Self-assembled DNA Monolayers Research Articles

Cross-linking of DNA monolayers by cisplatin examined using electrostatic denaturation

This paper reports on the electrostatic denaturation of double-stranded DNA monolayers in the presence of anti-cancer drug cis-Diamminedichloroplatinum(II) (cisplatin). Cisplatin binds DNA at purine bases, effecting its thermal stability and melting temperature. Here we show for the first time how cisplatin effects the stability of DNA monolayers in the presence of high electric fields generated at the electrode/solution interface, and how this behavior is effected by the method of monolayer preparation. A slight decrease in melting rate and a large decrease in the extent of melting are observed for electrodes prepared with methods that produce homogeneous DNA surface coverage where interstrand distances are maximized. On the other hand, heterogeneous and densely-packed monolayers are found to melt much slower and to a slightly greater extent with bound cisplatin. Three procedures for preparation of the mercaptohexanol/DNA mixed monolayers are compared, demonstrating the potential application of cisplatin, along with electrostatic denaturation analysis, to interrogate surface density and surface heterogeneity in self-assembled DNA monolayers.

Electrochemical Melting of DNA: Optimization and Application

A biosensor is an analytical device that can detect, quantitate, and/or characterize biologically-relevant molecules, often utilizing biologically-derived recognition elements, such as DNA oligomers or enzymes. Applications in point-of-care diagnostics, drug development, and forensic toxicology require portable, rapid, reliable, and easy-to-use devices. Electrochemistry provides a promising platform for such devices due to its potential for parallel measurements, its sensitivity, and its ease-of-miniaturization. In this work, gold electrodes modified with self-assembled monolayers of DNA are utilized to study the electric field assisted melting of double-stranded DNA (i.e. “e-melting”). We have previously demonstrated the ability to detect single mismatches in short DNA oligomers using this approach (J. Electrochem. Soc. 2019 volume 166, issue 4, B236-B242). Broadly, this project aims to (1) develop a better understanding of the mechanism of e-melting and (2) correlate e-melting behavior (kinetics, dependence on applied potential, ionic strength, etc.) to DNA structure and stability. Such analysis has the potential to provide fast and portable screening of biological samples for specific sequences, presence of mismatches, and other mutations. In addition, this method could provide a new platform for fundamental biophysical studies aimed at understanding the behavior of DNA in high electric fields. We will present several key improvements to the procedure for formation of the DNA monolayers using a potential pulse routine during electrode modification. This procedure has now replaced the “passive” method used in previous work and has decreased the amounts of reagents, decreased the reaction time, and increased reproducibility of the DNA surface coverage. This has made possible a correlation between the electrochemical signal (peak height obtained from voltammetric measurements) and the DNA surface coverage (DNA molecules per cm2). Additionally, improvements to the electrochemical melting routine itself will also be presented.These improvements in the methodology allows more controlled and reproducible studies, decreasing the signal-to-noise, and increasing sensitivity to small changes in DNA stability or structure. Results from the application of this method towards the study of interactions between DNA and small molecules, specifically cisplatin, will also be presented. Cisplatin has a strong affinity for DNA: it binds to the N7 of two neighboring purines with preference to neighboring guanines. This binding causes a kink in the DNA which destabilizes it. This research will contribute to a better understanding of the way in which small molecules, like cisplatin, affect the stability of DNA and how such interactions are affected by high electric fields. Ultimately, these studies may provide the foundation upon which future electrochemical biosensors can be developed.

Effect of Oxidative Damage on Charge and Spin Transport in DNA.

A Hall device was used for measuring spin polarization on electrons that are either reorganized within the molecules or transmitted through the self-assembled monolayers of DNA adsorbed on the device surface. We were able to observe spin-dependent charge polarization and charge transport through double-stranded DNA of various lengths and through double-stranded DNA containing oxidative damage. We found enhancement in the spin-dependent transport through oxidatively damaged DNA. This phenomenon can be rationalized either by assuming that the damaged DNA is characterized by a higher barrier for conduction or by charge transfer through the DNA being conducted through at least two channels, one involves the bases and is highly conductive but less spin selective, while the other pathway is mainly through the ribophosphate backbone and it is the minor one in terms of charge transmission efficiency, but it is highly spin selective.

