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Special-Purpose Modifications and Immobilized Functional Nucleic Acids for Biomolecular Interactions
Abstract
Immobilized oligonucleotides form the critical sensing element of many nucleic acid detection
methodologies. However, pressures on performance and versatility, along with an increasing desire
to expand the scope of targets and assay platforms has driven the integration of special modifications
to enhance stability, functionality and binding characteristics. Separately, aptamers, protein-binding
motifs and catalytic nucleic acids have been retailored for use in immobilized formats to exploit
the sensitivity, signalling and throughput capabilities of these novel assay platforms. Developments
in the field of nanotechnology have also utilized immobilized DNA, but as ascaffold for supramolecular
construction and in the synthesis of molecular bioelectronic components. This review endeavours to
examine the expanding capabilities of immobilized nucleic acids as sensing and structural componentry,
including the modifications required, and the technical advances made to utilize this molecule to
its fullest potential.
... DNA-templated nanowires) in a survey by Di Giusto and King. (1) In general, cDNA as a product of the PCR is available in relatively large amounts at low cost. On the contrary, ONDs are synthetic molecules produced in the laboratory and are, therefore, considerably more expensive than their cDNA counterparts, a fact that has to be taken into consideration concerning the expenses for techniques such as DNA array fabrication. ...
Immobilized DNA is required for the development of DNA chips and arrays, DNA sensors, or other sensing devices including microfluidics, in addition to gene delivery devices. The broad application range for all of these DNA-based systems is to a major extent found in the medical area, using the devices also in DNA sequencing and furthermore for food and environmental analyses. Therefore, strategies for DNA immobilization are described in this article, with focus on immobilization chemistry. Depending on the different surfaces, various immobilization techniques (e.g. via physical adsorption, covalent, affinity binding, and matrix entrapment) were developed and optimized, which are described for carbonaceous materials (e.g. carbon nanotubes), silica and silicon surfaces, gold surfaces, the same as for more recently complex biocompatible surfaces (e.g. polymeric gels). The ‘top-down techniques’ can be distinguished from the ‘bottom-up’ techniques started in the very beginning of DNA chip development. More recently, surfaces such as stents or biocompatible transplants are on focus to generate therapeutic devices. In the future, the power of DNA as a self-assembling material could be used to combine both the ‘bottom-up’ and ‘top-down’ strategies to generate complex nanostructures with multifunctional applications (e.g. three-dimensional DNA ‘origami’). Overall, the main aim of this article is to provide an overview of the methods currently used for DNA immobilization regarding the broad variety of the above mentioned potential applications.
... In particular, oligonucleotide microarrays have become an established tool to study e.g. gene expression profiling, SNP genotyping, and enzymatic reactions [1][2][3][4][5][6][7]. Although there is a wealth of studies investigating nearly every aspect of DNA microarray production, properties, and processing [8][9][10][11][12][13][14][15], little attention has been paid so far to the fact that the special conditions provided during drop drying processes have a crucial influence on the immobilization and further performance of oligonucleotides as probe molecules. ...
Background
Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface.
Results
We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface.
Conclusions
Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different surfaces in dependence of the applied spotting and reaction volume.
... 3 As a consequence of those synthetic efforts DNA analogs with improved stability, functionality and binding characteristics and with properties not present in natural nucleic acids have been produced. 4 The new nucleic acid analogs have been also produced with the aim to develop innovative therapeutic agents 5 or new tools for diagnostics. 6 Aptamers 7 and DNAzymes, 8 collectively referred to as functional nucleic acids, are RNA or DNA structures with binding and catalytic properties respectively. ...
The combined use of surface plasmon resonance (SPR) and modified or mimic oligonucleotides have expanded diagnostic capabilities of SPR-based biosensors and have allowed detailed studies of molecular recognition processes. This review summarizes the most significant advances made in this area over the past 15 years.
Functional and conformationally restricted DNA analogs (e.g., aptamers and PNAs) when used as components of SPR biosensors contribute to enhance the biosensor sensitivity and selectivity. At the same time, the SPR technology brings advantages that allows forbetter exploration of underlying properties of non-natural nucleic acid structures such us DNAzymes, LNA and HNA.
