Thiol-modified DNA Research Articles

Hybrid Surface Platform for the Simultaneous Detection of Proteins and DNAs Using a Surface Plasmon Resonance Imaging Sensor

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.

Binary monolayers of single-stranded oligonucleotides and blocking agent for hybridisation

Abstract 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 10 min. Binding of non-complementary and complementary DNA is dependent both on the blocking agent and on the assembling method used. The lowest non-complementary binding and the highest complementary binding of DNA was obtained with a layer assembled from a binary solution of SH-ssDNA and the lipoate blocking agent.

Monitoring the drown-out crystallization of sodium chloride on a DNA-modified surface

The drown-out crystallization of sodium chloride is conducted on a DNA-modified quartz crystal microbalance (QCM). To investigate the crystallization behavior of NaCl on a DNA template, a thiol-modified DNA oligomer is self-assembled on a gold electrode of QCM. The resonant frequency shift and resonant resistance of the DNA-modified QCM are measured with the continuous injection of ethanol in the NaCl saturated solution, and compared with those of a bare QCM without any modification. F– R diagram is interpreted to analyze a detailed mechanism of the QCM response change for the crystallization. An atomic force microscope (AFM) technique is also used to confirm the procedure of the NaCl crystallization on the DNA template. From these results, it is found that the QCM sensor system could be applied to monitor the NaCl crystallization in the aqueous solution, and to detect a super-saturation point or degree.

Electrochemical biosensors based on DNA strand exchange

Electrochemical biosensors for the detection of specific DNA sequences have been described. The redox-modified DNA sequences were hybridized with the 3'-thiol-modified DNA sequences as capture DNA strand. The resulting duplexes were assembled on a gold electrode surface to make DNA chip. Upon addition of fully complementary DNA or mismatch-containing DNA, the time-dependent changes in redox signal were monitored. Mismatched DNA gave less than 20% change when compared with fully matched DNA under the same conditions. The present assay format is thus applicable in discrimination of mutated DNA from fully matched DNA.

Measurements of thickness dispersion in biolayers by scanning force microscopy and comparison with spectroscopic ellipsometry analysis

Measuring the thickness of biological films remains a difficult task when using differential measurements by atomic force microscopy (AFM). The use of microstructured substrates combined with a selective adsorption constitutes an alternative to tribological measurements. The statistical thickness analysis of biological layers, especially via the dispersion measurements, can provide a way to quantify the molecular orientation. AFM thicknesses were then compared with those obtained optically by spectroscopic ellipsometry (SE) and surface plasmon resonance enhanced ellipsometry (SPREE). The biolayers could then be modeled using a vertical gradient of optical index, which reflects height dispersions. Thiol-modified DNA strands of various lengths account for a good biological model for the study of the strand motion in air and in liquid.

A Cleavable Amino−Thiol Linker for Reversible Linking of Amines to DNA

A cleavable heterobifunctional cross-linker for the reversible conjugation of amines to thiol-modified DNA has been developed and tested. The succinimidyl 2-(vinylsulfonyl)ethyl carbonate (SVEC) was prepared in three steps and tested for its ability to react with amines and thiols. The linker was efficient for binding leucine to a thiol-modified DNA sequence and for releasing the amino acid at pH 11.8.

Hybridisation of surface-immobilised single-stranded oligonucleotides and polymer monitored by surface plasmon resonance

We have investigated the hybridisation of thiol-modified single-stranded DNA embedded in a polyacrylamide layer through the technique of surface plasmon resonance (SPR). Kinetic studies were carried out by two different immobilisation methods: (a) SH-ssDNA was firstly attached on gold and the remaining free space was filled with polymer and (b) SH-ssDNA and the polymer was attached onto the surface from the same solution. The immobilisation methods were compared for various concentrations of SH-ssDNA. Hybridisation was dependent on both the immobilisation method and the concentration of the components. The highest hybridisation was obtained when SH-ssDNA and the polymer was immobilised from the same solution at low SH-ssDNA concentration or when high concentrations of oligos were spread onto the surface and the surface was post-treated with polymer. The target response corresponded to a surface coverage of 100+/-15 ng/cm2. The same surface coverage on hybridisation was also obtained when low concentration of SH-ssDNA and polymer was attached onto the surface from the same solution. The non-specific binding of sample DNA was very low at optimal concentrations due to the polymer and the hybridisation was linearly dependent on target concentration.

