Electroactive Indicator Research Articles

Interactions between DNA and a water-soluble C 60 derivative studied by surface-based electrochemical methods

A novel electrochemical micromethod for the investigation of the interactions between DNA and non-electroactive species is described. The method was developed using the system of double-stranded DNA (dsDNA) modified gold electrodes (dsDNA/Au), a synthesized water-soluble C 60 derivative as a model, and [Co(phen) 3] 3+/2+ (phen=1,10-phenanthroline) as an electroactive indicator. Electrochemical studies with dsDNA-modified gold electrodes suggest that the C 60 derivative can interact strongly with dsDNA, with binding sites of the major groove of the double helix and phosphate backbone of dsDNA, a binding constant of (1.6 ± 0.2) × 10 5 M −1 obtained in 5 mM NaCl in Tris–HCl buffer, and a dissociation rate constant from the dsDNA/Au surface of (1.2 ± 0.1) × 10 −2 min −1.

Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode

Sequence-known short-stranded hepatitis B virus (HBV) DNA fragment (181 bps) was obtained by PCR method. The strategy for its electrochemical detection was designed by covalently immobilizing single-stranded HBV DNA on gold electrode surface via carboxylate ester as a linkage between 3′-hydroxy end of DNA and carboxyl group of thioglycolic acid (TGA) self-assembled monolayer. The hybridization reaction on surface was evidenced by electrochemical methods using ferrocenium hexafluorophosphate (FcPF 6) as an electroactive indicator. The interactions of Fc + with single-stranded (ss) and double-stranded (ds) HBV DNA immobilized on TGA monolayer were studied. The difference between the responses of Fc + at ss- and ds-DNA/Au electrodes suggested that this hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. AC impedance and XPS techniques have been employed to characterize the immobilization of ss-DNA on the gold surface.

Hybridization biosensor using di(2,2 ′-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3 ′-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181 bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy) 2Cl 2] + as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy) 2Cl 2] + in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy) 2Cl 2] + at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.

DNA Sensor for Recognition of Native Yeast DNA Sequence with Methylene Blue as an Electrochemical Hybridization Indicator

An electrochemical DNA sensor based on the recognition of single-stranded yeast DNA (ssDNA) immobilized on gold electrode to its complementary ssDNA (cDNA) is presented for hybridization detection. The yeast single-stranded DNA was covalently bound on a self-assembled 3-mercaptopropionic acid monolayer by using water soluble N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as a linker. The self-assembled monolayer prevented the nonspecific adsorption of long DNA chain on the electrode surface. The covalently immobilized ssDNA could selectively hybridize with cDNA in solution to form double-stranded DNA (dsDNA) on the surface. The increase of peak currents of methylene blue (MB), an electroactive indicator, upon the hybridization of immobilized ssDNA with cDNA in the solution was observed, which was used to monitor the recognition of yeast DNA sequence. The interaction of immobilized ssDNA and dsDNA with MB also resulted in a transition of electrode process from diffusion-controlled to surface-controlled process. The adsorption constants of methylene blue on ssDNA and dsDNA modified gold electrode surface were found to be (3.3±0.3)×103 M−1 and (6.6±0.4)×103 M−1, respectively, indicating a higher affinity of dsDNA to MB.

An Electropolymerized Membrane Biosensor for Specific DNA Recognition

AbstractA sensitive electrochemical biosensor for detecting the sequence of short DNA oligomers is represented. The biosensor is based on a platinum electrode covered a polymerized membrane of conductive monomer N‐[6‐(thien‐3‐yl)acetoxy]‐pyrrolidine‐2, 5‐dione (TAPD). The membrane of TAPD immobilizes a probe DNA on the electrode. The hybridization of the probe with a sequence‐specific DNA in sample solutions is monitored by a self‐synthesized electroactive indicator, which specifically intercalates in the hybrids on the electrode surface. The current signal of the biosensor is proportional to the concentration of the target DNA in samples, and a very low detection limit of 5 × 10−10 mol/L is found. The biosensor has been used to detect the short oligomers containing of HTV‐1 and mycobacterrium nucleotide sequences.

Determination of Short DNA Oligomers Using an Electrochemical Biosensor with a Conductive Self-Assembled Membrane

A new conductive modifier N-[6-(thien-3-yl)acetoxy]-pyrrolidine-2,5-dione (TAPD), which can self-assemble on a gold surface and react with amino groups is synthesized to immobilize a probe DNA on gold electrodes. The hybridization of the probe with a target DNA in sample solutions is monitored by a self-synthesized electroactive indicator, which specifically intercalates in the hybrids on the electrode surface. The current signal of the modified electrode is proportional to the concentration of the target DNA in samples, and a detection limit of 2×10−9 mol L−1 was found. The biosensor has been used to detect short oligomers containing HIV-1 and mycobacterrium nucleotide sequences.