DNA orientation-specific adhesion and patterning of living mammalian cells on self-assembled DNA monolayers.

To better understand cell behaviors on substrates, the precise control of density and orientation of cell-specific ligands remains a great challenge. In this study, we established an easy-to-use approach to manipulate the adhesion and patterning of mammalian cells on gold substrates. We prepared DNA self-assembled monolayers (DNA-SAMs) on gold substrates and found that the sequence-specific orientation of DNA-SAMs played an important role in modulating cell adhesion. We also found that the DNA-SAMs on gold substrates could be used as a potentially universal cell culture substrate, which showed properties similar to cationic polymers (e.g. poly-l lysine, PLL) substrates. Furthermore, we could manipulate cell adhesion by tuning the length of poly adenine (polyA) in the DNA sequence. We also prepared a DNA aptamer-based SAM to regulate cell adhesion by exploiting stimuli-responsive conformational change of the aptamer. By using the well-established DNA spotting technology, we patterned cells on DNA-SAMs to form a spot matrix and four English letters "CELL". Our findings suggest that DNA-SAMs on gold substrates are potentially useful for making smart surfaces for cell studies, thus introducing a new platform for cell/tissue engineering research.

Electrochemistry of DNA Monolayers Modified With a Perylenediimide Base Surrogate

Electrochemistry of self-assembled DNA monolayers represents an attractive strategy for understanding the intrinsic properties of DNA and for developing DNA-based sensors. Thus, there is much interest in the discovery and characterization of new redox-active probes for application in DNA-based technologies. Herein, we report a detailed study of the electrochemical properties of a perylene-3,4,9,10-tetracarboxylic diimide base surrogate, when incorporated at various positions within a DNA monolayer. We demonstrate that the redox chemistry of this perylenediimide probe is mediated by the DNA base pair stack, dependent on its location within the DNA monolayer, and activated thermally. The electrochemical features and general synthetic flexibility of the perylenediimide base surrogate appear favorable for assays that leverage DNA-mediated charge transport.

Effects of Nanoscale Confinement on the Functionality of Nucleic Acids: Implications for Nanomedicine

The facile self-assembly and nanomanipulation of nucleic acids hold great promise in the design of innovative, programmable materials, with applications ranging from biosensing to cellular targeting and drug delivery. Little is known, however, of the effects of confinement on biochemical reactions within such systems, in which the level of packing and crowding is similar to that of intracellular environments. In this review article we outline novel, unexpected properties of nucleic acids that arise from nanoscale confinement, as mainly revealed by atomic force and electron microscopy, electrochemistry, fluorescence spectroscopy, and gel electrophoresis. We review selected scientific studies over the last decade that describe the novel behavior of nanoconfined nucleic acids with respect to hybridization, denaturation, conformation, stability, and enzyme accessibility. The nanoscale systems discussed include self-assembled, water-soluble, DNA or RNA nanostructures, ranging in width from a few to several tens of nm; gold nanoparticles coated with DNA monolayers; and self-assembled monolayers of DNA, from a few to several hundreds of bp in length. These studies reveal that the functionality of nucleic acid-based nanosystems is highly dependent upon the local density, molecular flexibility and network of weak interactions between adjacent molecules. These factors significantly affect steric hindrance, molecular crowding and hydration, which in turn control nucleic acid hybridization, denaturation, conformation, and enzyme accessibility. The findings discussed in this review article demonstrate that nucleic acids function in a qualitatively different manner within nanostructured systems, and suggest that these novel properties, if better understood, will enable the development of powerful molecular tools for nanomedicine.

