Development of disposable amperometric sulfur dioxide biosensors based on screen printed electrodes

The possibility of developing amperometric biosensors for the measurement of SO(2) in flowing gas streams has been examined. Screen-printed carbon electrodes (SPCEs) were tailored with the enzyme sulfite oxidase and cytochrome c and the response is generated through the resulting enzymatic and electrocatalytic reactions involving SO(3)(2-), formed when SO(2) gas is dissolved in the supporting electrolyte. Two methods of integrating the enzyme and cytochrome c with the SPCE were investigated. In one design (b-type biosensor), the components were mixed thoroughly with the same ink used to produce the SPCEs, then the modified ink was spread over the working electrode. In the second approach the bio-components were dissolved in the supporting electrolyte and simply deposited on top of the transducer (s-type biosensor). Both devices gave linear responses over the range 4--50 ppm but the sensitivity of the s-type was approximately twice that of the b-type biosensor. In addition, the time taken to reach 90% of the maximum response (t(90%)) was 110 s for the s-type biosensor compared with 200 s for the b-type biosensor. These studies illustrate the successful use of biosensors for the detection of sulfur dioxide at the relatively low potential of +0.3 V versus Ag.AgCl and should provide useful alternatives for decentralised environmental studies.     

Toward the development of smart and low cost point-of-care biosensors based on screen printed electrodes

Screen printing technology provides a cheap and easy means to fabricate disposable electrochemical devices in bulk quantities which are used for rapid, low-cost, on-site, real-time and recurrent industrial, pharmaceutical or environmental analyses. Recent developments in micro-fabrication and nano-characterization made it possible to screen print reproducible feature on materials including plastics, ceramics and metals. The processed features forms screen-printed disposable biochip (SPDB) upon the application of suitable bio-chemical recognition receptors following appropriate methods. Adequacy of biological and non-biological materials is the key to successful biochip development. We can further improve recognition ability of SPDBs by adopting new screen printed electrode (SPE) configurations. This review covers screen-printing theory with special emphasis on the technical impacts of SPE architectures, surface treatments, operational stability and signal sensitivity. The application of SPE in different areas has also been summarized. The article aims to highlight the state-of-the-art of SPDB at the laboratory scale to enable us in envisaging the deployment of emerging SPDB technology on the commercial scale.     

Bioelectrocatalysis-based application of quinoproteins and quinoprotein-containing bacterial cells in biosensors and biofuel cells

Electrochemical studies on the applied aspects of quinoproteins are briefly reviewed. Catalytic reactions of quinoprotein enzymes can be connected to electrochemical reactions directly or by the mediation of molecules functioning as electron acceptors of the enzymes. Such an enzyme-electrochemical reaction is called bioelectrocatalysis. It provides a novel method of kinetic analysis of enzyme catalysis and even whole bacterial cell catalysis. The principle of bioelectrocatalysis is first described, then, the bioelectrocatalysis-based application of quinoproteins in biosensors is mentioned. Characteristics and performance of this type of biosensor is explained by citing our own work. Possible application in bioreactors and biofuel cells is also mentioned.     

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.     

DNA based biosensors

Compared to advances in enzyme sensors, immunosensors, and microbial biosensors, relatively little work exists on DNA based biosensors. Here we review the DNA based biosensors that rely on nucleic acid hybridization. Major types DNA biosensors--electrochemical, optical, acoustic, and piezoelectric--are introduced and compared. The specificity and response characteristics of DNA biosensors are discussed. Overall, a promising future is foreseen for the DNA based sensor technology.     

RNA‐based fluorescent biosensors for live cell detection of bacterial sRNA

Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up‐ or downregulation of protein synthesis. In vivo imaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limited in vivo and do not provide real‐time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA‐based fluorescent biosensor for bacterial sRNA both in vitro and in vivo. We validated these sensors for three different bacterial sRNAs in Escherichia coli and demonstrated that the designs provide a bright, sequence‐specific signal output in response to exogenous and endogenous RNA targets.     

