A DNA biosensor based on the electrocatalytic oxidation of amine by a threading intercalator

An electrochemical biosensor for the detection of DNA based a peptide nucleic acid (PNA) capture probe (CP) modified indium tin oxide electrode (ITO) is described in this report. After hybridization, a threading intercalator, N,N′-bis[(3-propyl)-imidazole]-1,4,5,8-naphthalene diimide (PIND) imidazole complexed with Ru(bpy)₂Cl (PIND-Ru, bpy = 2,2′-bipyridine), was introduced to the biosensor. PIND-Ru selectively intercalated to double-stranded DNA (ds-DNA) and became immobilized on the biosensor surface. Voltammetric tests showed highly stable and reversible electrochemical oxidation/reduction processes and the peak currents can directly be utilized for DNA quantification. When the tests were conducted in an amine-containing medium, Tris-HCl buffer for example, a remarkable improvement in the voltammetric response and noticeable enhancements of voltammetric and amperometric sensitivities were observed due to the electrocatalytic activity of the [Ru(bpy)₂Cl] redox moieties. Electrocatalytic current was observed when as little as 3.0 attomoles of DNA was present in the sample solution.     

A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

Spectrophotometric determination of nitrite based on its catalytic effect on the oxidation of carminic acid by bromate

A highly sensitive and selective method is described for the determination of trace amounts of nitrite based on its effect on the oxidation of carminic acid with bromate. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of carminic acid at 490 nm after 3 min of mixing the reagents. The optimum reaction conditions were 1.8×10⁻¹ mol l⁻¹ H₂SO₄, 3.8×10⁻³ mol l⁻¹ KBrO₃, and 1.2×10⁻⁴ mol l⁻¹ carminic acid at 30°C. By using the recommended procedure, the calibration graph was linear from 0.2 to 14 ng ml⁻¹ of nitrite; the detection limit was 0.04 ng ml⁻¹; the R.S.D. for six replicate determinations of 6 ng ml⁻¹ was 1.7%. The method is mostly free from interference, especially from large amounts of nitrate and ammonium ions. The proposed method was applied to the determination of nitrite in rain and river water.     

Electrochemical microfluidic biosensor for the detection of nucleic acid sequences

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.     

Phenol biosensor based on electrochemically controlled integration of tyrosinase in a redox polymer

An amperometric biosensor for the detection of phenolic compounds was developed based on the immobilization of tyrosinase within an Os-complex functionalized electrodeposition polymer. Integration of tyrosinase within the redox polymer assures efficient catechol recycling between the enzyme and the polymer bound redox sites. The non-manual immobilization procedure improves the reproducibility of fabrication process, greatly reduces the desorption of the enzyme from the immobilization layer, and, most importantly prevents fast inactivation of the enzyme by its substrate due to fast redox cycling. A two-layer sensor architecture was developed involving ascorbic acid oxidase entrapped within an electrodeposition polymer in a second layer on top of the redox polymer/tyrosinase layer. Using this sensor architecture it was possible to eliminate the current interference arising from direct ascorbate oxidation up to a concentration of 630 μM ascorbic acid. The effects of the polymer thickness, the enzyme/polymer ratio, and the applied potential were evaluated with respect to optimal sensor properties. The sensitivity of the optimized sensors for catechol was 6.1 nA μM⁻¹ with a detection limit of 10 nM, and for phenol 0.15 nA μM⁻¹ with a detection limit of 100 nM.     

Spectrophotometric determination of osmium based on its catalytic effect on the oxidation of carminic acid by hydrogen peroxide

A highly sensitive spectrophotometric method is described for the determination of trace amounts of osmium(VIII), based on its catalytic effect on the oxidation of carminic acid by hydrogen peroxide. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of carminic acid at 540 nm after 3 min of mixing the reagents. The optimum reaction conditions were 1x10(-4) mol l(-1) carminic acid, 0.013 mol l(-1) hydrogen peroxide and pH 10 at 25 degrees C. By using the recommended procedure, the calibration graph was linear from 0.1 to 1.5 ng ml(-1) of osmium; the detection limit was 0.02 ng ml(-1); the RSD for five replicate determinations of 0.2-1.4 ng ml(-1) was in the range of 1.8-4.7%. The influence of several foreign ions on osmium determination were studied and the effect of interfering ions were removed by extracting osmium into isobuthyl methyl ketone and back extracting into sodium hydroxide solution.     

