DNA Surface Density Research Articles

On the Nature of DNA Self-Assembled Monolayers on Au: Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence Microscopy

The creation of gold surfaces modified by single- or double-stranded DNA self-assembled monolayers (SAMs) is shown to produce heterogeneous surface packing densities through the use of electrochemical studies coupled with fluorescence imaging. The modified surfaces created by direct adsorption of thiolate DNA [followed by passivation with mecaptohexanol (MCH)] resulted in regions covered by a monolayer of DNA SAM and other regions that were coated by large particles of DNA. The difference in fluorescence intensity measured from these regions was dramatic. More importantly, a regional variance in fluorescence intensity in response to electrochemical potential was observed: the large aggregates showing a significantly different modulation of fluorescence intensity than the monolayer-coated regions. Electrochemical desorption and detection of the fluorescently tagged DNA provided clear evidence of a complete surface modification. These studies have implications for biosensor/biochip development using DNA SAMs. A modification in the method used to produce the DNA SAMs resulted in a significantly different surface with much fewer aggregates and more significant electromodulation of the fluorescence intensity, though at much lower DNA surface density (ca. 1% of maximum theoretical coverage). This method for forming the modified surfaces has clear advantages over the currently accepted practice and emphasizes the importance of studying the nonaveraged nature of the sensor surface using in situ imaging tools like electrofluorescence microscopy.

Curvature Effects in DNA:Au Nanoparticle Conjugates

DNA-coated Au nanoparticles have myriad applications as versatile building blocks in nanomaterials assembly, powerful amplification tags for bioanalysis, and promising new approaches to medical therapeutics. Characterization, control, and a thorough understanding of the DNA surface interface are essential in the development of these conjugates. A new paper in this issue explores the impact of nanosphere diameter on DNA adsorption and demonstrates that particle curvature plays an important role in controlling the DNA surface density. The study proposes a model that can be used to predict DNA packing on nonspherical particles and validates it using Au nanorods. This work paves the way for improved understanding of the DNA:Au interface in these versatile bioconjugates.

Quantitative Study of the Effect of Coverage on the Hybridization Efficiency of Surface-Bound DNA Nanostructures

We demonstrate that, contrary to current understanding, the density of probe molecules is not responsible for the lack of hybridization in high density single-stranded DNA (ss-DNA) self-assembled monolayers (SAMs). To this end, we use nanografting to fabricate well packed ss-DNA nanopatches within a "carpet matrix" SAM of inert thiols on gold surfaces. The DNA surface density is varied by changing the "writing" parameters, for example, tip speed, and number of scan lines. Since ss-DNA is 50 times more flexible than ds-DNA, hybridization leads to a transition to a "standing up" phase. Therefore, accurate height and compressibility measurements of the nanopatches before and after hybridization allow reliable, sensitive, and label-free detection of hybridization. Side-by-side comparison of self-assembled and nanografted DNA-monolayers shows that the latter, while denser than the former, display higher hybridization efficiencies.

Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1–9) × 10 12/cm 2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 × 10 12/cm 2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.

Optimization of DNA Surface Density on Gold Electrodes for Label-Free Electrical Detection

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Controlling the surface density of DNA on gold by electrically induced desorption

We report on a method to control the packing density of sulfur-bound oligonucleotide layers on metal electrodes by electrical means. In a first step, a dense nucleic acid layer is deposited by self-assembly from solution; in a second step, defined fractions of DNA molecules are released from the surface by applying a series of negative voltage cycles. Systematic investigations of the influence of the applied electrode potentials and oligonucleotide length allow us to identify a sharp desorption onset at −0.65V versus Ag/AgCl, which is independent of the DNA length. Moreover, our results clearly show the pronounced influence of competitive adsorbents in solution on the desorption behavior, which can prevent the re-adsorption of released DNA molecules, thereby enhancing the desorption efficiency. The method is fully bio-compatible and can be employed to improve the functionality of DNA layers. This is demonstrated in hybridization experiments revealing almost perfect yields for electrically “diluted” DNA layers. The proposed control method is extremely beneficial to the field of DNA-based sensors.

Optical Study of DNA Surface Hybridization Reveals DNA Surface Density as a Key Parameter for Microarray Hybridization Kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.

DNA hybridization “turns on” electrocatalysis at gold electrodes

The efficiency of electro-catalysis occurring at DNA-modified gold electrodes is highly dependently on the density of DNA monolayers, as a result, DNA hybridization can "turn on" electro-catalysis by increasing the DNA surface density.

Electrochemical investigation of DNA-modified surfaces: From quantitation methods to experimental conditions

Abstract We have thoroughly compared cyclic voltammetry and chronocoulometry, the two commonly used electrochemical techniques in quantitative determination of DNA surface density on gold electrodes. The electrostatic binding of redox cations, such as [Ru(NH 3 ) 6 ] 3+ , to the anionic DNA phosphate backbone allows the determination of the number of phosphate residues and thereby the surface density of the oligonucleotide strands. The results obtained by the two methods agreed well with each other for thiol-terminated DNA immobilized on gold electrodes via Au–S linkages and for unmodified DNA physically adsorbed on the surface. An improved procedure has been developed for the preparation of DNA-modified gold chips with a relatively high surface coverage. The density of thiol-terminated single-stranded DNA probes was determined to be (2.2 ± 0.3) × 10 13 molecules/cm 2 upon reaching adsorption equilibrium, which was in good agreement with that obtained by phosphor imaging of 32 P-radiolabeled DNA.