Rapid detection of bacterial contamination in the food supply chain is critically important for food safety monitoring. Reliable extraction and concentration of bacteria from complex matrices is required to achieve high detection sensitivity, especially in situations of low contamination and infective dose. Carbohydrate ligands that attach to microbial cell-surface epitopes are promising economical and biocompatible substitutes for cell-targeting ligands and antibodies. Two different carbohydrate ligands immobilized onto magnetic nanoparticles (MNPs) were easily suspended in liquid food (milk) and allowed expedient extraction of microbes within minutes, without the need for centrifugation or loss in capture capacity. In this pilot study, 25-mL samples of undiluted milk were spiked with 5 mg of MNPs and artificially contaminated with bacteria at 3 to 5 log CFU/mL. MNPs and bacteria formed MNP-cell complexes, which were rapidly separated from the milk matrix with a simple magnet to allow supernatant removal. MNP-cell complexes were then concentrated by resuspension in 1 mL of fresh milk and plated per Bacteriological Analytical Manual procedures. Capture was carried out in vitamin D, 2% reduced fat, and fat-free milk spiked with Salmonella Enteritidis, Escherichia coli O157:H7, and Bacillus cereus for a combined total of 18 experiments (three replicates each). An additional eight experiments were conducted to investigate the effect of competitive bacteria on capture. All experiments were carried out over several months to account for environmental variations. Capture efficiency, on a log basis, for all combinations of milk and bacteria was 73 to 90%. Long-term exposure of the MNPs to milk did not markedly affect capture efficiency. These carbohydrate-functionalized MNPs have potential as nonspecific receptors for rapid extraction of bacteria from complex liquids, opening the door to discovery of biocompatible ligands that can reliably target pathogens in our food.
The past 15 years have seen a revolution in the area of functional nucleic acid (FNA) research since the demonstration that
single-stranded RNA and DNA species can be used for both ligand binding and catalysis. An emerging area of application for
such species is in the development of solid-phase fluorimetric assays for biosensing, proteomics, and drug screening purposes.
In this chapter, the methods for immobilization of functional nucleic acids are briefly reviewed, with emphasis on emerging
technologies such as sol-gel encapsulation. Methods for generating fluorescence signals from aptamers and nucleic acid enzymes
are then described, and the use of such species in solid-phase fluorimetric assays is then discussed. Unique features of sol-gel
based materials for the development of solid-phase assays are highlighted, and some emerging applications of immobilized FNA
species are discussed.
Advances in the development and the applications of optical biosensing systems based on immobilized aptamers are presented. These nucleic acid sequences have been used as new molecular recognition elements to develop heterogeneous assays, biosensors and microarrays. Among different detection modes that have been employed, optical ones which are described here are among the most used. Since their first report in 1996, numerous optical detection systems using aptamers and mainly based on fluorescence have been developed. Two main approaches have been used: label-based (using fluorophore, luminophore, enzyme, nanoparticles) or aptamer label-free detection systems (e.g. surface plasmon resonance, optical resonance). Most methods are based on a labeling approach. Some targets can be optically detected using not only colorimetry, chemiluminescence or the most developed fluorescence mode but also more recent non conventional optical methods such as surface plasmon-coupled directional emission (SPCDE). The first SPCDE-based aptasensor for thrombin detection has recently been reported in 2009. Aptasensors based on surface-enhanced Raman scattering spectroscopy (SERS) which presents advantages compared to fluorescence have also been described. Different label-free techniques have recently been shown to be suitable for developing performant aptasensors or aptamer-based microarrays, such as surface plasmon resonance (SPR), diffraction grating, evanescent-field-coupled (EFC) waveguide-mode, optical resonance or Brewster angle straddle interferometry (BASI). Important advances have been realized on optical aptamer-based detection systems that appear as highly efficient devices with enormous potential.
A novel DNA microarrying platform based on oxygen plasma activation of polycarbonate surface of compact disks (DVD) is presented. Carboxylic acid groups are generated in few seconds on polycarbonate in an efficient, fast, and clean way. Following this surface activation strategy, amino-modified oligonucleotide probes were covalently attached, reaching an immobilization density of 2 pmol cm(-2). Atomic force microscopy imaging revealed the nondestructive character of this treatment when applied for short times, allowing for disk scanning in standard DVD drives. DNA assays performed on oxygen plasma treated disks resulted very efficient with maximum hybridization yield of 93% and reaching a low limit of detection (200 pM) for perfect match synthetic oligonucleotide targets when reading the disk with a standard drive as detector. The approach was also evaluated by scoring single nucleotide polymorphisms with a discrimination ratio of 12.8. As proof of concept, the oxygen plasma treated interactive polycarbonate DNA microarraying platform was applied to the detection of PCR products of Salmonella spp., reaching a detection limit of 2 nM that corresponds to a DNA concentration of only 1 c.f.u./mL. The results confirm the suitability of the microarray platform for analysis of biological samples with high sensitivity.