An amperometric biosensor for glucose based on electrodeposited redox polymer/glucose oxidase film on a gold electrode.

In this paper, we described a glucose biosensor based on the co-electrodeposition of a poly(vinylimidazole) complex of [Os(bpy)2Cl](+/2+) (PVI-Os) and glucose oxidase (GOX) on a gold electrode surface. The one-step co-electrodeposition method provided a better control on the sensor construction, especially when it was applied to microsensor construction. The modified electrode exhibited the classical features of a kinetically fast redox couple bound to an electrode surface and the redox potential of the redox polymer/enzyme film was 0.14 V (vs. SCE). For a scan rate of up to 200 mV s(-1), the peak-to-peak potential separation was less than 25 mV. In the presence of glucose, a typical catalytic oxidation current was observed, which reached a plateau at 0.25 V (vs. SCE). Under the optimal experimental conditions, the steady-state electrooxidation current measured at 0.30 V (vs. SCE) was linear to the glucose concentration in the range of 0-30 mM. Successful attempts were made in blood sample analysis.     

Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator

The natural dye hematein in water solution was used as a pH-sensitive redox-active mediator for amperometric pH-sensing. The electrochemical characteristics were studied using cyclic voltammetry and chronoamperometry. Several types of urea biosensors were constructed with urease on the surface of platinum and graphite composite electrodes or in the bulk of the graphite composite. They were used for the amperometric urea determination at a working potential of 0 mV (versus SCE) using 0.5 mM hematein. Detection limits and response linearity was in the micromolar range depending on the biosensor type, concentration and pH of buffers used. An interference study of various cations, anions, and substances, which may be present in real samples demonstrated good selectivity for the determination of urea. The biosensors showed good operational (>3 h) and storage (>3 months) stability. The results of urea determination in blood and urine obtained by biosensor correlated well with those obtained by a spectrophotometric reference method.     

Characterization of an oxaluric acid derivative as a guanine oxidation product

The oxidation of guanine in the dinucleoside monophosphate d(GpT) by an oxo-metalloporphyrin generates a linear oxaluric acid derivative after heating at 65 degrees C for 30 min and at neutral pH.     

Combined redox couples for catalytic oxidation of methane by dioxygen at low temperatures

The combination of three redox couples Pd2+/Pd0-quinone/hydroquinone-NO2/NO creates an electron-transfer loop, which facilitates one-pot catalytic oxidation of methane selectively to methanol by dioxygen in CF3COOH at a temperature as low as 80 degrees C. This is the first example where an organic cocatalyst significantly increases the catalytic efficiency of a transition metal in the selective oxidation of methane in the presence of dioxygen.     

Simultaneous determination of trace iron and aluminum by catalytic spectrophotometry based on a novel oxidation reaction of xylene cyanol FF.

A new, simple, sensitive and selective method for the simultaneous determination of trace iron and aluminum by catalytic spectrophotometry was presented, based on the catalytic effects of iron and aluminum on the discoloring reaction of xylene cyanol FF proceeded by hydrogen peroxide and potassium periodate in weak nitric acid medium. No catalytic effect was obtained in the presence of hydrogen peroxide or potassium periodate only. With the conditional rate constants determined in reaction systems catalyzed by Al or Fe only, the concentrations of Fe and Al in the samples can be calculated. The method was applied to the simultaneous determination of trace Fe and Al in tap water, lake water, river water and tea leaves without separation and preconcentration.     

Regioselective alkylation of guanine derivatives in the synthesis of peptide nucleic acid monomers

A method for the synthesis of 6-O-benzyl- and 6-O-benzyl-2-N-benzyloxycarbonyl-protected guanine derivatives starting from 2-amino-6-chloropurin is described. A regioselective alkylation of these N(9)-protected guanine derivatives gave the corresponding α-monomers of chiral peptide nucleic acids, the L-glutamic acid derivatives. It was shown that these compound do not inhibit (in the concentrations <20 µmol L^–1) the topoisomerase I activity.     