Nucleic Acids and Their Analogs as Nanomaterials for Biosensor Development

Nucleic acids are natural biopolymers that store the genetic information of organisms. This makes the detection and characterization of DNA and RNA a relevant task in biotechnology, with applications ranging from medicine to environmental control. During the last decades, a large effort has been focused on the development of biosensors, among them those devoted to the detection of nucleic acids in natural samples and those that include nucleic acids as nanosized capture probes for different biomolecules. DNA microarray technology has been successfully used in biotechnological applications including genotyping and gene expression studies. Nevertheless, the performance of DNA microarrays has a limitation imposed by the need of a previous fluorescent labeling of the target molecule to be analyzed. This encouraged the use of alternative detection methods, such as optical and electrochemical ones, and recently others based on surface characterization techniques. New trends in nanotechnology point towards new tools for manipulating molecules and macromolecules that could be developed as high performance biosensors. This interdisciplinary approach towards the integration of novel biosensors can benefit from the capability of certain polymers to form self-assembled monolayers (SAMs) on different surfaces. Thiol-modified DNA can form SAMs on gold surfaces with reduced efficiency, and the biological activity of the probe is decreased upon adsorption. Therefore, thiolated DNA has a very limited use in biosensor development. These constraints have been successfully by-passed using uncharged, artificial analogs of natural nucleic acids, such as peptide nucleic acids (PNAs), as molecular probes. This contribution reviews the state of the art in the use of nucleic acids and their analogs as biosensor nanomaterials, and summarizes the novel approach towards the development of biosensors based on SAMs of PNAs. Finally, we present the current trends in this promising aspect of nanobiotechnology.

Periodically arranged benzene-linker molecules on boron-doped single-crystalline diamond films for DNA sensing

Electrochemical surface modification via reduction of 4-nitrobenzene diazonium tetrafluoroborate in acetonitrile solution has been performed on highly boron-doped homoepitaxial single-crystalline CVD diamond films to build up surface functionalization for DNA hybridization. Due to significantly improved voltammetric resolution and regularly oriented arrangements, covalently bonded 4-nitrophenyl groups on single-crystalline diamond are characterized to have two successive reversible one-electron transfer reactions interacting with stable radical anion and dianion species. This is new and has not been detected on other electrode materials. Their surface density is in the range of 7.9 × 10 13 molecules/cm 2 or 1.3 × 10 −10 mol/cm 2 as detected by cyclic voltammetric measurements. The aminophenyl-modified diamond surface was further modified by cross linker molecules and thiol-modified DNA oligonucleotides. The resulting DNA immobilization on diamond surface is confirmed by DNA hybridization reaction, using fluorescein-labeled complementary/non-complementary target DNA oligonucleotides. We characterize the DNA film using AFM experiments which reveal a closed and dense film with a height of about 90 Å. Finally, we compare the attachment efficiency on initially H-terminated and oxidized diamond surfaces.

Glass Surfaces Grafted with High-Density Poly(ethylene glycol) as Substrates for DNA Oligonucleotide Microarrays

Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.

Synthesis of Covalent Oligonucleotide‐Streptavidin Conjugates and Their Application in DNA‐Directed Immobilization (DDI) of Proteins

Semisynthetic DNA-streptavidin conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin (STV). The resulting conjugates have binding capacities for four equivalents of biotin and a complementary nucleic acid sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography, and are characterized by spectrophotometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular connectors in the DNA-directed immobilization (DDI) of biotinylated antibodies using DNA microarrays as immobilization matrices. The results are protein microarrays that can be used for the multiplexed detection of various antigens.

Chemically homogeneous, silylated surface for effective DNA binding and hybridization

We report on a method for covalent immobilization of 5′-thiol-modified single strand DNA probes, onto oxygen exposing surfaces by exploiting surface derivatization by 3-mercaptopropyltrimethoxysilane and subsequent intermolecular disulfide bond formation. The various steps in the formation of the molecular edifices have been characterized by X-ray photoelectron spectroscopy, quartz crystal microbalance and atomic force microscopy under liquid. Surface reaction kinetics of thiol-modified DNA probes with thiol-bearing silanes turned out to be a second-order one, possibly due to the presence of both free thiol and S–S dimers in solution. The ability of immobilized single strand DNA to bind the complementary strand has been tested and confirmed by quartz crystal microbalance measurements. The presented DNA immobilization method appears to be applicable to any surface bearing exposed hydroxyl moieties.