1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.
Three fluorescence signaling DNA enzymes (deoxyribozymes or DNAzymes) were successfully immobilized within a series of sol-gel-derived matrixes and used for sensing of various metal ions. The DNAzymes are designed such that binding of appropriate metal ions induces the formation of a catalytic site that cleaves a ribonucleotide linkage within a DNA substrate. A fluorophore (fluorescein) and a quencher (DABCYL, [4-(4-dimethylaminophenylazo)benzoic acid]) were placed on the two deoxythymidines flanking the ribonucleotide to allow the generation of fluorescence upon the catalytic cleavage at the RNA linkage. In general, all DNAzymes retained at least partial catalytic function when entrapped in either hydrophilic or hydrophobic silica-based materials, but displayed slower response times and lower overall signal changes relative to solution. Interestingly, it was determined that maximum sensitivity toward metal ions was obtained when DNAzymes were entrapped into composite materials containing approximately 40% of methyltrimethoxysilane (MTMS) and approximately 60% tetramethoxysilane (TMOS). Highly polar materials derived from sodium silicate, diglycerylsilane, or TMOS had relatively low signal enhancements, while materials with very high levels of MTMS showed significant leaching and low signal enhancements. Entrapment into the hybrid silica material also reduced signal interferences that were related to metal-induced quenching; such interferences were a significant problem for solution-based assays and for polar materials. Extension of the solid-phase DNAzyme assay toward a multiplexed assay format for metal detection is demonstrated, and shows that sol-gel technology can provide new opportunities for the development of DNAzyme-based biosensors.
DNAzyme-based catalytic beacons have the potential for sensing a large number of relevant analytes. Thus, a systematic investigation of factors affecting their performance when immobilized into gold-coated nanocapillary array membranes (NCAMs) was undertaken. Enzyme immobilization times were varied to determine that as little as 15 min was sufficient for ratiometric detection of Pb2+-specific activity, while immobilization density saturated after 1.5 h. Immobilization of the DNAzymes into NCAMs with 600 nm pore size resulted in higher immobilization efficiency and higher enzymatic activity than that with 200 nm pore size. A poly-T linker length between the tethering thiol and first oligonucleotide, used to extend the DNAzyme above the backfilling mercaptohexanol (MCH) monolayer, had no effect on DNAzyme activity. The backfilling method of immobilization, involving backfilling followed by hybridization, was found most effective for DNAzyme activity compared to immobilization of hybridized DNAzyme complex (a 67% loss of activity) or concurrent enzyme and MCH immobilization (75% loss of activity). The backfilling MCH monolayer provided approximately 3.5 times increase in activity compared to DNAzyme assembled without MCH, and was over 5 times more active than shorter and longer backfilling molecules tested. The immobilized DNAzyme retained its optimized performance at 50 mM NaCl. Finally, the generalized immobilization and ratiometric procedure was employed for a uranyl-specific DNAzyme with 25 +/- 15 times increase in ratio observed. These findings form a firm basis on which practical applications of catalytic beacons can be realized, including sensors for both Pb2+ and UO22+ ions.
A new methodology for the preparation of adressed DNA matrices is described. The process includes an electrochemically directed
copolymerization of pyrrole and oligonucleotides bearing on their 5′ end a pyrrole moiety introduced by phosphoramidite chemistry.
The electro-controlledsynthesis of the copolymer (polypyrrole) gives, in one step, a solid conducting film deposited on the
surface of an electrode. The resulting polymer consists of pyrrole chains bearing covalently linked oligonucleotide. The polymer
growth is limited to the electrode surface, so that it is possible to prepare a DNA matrix on a multiple electrode device
by successive copolymerizations. A support bearing four oligonucleotides was used to detect three ras mutations on asynthetic
DNA fragment
Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties
to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like
DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to
phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain.
LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living
rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.
An ambitious goal in the area of nanobiosciences is to combine the functionality and stability of nanostructured inorganic solids with the structural variety and the self‐organizing abilities of biochemical molecules. How the ligand shell of nanoparticles needs to be built up to achieve a stable and specific conjugation with biological molecules and to coordinate the chemical properties of nanoparticles and biomolecules is exemplified for gold nanoparticles conjugated with avidin (see picture).
A crop of gold circles and lines: Psoralen‐functionalized Au nanoparticles incorporated into DNA, and Au‐nanoparticle‐functionalized poly‐L‐lysine, yield linear and circular nanowires, respectively, on mica surfaces (see atomic force microscopy images).
