Surface Structure and Coverage of an Oligonucleotide Probe Tethered onto a Gold Substrate and Its Hybridization Efficiency for a Polynucleotide Target (original) (raw)
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Bioelectrochemistry, 2001
We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3 X or both 3 X and 5 X terminal. After Ž . immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid MUA solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary Ž oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support DNA with one thiol . Ž group , while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support with DNA X X . modified by thiol groups at both 3 and 5 terminals . Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique. q
ChemElectroChem, 2015
Mixed self-assembled monolayers composed of a fluorescently labeled DNA and a mercaptobutanol diluent immobilized on gold electrodes were characterized by electrochemical mea- surements coupled with in situ fluorescence microscopy. The reductive desorption of the self-assembled monolayers was monitored in real time through variations in the capacitance and fluorescence intensity. Desorption occurred in several steps, which was related to substrate crystallinity. Fluorescence microscopy revealed the presence of spatial heterogeneities in the form of highly fluorescent aggregates that remained at the electrode surface even after a reductive desorption step. This in situ electrofluorescence microscopy technique is useful to optimize the formation of the mixed layer to obtain a homoge- neous distribution of the probes, which thus improves the effi- ciency of the recognition process in the development of bio- sensors.
XPS and AFM Characterization of Oligonucleotides Immobilized on Gold Substrates
Langmuir, 2003
The adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous phosphate buffer solutions using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) in conjunction with the quartz crystal microbalance. The hybridization of a thiol-modified, singlestranded oligonucleotide (HS-polyA), anchored to a gold surface via the thiol group, with its complementary sequence (polyT) has been observed and characterized via XPS and AFM techniques, which provide semiquantitative information about the amount of the material deposited and its surface morphology, making possible the monitoring of the hybridization process. The use of these techniques represents a complementary approach to those generally employed, such as spectrophotometry and surface plasmon resonance.
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2018
Adsorption of molecules of DNA (deoxyribonucleic acid) or modified DNA on gold surfaces is often the first step in construction of many various biosensors, including biosensors for detection of DNA with a particular sequence. In this work we study the influence of amine and thiol modifications at the 3' ends of single stranded DNA (ssDNA) molecules on their adsorption on the surface of gold substrates and on the efficiency of hybridization of immobilized DNA with the complementary single stranded DNA. The characterization of formed layers has been carried out using infrared spectroscopy and atomic force microscopy. As model single stranded DNA we used DNA containing 20 adenine bases, whereas the complementary DNA contained 20 thymine bases. We found that the bands in polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) spectra of layers formed from thiol-modified DNA are significantly narrower and sharper, indicating their higher regularity in the orien...
Journal of Biotechnology, 2005
The immobilization of short ss-DNA (18-and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (∼12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (×10 11) globules/cm 2 for the 18-and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (×10 11) average pore density corresponding to the 18-and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.
Fabrication of DNA monolayers on gold substrates and guiding of DNA with electric field
2003
We report electrically controlled selective coating of gold electrodes with mixed monolayers of oligonucleotides and alkanethiol passivation molecules. Gold nanoparticles are used as labels for visualization and voltage between the electrodes is applied for guiding the oligonucleotides. We discuss the efficiency of the guiding and monolayer preparation procedure.
Binary monolayers of single-stranded oligonucleotides and blocking agent for hybridisation
Sensors and Actuators B-chemical, 2008
The hybridisation of thiol-modified single-stranded DNA (SH-ssDNA) embedded in lipoate or hydrophilic polymer monolayers on gold have been studied through the technique of surface plasmon resonance (SPR). SH-ssDNA and the blocking agents were co-adsorbed on gold from the same solution or the surface was post-treated with the blocking agents. The assembling process is allowed to take place for only 10min.
Spatial and Mechanical Properties of Dilute DNA Monolayers on Gold Imaged by AFM
The Journal of Physical Chemistry B, 2003
Spatially distributed DNA oligomer arrays on Au(111) surfaces were created by one-step co-assembly of mixed monolayers of alkanethiol-conjugated DNA and mercaptohexanol (MCH). Tapping-mode AFM was used to visualize the distribution of DNA molecules on the surface and to study the mechanical properties of individual molecules. The DNA coating density increased nonlinearly with increasing mole fraction of DNA oligomer to MCH in the coating solution. For imaging in air, where surfaces are coated with a thin layer of water, the interaction between the AFM tip and the different structures on the monolayer varies between attractive and repulsive depending on the tapping amplitude, set-point ratio, and tip shape. It was found that both duplex and single-stranded DNA molecules extend approximately vertically upward from the surface.
2012
Optimization of the probe adsorption has a major key in the preparation of electrochemical sensors for the detection of oligonucleotide sequences hybridization. The role of a mixed monolayer of ssDNA sequences and MCH coadsorbed on a gold electrode surface was studied in this work. The working electrode was modified by chemisorption using a solution of thiol-tethered 33-mer DNA probe and mercaptohexanol (MCH), in a concentration range from 2 nM to 20 lM. The probe surface density was monitored by means of electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronocoulometry. From EIS measurements, the charge transfer resistance was obtained as a function of the MCH concentration in the immobilization solution. The time dependence of mixed SAM adsorption was also investigated. The SAM adsorption was characterized regarding the electrode surface coverage with DPV and EIS measurements. Moreover, the probe surface density was investigated with chronocoulometry in RuðNH 3 Þ 3þ 6 solution. Sensor behavior and sensitivity showed significant differences as a function of ssDNA/MCH concentration ratio as hybridization detection efficiency decreases while increasing the MCH concentration. The effect of different probe density in the hybridization detection efficiency was determined. Results demonstrated the effective of the coadsorption of ssDNA and thiols to control the SAM property and the probe density. It was therefore shown the importance to identify the correct density of probes on the electrode, below the saturation value, to ensure both a proper hybridization process and having a high hybridization signal.
Biochemical and Biophysical Research Communications, 2000
A novel assay for selective determination of polynucleotides using atomic force microscopy in conjunction with the formation of the probe/target/DNA-gold nanoparticle sandwich structure at a gold surface is described. A 17-mer probe was attached to the surface for subsequent hybridization with a polynucleotide target. Due to the flat orientation of the probe-target hybrid with respect to the surface and the spatial obstruction of the unhybridized probes near the hybrids, the AFM images are not clear. The hybridization efficiency was estimated to be about 1.1% since certain surface features could not be resolved. The utilization of 30-mer-capped gold nanoparticles not only provides another dimension of selectivity, but also reorients the previously formed probe-target hybrid in such a way that the strands of the target become tethered with respect to the surface. This reorientation improves the resolution in imaging the hybridized target molecules and provides an accurate determination of the hybridization efficiency (16%).