructs for examining the thermodynamics and kinetics of biological electron transfer.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Towards the protein phosphatase-based biosensor for microcystin detection

A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol-gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and alpha-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach.     

SOI-nanowire biosensor for detection of D-NFATc1 protein

The nanowire (NW) detection is one of the fast-acting and high-sensitive methods, which can recognize potentially relevant protein molecules. A NW-biosensor based on the silicon-on-insulator (SOI)-structures has been used for biospecific label-free real time detection of the NFATc1 (D-NFATc1) oncomarker. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFATc1 used as molecular probes. It was shown that using this biosensor it is possible to reach sensitivity of 10^?15 M. This sensitivity was comparable to that of the NW-biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate that approaches used in this study are promising for development of sensor elements for high-sensitive diagnostics of diseases.     

Aptamer-based biosensor arrays for detection and quantification of biological macromolecules

We have developed a chip-based biosensor for multiplex analysis of protein analytes. The biosensor utilizes immobilized DNA and RNA aptamers, selected against several different protein targets, to simultaneously detect and quantify levels of individual proteins in complex biological mixtures. Aptamers were each fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding. We show that solid-phase aptamer-protein interactions recapitulate binding interactions seen in solution. Furthermore, we demonstrate specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts. It is expected that this technology could speed diagnosis of cancer by enabling direct detection of the expression and modification of proteins closely correlated with disease.     

Laccase immobilization in redox active layered double hydroxides: a reagentless amperometric biosensor

This paper describes a new system for amperometric determination of dissolved oxygen and its application for the detection of anionic toxic substances, which are known as enzyme inhibitors. This biosensor is based on the co-immobilization of laccase from Trametes versicolor and a redox active layered double hydroxide [Zn-Cr-ABTS] on a glassy carbon electrode. The electrochemical transduction step corresponds to the electrocatalytic reduction of O2 at 0.2V by laccase as catalyst and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator. Such device provides a fast and a sensitive response for dissolved oxygen determination between 6 x 10(-8) and 4 x 10(-6)M and very low detection limits for azide (5.5 nM), fluoride (6.9 nM) and cyanide (6.2 nM).     

Optical biosensor for simultaneous detection of captan and organophosphorus compounds

The optical biosensor consisting of GST and acetylcholinesterase (AChE)-immobilized gel film was developed to detect captan and organophosphorus compounds simultaneously in contaminated water. The sensing scheme was based on the measurement of decrease of products formation (s-(2,4-dinitrobenzene) glutathione and alpha-naphthol by GST and AChE, respectively) due to the inhibition by captan and organophosphorus compounds. The absorbance of s-(2,4-dinitrobenzene) glutathione and alpha-naphthol was detected at 400 and 500 nm, respectively, by a proposed optical biosensor system. It was observed that AChE was inhibited by both captan and organophosphorus compounds, and GST was inhibited only by captan. The simultaneous detection and quantification of captan and organophosphorus compounds could be successfully achieved by the proposed sensor system. The proposed biosensor could successfully detect the captan and organophosphorus compounds concentration from 0 to 2 ppm.     

Boron nitride nanotube-based biosensor for acetone detection: molecular structural mechanics-based simulation

In this study, an ultra-sensitive biosensor based on a single-walled boron nitride nanotube (SWBNNT) structure is proposed for acetone detection. The molecular structural mechanics-based simulation approach has been used to model the atomic structure of SWBNNTs. The cantilevered and bridged configurations of SWBNNT-based biosensor have been considered for analysis. The resonant frequency shift due to attached mass has been analysed for the mass-based detection of acetone molecules. The present simulation approach is validated by comparing obtained simulated results with the continuum mechanics-based analytical results. Along with detection of the attached molecule, identification of its intermediate landing position along the length of the nanotube is equally important for the better performance of the biosensor systems. The frequency shift-based analysis has been reported for the mass-based detection of acetone molecules as well as its intermediate landing position along the length of the nanotube. The resonant frequency shift variations of the higher order modes of vibration for both the considered configurations of SWBNNTs have been assistive for the identification of intermediate landing position of the acetone molecule. The proposed molecular structural mechanics-based simulation approach is found to be very effectual in terms of simulation of the real atomic structures of the nanotube. The proposed biosensor can achieve extremely high sensitivity at molecular level and it can be potentially used for real-time sensing capability for the acetone concentration for future health monitoring.