Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

Improved sensitivity for the electrochemical biosensor with an adjunct probe

Despite their promising applications in the biomedical research, the development of electrochemical biosensors with improved sensitivity and low detection limit has remained a great challenge. Here, we demonstrate a new approach to improve the sensitivity of the electrochemical biosensor by simply introducing an adjunct probe into its construction. This signal-on biosensor consists of a thiol-functionalized capture probe attached on the gold electrode surface, an electrochemical sign (methyl blue, MB)-modified reporter probe which is complementary to the capture probe, and an adjunct probe attached nearby the capture probe. The adjunct probe functions as a fixer to immobilize the element of reporter probe which is displaced by the target DNA and protein, increasing the chance of the dissociative reporter probe to collide with the electrode surface and facilitating the electron transfer. The biosensor with an adjunct probe exhibits improved sensitivity and a large dynamic range for DNA and the thrombin assay and can even distinguish 1-base mismatched target DNA. Importantly, the use of this biosensor is not limited to such and is viable for sensitive detection of numerous biomolecules, including RNA, proteins, and small molecules such as cocaine.     

Probing trace phenols based on mediator-free alumina sol--gel-derived tyrosinase biosensor

A novel tyrosinase biosensor has been developed for the subnanomolar detection of phenols, based on the immobilization of tyrosinase in a positively charged Al2O3 sol-gel membrane on a glassy carbon electrode. It has been found that Al2O3 sol-gel is perfectly beneficial to the immobilization of tyrosinase, because it not only possesses the general advantages of sol-gel but it also is an effective promoter of the biosensor. The large microscopic surface area, porous morphology, and hydrophilic property of the sol-gel matrix result in high enzyme loading, and the enzyme entrapped in this matrix retains its activity to a large extent. The Al2O3 sol-gel-containing surface also displays an intrinsic electrocatalytic o-quinone response and, hence, offers a high-sensitivity (127 microA mM(-1)) monitoring of phenols. The detection limit is 0.2 nM at a signal-to-noise ratio of 3, the response time is less than 4 s reaching 95% of the steady-state value, and 70% of the activity is retained after 3 months.     

Carbon paste biosensor for phenol detection of impregnated tissue: modification of selectivity by using β-cyclodextrin-containing PVA membrane

In order to detect and quantify with a biosensor the phenol contents in animal tissue (salmon flesh) by simple contact, we have undertaken experimental study on nonwoven cellulose fibers soaked with phenolic compounds. An electrochemical biosensor made of carbon paste and tyrosinase mixed together with electropolymerized pyrrole was elaborated. Phenol detection was realized by electrochemical reduction of quinones produced by the tyrosinase activity. The biosensor was first optimized based on enzyme loading and nature of the carbon paste. A semipermeable membrane containing cyclodextrin moieties was deposited on the biosensor in order to test its sensitivity for phenol detection. Finally, the biosensor was put in contact with phenol absorbing cellulose fibers. Results showed that the relaxation time response of the sensor was relevant of phenol concentration.     

Portable surface-enhanced Raman scattering sensor for rapid detection of aniline and phenol derivatives by on-site electrostatic preconcentration

A portable surface-enhanced Raman scattering (SERS) sensor is developed and applied to simultaneous detection of aniline and phenol derivatives in a label-free way with an electrostatic preconcentration technique to amplify the signals. A SERS-active substrate, silver-electrodeposited screen-printed electrodes (Ag-SPEs), is used for qualification and quantification of polar organic pollutants. Observation of SERS spectra at different potentials indicates that polar pollutants are selectively adsorbed on the Ag-SPEs at a given potential, suggesting that Ag-SPEs could selectively attract polar pollutants to an oppositely charged electrode at different potentials. Optimum SERS-active substrate was obtained when a potential of -0.15 V vs Ag/AgCl was applied on the SPEs in 0.1 M AgNO(3) solution for 10 min. Moreover, the effects of experimental variables such as the electrodeposition time and potential of Ag and preconcentration time of polar molecules on the SERS signals are presented. Under optimum conditions and with a 785 nm laser, the method is effective over a wide range of concentration (1 nM to 1 μM) for aniline and phenol derivatives. The novel method described herein presents a new detection regime for environmental pollutant analysis and also demonstrates simultaneous multiplexed detection of polar organic pollutants using convenient Ag-SPEs.     

Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene-streptavidin-magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

Combinations of PCR-based amplification platform using 5′ thiolated and biotinylated specific primers, S1 nuclease-PCR products treatment, ferrocene-streptavidin (Fc-Stv)-magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5′ terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene-streptavidin-magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA) compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA) were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.     

