Direct and label-free monitoring oligonucleotide immobilization, non-specific binding and DNA biorecognition (original) (raw)
Related papers
Ultramicroscopy, 2002
The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.9770.15 ( A per base pair (bp) (mean7standard deviation) by imaging a 648-bp DNA fragment and 2.9570.14 ( A per bp for a 1115-bp fragment. This confirms earlier observations suggesting that drying DNA fragments on mica in the presence of nickel induces limited conformational changes. At this point the exact nature of these conformational changes remains unknown. Simple hypotheses are the transconformation of stretches of the DNA molecules to the A-form of the double helix or alteration of the helix structure at the points of contact between DNA and mica. By contrast, in aqueous buffer, the measured helical rise was 3.1470.15 ( A per bp for the 648bp fragment and 3.1770.13 ( A per bp for the 1115-bp fragment. Thus, measured helical rises do not depend on the fragment length and are significantly shorter than the 3.38 ( A per bp measured by crystallography, but close to the 3.18 ( A per bp found in NMR studies. These findings are discussed with respect to discrepancies in earlier results published in the literature. r (J. Barbet). 0304-3991/02/$ -see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 -3 9 9 1 ( 0 2 ) 0 0 1 2 8 -6
Langmuir, 2001
A single-stranded oligonucleotide whose 5′ end is derivatized with a mercaptohexyl tether group was either directly anchored onto a gold surface or attached to a gold surface as part of a mixed self-assembled monolayer that contains mercaptohexanol. The application of these surface-confined DNA oligomers as heterogeneous probes for the detection of polynucleotides (e.g., M13 phage DNA) is considered, with an emphasis on the elucidation of the relationship between the hybridization efficiency and the surface coverage and orientation of the probe molecules. Atomic force microscopy (AFM) and flow-injection quartz crystal microbalance (FI-QCM) were used in tandem to study the immobilization of the probe, to estimate the extent and efficiency of the hybridization of M13 phage DNA (7249 bases), and to examine the effect of using a different alkanethiol to reorient the preformed film for a higher hybridization efficiency. The surface density and the resultant hybridization efficiency were found to be highly dependent on the morphology and surface structure of the gold substrate as well as on the concentration of the solution used for the probe fabrication but much less dependent on the probe immobilization time. The lower limit of the hybridization efficiency was estimated to be about 1.1% which is an underestimate because only the resolvable circular features were included in the estimation. Although the duplex formed at the gold surface covered with only the thiolated DNA probe adopts exclusively the orientation in which the target loop is parallel with respect to the substrate surface, the predominant duplex orientation at the gold substrate modified with mixed self-assembled monolayers is tethered to the surface with a small tilt angle versus the surface normal. Visualization of the duplex orientation allows one to understand whether the Sauerbrey equation is valid for the interpretation of certain FI-QCM results. Although it is probably valid to use the Sauerbrey equation to calculate the amount of a polynucleotide at a surface covered only by the thiolated DNA probe, the practice might be questionable for that at the surface with the DNA/alkanethiol mixed SAM on the basis of our AFM images of the target orientations.
J am Chem Soc 2005, 127, 15071
To broaden the applicability of chemically modified DNAs in nano-and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.
Applied Surface Science, 2014
To immobilize effectively oligonucleotide probes on SiO 2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin-biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin-b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin-b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m 2) and second (1.31(±0.22) mg/m 2) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m 2) and fourth (0.41(±0.11) mg/m 2) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.
Analytical Chemistry, 2010
and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).
Biosensors and Bioelectronics, 2006
Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was ∼35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was ∼85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.
Biosensors and Bioelectronics, 1996
Langmuir-Blodgett (LB) and film technologies based on electrostatic attraction self-assembly (SA) are shown to be useful for immobilization of nucleic acids (DNA, polynucleotides) onto solid supports in sensor devices. The nucleic acids were immobilized in complexes with cationic surfactants (for LB) and polycations (for SA). Infrared spectral studies showed that DNA unfolds in multilayer LB films with octadecylamine and conserves its double helical structure in the LB films with dioctadecyldimethylammonium and in the SA films with polyallylamine, polyethylenimine and poly-L-lysine. Atomic groups and the types of interactions determining the complex formation of these films have been identified. The hydration of LB and SA films was studied to find out binding sites of water molecules and to evaluate the flexibility of nucleic acid compounds in the multilayer films. The possibilities of biosensor applications of these LB and SA films were monitored on binding of specific reagents for DNA by DNA-containing films and mononucleotides by a complementary single-stranded polynucleotide immobilized on a positively charged solid support.
Four oligopeptides consisting of a sequence of R-aminoisobutyric acid (Aib) residues, thiolated at either the N-or C-terminus by means of a -(CH 2 ) 2 -SH anchor, were self-assembled on mercury, which is a substrate known to impart a high fluidity to self-assembled monolayers (SAMs). The surface dipole potential of these peptide SAMs was estimated in 0.1 M KCl aqueous solution at a negatively charged electrode, where the interfacial electric field is directed toward the metal. To the best of our knowledge, this is the first estimate of the surface dipole potential of peptide SAMs in aqueous solution. The procedure adopted consisted in measuring the charge involved in the gradual expansion of a peptide-coated mercury drop and then combining the resulting information with an estimate of the charge density experienced by diffuse layer ions. The dipole moment of the tethered thiolated peptides was found to be directed toward the metal, independent of whether they were thiolated at the C-or N-terminus. This result was confirmed by the effect of these SAMs on the kinetics and thermodynamics of the Eu(III)/Eu(II) redox couple. The combined outcome of these studies indicates that a strong interfacial electric field orients the dipole moment of peptide SAMs tethered to mercury, even against their "natural" dipole moment. (4) Imanishi, Y.; Miura, Y.; Iwamoto, M.; Kimura, S.; Umemura, J. Proc. Jpn. Acad. Ser. B 1999, 75, 287-290. (5) Ray, S. G.; Cohen, H.; Naaman, R.; Liu, H.; Waldeck, D. H. Hofmann, M.; Smith, D. L.; Gruhn, N. E.; Graham, A. L.; Colorado, R., Jr.; Wysocki, V. H.; Lee, T. R.; Lee, P. A.; Armstrong, N. R.
Surface characterization of oligonucleotides immobilized on polymer surfaces
Biomedical Applications of Micro- and Nanoengineering, 2002
The immobilization and hybridization of amino-terminated oligonucleotide strands to cyclo-olefin-copolymer (COC) and polycarbonate (PC) surfaces have been investigated for potential application in micro-PCR devices. The oligonucleotides were covalently bound to the plasma-treated COC and PC surfaces via an N-hydroxysulfosuccinimide (NHSS) intermediate. Analysis by AFM showed that the oligonucleotides were present on the surfaces as lumps, and that the size, both vertically and laterally, of these lumps on the COC surface was larger compared to the PC surface. The immobilization efficiency of the former was also higher (15.8 x 10 12 molecules / cm 2) compared to the latter (3.3 x 10 12 molecules / cm 2). The higher efficiency of the COC surface is attributed to the more effective NHSS-functionalization and its higher surface roughness. Subsequent hybridization doubled the height of the lumps, while the lateral dimensions remained essentially unchanged. This is explained in terms of organization of the long probe strands used on the surface as flexible, coil-like polymer chains, which allow the complementary oligonucleotides to bind and increase the height of the lumps. The AFM frictional images showed that the hybridization had the effect of reversing hydrophilicity of the oligonucleotide lumps from being more hydrophilic to more hydrophobic, consistent with the hydrophilic bases of the probe strands being shielded as a result of hybridization.