Scanning Electrochemical Microscopy. 51. Studies of Self-Assembled Monolayers of DNA in the Absence and Presence of Metal Ions

Scanning electrochemical microscopy was used to examine electron transfer across a self-assembled monolayer of thiol-modified DNA duplexes on a gold electrode. The apparent rate constant for heterogeneous ET from a solution redox probe, Fe(CN)6(3-/4-), to the gold surface through ds-DNA was 4.6 (+/-0.2) x 10(-7) cm/s. With the addition of Zn2+, which resulted in the formation of a metalated DNA (M-DNA) monolayer, the rate constant increased to 5.0 (+/-0.3) x 10(-6) cm/s. Upon treating M-DNA with EDTA, the zinc ions were released from the monolayer and the original rate constant for the DNA duplexes was restored. The enhanced ET rate was also observed at a DNA monolayer treated with Ca2+ or Mg2+, which does not complex by the DNA bases to form M-DNA. The binding of these cations facilitated the monolayer penetration by the probe mediator Fe(CN)6(3-/4-) and accordingly caused an increased redox signal of the mediator at the ds-DNA-modified electrode. Cationic or neutral mediators were not blocked by the ds-DNA monolayer. These results suggest that although the increased electron transport through M-DNA could partially be ascribed to the intrinsic enhancement of electric conductivity of M-DNA, which has been confirmed by photochemical studies, the change in the surface charge of DNA monolayers on the electrode caused by the binding of metal ions to DNA molecules may play a more important role in the enhancement of current with M-DNA.

An ultra sensitive DNA detection by using gold nanoparticle multilayer in nano-gap electrodes

This paper reports an electrical DNA detection method that detects target DNA at concentrations as low as 1 fM by using self-assembly multilayer gold nanoparticle structure between nano-gap electrodes. The distance of the gap between the electrodes is 300 nm and the height of the electrodes is 65 nm. A multilayer gold nanoparticle structure can be built on thin film of SiO 2 silicon wafer. Bifunctional organic molecules are used to build up gold nanoparticle monolayer on the wafer substrate. After hybridization among target DNA, 5′-end thiol-modified probe DNA and 3′-end thiol-modified capture DNA; a second layer of gold nanoparticle is built up through a self-assembly process between gold nanoparticles and thiol-modified end of probe DNA. When the applied voltage is the same, electrical current through multilayer gold nanoparticle structure is much greater than that through monolayered gold nanoparticle structure. Therefore, target DNA in sample solution can be detected through a significant degradation in electrical resistance from monolayered gold nanoparticle to multilayer gold nanoparticle structures. The concentration of target DNA in tested sample solutions ranges from 1 fM to 100 pM. The linear I– V curves of multilayer gold nanoparticle structures indicate the device proposed in this study can detect target DNA concentrations as low as 1 fM. Additional approaches are also suggested in this study to improve the sensitivity in DNA detection by using I– V measurements through multilayer gold nanoparticle structures.

Electrochemical behavior of gold electrodes modified with photosensitizer-tethered DNA

We investigated electrochemical characteristics of gold electrodes modified with photosensitizer-tethered DNA. We synthesized 3'-thiol-modified DNA strands (ODN-SH) and 3'-anthraquinone-modified DNA strands (AQ-ODN). We prepared an AQ-ODN/ODN-SH duplex-modified electrode through hybridization of AQ-ODN with ODN-SH on the gold electrode. We used an amperometric technique for its photocurrent measurement. The large photocurrent was observed by irradiation of a DNA modified electrode at 365 nm. We also measured photocurrent with different DNA sequences on gold electrodes.