We have already shown that introduction of anthraquinone via a one-carbon linker into the sugar 2′-position of oligonucleotide provides a probe with high affinity and specificity in hybridization to DNA In this paper, electrochemical properties of the anthraquinone-modified oligonucleotides (AQ-ODN) in an aqueous solution have been investigated to clarify the potential of AQ-ODN as an electrochemical probe of DNA. Positive shift of E1/2 and decrease in the peak current intensity of AQ-ODN were observed upon hybridization. Intercalation of the anthraquinone moiety and increase of the molecular size due to the hybridization are most likely responsible elements for these electrochemical changes.
Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active
oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5′-amino-hexyl-terminated synthetic oligonucleotides.
Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels
by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436–443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The
thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2–3 kcal mol−1 (1–2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several
base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational
change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe
was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent
(with a 10–30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence
is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small
potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using
ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids
due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and
RNA; single- and double strands) and the sequences.
A method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional
flatbed scanner is described here. Labeling oligonucleotide targets with nanoparticle rather than fluorophore probes substantially
alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide
sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled
targets. In addition, when coupled with a signal amplification method based on nanoparticle-promoted reduction of silver(I),
the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders
of magnitude.
2'-Anthraquinone-modified oligonucleotide (AQ-ODN) possessing a disulfide terminus has been immobilized on gold surface. The AQ-ODN modified electrode exhibited faster electron transferability in double-stranded form than that in single-stranded form, providing a new type of electrochemical DNA sensing device.
Nanometre-sized CdS semiconductor particles were synthesized in the presence of sodium citrate, and subsequently surrounded by a homogeneous silica shell. The coating procedure makes use of 3-(mercaptopropyl) trimethoxy silane (MPS) as a surface primer to deposit a thin silica shell in water. The dispersion is then transferred into ethanol, where thicker shells can be grown. The citrate-stabilized particles are slowly degraded through photochemical oxidation in the presence of dissolved oxygen. This destabilizing process is suppressed when a homogeneous, microporous silica shell is built up around the particles, through a limited access of O2 molecules to the CdS surface.
Proton NMR spectroscopy and molecular dynamics simulations are employed to investigate the conformations of PNA monomers, a dimer and an octamer. The monomers exist as a 70:30 mixture of two amide rotamers interconverting slowly on the NMR time scale at 20 °C. In the major form, the side chain carbonyl group points toward the glycine, which places the methylene protons in proximity to the 2-aminoethyl protons. The minor form places its side chain carbonyl group away from the glycine, and the methylene protons are close in space to the glycine α protons. The PNA CT-dimer has multiple rotamers at 20 °C. In contrast, a NOESY spectrum taken from an octamer indicates only a single conformer in solution at 40 °C.
A novel class of nucleic acid analogues, termed LNA (locked nucleic acids), is introduced. Following the Watson–Crick base pairing rules, LNA forms duplexes with complementary DNA and RNA with remarkably increased thermal stabilities and generally improved selectivities.
We would like to acknowledge the contribution of R Seidel, M Mertig and W Pompe to this work by adding their names as co-authors of the published article.
The correct list of authors for the paper `Controlled positioning of a DNA molecule in an electrode setup based on self-assembly and microstructuring' is
G Maubach1, A Csáki1, R Seidel2, M Mertig2, W Pompe2, D Born1 and W Fritzsche1
1Institute for Physical High Technology, PO Box 100239, 07702 Jena, Germany2Max-Bergmann-Center of Biomaterials and Institute of Materials Science, Technical University Dresden, D-01169 Dresden, Germany.
The controlled conjugation of Au nanoparticles using single-stranded DNA has been achieved by Watson–Crick base pairing interaction and a biotin-streptavidin reaction. Observation by atomic force microscopy have confirmed the one-to-one binding of Au nanoparticles to plasmid DNA. The results suggest that this method could be utilized to achieve nanoscale arrangements in a wide range of applications. © 1999 American Vacuum Society.
A method was established for the nanoscale Pd metallization of DNA by electroless deposition of a metal. The surface of the DNA was activated with Pd ions before a reduction bath was applied to form nanoscale clusters on the DNA. Different cluster sizes were observed according to the duration of the reduction process.
A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidin-biotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 μg/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.