FluRox based biosensors

A biosensor is an integrated device of biomaterials and electronic components which detects physiological change or physico-chemical response. The efforts towards the development of a supersensitive FluoRox biosensor are discussed in this paper. FluoRox principle is based on the novel concept of monitoring redox events in vitro and in vivo by fluorescence detection based on forster resonance energy transfer (FRET). Unlike conventional electrochemical biosensors fluorescence based sensors has the advantage of higher sensitivity which under suitable conditions can detect single molecules. Thus a highly sensitive and a miniaturized device is aimed at, which will enable the detection of trace amounts of pollutants and the detection of diseases at an early stage. Think of a biosensor and youwould conjure up with the question of sensitivity. Nusrat Sanghamitra, Fellow of EdRox network explains how a simple but yet novel concept of detecting redox reaction induced fluorescence change shoots up the sensitivity.     

Ethanol biosensors based on alcohol oxidase

The detection and quantification of ethanol with high sensitivity, selectivity and accuracy is required in many different areas. A variety of methods and strategies have been reported for the determination of this analyte including gas chromatography, liquid chromatography, refractometry and spectrophotometry, among other. The use of the enzyme alcohol oxidase (AOX) on the analysis of ethanol in complex samples allows a considerable enhancement in specificity. This paper reviews the state of the art on ethanol determination based on AOX sensors, using either electrochemical electrodes or immobilised enzyme reactors. Almost all AOX-based ethanol sensors developed so far are based on the monitoring of O2 consumption or H2O2 formation. This has been mostly achieved using amperometric electrodes set at appropriate potentials namely, -600 mV for O2 monitoring or +600 mV for H2O2 monitoring. Mediated and non-mediated bienzymatic systems have also been assembled using AOX coupled to horseradish peroxidase (HRP). Different types of electrodes have been proposed for the detection of ethanol, namely, membrane electrode, carbon paste electrodes, screen-printed electrodes and self-assembled monolayers. Another approach to work with this sensitive enzyme is to use high amounts of AOX in order to create an enzyme reservoir, a strategy which can be implemented using immobilised enzyme reactors. These reactors can be combined with a colorimetric detection in a flow-injection analysis system or with electrochemical transducers.     

Properties of modified amperometric biosensors based on methanol dehydrogenase and cells Methylobacterium nodulans

The properties of amperometric biosensors based on methanol dehydrogenase (MDH) Methylobacterium nodulans, cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition of hydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135–0.5 and 0.032–1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 μM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.     

DNA biosensors based on self-assembled carbon nanotubes

DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.     

DNA biosensors based on water-soluble conjugated polymers

Conjugated polymers (CPs) with large, delocalised molecular structures exhibit unique optical and electrochemical characteristics that can be used as excellent sensing elements. Recently, research on chemical and biological sensors that use water-soluble CPs as transducers has generated intense interest. Two main sensing mechanisms are used for the detection of DNA-related events, such as hybridisation, mismatch, single nucleotide polymorphism (SNP), SNP genotyping, conformational changes, and cleavage of the nucleic acids. One mechanism takes advantage of the fluorescence resonance energy transfer (FRET) between CPs and a chromophore label on the nucleic acid probes in which a series of cationic polyfluorene, polythiophene and polyarylene derivatives are frequently used. The other mechanism relies on the conformational effects of CPs, which is induced by combination of the specific targets in which cationic polythiophene derivatives are often used. The electron transfer property of CPs are always used to design high sensitive electrochemical DNA biosensors. Here we review recent progress in the development of optical and electrochemical DNA biosensors based on water-soluble CPs.     