Electrochemical Detection of Nonlabeled Oligonucleotide DNA Using Biotin-Modified DNA(ss) on a Streptavidin-Modified Gold Electrode

In the electrochemical detection of nonlabeled DNA, it is important to control the bonding at the interface between the DNA and the electrode. Atomic force microscope (AFM) was taken for the commonly used thiol-modified DNA on a gold surface. It was found that the coverage of the DNA was very low. On the other hand, a streptavidin-modified gold electrode provided a much better alternative where DNA hybridization resulted in large changes in the electrochemical reaction responses. This work demonstrates that streptavidin-modified gold electrodes could be used in the development of a new electrochemical protocol for the detection of nonlabeled DNA.

Covalent coupling of DNA oligonucleotides and streptavidin.

Semisynthetic DNA-protein conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin. The resulting conjugates have a binding capacity for four equivalents of biotin and one nucleic acid of complementary sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography and characterized by photometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular linkers in the DNA-directed immobilization of a biotinylated enzyme on a microplate, containing complementary capture oligonucleotides.

Towards multistep nanostructure synthesis: programmed enzymatic self-assembly of DNA/gold systems.

The programmed self-assembly of nanostructures from well-defined units is an important aim in nanoscience.[1], [2] The use of gold nanoparticles stabilized by thiol-modified DNA is a promising approach towards this goal.[3]-[8] The specificity of DNA base-pairing provides a precise means of programming interactions between particles by hybridization with specifically designed linker strands. To introduce an additional level of control, we have developed a general method by which the reactivity of initially latent DNA linking sites can be switched on deliberately. We have adapted well-developed methods of molecular biology to produce a nanoscale analogue of protecting groups. We show that linking sites can be protected by hybridization with complementary strands and deprotected by cleaving these double strands at predetermined sites with restriction enzymes. This results in cohesive ends of single-stranded DNA, which can bind by hybridization to complementary sequences present in the system. In a second enzymatic step the DNA phosphodiester backbones at the hybridization sites are covalently joined using a DNA ligase. This approach represents a generic protocol that will enable multistep nanostructure syntheses.

Meniscus Force Nanografting: Nanoscopic Patterning of DNA

Thiol-modified DNA is patterned with an atomic force microscope (AFM) on a resist-covered gold surface with line widths as small as 15 nm at speeds as high as 320 μm/s. In meniscus force nanografting, a drop of patterning solution simultaneously wets a hydrophilic resist on gold and an AFM tip. The surface tension supplies enough normal force for the tip to selectively remove the resist molecules and allow the DNA to attach to the gold surface. Different DNA-derivatized nanostructures can then hybridize to the complementary, surface-bound DNA in a demonstration of surface initiated orthogonal self-assembly.

Formation, Spectroscopic Characterization, and Application of Sulfhydryl-Terminated Alkanethiol Monolayers for the Chemical Attachment of DNA onto Gold Surfaces

A novel surface modification procedure for the creation of sulfhydryl-terminated alkanethiol monolayers that can be used for the attachment of biomolecules onto gold surfaces is described. A self-assembled monolayer of the amine-terminated alkanethiol 11-mercaptoundecylamine (MUAM) is reacted with the heterobifunctional cross-linker N-succinimidyl S-acetylthiopropionate (SATP) in order to create a protected sulfhydryl-terminated monolayer. This monolayer can then be deprotected in an alkaline solution to create an active sulfhydryl surface. Compounds that have been modified to contain a maleimide moiety can be easily attached onto the sulfhydryl-derivatized gold surface. In a second attachment strategy, the sulfhydryl-terminated monolayer is reacted with 2,2‘-dipyridyl disulfide to form disulfide bonds on the surface. These disulfide bonds are then used in a thiol−disulfide exchange reaction with free sulfhydryls in order to attach biomolecules, such as thiol-modified DNA or cysteine-containing polypeptides, onto the surface. In contrast to the maleimide-attached monolayers, the disulfide-immobilized species can be cleaved in the presence of dithiothreitol (DTT) in order to regenerate the free sulfhydryl surface. Polarization modulation FTIR reflection−absorption spectroscopy (PM-FTIRRAS) has been used to characterize these surface reactions, and fluorescence “wash off” measurements provided an estimate of 1.5 × 1012 molecules/cm2 for the surface coverage of DNA immobilized using a thiol−disulfide exchange reaction. Surface plasmon resonance (SPR) imaging measurements were employed to monitor in situ hybridization onto DNA arrays fabricated using this surface immobilization reaction.