DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages.

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA(21)) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADH measured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50 nM of AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Electrochemical sensing with electrodes modified with molecularly imprinted polymer films.

This article summarises our work on the development of voltammetric sensors based on molecularly imprinted polymers. Several recognition elements and integration strategies were used:1.membranes electropolymerised at the electrode surface; 2.casting of polymeric membranes by drop-coating a solution of pre-formed polymer (polyphosphazene) and template in a low-boiling-point solvent on to the electrode surface; 3.preparation of composite membranes containing conductive material (graphite or carbon black), acrylic-type molecularly imprinted polymers (small particle size), and PVC as binder; and 4.in-situ polymerisation of a thin layer of acrylic imprinted polymer deposited on the electrode surface by spin coating. All the options evaluated offer the possibility of controlling electrode characteristics such as hydrophobic/hydrophilic character, permeability, or film thickness, which are essential for obtaining good sensor performance.

Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes.

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes.

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.

Amperometric glutamate biosensor based on poly(o-phenylenediamine) film electrogenerated onto modified carbon paste electrodes.

A glutamate biosensor was developed by electropolymerizing o-phenylenediamine on a dehydrogenase-NAD+ modified carbon paste electrode. The amperometric response to glutamate was obtained by means of the electrocatalytic oxidation of the enzymatically produced NADH, at an applied potential close to 0 V (Ag/AgCl). After optimizing carbon paste composition, polymer thickness and operating variables, a linear amperometric response to glutamate was obtained within the concentration range 5.0 x 10(-6)-7.8 x 10(-5) M with a detection limit of 3.8 x 10(-6) M. The biosensor was applied to the determination of glutamate in chicken bouillon cubes. Good accuracy was found with respect to a reference enzymatic spectrophotometric method.

Poly(o-aminophenol)-modified bienzyme carbon paste electrode for the detection of uric acid.

A reagentless uric acid selective biosensor constructed by immobilising uricase and horseradish peroxidase (HRP) in carbon paste without the addition of an electron transfer mediator is described. The response of the electrode is based on the enzymatic reduction of hydrogen peroxide in the presence of uric acid. Uricase and HRP were dispersed in the carbon paste and the optimum paste mixture was determined. Poly(o-aminophenol) was electropolymerised at the working surface area of the electrode acting as a conducting polymer layer. Cyclic voltammetry was used to characterise the permselective characteristics of the polymer layer. At an applied potential of 50 mV vs. Ag/AgCl, a linear response was obtained up to 1 x 10(-4) M, with a limit of detection of 3 x 10(-6) M. The sensor had a response time of 37 s. a calibration precision of 2.2% (n = 4) and an estimated sample frequency of 20 h(-1). Responses to the analyte of interest were pH dependent. The sensor was incorporated into a flow injection system for the qualification of uric acid in human serum. Results compared favourably with a standard spectrophotometric method.

Electrochemical behaviour of clenbuterol at Nafion-modified carbon-paste electrodes.

A detailed study of the electrochemistry of clenbuterol at bare carbon-paste electrodes (CPEs) has been carried out. Results showed that clenbuterol undergoes an ECE process. This compound is irreversibly oxidised at high potentials, resulting in the formation of a product which demonstrates quasi-reversible electrochemical behaviour at less positive potentials. The amount of this chemical product formed is very pH-dependent. Investigations into the electrochemical behaviour of clenbuterol at Nafion-modified CPEs were also made. The use of a thin Nafion film cast over the CPE resulted in a large increase in peak current over bare electrodes. Linear accumulation occurred with time, the linear range increasing with decreasing concentration. This allowed the detection of low concentrations of clenbuterol. Diffusion proved to be the rate-controlling process of clenbuterol through the Nafion membrane.

Application of a Nafion-modified carbon paste electrode for the absorptive stripping voltammetric determination of fenoterol in pharmaceutical preparations and biological fluids.

The preconcentration of fenoterol on a Nafion-modified carbon paste electrode and its subsequent determination using differential pulse voltammetry is described. The effect of pH and percentage Nafion concentration on the accumulation behaviour of fenoterol was studied, and accumulation curves, calibration graphs and reproducibility studies at two different Nafion concentrations have been carried out in the range 2.5 x 10(-8)-5.0 x 10(-7) M fenoterol. A limit of detection in aqueous solutions, calculated using a signal-to-noise ratio (S/N) of 3, was 9.0 x 10(-9) M. Application of the electrode to pharmaceutical preparations, without sample pretreatment, resulted in acceptable deviation from the stated concentration (RSD = +/- 3.81%, n = 4). For more complex matrices, a suitable extraction procedure was developed, resulting in recoveries of > 90% (urine) and > 75% (serum).

Comparative voltammetric study of 2,4-dinitrophenol (DNP), albumin and DNP-albumin as an analytical approach to the use of DNP as a universal label in immunoelectrochemical assays.

A comparative electrochemical study of 2,4-dinitrophenol (DNP), albumin and DNP-albumin has been carried out at a hanging mercury drop electrode, in order to use DNP as a universal label in immunoelectrical assays. Several electrochemical techniques have been used. Differential pulse voltammetry has proved to be the most suitable. Wide dynamic linear ranges (more than three orders of magnitude for DNP-albumin) and low detection limits have been achieved (5 x 10(-10)M, 2 x 10(-10)M, 3 x 10(-12)M for DNP, albumin and DNP-albumin, respectively). Good reproducibility has been obtained in all cases (R.S.D. < 2.2%).