Inhibitive detection of benzoic acid using a novel phenols biosensor based on polyaniline–polyacrylonitrile composite matrix

A novel sensitive and stable phenols amperometric biosensor, based on polyaniline–polyacrylonitrile composite matrix, was applied for determination of benzoic acid. The electrochemical biosensor functioning was based on the inhibition effect of benzoic acid on the biocatalytic activity of the polyphenol oxidase (PPO) to its substrate (catechol) in 0.1 M phosphate buffer solution (pH 6.5). A potential value of −50 mV versus SCE, and a constant catechol concentration of 20 μM were selective to carry out the amperometric inhibition measurement. The kinetic parameters Michaelis-Menten constant and maximum current (I_max) in the absence and in the presence of benzoic acid were also evaluated and the possible inhibition mechanism was deduced. The inhibiting action of benzoic acid on the polyphenol oxidase electrode was reversible and of the typical competitive type, with an apparent inhibition constant of 38 μM. This proposed biosensor detected levels of benzoic acid as low as 2 × 10⁻⁷ M in solution. In addition, the effects of temperature, pH value of solution on the inhibition and the interferences were investigated and discussed herein. Inhibition studies revealed that the proposed electrochemical biosensor was applicable for monitoring benzoic acid in real sample such as milk, yoghurt, sprite and cola.     

The development of a reagentless lactate biosensor based on a novel conducting polymer

A reagentless lactate biosensor is described, based on an electropolymerized copolymer film poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-acetic acid-1,4-naphthoquinone). The quinone group, as part of the polymer backbone, is electroactive and very stable in neutral aqueous medium. It can therefore act as an immobilized mediator for the enzyme recycling, at a working potential much lower than those commonly reported in the literature for other mediators. Experimental conditions for amperometric measurements (temperature, pH) are studied, especially the interference between quinone and molecular oxygen to investigate the enzyme/quinone recycling kinetic. Some well-known interferents are shown to have no measurable effect on the amperometric curves.     

Evaluation of an amperometric glucose biosensor based on a ruthenium complex mediator of low redox potential

An amperometric glucose ring-disk biosensor based on a ruthenium complex mediator of low redox potential was fabricated and evaluated. This thin-layer radial flow microsensor (10 μl) with ring-disk working electrode displayed remarkable amperometric sensitivity. For Ru₃(μ₃-O)(AcO)₆(Py)₃(ClO₄) (Ru-Py), a trinuclear oxo-acetate bridged cluster, a reversible redox curve of low redox potential and narrow potential window (redox potentials were −0.190 and −0.106 V versus Ag/AgCl wire, respectively) was observed, which is comparable to many reported mediators such as ferrocene derivatives and other ruthenium complexes. The glucose and hydrogen peroxide assays were carried out with this complex-modified electrode Ru-Py-HRP-GOx/Nafion. The sensitivity was obtained 24 nA (15.4 mA M⁻¹ cm⁻²) for 10 μM glucose and 126 nA (160 mA M⁻¹ cm⁻²) for 5 μM H₂O₂, respectively with a working potential at 0 V versus Ag/AgCl. Ascorbic acid was studied as interference to the glucose assay. The application of 0 V potential versus Ag/AgCl did not avoid the occurrence of the oxidation of ascorbic acid, however, the pre-coating of ascorbate oxidase on the disk part of the ring-disk working electrode efficiently pre-oxidized the ascorbic acid and hence eliminated its interference on the glucose response. The practical reliability was also evaluated by assaying the dialysate from the prefrontal cortex of Wistar rats.     

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

An ultrasensitive electrochemical biosensor for detection of DNA species related to oral cancer based on nuclease-assisted target recycling and amplification of DNAzyme

A simple, label-free, ultra-highly sensitive and selective electrochemical sensor based on nuclease-assisted target recycling and DNAzyme for the detection of DNA species related to oral cancer in saliva is developed.     

Spectrofluorimetric determination of uric acid based on its activation of catalytic oxidation of pyronine Y

A novel kinetic spectrofluorimetric method for the determination of uric acid based on the activation effect of uric acid on the Cu(II) ion catalyzed oxidation of pyronine Y by hydrogen peroxide was developed. The influence of different buffer solutions was tested and the Britton-Robinson buffer solution with pH 2.2 was found to be the optimum. The detection limit and the linear range for uric acid are 0.09 μg mL⁻¹ and 0.3–3.0 μg mL⁻¹, respectively. The RSD for eleven determinations of 1.6 μg mL⁻¹ uric acid was 1.6 %. Satisfactory results were obtained when using this method of uric acid determination in human urine.