Interactions of benzyl viologen with surface-bound single- and double-stranded DNA.

The interactions of benzyl viologen (BV) with single- and double-stranded calf-thymus DNA immobilized onto gold electrodes have been studied by electrochemical methods. Benzyl viologen interacts electrostatically with both double-stranded (ds) and single-stranded (ss) DNA, and the strength of the interactions is dependent on ionic strength (mu). The dicationic form (BV2+) binds to dsDNA 9 times more strongly than the singly reduced form, BV*+, in a pH 7.4 Tris-HCl buffer solution at mu = 8.4 mM. BV2+ binds to ssDNA 5 times more strongly than the BV*+ form. From measurements at mu = 8.4 mM, a binding constant (K2+) of 2.0 (+/-0.2) x 10(4) M(-1) and a binding site size (s) of 1 base pair were obtained, respectively, for dsDNA. For ssDNA, at the same ionic strength, the values obtained for K and s were 3.6 (+/-0.4) x10(4) M(-1) and 2 nucleotides, respectively. The amount of BV bound, whether to dsDNA or ssDNA, decreased with increasing ionic strength. Whereas the binding rate of BV to both dsDNA and ssDNA immobilized onto gold electrodes is relatively low, once immobilized, it dissociates rapidly away from the electrode surface. The electron-transfer rate constant for BV is moderately fast at both dsDNA- and ssDNA-modified gold electrodes. The application of benzyl viologen as an electroactive indicator capable of differentiating between surface-immobilized single- and double-stranded DNA in denaturation/regeneration cycles has been explored.

Detection of Interaction Between Metal Complex Indicator and DNA by Using Electrochemical Biosensor

There has been extensive research on binding of transition metal complexes to DNA via electrostatic and hydrophobic interactions. Most indicator based electrochemical DNA biosensors have used cationic metal complexes that interact in a different way with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Described here are the electrochemical parameters for a mixed-ligand complex, [Co(phen)33+] (phen: 1,10-phenanthroline), on binding to DNA. The milimolar quantities of [Co(phen)33+], which associates reversibly with immobilized calf thymus DNA was detected by using dsDNA-modified carbon paste electrode (dsDNA-modified CPE), ssDNA-modified carbon paste electrode (ssDNA-modified CPE) and bare carbon paste electrode (bare CPE), voltammetrically and the decreased peak currents were observed, respectively. The extend of hybridization between the complementary sequences is determined by the enhancement of the voltammetric peak of the [Co(phen)33+] indicator. Numerous factors affecting the DNA immobilization and indicator were investigated. Experiments were also performed at various salt concentrations and the optimum salt concentration was determined. The difference between the peak currents of denaturated calf thymus DNA (ssDNA)-modified CPE and dsDNA-modified CPE was also observed. These results demonstrated the use of the electroactive hybridization indicator, [Co(phen)33+] for DNA biosensors.

Electrochemical DNA Probes

A report on the status of the electrochemical DNA probes for the detection of a DNA sequence for environmental and clinical studies is presented. The literature on the electrochemistry of DNA in the last ten years is reviewed. Results obtained in this laboratory using an electroactive hybridization indicator for constructing an electrochemical DNA probe are reported.

Sulfonyl groups as linking agents for chemical modification of electrodes

SnO 2 electrodes have been chemically modified using p-carboxybenzenesulfonylchloride and 4,4′-biphenyldisulfonylchloride as linking agents. Hydroxymethylferrocene was then reacted with these surface bound species to serve as an electroactive indicator. The electrochemical response of these electrodes is characteristic of surface processes. Electrodes with hydroxymethylferrocene linked through p-carboxybenzenesulfonylchloride gave anodic peak potentials between 0.37 and 0.39 V vs. Ag/AgCl in 0.10 M tris(hydroxymethyl)aminomethane perchlorate, pH 7 supporting electrolyte with half-peak widths between 70 and 100 mV. Hydroxymethylferrocene linked through 4,4′-biphenyldisulfonylchloride gave the same peak potentials with half-peak widths of 120 to 167 mV. Surface coverage of hydroxymethylferrocene was 6.4×10 −10 and 1.3×10 −9 mol cm −2 respectively. Attachment of these sulfonyl compounds to electrodes involves simpler procedures and techniques than many other modification schemes.