Quantitative Analysis and Characterization of Self-Assembled DNA on a Silver Surface

Self-assembled monolayers of DNA on a silver surface were prepared and characterized by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), fluorescence imaging, and (32)P radioactive labeling. The buffer concentration of the DNA solution and the surface roughness of the silver substrate were found to affect the surface coverage of DNA and its hybridization. At low buffer concentrations, surface coverage and hybridization were greatly reduced. Ethidium bromide intercalated into the adsorbed dsDNA clearly indicates the presence of dsDNA.

Monitoring the hydration of DNA self-assembled monolayers using an extensional nanomechanical resonator

We have fabricated an ultrasensitive nanomechanical resonator based on the extensional vibration mode to weigh the adsorbed water on self-assembled monolayers of DNA as a function of the relative humidity. The water adsorption isotherms provide the number of adsorbed water molecules per nucleotide for monolayers of single stranded (ss) DNA and after hybridization with the complementary DNA strand. Our results differ from previous data obtained with bulk samples, showing the genuine behavior of these self-assembled monolayers. The hybridization cannot be inferred from the water adsorption isotherms due to the low hybridization efficiency of these highly packed monolayers. Strikingly, we efficiently detect the hybridization by measuring the thermal desorption of water at constant relativity humidity. This finding adds a new nanomechanical tool for developing a label-free nucleic acid sensor based on the interaction between water and self-assembled monolayers of nucleic acids.

Optoelectronic Signatures of DNA-Based Hybrid Nanostructures

This paper presents a characterization of the vibrational modes of nanostructure–DNA complexes immobilized on substrates, such as silver-coated microspheres and silver nanostructure array DNA strands end terminated with titanium dioxide ( <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">${\rm TiO}_{2}$</tex></formula> ) nanoparticles are used to study UV-induced cleaving of DNA molecules functionalized with indirect-bandgap semiconductors. In addition, conventional DNA-based molecular beacons were designed and applied in the detection of DNA of selected organisms. Micro-Raman (μRaman) measurements of DNA in water have proven to be a major challenge because of: 1) weak DNA signatures in solution; 2) changes in structural conformations of the DNA; and 3) environmental effects, such as temperature and pH of the solution in which DNA is suspended. We have studied optoelectronic properties of nanostructure–DNA complexes immobilized on silver nanosphere substrates as well as on Ag-coated micro- and nanostructures. In this research, self-assembled monolayers of DNA formed on these substrates were studied using μRaman techniques. These Raman spectra were used to identify prominent vibrational modes of DNA, and to characterize DNA Raman spectra for both B-DNA with a right-handed double helix, and Z-DNA with a left-handed double helix (S. C. Ha, K. Lowenhaupt, A. Rich, Y. Kim, and K. Kim, “Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases,” Nature, Vol. 437, pp. 1183–1186, 2005). These Raman-based studies of the conformational states of DNA employ pH-changing trivalent salts, methylation of cytosine bases, and alternating GC bases. Moreover, DNA strands terminated with titanium dioxide ( <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex Notation="TeX">${\rm TiO}_{2}$</tex></formula> ) nanoparticles were observed to undergo cleaving upon UV illumination.

Electron stimulated desorption of anions containing Oxygen and Nitrogen from self-assembled monolayers of DNA

The electron stimulated desorption of anions from self-assembled monolayers of double stranded DNA is reported. Desorption of the oxygen and nitrogen containing anions O−, OH−, CN−, OCN−, and OCNH− is induced by the impact of 0.1−20 eV electrons. The anion desorption yields, measured as a function of incident electron energy exhibit pronounced maxima that can be attributed to dissociative electron attachment (DEA) to basic DNA units. Above 15 eV, desorption is attributed to dipolar dissociation (DD). This study further indicates that electrons with energy as low as 2.5 ± 0.3 eV can not only cause damage to DNA but also produce fragments with considerable kinetic energy.

