Gold nanoparticle/streptavidin conjugates covered with 6-ferrocenylhexanethiol were attached onto a biotinylated DNA detection probe of a sandwich DNA complex. Due to the elasticity of the DNA strands, the ferrocene caps on gold nanoparticle/streptavidin conjugates are positioned in close proximity to the underlying electrode modified with a mixed DNA capture probe/hexanethiol self-assembled monolayer and can undergo reversible electron-transfer reactions. A detection level, down to 2.0pM (10amol for the 5μL of sample needed) for oligodeoxynucleotide samples was obtained. The amplification of the voltammetric signals was attributed to the attachment of a large number of redox (ferrocene) markers per DNA duplex formed. The ferrocene oxidation current increased with the target concentration and began to level off at a target concentration of 10nM. An Excellent linearity was found within the range between 6.9 and 150.0pM and reasonable relative standard deviations (between 3.0 and 13.0%) were obtained. The amenability of this method to the analyses of polynucleotides (i.e., PCR products of the pre-S gene of hepatitis B virus in serum samples) was also demonstrated. The method is shown to be simple, selective, reproducible, and costeffective and does not require labeling of the DNA targets.

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 doublestranded (ds) and single-stranded (ss) DNA, and the strength of the interactions is dependent on ionic strength (μ). 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 μ=8.4mM. BV2+ binds to ssDNA 5 times more strongly than the BV·+ form. From measurements atμ=8.4mM, a binding constant (K2+) of 2.0 ((0.2)×104M-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) ×104M-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.

A visual gene-detecting technique using nanoparticle-supported gene probes is described. With the aid of gold nanoparticle-supported 30-end-mercapto-derivatized oligonucleotide serving as detection probe, and 50-end-amino-derivatized oligonucleotide immobilized on glass surface acting as capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nano-amplification" and silver staining. Different genotypes of Hepatitis B and C viruses in the serum samples from infected patients were detected using homemade HBV, HCV, and HBV/HCV gene chips by the gold/silver nanoparticle staining amplification method. The present visual gene-detecting technique may avoid limitations with the reported methods, for its high sensitivity, good specificity, simplicity, speed, and cheapness. This technique has potential applications in many fields, especially in multi-gene detection gene chips coupled with the detection will find applications in clinic. Additionally, resonance Rayleigh light scattering (RLS) spectroscopy is used, for the first time, to judge and monitor the immobilization of gene probes on gold nanoparticle surfaces.

The modification of glassy carbon and gold electrodes with DNA by adsorption or covalent immobilization in a mono-or submonolayer has been investigated using the couple Co(bpy)3+/2+ as an indicator. It has been found that when the solution containing double stranded or single stranded DNA is evaporated to dryness, dehydrated DNA molecules can be irreversibly adsorbed on the surfaces of glassy carbon electrodes, in an amount close to that of the saturated adsorptive monolayer. The DNA-adsorbed layer on glassy carbon electrodes is unstable to bases, but stable to 1 M HCl solution. The adsorption of DNA on the electrodes can be evaluated from the increase in the peak current, the decrease in the value of △Ep, and the negative shift in the value of E for the Co(bpy)3+/2+ couple. DNA is very strongly adsorbed on the oxidized surfaces of glassy carbon electrodes, and the adsorptive layer is very stable towards heating. The covalent immobilization of DNA directly onto the electrode surfaces is impossible due to considerable steric hindrance; but if the active groups (sites) on the electrode surfaces are elongated with other suitable molecules, the covalent immobilization of DNA becomes possible on the electrode surfaces. The quantity of covalently immobilized DNA at the electrodes reported in the paper is about 31% of the saturated monolayer.

Gold electrodes were modified with DNA by adsorption. The DNA-modified electrodes were electrochemically characterized with Co(bpy)3+, a electroactive DNA-binding complex, as an indicator. It is interesting that the pair of redox peaks of Co(bpy)3+ split into two pairs at dsDNA-modified gold electrodes. One pair of peaks shifts negative, and the peak current increases notably; another pair of peaks shifts positive. These suggest that dsDNA has been immobilized onto gold electrode surfaces and the layer of dsDNA on the surfaces can bind Co(bpy)3+ in two different ways. Gold electrodes can be modified also with ssDNA by adsorption but only one pair of peaks of Co(bpy)3+ appears at ssDNA-modified gold electrodes. The amount of Co(bpy)3+ enriched by the layers of dsDNA or ssDNA adsorbed at gold electrodes was estimated from the peak charge of Co(bpy)3+ reduction at the electrodes obtained by CV. The stability of the DNA-modified electrodes was investigated. The DNA modification layer on gold surfaces is unstable to alkali and to heat, but stable to acid solutions and very stable in long stock in a dry. state. A comparison of modifications of gold, platinum and glassy carbon with DNA was carried out.