Determination of active diaphorase immobilized at electrode surfaces

The activity of diaphorase (from Bacillus stearothermophilus) immobilized on glassy carbon (GC) electrodes was determined by analyzing the catalytic currents for oxidation of the immobilized enzyme using digital simulation techniques, which gives the concentration of the active enzyme at the electrode surface. Results show that the immobilization by the cross-linking reaction with glutaraldehyde deactivates the enzyme and only about 10% of the total enzyme remains active at the electrode surface.     

Hydrosilylation of alkenes catalysed by rhodium complexes immobilized on silica via a pyridine group

2-(2-Trimethoxysilylethyl)pyridine, together with 3-methcryloxypropyltrimethoxysilane, was used to prepare a series of rhodium carbonyl complexes bound to silica via a pyridine group. The rhodium complex Rh₂(CO)₄Cl₂ (Rh₂) was used as the starting compound, and the immobilized complexes were prepared by four different routes which yielded both surface-bonded complexes and complexes bonded within the silicate matrix. These complexes were efficient catalysts of hydrosilylation of octene by triethxysilane. All the immobilized complexes were more than their homogeneous analogues and some could be re-used.     

Amperometric immunosensor for direct detection based upon functional lipid vesicles immobilized on nanowell array electrode

An original electrochemical immunosensor has now been developed that is based upon the spontaneous immobilization of biotinylated, functional lipid vesicles (FLVs) on a polymeric resist layer. An electrode was fabricated utilizing a form of electron-beam (e-beam) that has been used to fabricate 200 nm (nanoscale) wells in the resist layer covering of the gold electrode. The stability of adhered FLVs upon the nanowell (NW) electrode was observed by atomic force microscopy (AFM). From these observations, we were able to determine that the assembled FLVs primarily adhered as individual molecules, that is, without the aggregation or fusion noted in earlier designs. Additionally, these immobilized FLVs demonstrated clearly defined redox activity in electrochemical measurements. Streptavidin, biotinylated capture antibody, and target proteins were consequently injected in order to set up the immunoassay environment. Electrochemical immunoassay experimentation was performed on the NW array electrode with model proteins, such as human serum albumin (HSA) and carbonic anhydrase from bovine (CAB). We observed a notable current decrease, following the redox path, interrupted by the target HSA, indicating the binding of the capture antibody with the target antigen. On the basis of these results, we propose a new type of immunosensor incorporating this mechanism of spontaneous immobilization of FLVs.     

Amperometric detection of acetylcholine and choline in a liquid chromatographic system with an immobilized enzyme reactor

Acetylcholinesterase and choline oxidase were co-immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, which was incorporated as the enzyme reactor in an h.p.l.c. system for the determination of acetylcholine and choline. The hydrogen peroxide produced enzymatically in the enzyme reactor, after the separation of acetylcholine and choline by the reverse-phase column, was monitored amperometrically. The detection limits were 1.2 pmol for choline and 1.8 pmol for acetylcholine.     

Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes

In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.     

Amperometric determination of cholinesterase with use of an immobilized enzyme electrode

A choline-sensitive electrode consisting of an immobilized choline oxidase layer and an oxygen electrode is described. Cholinesterase (0.5–60 I.U. l^-1) is measured by addition of acetylcholine, and detection of the choline produced. The precision is 3%, and the electrode is stable for more than 2 weeks (140 assays).     

Amperometric determination of acetic acid with immobilized Trichosporon brassicae

A microbial sensor consisting of immobilized Trichosporon brassicae, a gas-permeable Teflon membrane and an oxygen electrode is suitable for the continuous determination of acetic acid in fermentation broths. When an acetic acid solution is pumped through the flow system, the current decreases to a steady state with a response time of 8 min; shorter pumping times give peaks which can also be measured. The relationship between the current decrease and the acetic acid concentration is linear up to 54 mg l^-1, with a relative standard deviation of about 6% at the higher concentrations. Selectivity is satisfactory. Results obtained with this sensor and by gas chromatography for a glutamic acid fermentation broth were in good agreement (regression coefficient 1.04). The sensor was stable for more than 3 weeks and 1500 assays.     

Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution.     

Direct electron transfer from modified glassy carbon electrodes carrying covalently immobilised mediators to a dissolved viologen accepting pyridine nucleotide oxidoreductase and dihydrolipoamide dehydrogenase

N,N′-Diaminopropyl-4,4′-bipyridinium dication (DAPV) and cobalt diaminosarcophagine (Co—diamsar) were covalently bound on functionalised glassy carbon electrodes. The electron transfer from the modified electrodes to dissolved dihydrolipoamide dehydrogenase (Lip-DH) or viologen accepting pyridine nucleotide oxidoreductase (VAPOR) was tested by NADH formation in potentiostatically controlled electrochemical cells. The NADH formed was used to reduce pyruvate to -lactate catalysed by -lactate dehydrogenase ( -LDH). The reaction kinetics between the modified electrodes and the dissolved enzymes have been examined.     

Amperometric detection of morphine based on poly(3,4-ethylenedioxythiophene) immobilized molecularly imprinted polymer particles prepared by precipitation polymerization

Molecular imprinting is a novel technique used for chiral separation, artificial antibodies, sensors, and assays. Typically, molecular imprinted polymers (MIPs) are monoliths with irregular shapes. However, microspherical shapes with more uniform size can be obtained by the method of precipitation polymerization, which offers a higher active surface area by manipulating its compositions. In this study, MIP particles for the target molecule, morphine, were synthesized using a precipitation polymerization method that is more facile than the previous one that produced a thermally polymerized bulk. The conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), was utilized to immobilize the MIP particles onto the indium tin oxide (ITO) glass as a MIP/PEDOT-modified electrode. The sensitivity for the MIP/PEDOT-modified electrode with MIP particles was 41.63 μA/cm² mM, which is more sensitive than that with non-MIP particles or that of a single PEDOT film with no incorporated particles in detecting morphine ranging from 0.1 to 2 mM. The detection limit was 0.3 mM (S/N = 3). In addition, we presented that the modified electrode can discriminate codeine that plays an interfering species.     

Amperometric determination of choline with enzyme immobilized in a hybrid mesoporous membrane

Choline sensor is successfully prepared by using immobilized enzyme, i.e., choline oxidase (ChOx) within a hybrid mesoporous membrane with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide, which is the co-product of the enzymatic choline oxidation. The determination range and the response time are 5.0-800 μM and approximately 2 min, respectively. The sensor is very stable compared to the native enzyme sensor and 85% of the initial response was maintained even after storage for 80 days. These results indicate that ChOx is successfully immobilized and well stabilized, and at the same time, enzyme reaction proceeds efficiently. Such ability of hybrid mesoporous membrane F127M suggests great promise for effective immobilization of enzyme useful for electrochemical biosensors.     

Amperometric flow-injection system with an immobilized enzyme reactor for the highly selective detection of phosphate and on-line amplification by substrate recycling

Bio-amperometric flow-injection systems are proposed for the highly selective and sensitive determination of phosphate. One system studied is based on the use of a co-immobilized purine nucleoside phosphorylase-xanthine oxidase reactor, which responds to phosphate with high selectivity, and a detection limit of 3 × 10^?7 M for a 20-μl injection. Another system with a co-immobilized purine nucleoside phosphorylase-xanthine oxidase-alkaline phosphatase reactor gives responses amplified by substrate recycling during passage through the enzyme reactor. Phosphate can be determined with 12 times the sensitivity in the latter system compared with the former, but the latter system responds to nucleotides and pyrophosphate in addition to orthophosphate.     

Amperometric detection of sulfur-containing compounds in alkaline media

A copper-based chemically modified glassy carbon electrode (GC-Cu) was developed to be used as an amperometric sensor for electrochemically detecting several sulfur-containing compounds in alkaline media. Under optimised flow injection conditions the calibration curves for sulfite, sulfide, thiosulfate, cysteine, cystine, etc., were linear over three orders of magnitude of concentration. Detection limits were of the order of 0.04-1.5 microM. A simple and rapid method for determining sulfite in red and white wines by anion-exchange chromatography with electrochemical detection is described.     