Self‐assembled monolayers of DNA on cysteamine modified Au(111) surface: Atomic force microscopy study

The lambda-DNA molecules self-assemble on cysteamine-modified gold (111) surface to form flat-lying self-assembled monolayers (SAMs). The formation kinetics of such DNA SAMs is studied by atomic force microscopy (AFM). AFM results show that DNA molecules do not arrange themselves on cysteamine-modified gold (111) surface into a well-ordered monolayer. It is also found that the surface density of DNA monolayer does not increase as the DNA concentration increases. The high temperature of DNA solution and the immersing in ultrapure water produce some obvious DNA bundles. Whereas divalent cations in DNA solution result in the formation of more compact DNA films. The obtained information may be useful for practical application of the DNA films and further theoretical studies.

DNA-Mediated Electrochemistry

The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry.

Interaction of Self-Assembled Monolayers of DNA with Electrons: HREELS and XPS Studies

We present results from high-resolution electron energy loss spectroscopy (HREELS) and XPS studies of self-assembled monolayers of DNA. The monolayers are well-organized and display sharp vibrational peaks in the HREEL spectra. The electrons interact mainly with the backbone of the DNA. The XPS results indicate that, in most of the samples studied, the phosphates on the DNA are not charged.

Electrochemistry Using Self-Assembled DNA Monolayers on Highly Oriented Pyrolytic Graphite

Duplex DNA functionalized with pyrene has been utilized to fabricate DNA-modified electrodes on highly oriented pyrolytic graphite (HOPG). Films have been characterized using AFM and radioactive labeling as well as electrochemically. The data obtained are consistent with a close-packed structure in the film with helices oriented in a nearly upright orientation, as seen earlier with the fabrication of thiol-tethered duplexes on gold. Also as on gold, we observe the reduction of DNA-bound intercalators in a DNA-mediated reaction. The reduction of the intercalator is attenuated in the presence of the single-base mismatches, CA and GT, independent of the sequence composition of the oligonucleotide. This sensitivity to single-base mismatches is enhanced when methylene blue reduction is coupled in an electrocatalytic cycle with ferricyanide. The extended potential range afforded by the HOPG surface has allowed us also to investigate the electrochemistry of previously inaccessible metallointercalators, Ru(bpy)2dppz2+ and Os(phen)2dppz2+, at the DNA-modified HOPG surface. These results support the application of DNA-modified HOPG as a convenient and reproducible surface for electrochemical DNA sensors using DNA-mediated charge transport.

Surface Coverage and Structure of Mixed DNA/Alkylthiol Monolayers on Gold: Characterization by XPS, NEXAFS, and Fluorescence Intensity Measurements

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).

Effect Of Electric Potential On Self-Assembled DNA Monolayers. Infrared Ellipsometric And Surface Plasmon Resonance Spectroscopic Investigations.

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Chirality-Induced Spin-Selective Properties of Self-Assembled Monolayers of DNA on Gold

Here we show that self-assembled monolayers on gold of double-stranded DNA oligomers interact with polarized electrons similarly to a strong and oriented magnetic field. The direction of the field for right-handed DNA is away from the substrate. Moreover, the layer shows very high paramagnetic susceptibility. Interestingly, thiolated single-stranded DNA oligomers on gold do not show this effect. The new findings are rationalized based on recent results in which high paramagnetism was measured for diamagnetic films adsorbed on diamagnetic substrates.

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.

Conformationally gated electrochemical gene detection.

The synthesis and characterization of a 26-base DNA hairpin containing both a redox-active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron-transfer dynamics that alter as a consequence of a large structural rearrangement (hairpin-to-duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26-mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single-stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self-assembled DNA monolayers suggest that hybridization with complementary DNA affords a conformational change that alters the electrochemical response.

Development of electrochemically gene-analyzing method using DNA-modified electrodes.

For electrochemical DNA sequencing, several redox-responsive uridines modified at C5 were synthesized and site-specifically incorporated into DNA. Self-assembled DNA monolayers were prepared on a gold electrode using the dsDNA containing modified uridines. By measuring differential pulse voltammetry (DPV) using DNA-modified electrodes, the electrochemical response was strongly affected by the site of the redox-responsive uridine in dsDNA.