Phase-selective AC adsorptive stripping voltammetric assay for aminopterin and 10-Edam in human serum.

Aminopterin was studied as a model compound for its analogues which maintain the pteridine ring in their structure. Its adsorptive behaviour on mercury was studied and the DC adsorptive stripping and phase-selective AC adsorptive stripping conditions were optimized. 10-Edam, an aminopterin analogue, was studied and shown to behave similarly to aminopterin. Phase-selective AC voltammetry provided the best signal and gave a detection limit of 4 x 10(-12) M aminopterin in aqueous solution employing an accumulation time of 10 min. The optimized method was applied to the analysis of both aminopterin and 10-Edam respectively in human serum. After extraction with a C18 reversed-phase cartridge the detection limit of the method was 1 x 10(-8) M aminopterin and the overall assay percentage recovery was 73.5% (n = 5) at a concentration of 5 x 10(-7) M aminopterin in serum. The analysis of 10-Edam at the same concentration in serum yielded the higher percentage recovery of 94.46% (n = 5) following the same procedure.

Determination of mitoxantrone by flow injection analysis using an amperometric detector.

Mitoxantrone was determined by flow injection analysis using a flow cell modified in the laboratory and fitted with carbon paste as an amperometric detector. The sample solution (100 microliters, 5 x 10(-8)-1 x 10(-5) M) was injected into the carrier stream of 0.1 M perchloric acid (pH 1.12). Mitoxantrone was determined by oxidation at the carbon paste electrode (CPE) at +0.90 V. A 60-cm delay coil (0.5 mm i.d.) was incorporated just before the detector (a canal thin layer) and a flow rate of about 4 ml min-1 was used. The system was successfully applied to the determination of mitoxantrone in a pharmaceutical preparation; the method was fast and reproducible.

Adsorptive stripping voltammetric determination of pipemidic acid in human urine.

The electrochemical behaviour of pipemidic acid (8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)-pyrido[2,3-d]pyrimidine-6- carboxylic acid), a well known antimicrobial agent used for urinary infections, was investigated by linear-sweep, differential-pulse and square-wave voltammetry at a hanging mercury drop electrode. Two reduction processes were observed in Britton-Robinson buffers at acid pH, whereas only one or two processes were observed in alkaline solutions, dependent on the pH of the buffer employed. Adsorptive effects were used to accumulate the drug on to the electrode. The adsorbed species were measured voltammetrically by using a cathodic process appearing at -0.76 V in 0.1 M HCIO4. Linear calibration graphs were obtained in the range 2.5 x 10(-9)-2.0 x 10(-7) M. A simple procedure of extraction was employed for the determination of the drug in urine samples.

Voltammetric assay of heroin in illicit dosage forms.

The oxidation of heroin on a carbon paste electrode has been studied by using voltammetric techniques under both semi-infinite linear diffusion and hydrodynamic conditions. By employing a simple and rapid in situ pre-treatment of the electrode, excellent reproducibility of the current signal was obtained. Subsequently, the current was measured and the concentration of total heroin present could be determined. The method compared favourably with instrumental methods that are more commonly used for the quantification of heroin in clinical laboratories such as chromatographic, spectrophotometric or radiometric techniques. Moreover, the proposed method showed good selectivity and was superior with respect to cost and time of analysis, permitting the determination of heroin in seized drug samples over a wide concentration range.

Voltammetric assay of methadone in urine.

A simple, rapid and sensitive voltammetric method for the determination of methadone in human urine is proposed, which permits the detection of concentrations of methadone as low as 0.3-0.4 microgram ml-1. The proposed method was compared with methods normally used in clinical trials, such as the enzyme multiplied immunoassay technique, and was found to be superior with regard to cost and to possess high sensitivity.

Adsorptive stripping voltammetric analysis of some pterines in human urine.

The combined content of biopterin and he separate folic acid content of human urine have been quantitated by phase-selective alternating-current stripping voltammetry at the static mercury drop electrode. The use of disposable C18 cartridge allowed the samples to be separated into two fractions, one containing biopterin and neopterin and the other folic acid. The average concentration of biopterin and neopterin together was 704 ng ml-1; precision, 6.6% (RSD, n=7); mean recovery, 96%. The average concentration of folic acid was 836 ng ml-1; precision, 8.2% (RSD, n=8); mean recovery, 91%.

Adsorptive stripping voltammetric assay of folic acid in human serum.

Folic acid was voltammetrically measured after preconcentration by adsorption at the static mercury drop electrode. Phase selective AC voltammetry provided the most sensitive stripping signal. After previous analyte extraction, using liquid-solid extraction with a C-18 reversed phase cartridge, the method was suitable for folic acid measurement in human serum. The detection limit was 5.9 x 10(-9) M with an overall precision of 9.9% (R.S.D.; n = 7) at the concentration level of 1.0 x 10(-7) M with a mean recovery of 57%.