Detection of toxic compounds in real water samples using a conductometric tyrosinase biosensor

A conductometric tyrosinase biosensor for the detection of some toxic compounds including diuron, atrazine, and copper ions was developed. The work of this biosensor is based on the principle of change of conductivity of the enzyme membrane when tyrosinase either interacts with 4-chlorophenol substrate or is inhibited by pollutants. The different samples tested were solutions containing diuron, atrazine, copper, lead and zinc ions, mixtures of copper/atrazine or copper/diuron and real water samples coming from a Vietnamese river. In the last case, classical techniques such as GC-MS or atomic absorption spectrometry were used in order to estimate exact concentration of these species in real water samples. Results have shown that such a biosensor could be used as an early warning system for the detection of these pollutants, as no matrix effect coming from the real sample was observed and no synergetic or antagonist effects were found for the mixture of toxic compounds. In addition, results were coherent with the content of the tyrosinase inhibitors.     

Enzyme electrodes with conductive polymer membranes and Langmuir-Blodgett films

A glucose sensor consisting of a conductive polypyrrole membrane and a lipid Langmuir-Blodgett (LB) film has been investigated. Different arrangements of the biosensor on the electrodes examined were (1) electrode with glucose oxidase (GOD)-immobilized lipid LB films; (2) GOD-immobilized LB film coated on polypyrrole-modified electrode; (3) electrode with a GOD-immobilized polypyrrole membrane; (4) GOD-immobilized LB film coated on a GOD-polypyrrole-modified electrode. It was shown that the quality of the biosensor was apparently improved in case (4) with respect to both the detectable concentration range of glucose and the lifetime over which it could be used. The number of layers of the LB film has a marked influence on the sensitivity of the biosensor, an optimum number of layers existing for the best response. The mechanism of such an improvement is discussed.     

Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode

In this study, an electrochemical DNA biosensor was developed for detection of the breakpoint cluster region gene and the cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia by using 18-mer locked, nucleic acid-modified, single-stranded DNA as the capture probe. The capture probe was covalently attached on the sulfonic-terminated aminobenzenesulfonic acid monolayer-modified glassy carbon electrode through the free amines of DNA bases based on the acyl chloride cross-linking reaction. The covalently immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on the LNA/4-ABSA/GCE surface. Differential pulse voltammetry was used to monitor the hybridization reaction on the capture probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated that, in pH 7.0 Tris-HCl buffer solution, the peak current was linear with the concentration of complementary strand in the range of 1.0 x 10 (-12)1.1 x 10 (-11) M with a detection limit of 9.4 x 10 (-13) M. This new method demonstrates its excellent specificity for single-base mismatch and complementary dsDNA after hybridization, and this probe has been used for assay of PCR real sample with satisfactory results.     

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.     

DNA as a support for glucose oxidase immobilization at Prussian blue-modified glassy carbon electrode in biosensor preparation

An amperometric glucose biosensor has been developed using DNA as a matrix of Glucose oxidase (GOx) at Prussian-blue (PB)-modified glassy carbon (GC) electrode. GC electrode was chemically modified by the PB. GOx was immobilized together with DNA at the working area of the PB-modified electrode by placing a drop of the mixture of DNA and GOx. The response of the biosensor for glucose was evaluated amperometrically. Upon immobilization of glucose oxidase with DNA, the biosensor showed rapid response toward the glucose. On the other hand, no significant response was obtained in the absence of DNA. Experimental conditions influencing the biosensor performance were optimized and assessed. This biosensor offered an excellent electrochemical response for glucose concentration in micro mol level with high sensitivity and selectivity and short response time. The levels of the relative standard deviation (RSDs), (<4%) for the entire analyses reflected a highly reproducible sensor performance. Through the use of optimized conditions, a linear relationship between current and glucose concentration was obtained up to 4 x 10(-4) M. In addition, this biosensor showed high reproducibility and stability.     

Accumulation of amplified target DNAs using thiol/biotin labeling,S1 nuclease,and ferrocene–streptavidin–magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

Combinations of PCR-based amplification platform using 5’ thiolated and biotinylated specific primers, S1 nuclease–PCR products treatment, ferrocene–streptavidin (Fc–Stv)–magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5’ terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene–streptavidin–magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA) compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA) were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.     

Thionine covalently tethered to multilayer horseradish peroxidase in a self-assembled monolayer as an electron-transfer mediator

A new approach to construct a reagentless enzyme biosensor is described. Based on multilayer horseradish peroxidase in a self-assembled monolayer configuration, the biosensor was constructed using multilayer thionine covalently tethered to the enzyme as an electron-transfer mediator. The multilayer enzyme and the multilayer mediator were stepwisely synthesized onto an l-cysteine-assembled gold electrode using glutaraldehyde as a bifunctional reagent. The multilayer mediator tethered to the multilayer enzyme could effectively and stably shuttle electrons between the electrode and the multilayer enzyme linked onto the monolayer. The sensitivity of the resulting enzyme biosensor with eight layers of enzyme and three layers of mediator was more than 250 μA cm(-)(2) for 1.0 × 10(-)(4) mol/L hydrogen peroxide under optimal conditions, whereas such a modified electrode with one layer of enzyme and one layer of mediator did not yield a detectable response to 1.0 × 10(-)(4) mol/L hydrogen peroxide.     

Thermometric sensing of peroxide in organic media. Application to monitor the stability of RBP-retinol-HRP complex

The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.     

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.     

