Percio A. M. Farias

DNA electrochemical biosensors for environmental monitoring. A review

Abstract DNA sensing protocols, based on different modes of nucleic acid interaction, possess an enormous potential for environmental monitoring. This review describes recent efforts aimed at coupling nucleic acid recognition layers with electrochemical transducers. It considers DNA hybridization sensors for sequences related to microbial or viral pathogens, and DNA-modified carbon electrodes for monitoring low molecular weight priority pollutants interacting with the surfaceconfined DNA. Carbon strip or paste electrode transducers, supporting the DNA recognition layer, are used with a highly sensitive chronopotentiometric transduction of the DNA analyte recognition event. Factors influencing the performance of these new environmental biosensors are discussed, and their environmental utility is illustrated. While the use of DNA biosensors is at a very early stage, these and similar developments are expected to have a profound effect on environmental analysis.

DNA biosensor for the detection of hydrazines.

A double-stranded (ds) DNA-coated carbon paste electrode is employed as a remarkably sensitive biosensor for the detection of hydrazine compounds. The sensor relies on monitoring changes in the intrinsic anodic response of the surface-confined DNA resulting from its interaction with hydrazine compounds and requires no label or indicator. Short reaction times (1-10 min) are sufficient for monitoring part-per-billion levels of different hydrazines. Applicability to untreated natural water samples is illustrated. The response mechanism is discussed, along with prospects of using DNA biosensors for quantitaing other important molecules and elucidating DNA interactions and damage.

Accumulation and trace measurements of phenothiazine drugs at DNA-modified electrodes

Abstract The DNA-phenothiazine intercalative association is utilized for designing a highly sensitive electrochemical biosensor for phenothiazine drugs. The DNA-modified carbon paste electrode permits convenient measurements of nanomolar concentrations of various phenothiazines following short accumulation times. The extent and rate of the accumulation depend upon the structure of the drug. The chronopotentiometric response is evaluated with respect to the preconcentration potential and time, concentration dependence, detection limits, reproducibility and other variables. Detection limits of 5 nM promethazine, 7 nM chlorpromazine and 12 nM phenothiazine are obtained after 10 min accumulation. Similar results are reported for DNA-coated microfabricated thick-film electrodes. The profound structural effect upon the response holds a great promise for using DNA-modified electrodes for studying drug-DNA interactions.

Stripping voltammetry of aluminum based on adsorptive accumulation of its solochrome violet RS complex at the static mercury drop electrode

Abstract A very sensitive electrochemical stripping procedure for aluminum is reported. Accumulation is achieved by controlled adsorption of the aluminum/solochrome violet RS complex on the static mercury drop electrode. Optimal experimental parameters include an accumulation potential of −0.45 V, solochrome violet RS concentration of 1 × 10 −6 M, and a linear-scan stripping mode. The detection limit is 0.15 μg l −1 , the response is linear over the 0–30 μg l −1 concentration range, and the relative standard deviation (at the 10 μg l −1 level) is 2%. Most cations do not interfere in the determination of aluminum. The interference of iron(III) is eliminated by addition of ascorbic acid. Results are reported for snow samples.

Potentiometric stripping analysis of bioactive peptides at carbon electrodes down to subnanomolar concentrations

Abstract The bioactive peptides bombesin, neurotensin and luteinizing hormone releasing hormone (LH-RH) can be adsorbed and accumulated at carbon paste electrodes and determined at low solution concentrations by potentiometric stripping analysis (PSA). The determination is based on the oxidation peaks of tryptophan (at about 0.7 V, against Ag/AgCl reference electrode) and/or of tyrosine (at about 0.55 V). Neurotensin and bombesin containing only tyrosine or tryptophan, respectively, produce single peaks at the corresponding potentials, while LH-RH, which contains both tyrosine and tryptophan residues, produces two well-resolved peaks. The coupling of the effective adsorptive preconcentration step with the advanced PSA operation allows peptide measurements down to subnanomolar and nanomolar concentrations. Analogous voltammetric runs produce no measurable signals at these levels. With 10 min accumulation, the PSA detection limit for bombesin is 2 × 10 −10 M (370pg). The peptides can be immobilized at the electrode by strong adsorption forces. If the electrode with the immobilized peptide is washed and transferred into the blank electrolyte to perform PSA, interferences of a number of low molecular mass substances (which are not attached to the electrode or are removed from it by washing) can be avoided. The peptides can be analyzed in the presence of an excess of compounds frequently present in the protein and peptide samples. In addition, large (75-fold) excess of the monomeric tyrosine and tryptophan show no interference.