Rhizopine biosensors for plant-dependent control of bacterial gene expression

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl^?, SO ₄ ^2? , HCO ₃ ^? , and CO ₃ ^2? on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Cholesterol biosensors based on oxygen sensing alginate-silica microspheres

Cholesterol determination in body is important in diagnosis of diseases like coronary heart disease, arteriosclerosis, diabetes, and obstructive jaundice. This research aims at developing fluorimetric cholesterol biosensors based on self-assembled mesoporous alginate-silica (Algilica) microspheres. For preparing the biosensor, Pt-(II)-octaethylporphine (PtOEP; oxygen sensitive metalloporphyrin) dye has been loaded in the Algilica microspheres using the solvent-mediated precipitation method. Cholesterol oxidase (ChOx) was then covalently conjugated to PtOEP/Algilica microspheres using EDC and NHS reagents. PtOEP dye and enzyme encapsulation, activity and stability were then analyzed. Layer-by-layer self-assembly was finally performed using PAH and PSS polyelectrolytes to minimize leaching of the biosensor components. The prepared biosensor exhibited linearity over a range of 0.77-2.5 mM O(2) (K(SV) : 0.097/mM of O(2) ) obtained using from Stern-Volmer plots. The biosensor response to standard cholesterol displayed a linear analytical range from 1.25 to 10 mM of cholesterol with regression coefficient of 0.996 (1.25-3.75 mM), 0.976 (1.25-6 mM), and 0.959 (1.25-10 mM) and response time of 10 min. Thus, the prepared cholesterol biosensor shows great potential in the diagnosis of hypercholesterolemia.     

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.     

Recent advances in polyaniline based biosensors

The present paper contains a detailed overview of recent advances relating to polyaniline (PANI) as a transducer material for biosensor applications. This conducting polymer provides enormous opportunities for binding biomolecules, tuning their bio-catalytic properties, rapid electron transfer and direct communication to produce a range of analytical signals and new analytical applications. Merging the specific nature of different biomolecules (enzymes, nucleic acids, antibodies, etc.) and the key properties of this modern conducting matrix, possible biosensor designs and their biosensing characteristics have been discussed. Efforts have been made to discuss and explore various characteristics of PANI responsible for direct electron transfer leading towards fabrication of mediator-less biosensors.     

Highly sensitive dopamine biosensors based on organic electrochemical transistors

Organic electrochemical transistors (OECTs) based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonic acid) (PEDOT:PSS) with different gate electrodes, including graphite, Au and Pt electrode, etc., have been used as dopamine sensor for the first time. The sensitivity of the OECT to dopamine depends on its gate electrode and operation voltage. We find that the device with a Pt gate electrode characterized at the gate voltage of 0.6 V shows the highest sensitivity. The detection limit of the device to dopamine is lower than 5 nM, which is one order of magnitude better than a conventional electrochemical measurement with the same Pt electrode. It is expected that OECT is a good candidate for low cost and highly sensitive biosensor for the detection of dopamine.     

Amperometric biosensors based on recombinant laccases for phenols determination

Graphite (GE) or printed graphite electrode (PGE) based biosensors containing recombinant fungal laccase Polyporus pinsitus (rPpL), and Myceliophthora thermophila (rMtL) were developed. The enzymes were immobilized using bovine serum albumin and glutaraldehyde. At pH 5.5 and -0.1 V, the calibration graphs of GE based biosensors were hyperbolic if pyrocatechol was used. The concentration of substrate that results in 50% of steady-state response (EC(50)) was 0.7 mM and sensitivity (S) was 3.8 mA/M. The sensitivity increased up to 4 A/M if larger amount of rPpL was used. The sensitivity of biosensors changed little during 9 days of exploitation, but decreased at longer time. The PGE based biosensors were mounted into the flow-through cell and calibrated under kinetic regime. EC(50) of the biosensors containing rPpL varied from 0.6 to 4.0 mM and sensitivity varied from 0.11 to 1.9 mA/M. The response of biosensor containing thermostable laccase rMtL was less, but response saturated at larger pyrocatechol concentration. The sensitivity changed little during 6 days. Both type of biosensors responded also to 1-naphthol, o-phenylenediamine, guaiacol, o-anizidine, benzidine. The experiments demonstrate recombinant laccases application to biosensor engineering and their use to phenol and related compound determination under steady-state and flow-through regimes.