The effect of the shape of single, sub-ms voltage pulses on the rates of surface immobilization and hybridization of DNA

Abstract

Electric fields generated by single square and sinusoidal voltage pulses with amplitudes below 2 V were used to assist the covalent immobilization of single-stranded, thiolated DNA probes, onto a chemically functionalized SiO2 surface and to assist the specific hybridization of single-stranded DNA targets with immobilized complementary probes. The single-stranded immobilized DNA probes were either covalently immobilized (chemisorption) or electrostatically adsorbed (physisorption) to a chemically functionalized surface. Comparing the speed of electric field assisted immobilization and hybridization with the corresponding control reactions (without electric field), an increase of several orders of magnitude is observed, with the reaction timescaled down from 1 to 2 h to a range between 100 ns and 1 ms. The influence of the shape of the voltage pulse (square versus sinusoidal) and its duration were studied for both immobilization and hybridization reactions. The results show that pulsed electric fields are a useful tool to achieve temporal and spatial control of surface immobilization and hybridization reactions of DNA.

Electrokinetic techniques applied to electrochemical DNA biosensors

“…In solution the phosphate groups on the sugar-phosphate backbone dissociate endowing to the strands a negative charge surrounded radially by a cloud of positively charged counter-ions from the solution [17][18][19][20]. DNA strands behave in solution as stiff rod-like polymers with a linear charge distribution of 4.7 Â 10 À19 C/nm along the phosphate backbone [21]. The negatively charged DNA backbone makes the DNA highly polarizable allowing its handling and manipulation with electric fields by means of EP and DEP [22].…”

“…The application of these manipulation techniques to the biosensors field involves the specific and controlled attachment of single-stranded oligonucleotides onto a surface, facilitating the rapid transport and selective addressing of DNA probes to any position [21,[26][27][28][29][30], the acceleration of the basic hybridization process and the deattachment of DNA from the sensors for its regeneration.…”

“…In order to further improve the technique, new variations were introduced by Fixe et al [34] in 2003, which contemplated the use of nano-and micro-second pulses in order to induce mobility in the DNA strands, reducing the capacity effect. Fixe's group [21,30,34] also changed the permeation layer, using Al electrodes passivated with 200 nm Al 2 O 3 acting as insulator against adverse electrochemical reactions, and 200 nm SiO 2 , facilitating the functionalization of the electrodes. This surface was silanized with 3-aminopropyltriethosylane (APTES) in 2% of acetone obtaining a surface with reactive primary amines (-NH 2 ).…”

“…With this improved technique, fluorescence measurements showed that immobilization and hybridization rates are 10 9 and 10 7 times faster, respectively, than in the passive assays without applying the electric field pulse of 1 V (electric field density of 10-0.1 V/mm). The effect of the shape of single voltage pulses, on the rates of the surface immobilization and hybridization of DNA, was later studied by Cabec -a et al [21]. Two types of capture probe attachment; covalent immobilization (chemisorption) and electrostatic adsorption (physisorption) were compared, as well as the role and behaviour of the double-layer capacity at the electrode interphase.…”

“…A standard deviation of 25% was obtained for samples within the same experimental run and 38% for samples from different assays [21].…”