Diluting thiol-derivatized oligonucleotide monolayers on Au(1 1 1) by mercaptohexanol replacement reaction

Surface replacement reaction of thiol-derivatized, single-stranded oligonucleotide (HS-ssDNA) by mercaptohexanol (MCH) is investigated in order to reduce surface density of the HS-ssDNA adsorbed to Au(1 1 1) surface. Cyclic voltammograms (CVs) and scanning tunneling microscopy (STM) are employed to assess the composition and state of these mixed monolayers. It is found that each CV of mixed self-assembled monolayers (SAMs) only shows a single reductive desorption peak, which suggests that the resulted, mixed SAMs do not form discernable phase-separated domains. The peak potential gradually shifts to negative direction and the peak area increases step by step over the whole replacement process. By analyzing these peak areas, it is concluded that two MCH molecules will replace one HS-ssDNA molecule and relative coverage can also be estimated as a function of exposing time. The possible mechanism of the replacement reaction is also proposed. The DNA surface density exponentially reduces with the exposing time increasing, in other words, the replacement reaction is very fast in the first several hours and then gradually slows down. Moreover, the morphological change in the process is also followed by STM.

“Direct” Detection and Separation of DNA Using Nanoporous Alumina Filters

The concept of using alumina nanoporous filters (AAO) modified with DNA for "label-free" detection and separation/purification of the target ss-DNA is demonstrated. The high surface density of DNA (4 x 10(12) cm(-2)) and high efficiency of hybridization (ca. 70%) in combination with increased effective surface area make this system very attractive for development of various ss-DNA (or RNA) detection methods. Moderate transparency of AAO in the UV and IR regions allows direct detection of DNA hybridization by optical and IR absorption. Close to the quantitative efficiency of binding the target ss-DNA from solution using a single pass through the modified filter is observed.

Structural Characterization of Multilayered DNA and Polylysine Composite Films: Influence of Ionic Strength of DNA Solutions on the Extent of DNA Incorporation

Thin films of alternating DNA and poly-l-lysine (PLL) layers were fabricated onto various planar surfaces by the layer-by-layer (LbL) self-assembly technique. For both the polydeoxynucleotide (PDN, polyguanylic acid or poly[G]) and the oligodeoxynucleotide (ODN, a 30-mer) investigated, UV−visible spectrometry and contact angle measurements showed that a uniform layer of DNA can be fully adsorbed onto each alternate PLL layer. Various parameters affecting the DNA loading into the composite film were investigated with a particular emphasis placed on the effect of the ionic strength in the DNA solution used for the film preparation. For the PLL/poly[G] film, it was found that 150 μg/mL of poly[G] solution containing 0.5 M NaCl attains the maximum loading for every poly[G] layer. Atomic force microscopy was utilized to measure the DNA surface density and structure at the topmost layers of several representative composite films. While the DNA film thickness increases with the ionic strength of the DNA solution...

Selective Desorption of Alkanethiols in Mixed Self-Assembled Monolayers for Subsequent Oligonucleotide Attachment and DNA Hybridization

Oligonucleotides whose 5‘ ends are linked to mercaptohexyl tether groups (HS-ss-DNA) are attached onto gold regions freshly exposed by reductive desorption of the mercaptopropionic acid (MPA) in a mixed MPA/hexanethiol (HT) or MPA/mercaptohexanol (MCH) self-assembled monolayer (SAM). The size of the bare Au region was shown to be controllable by using various MPA/HT solution compositions during the fabrication of the mixed SAMs. Atomic force microscopy shows that the size of the segregated gold regions has a profound effect on the surface density and orientation of the subsequently immobilized HS-ss-DNA molecules. The DNA surface density begins to increase when the bare gold domain (12−19 nm wide, prepared by stripping MPA off a mixed SAM formed from a solution with an alkanethiol molar ratio of χMPA/χΗΤ = 1/4) approaches the size of the HS-ss-DNA (ca. 5−6 nm for a 17mer). The surface density of the HS-ss-DNA in the mixed HS-ss-DNA/HT SAM or the mixed HS-ss-DNA/MCH SAM is slightly greater than SAMs contai...

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 microg/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.

Synthesis and Characterization of DNA-Modified Silicon (111) Surfaces

Hydrogen-terminated Si(111) surfaces are modified by attachment of oligodeoxynucleotides and characterized with respect to DNA surface density, chemical stability, and DNA hybridization binding specificity. Surface functionalization employs the reaction of ω-unsaturated alkyl esters with the Si(111) surface using UV irradiation. Cleavage of the ester using potassium tert-butoxide yields a carboxyl-modified surface, which serves as a substrate for the attachment of DNA by means of an electrostatically adsorbed layer of polylysine and attachment of thiol-modified DNA using a heterobifunctional cross-linker. The resultant DNA-modified surfaces are shown to exhibit excellent specificity and chemical stability under the conditions of DNA hybridization. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to semiconductor devices.

Electrochemical quantitation of DNA immobilized on gold.

We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensation redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1-10) x 10(12) molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density.