Gustavo Rivas

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.

Indicator-free electrochemical DNA hybridization biosensor

A new electrochemical hybridization biosensor protocol without an external indicator is described. The biosensor format involves the immobilization of inosine-substituted (guanine-free) probe onto the carbon paste transducer, and a direct chronopotentiometric detection of the duplex formation by the appearance of the guanine oxidation peak of the target. Such a use of the intrinsic DNA electrochemical response for monitoring hybridization events offers several advantages (over the common use of external indicators), including the appearance of a new peak, a flat background, or simplicity. A 4 min short hybridization period allows a detection limit around 120 ng/ml. Performance characteristics of the sensor are described along with future prospects.

Interactions of antitumor drug daunomycin with DNA in solution and at the surface

Abstract The interaction of the antitumor drug daunomycin with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of cyclic voltammetry and particularly by constant-current chronopotentiometric stripping analysis (CPSA) with the carbon paste electrodes (CPE). As a result of intercalation of this drug between the base pairs in dsDNA, the CPSA daunomycin peak δ decreased and a new more positive shoulder δb appeared. This shoulder was attributed to the oxidation of the drug intercalated in DNA. Under the same conditions almost no changes in the DNA peak Gox (due to oxidation of guanine residues) were observed. It was shown that daunomycin adheres strongly to the bare CPE (resisting washing) so that a daunomycin-modified electrode can be easily prepared. Daunomycin immobilized at CPE interacted with DNA on immersion of the modified electrode into the dsDNA solution, showing a decrease of peak δ and a well-separated peak δb instead of the shoulder (which resulted from the interaction of DNA with daunomycin in solution). When the DNA-modified CPE was immersed into a daunomycin solution the peak Gox increased in dependence on daunomycin concentration or on the time of interaction of daunomycin with dsDNA at the electrode surface. Such changes in peak Gox were observed only at submicromolar concentrations of daunomycin. At higher daunomycin concentrations or at longer interaction time intervals a daunomycin peak appeared, which was substantially smaller and more positive than the peak of free daunomycin. The increase of the DNA peak Gox was attributed to interaction of daunomycin from the side of the DNA double helix not contacting the electrode surface. Such binding may induce changes in the DNA structure including bending of the DNA molecule which may result in the increase of peak Gox. Our results thus suggest that the interaction of daunomycin with DNA anchored at the surface may significantly differ from that with DNA in solution. The prospects of using of electroanalytical methods in studies of DNA–drug interactions are discussed.

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.

Nucleic-acid immobilization, recognition and detection at chronopotentiometric DNA chips

Wide-scale DNA testing requires the development of fast, small, easy-to-use biosensing devices. Various synthetic oligonucleotides and DNA have thus been immobilized onto microfabricated thick-film carbon transducers for performing several new nucleic-acid assay protocols. These include hybridization detection of nucleic acid sequences, determination of small molecules (drugs, pollutants) based on their collection into the dsDNA layer or via monitoring their effect upon the intrinsic DNA oxidation signal, and direct adsorptive stripping measurements of ultratrace levels of nucleic acids. Transduction of these DNA recognition processes is accomplished by a new highly-sensitive constant-current stripping chronopotentiometric operation. Comparison to traditional electrodes indicates that the biosensing performance is not compromised by the use of mass-producible disposable transducers. Such thick-film DNA biosensors have been coupled to a compact, user-friendly, hand-held analyzer. Applicability for the detection of sequences from M. tuberculosis and HIV-1 DNAs is illustrated. Such activity in the authors laboratory, aimed at developing DNA-coated screen-printed electrodes, is reviewed.

Detection of point mutation in the p53 gene using a peptide nucleic acid biosensor

A 17-mer peptide nucleic acid (PNA) is used as the recognition layer of an electrochemical biosensor for detecting a specific mutation in the p53 gene. The performance of the PNA-derived biosensor is compared with that of its DNA counterpart. The significantly higher specificity of the PNA probe greatly improves the detection of a single point mutation, found in many types of cancer. Factors influencing the surface immobilization of the PNA probe, its hybridization to the p53 target sequence, and the chronopotentiometric detection step, are explored and optimized. This and similar developments hold promise for the diagnosis and management of cancer.

Stripping potentiometric transduction of DNA hybridization processes

Abstract DNA hybridization on carbon paste electrodes was followed by stripping potentiometric measurement of electroactive metal complexes associated with surface hybrids. The chronopotentiometric transduction mode offers high sensitivity and effective background compensation, and improves the sequence detection compared to analogous (cyclic- and square-wave) voltammetric measurements. Immobilization of the single-stranded nucleic-acid probe is accomplished by adsorptive accumulation onto an anodically activated carbon paste transducer. Variables of the probe immobilization, hybridization and intercalation steps, and chronopotentiometric transduction have been examined and optimized. The resulting assay protocol offers convenient quantitation of nanogram quantities of the target strand in connection with short (5–20 min) hybridization times.

Sequence-specific electrochemical biosensing of M. tuberculosis DNA

Abstract An electrochemical biosensor for the determination of short sequences from the Mycobacterium tuberculosis (MTB) DNA is described. The sensor relies on the modification of the carbon-paste transducer with 27- or 36-mer oligonucleotide probes and their hybridization to complementary strands from the MTB DNA direct repeat region. Chronopotentiometry is employed to transduce the hybridization event, in connection with a Co(phen) 3 3+ indicator. Short (5–15 min) hybridization periods permit convenient quantitation of ng ml −1 levels of the MTB DNA target. Similar results are observed using microfabricated carbon-strip transducers. The new sensor holds great promise for rapid screening for MTB infections. Direct adsorptive stripping potentiometric measurements of ultratrace levels of MTB DNA are also reported.

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.

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.