Amperometric detection of indapamide on glassy carbon electrode

This work describes the development of a fast, precise and reliable voltammetric method for the quantification of indapamide, an orally active diuretic sulfonamide used for hypertensive treatment. This compound acts inhibiting sodium reabsorption and increasing the elimination of water. This characteristic was responsible for its banishment by the International Olympic Committee since 1999. The study begins by finding an adequate potential range (−0.20 to 0.30 V) to avoid poisoning the working glassy carbon electrode (GCE) in phosphate buffer 0.10 mol L⁻¹ (pH=12.0). Utilizing flow injection analysis, linear responses between 2.0 × 10⁻⁶ mol L⁻¹ to 2.5 × 10⁻⁵ mol L⁻¹ of indapamide (R²=0.995), and detection limit (LOD) 3.0 × 10⁻⁷ mol L⁻¹ were obtained. This method was applied for the quantification of indapamide in tablets and in synthetic urine. The same flow system was used for the analysis of commercial drugs and the response obtained corresponded to 98 % of the concentration indicated on the drug label. These tablets were also analyzed by high performance liquid chromatography (HPLC), obtaining a recovery of 103 % and LOD 4.0 × 10⁻⁷ mol L⁻¹. The velocity of analysis using flow methods compares advantageously to the classical chromatographic methods. For synthetic urine, linear responses were obtained in samples spiked in the region from 5.0 × 10⁻⁶ mol L⁻¹ to 30 × 10⁻⁶ mol L⁻¹ (R²=0.991) and LOD 3.0 × 10⁻⁷ mol L⁻¹.     

Diazonium-functionalized horseradish peroxidase immobilized via addressable electrodeposition: direct electron transfer and electrochemical detection

A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 +/- 0.7 s-1 and a DeltaEp of 8 mV (v = 150 mV/s). The large ks and low DeltaEp are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.     

Amperometric determination of sulfite with sulfite oxidase immobilized at a platinum electrode surface

Sulfite oxidase is immobilized on collagen membrane at the surface of a platinum electrode and catalyzes the oxidation of sulfite to sulfate with stoichiometric production of hydrogen peroxide. The hydrogen peroxide is detected amperometically at the platinum electrode at an applied potential of 700 mV. The system responds linearly to sulfite in the range 1–150 μM, with a detection limit of 0.2 μM. The enzyme retains over 95% of its activity for three weeks if stored at ?20° C when the probe is not in use.     

Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer

Choline and acetylcholine sensors were prepared by using choline oxidase and acetylcholinesterase, entrapped in photocross- linkable poly(vinyl alcohol) bearing styrylpyridinium (PVA-SbQ). The measurements were based on the detection of hydrogen peroxide liberated by an enzyme reaction (choline oxidase) or two sequential enzyme reactions (acetylcholine esterase and choline oxidase). The determination range for choline was 2.5-2-150 αmol 1^-1 and for acetylcholine 20-2-750 αmol 1^-1. The response times were 2-2-4 min. The immobilized enzyme membranes stored in a dry state were very stable and no loss of activity was observed after storage for 60 days.     

Amperometric detection of uric acid and hypoxanthine with Xanthine oxidase immobilized and carbon based screen-printed electrode. Application for fish freshness determination

Carbon-based screen-printed electrodes are suitable for uric acid detection. Xanthine oxidase (XO) was immobilized either directly on the surface of the electrode or in a reactor with CPG aminopropylsilane in a FIA assembly. Higher reproducibility and lifetime was obtained with the reactor. Optimum conditions were found for the determination of Hypoxanthine (Hx), Inosine (HxR) and Inosine monophosphate (IMP). Calibration curves for IMP, HxR and Hx are linear up to 50 muM with detection limit of 1 muM for 50 mul injection. One assay is completed within 30 s. The reproducibility of 20 muM of Hx was obtained with CV 2%.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.