Electrochemiluminescent biosensor for hypoxanthine based on the electrically heated carbon paste electrode modified with xanthine oxidase

A new electrochemiluminescent (ECL) biosensor based on an electrically heated carbon paste electrode (HCPE) that was surface modified by xanthine oxidase (XOD) was designed and constructed in this work. It was found that the ECL intensity of luminol could be enhanced at the surface of XOD/HCPE by adding hypoxanthine (HX) to the solution, and there was a linear relationship between the ECL intensity and the concentration of HX. On the basis of this, an ECL enzyme biosensor can thus be developed to detect HX. However, because the activity of XOD is highly dependent on temperature, the biosensor is very sensitive to the temperature of the electrode. Also, because the temperature of the electrode may also affect the diffusion and convection of the luminescent compounds near the electrode surface, a suitable temperature for XOD/HCPE has to be controlled to achieve the best ECL signal. The key feature of the designed biosensor is that the temperature of the electrode is controllable so the most suitable temperature for the enzyme reaction can be obtained. The obtained results showed that the ECL enzyme biosensor exhibited the best sensitivity at an electrode temperature of 35 degrees C for the detection of HX. The detection limit was 30-fold lower than that at room temperature (25 degrees C).     

An enzyme flow immunoassay that uses beta-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector

The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for colorimetric and CDH detection, respectively. Matrix effects were less pronounced with the CDH biosensor than with colorimetric detection.     

Application of graphene–pyrenebutyric acid nanocomposite as probe oligonucleotide immobilization platform in a DNA biosensor

A stable and uniform organic–inorganic nanocomposite that consists of graphene (GR) and pyrenebutyric acid (PBA) was obtained by ultrasonication, which was characterized by scanning electron microscopy (SEM) and UV–vis absorption spectra. The dispersion was dropped onto a gold electrode surface to obtain GR–PBA modified electrode (GR–PBA/Au). Electrochemical behaviors of the modified electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy using [Fe(CN)₆]^3 ?/4 ? as the electroactive probe. A novel DNA biosensor was constructed based on the covalent coupling of amino modified oligonucleotides with the carboxylic group on PBA. By using methylene blue (MB) as a redox-active hybridization indicator, the biosensor was applied to electrochemically detect the complementary sequence, and the results suggested that the peak currents of MB showed a good linear relationship with the logarithm values of target DNA concentrations in the range from 1.0 × 10^? 15 to 5.0 × 10^? 12 M with a detection limit of 3.8 × 10^? 16 M. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences.     

Hydrogel network entrapping cholesterol oxidase and octadecylsilica for optical biosensing in hydrophobic organic or aqueous micelle solvents

Two optical cholesterol biosensors have been fabricated by immobilizing cholesterol oxidase (ChOx) and octadecylsilica (ODS) particles in hydrogel network matrixes of copolymer of poly(vinyl alcohol) (PVA)/hydroxyethyl carboxymethyl cellulose (HECMC), and sol-gel, respectively. In conjunction with an optical oxygen transducer, the immobilized ChOx in the sol-gel/ODS matrix was assembled as an optical cholesterol biosensor to continuously detect free cholesterol in aqueous micelle solution, while the immobilized ChOx in the PVA/HECMC/ODS matrix was constructed as an organic-phase optical cholesterol biosensor for the continuous analysis of free cholesterol in hydrophobic organic solvent. The compositions and properties of the immobilization matrixes, the effects of solvents and the analytical features were studied in detail. Both biosensors showed stable and reliable responses toward free cholesterol. For the aqueous micelle cholesterol biosensor, the analytical working range was from 0.05 to 8.0 mM cholesterol, the response time was 7-12 min, the operation life was more than 35 assays, and the shelf life was approximately 4 months. For the organic-phase cholesterol biosensor, the analytical working range was from 0.07 to 18.0 mM cholesterol, the response time was 4-8 min, the operation life was more than 120 assays, and the shelf life was longer than 5 months. The organic-phase cholesterol biosensor has been successfully applied to determine the free cholesterol content in commercial butter samples.     

A disposable,reagentless, third-generation glucose biosensor based on overoxidized poly(pyrrole)/tetrathiafulvalene-tetracyanoquinodimethane composite

A disposable glucose biosensor based on glucose oxidase immobilized on tetrathiafulvalene-tetracyanoquinodimethane (ITF-TCNQ) conducting organic salt synthesized in situ onto an overoxidized poly(pyrrole) (PPy(ox).) film is described. The TIF-TCNQ crystals grow through the nonconducting polypyrrole film (ensuring electrical connection to the underlying Pt electrode) and emerge from the film forming a treelike structure. The PPy(ox) film prevents the interfering substances from reaching the electrode surface. The sensor behavior can be modeled by assuming a direct reoxidation of the enzyme at the surface of the TTF-TCNQ crystals. A heterogeneous rate constant around 10(-6) - 10(-7) cm s(-1) has been estimated. The biosensor is nearly oxygen- and interference-free and when integrated in a flow injection system displays a remarkable sensitivity (70 nA/mM) and stability.