Trace measurements of plasmid DNAs by adsorptive stripping potentiometry at carbon paste electrodes

Supercoiled, linear and denatured forms of plasmid pUC19 DNA can be adsorbed at carbon paste (CPE) and pyrolytic graphite (PGE) electrodes and detected by constant current chronopotentiometric stripping analysis (PSA) at low bulk concentrations. The determination is based on the PSA peak at about 1.0 V (against Ag|AgCl) due to the oxidation of guanine residues in the DNA. In agreement with earlier voltammetric data obtained with chromosomal DNAs at carbon electrodes, the denatured DNA produces a higher signal than linear and supercoiled DNAs. Compared with voltammetry, the sensitivity of PSA is higher by several orders of magnitude. PSA detection limits (obtained with a CPE) are about 100 ng ml−1 for supercoiled and linear DNAs and 15 ng ml−1 for denatured DNA. This great increase in sensitivity of the analysis at carbon electrodes offers new possibilities for electrochemical research, involving various forms of plasmid DNA.

Ultratrace determination of adenine in the presence of copper by adsorptive stripping voltammetry.

An electrochemical stripping procedure for ultra-trace measurements of adenine is described based on the adsorption of the adenine-copper complex on a static mercury drop electrode. Cyclic voltammetry was used to characterize the interfacial and redox behavior. Optimum experimental conditions were found when using a 0.005 M NaOH solution containing 0.4 ppm of copper, an accumulation potential of -0.30 V, a scan rate of 100 mV s(-1) and a linear scan mode. There is a linear response to adenine concentration in the range of 0.1-1.0 ppb and the detection limit for 6 min accumulation time was of 4.0 ppt (3.0x10(-11) M). Proper conditions for measuring the adenine in presence of guanine, thymine and cytosine were also investigated. The method was applied for the determination of adenine in a sample of single-stranded calf thymus DNA.

Electrochemical analysis of formation of polynucleotide complexes in solution and at electrode surfaces

Abstract The formation of double- and triple-stranded complexes between the synthetic homopolyribonucleotides in solution and at the surfaces of the hanging mercury drop (HMDE) and carbon paste (CPE) electrodes was studied by means of voltammetric and chronopotentiometric methods. It was shown that the cyclic voltammetry reduction signal of poly(C) and the anodic signal of poly(G) obtained with HMDE, as well as the chronopotentiometric oxidation signal of poly(G) obtained with CPE can be used to detect the duplex and triplex formation in solution. In addition to these intrinsic polynucleotide signals, the indicator [Co(phen) 3 ] 3+ was used to differentiate between single-stranded and ordered structures at CPE. It was shown that the indicator binds preferentially to the duplex poly(G) · poly(C) (formed in solution followed by adsorption at CPE) displaying, however, no preferential binding to the triplex structures. If poly(C) or poly(G) were immobilized at the electrode surface (by adsorption forces) and hybridized with the complementary polynucleotide in solution (at neutral pH), only a small extent of complexation could be detected at HMDE under the given conditions. On the other hand, analogous experiments with CPE suggested formation of ordered polynucleotide structures at the carbon electrode surface. As a result of complexation of poly(C) with poly(G) at neutral pH, formation of more than one ordered structure, including the duplex, was indicated. Polynucleotide complexation performed at pH 3.3 (optimum for formation of the protonated triplex poly(C + ) · poly(G) · poly(C) in solution) resulted in the formation of a mixture of structures including triplex and duplex structures. Binding of [Co(phen) 3 ] 3+ to the product of poly(A) complexation with poly(U) suggested that the yield of the duplex formed at the CPE surface may be influenced by the conformation of the single-stranded polynucleotide anchored at the surface prior to the hybridization. The implications of these findings to DNA biosensor development are discussed.

Determination of bilirubin by adsorptive stripping voltammetry

Abstract A stripping method for the determination of bilirubin at the submicromolar and nanomolar concentration levels is described. The method is based on controlled adsorptive accumulation of bilirubin at the static mercury drop electrode followed by differential pulse measurement of the surface species. After 15 min preconcentration, a detection limit near 5 × 10 −10 M bilirubin is obtained. The adsorptive stripping response is evaluated with respect to concentration dependence, preconcentration time and potential, pH, ionic strength, the presence of surfactants and other variables. Best results are obtained using sodium acetate (pH 8.2) solution. A signal enhancement factor of 11 is observed using 5 min preconcentration (compared to the response of solution-phase voltammetry). The relative standard deviation at the 2 × 10 −7 M level is 2.7%.

Trace determination of lanthanum, cerium, and praseodymium based on adsorptive stripping voltammetry

Abstract A sensitive stripping procedure is described for quantifying lanthanum, cerium and praseodymium ions, based on the controlled adsorptive accumulation of the lanthanide/ o - cresolphthalexon complex onto the static mercury drop electrode. The effect of various operational parameters on the stripping response is discussed. A 20-min accumulation period coupled with differential pulse measurement of the current resulting from the adsorbed complex permits quantitation down to the 1 × 10 −10 M level. For concentrations ranging from 2.5 × 10 −8 M to 2.5 × 10 −9 M, a 0.5- to 4-min accumulation period is sufficient. The relative standard deviation ar the 7 × 10 −8 M level ranges from 1 to 6%.