Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

Abstract

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surfaceimmobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.electrochemistry ͉ DNA hybridization ͉ DNA mismatch discrimination I ncreasing research efforts are directed toward the detection of nucleic acid interactions with immobilized oligonucleotide probes for DNA microarray applications (1, 2). Limitations of most current technologies include complex DNA immobilization procedures, the need for fluorescence or other labeling, and slow hybridization kinetics that require long incubation times. In addition, those experimental conditions that optimize DNA duplex formation often also reduce the stringency of hybridization. That is, for oligonucleotides that are sufficiently long to distinguish a particular sequence in the presence of unrelated DNA, a mismatched base pair has only a marginal effect on the stability of the duplex (3). In DNA microarray applications, mismatched hybrids lead to ''false positives.''The use of an electric field to easily control the electrostatic forces on surface-immobilized polyelectrolytes such as DNA has not yet been fully exploited as a means for improving the speed and stringency of biomolecular interactions at interfaces.In this paper, we investigate the effect of simple electrostatic charging on the interactions of surface-bound monolayer nucleic acid films with unlabeled DNA target oligonucleotides by using a combination of electrochemical control and in situ real-time surface plasmon resonance (SPR) spectroscopy detection.The monolayer DNA thiol films used in this work are tethered directly to the SPR metal sensor surface through a gold-thiol covalent attachment. Therefore, the immobilized DNA hybrids are exposed to a field gradient at the metal͞electrolyte interface on the order of 10 9 V͞m. We show that this field can be used, in a reversible manner, to increase or decrease the rate of oligonucleotide hybridization. In addition, we show that a repulsive potential preferentially denatures mismatched DNA hybrids within a few minutes, while leaving the fully complementary hybrids largely intact. This sequence selectivity in hybrid denaturation imparts an extr...

SERS-Melting: A New Method for Discriminating Mutations in DNA Sequences

“…A significant increase in the rate of hybridization was found by Heaton et al 64 in a study of the reaction of target DNA sequences with probe sequences attached to a gold surface through a thiol link. In the same paper, they also show that denaturation of dsDNA at the surface can be driven by the potential.…”

“…71,72 However, in these experiments, the authors were unable to generate sufficient difference to distinguish SNP targets from the perfect match when using linear DNA probes. There are also a few reports on monitoring electrochemical denaturation with other techniques such as SPR 64 and chemiluminescence 73 in which a constant potential was applied 68 and denaturation was monitored with time.…”

Formation and Investigations of Bioselective Element of Surface Plasmon Resonance Sensor for Recognition of Specific Oligonucleotide Sequences

“…²íø³ àâòîðè äîñÿãàëè óñï³øíî¿ ã³áðèäèçàö³¿ â 1 M NaCl ïðîòÿãîì 14 ãîä. [27], àáî â 50 ìM ôîñôàòíîìó áóôåð³ (pH 7.0), ùî ì³ñòèâ 100 ìM K 2 SO 4 [26], àáî â 10 ìM Tris (pH 7.3), ùî ì³ñòèâ 50 ìM NaCl, ïðîòÿ-ãîì 1 ãîä. [28].…”

Electrochemical Biosensors - Sensor Principles and Architectures

“…reaction kinetics of biomolecules in the presence of electric fields. Such experiments were performed by Georgiadis et al and Heaton et al to monitor the in situ hybridization of DNA in the presence of different electrochemical fields [114,120,121]. A further and recent use of EC-SPR was the characterization Figure 13.…”

“…The surface concentration or mass coverage of which can then be calculated using the de Feijter formula [111,112]. SPR biosensors can be used in the determination of a number of surface binding interactions, such as: small molecule adsorption [109], protein adsorption on self-assembled monolayers, antibody-antigen binding, DNA and RNA hybridization [113,114], protein-DNA interactions [106], binding kinetics, affinity constants, equilibrium constants, as well as receptor-ligand interactions in immunosensing [105,115] and many more.…”