Label-Free Determination of Picogram Quantities of DNA by Stripping Voltammetry with Solid Copper Amalgam or Mercury Electrodes in the Presence of Copper

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of the DNA sensors. We show that subnanomolar concentrations (related to monomer content) of unlabeled DNA can be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the presence of copper. DNA is first treated with acid (e.g., 0.5 M perchloric acid), and the acid-released purine bases are directly determined by the cathodic stripping voltammetry. Volumes of 5-3 microL of acid-treated DNA can easily be analyzed, thus making possible the determination of picogram and subpicogram amounts of DNA corresponding to attomole and subattomole quantities of 1000-base pair DNA. Application of this determination in DNA hybridization detection is demonstrated using surface H for the hybridization (superparamagnetic beads with covalently attached DNA probe) and the mercury electrodes only for the determination of DNA selectively captured at surface H.

Electrochemical DNA sensors

“…Numerical methods to improve the signal-to-noise ratio have been developed, but more recent designs employ physical separation techniques to remove the sources of background interference. For example, Palecek and colleagues 22 and Wang and Kawde 23 have separately reported a two-step strategy for capturing target sequences using probe DNA immobilized onto magnetic beads. After target hybridization, the beads are magnetically separated from the pool of analytes.…”

DNA Electrochemical Behaviors, Recognition and Sensing by Combining with PCR Technique

“…Many works have been carried out at various mercury electrodes [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Recently, Paleček et al [27][28][29] introduced a new approaches in development of DNA sensors by detecting hybridization and electrochemical signals of DNA and RNA at two different surfaces: mercury or solid mercury amalgam electrode. Mercury electrodes have shown a remarkable sensitivity for small changes in the DNA structure and produced an early evidence of DNA premelting and ploymorphy of the DNA double helix.…”

Two‐Surface Strategy in Electrochemical DNA Hybridization Assays: Detection of Osmium‐Labeled Target DNA at Carbon Electrodes

“…1). These techniques may include, for example, measurements of intrinsic electrochemical signals of the target DNA [27,29], acid DNA hydrolysis followed by cathodic stripping voltammetry [26,31], labeling of target DNA with osmium tetroxide, 2,2'-bipyridine (Os, bipy), use of second hybridization (reporter) probes labeled with an electroactive marker [M. Fojta et al, unpublished] or with biotin [28], etc. Among these approaches, DNA osmium labeling appears to be most suitable for pyrimidine-rich DNAs because of the reactivity of Os, bipy towards (predominantly) thymine (while direct DNA voltammetry at carbon electrodes and CSV are better suited for purine-rich DNA strands).…”

“…This system allows much easier finding of optimal conditions for both the hybridization and the detection steps [3,25,26] than the one-surface systems. In our work we used commercially available magnetic Dynabeads oligo(dT) 25 as surface H, in connection with various detection techniques including i) cathodic stripping voltammetry of purine bases (released from captured target DNAs by acid treatment) at HMDE [26,31] or at a solid copper amalgam electrode [31] , or ii) enzyme-linked electrochemical immunoassay of Os, bipy-labeled DNA [3]. In this paper we utilize a recently developed [22] technique of analysis of the Os, bipy-modified DNA at carbon electrodes as the detection technique in two-surface DNA hybridization assays involving osmium labeling of target DNAs.…”