The electron/hole conduction of disordered bulk double-stranded (ds) calf thymus DNA and of one-dimensionally aligned 12-base pair single- and double-stranded oligonucleotide monolayers on gold was probed by testing for the occurrence of Faradaic processes. The disordered ds-DNA film was probed by doping it with soybean peroxidase, an easy to “wire” thermostable polycationic enzyme and measuring the current density of electroreduction of H2O2 to water at SCE potential. Although the current density in films with hydrophilic electron-conducting polymeric “wires” is ∼0.5 mAcm-2, when ds-DNA was used to “wire” soybean peroxidase, the current density was only 0.1 μA cm-2, similar to that in the absence of an electron-conducting enzyme-“wiring” polymer. We conclude that the diffusivity of electrons in unaligned and unstretched calf thymus DNA is less than 10-11 cm2 s-1. Nevertheless, the occurrence of a Faradaic reaction was observed in the Au−S−(CH2)2−ds-oligo-NH−PQQ/Au−S−CH2−CH2−OH monolayer on gold, in which the helices were one-dimensionally aligned and comprised a >30 Å ds-oligonucleotide segment. In these the rate constant for PQQ electrooxidation−electroreduction was 1.5 ± 0.2 s-1, only about 4-fold less than the 5 ± 1 s-1 constant for the reference Au−S−(CH2)2−NH−PQQ monolayer. When two mismatches were introduced in the 12 base-pair ds-oligonucleotide (by C → A and C → T substitutions) the constant decreased to 0.6 ± 0.2 s -1. In contrast, the rate for the Au−S−(CH2)2−ss-oligo-NH−PQQ/Au−S−CH2−CH2−OH monolayer was too small to be measured; no voltammetric waves were detected at a scan rate of 10 mV s-1. The anisotropic conduction in the one-dimensionally ordered solid ds-DNA films is attributed to the concerted movement of cations in the direction of the main axes of the ds-helices when an electric field is applied. Such movement causes the high-frequency longitudinal (not the high-frequency transverse optical) polarizability to be high and thereby makes the resolved component of the high-frequency dielectric constant high. The solid ds-DNA films also contain less water than their solutions, which reduces the static dielectric constant relative to that of water. As shown by Mott and Gurney, reduction of the difference between the static dielectric constant and the high-frequency longitudinal dielectric constant increases the mean free path and the mobility of electrons in an ionic solid and makes ds-DNA a one-dimensional semiconductor. The high frequency dielectric constant, as described in textbooks on solid-state physics, also decreases the ionization energies of donors and greatly extends their Bohr-radii, which are sausage-shaped in ds-DNA. A likely n-type dopant is the G-base in the GC base pair, a dopant ionized (“oxidized”) in the high-frequency dielectric constant medium. The proposed biological function of the insulator-to-semiconductor transition upon parallel alignment of the ds-DNA is protection against irreversible chemical change by oxidation or reduction. Removing or adding of an electron produces...

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically "wiring" horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the H2O2 electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, converting the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of H2O2 to water. Flow of H2O2 electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 microA cm(-2), exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in approximately 30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.

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Amplification of an 800-base template was verified in a 10-min test on a 2-microL sample of the PCR product solution. For verification, digoxigeninylated primers and biotinylated d-UTP-16-biotin were added to the amplification solution. The resulting amplified product was digoxigeninlabeled at its 3'-end and was also labeled with multiple biotin functions along its chain. The detecting electrode was coated with an electron-conducting redox hydrogel to which anti-digoxin monoclonal antibody was covalently bound. The amplified DNA was captured by the electrode through conjugation of its 3'-digoxigenin with the antibody. Exposure to a solution of horseradish peroxidase-labeled avidin led to capture of the enzyme and switched the redox hydrogel from a noncatalyst to catalyst for H2O2 electroreduction. The switching resulted in an H2O2 electroreduction current density of 2.1 +/- 0.9 microA cm-2 in 10-4 M H2O2 at Ag/AgCl potential and at 25 degrees C.

The hybridization of 10 5 copies of 25-30-base singlestranded poly(deoxythymidine)-5′-phosphate [ss-pd(T) 25-30 ] was detected amperometrically with a 10 µm-diameter microelectrode. The melting of 10 5 copies of the hybrid with horseradish peroxidase (HRP)-labeled poly(deoxyadenine)-5′-phosphate [ss-pd(A) 25-30 -HRP] was also tracked by amperometry. The microelectrode was coated with its hybridization-sensing layer in a two-step process involving electrophoretic deposition, which yielded reproducible electrode coatings. In the first step, a thin film of an electron-conducting redox polymer was deposited electrophoretically at constant current on the vitreous carbon surface of the microelectrode. In the second step, carbodiimide-activated 5′-phosporylated ss-pd(T) 25-30 was reactively electrophoretically deposited and covalently attached to the redox polymer film. Subsequent hybridization led to electrical contact between the HRP label of sspd(A) 25-30 and the conducting redox polymer. This contact resulted in catalysis of H 2 O 2 electroreduction to water at 0.0 V vs Ag/AgCl. The 20 ( 2 pA current produced by 10 5 copies of hybridized pd(A) 25-30 -HRP was at least 8 times greater than the 2.5 ( 2.5 pA current measured with noncomplementary HRP-labeled poly(deoxyguanidine)-5′-phosphate and 40 times greater than the 0.5 pA electrical background noise.