A new methodology, sandwich-type monitoring assay system using an estrogen response element (ERE)-immobilized piezoelectric biosensor in combination with flow injection technique, has been developed for analysis of estrogen (E). The principle of the assay is that the estrogen receptor (ER) captures estrogen and then the complex is bound with ERE immobilized on the sensor. It was confirmed that initial binding of the ERE with ER is significantly accelerated by the formation of an E–ER complex. This kinetic difference was monitored by real-time measurement of the resonance frequency of the ERE piezoelectric biosensor and applied to detect 17β-estradiol. The calibration graph was linear in the range 10–100 nmol l–1 with a detection limit of 7.8 nmol l–1, an RSD of 5.9% for 50 nmol l–1 (6 replicates), and one run time of 4 min. Gradient flow injection on-line connection to an array of ERE piezoelectric biosensors is in progress for the simultaneous determination of endogenous and synthetic estrogens in drinking water and urine. In comparison with present chromatographic methods coupled with MS, the proposed technique is simple, flexible and cheap and has great potential in applications such as field and on-site monitoring and screen testing of endocrine disruptors.
A novel microgravimetric gene sensing system has been
developed using an oligonucleotide anchored on the gold electrode of a
quartz crystal microbalance and an Au nanoparticle modified
oligonucleotide, both of which formed a sandwich-type ternary complex with
the target DNA to give an amplified frequency response.
The synthesis of a biotinylated pyrrole and its copolymerization with pyrrole for the purpose of constructing an electronic conducting polymer (ECP) avidin sensor is reported, using fluorescence detection to determine the efficiency of the sensing process.
We have studied electron transport in DNA duplexes, covalently bonded to two electrodes in aqueous buffer solutions, by repeatedly forming a large number of DNA junctions. The histogram of conductances reveals peaks at integer multiples of a fundamental value, which is used to identify with the conductance of a single DNA molecule. The measured conductance depends on the DNA sequence and length. For (GC)(n) sequences, the conductance is inversely proportional to the length (greater than eight base pairs). When inserting (A:T)n, into GC-rich domains, it decreases exponentially with the length of A:T base pairs (m) with a decay constant of 0.43 Angstrom(-1). This work provides an unambiguous determination of single DNA conductance and demonstrates different conduction mechanisms for different sequences.
We report on the detection of DNA hybridization in connection to cadmium sulfide nanoparticle tracers and electrochemical stripping measurements of the cadmium. A nanoparticle-promoted cadmium precipitation is used to enlarge the nanoparticle tag and amplify the stripping DNA hybridization signal. In addition to measurements of the dissolved cadmium ion we demonstrate solid-state measurements following a ‘magnetic’ collection of the magnetic-bead/DNA-hybrid/CdS-tracer assembly onto a thick-film electrode transducer. The new protocol combines the amplification features of nanoparticle/polynucleotides assemblies and highly sensitive stripping potentiometric detection of cadmium, with an effective magnetic isolation of the duplex. The low detection limit (100 fmol) is coupled to good reproducibility (RSD=6%). Prospects for using binary inorganic colloids for multi-target detection are discussed.
DNA analytics is a growing field based on the increasing knowledge about the genome with special implications for the understanding of molecular bases for diseases. Driven by the need for cost-effective and high-throughput methods for molecular detection, DNA chips are an interesting alternative to more traditional analytical methods in this field. The standard readout principle for DNA chips is fluorescence based. Fluorescence is highly sensitive and broadly established, but shows limitations regarding quantification (due to signal and/or dye instability) and the need for sophisticated (and therefore high-cost) equipment. This article introduces a readout system for an alternative detection scheme based on electrical detection of nanoparticle-labeled DNA. If labeled DNA is present in the analyte solution, it will bind on complementary capture DNA immobilized in a microelectrode gap. A subsequent metal enhancement step leads to a deposition of conductive material on the nanoparticles, and finally an electrical contact between the electrodes. This detection scheme offers the potential for a simple (low-cost as well as robust) and highly miniaturizable method, which could be well-suited for point-of-care applications in the context of lab-on-a-chip technologies. The demonstrated apparatus allows a parallel readout of an entire array of microstructured measurement sites. The readout is combined with data-processing by an embedded personal computer, resulting in an autonomous instrument that measures and presents the results. The design and realization of such a system is described, and first measurements are presented. © 2003 American Institute of Physics.
We present measurements of the electrical conductivity of metallic nanowires which have been fabricated by chemical deposition of a thin continuous palladium film onto single DNA molecules to install electrical functionality. The DNA molecules have been positioned between macroscopic Au electrodes and are metallized afterwards. Low-resistance electrical interfacing was obtained by pinning the nanowires at the electrodes with electron-beam-induced carbon lines. The investigated nanowires exhibit ohmic transport behavior at room temperature. Their specific conductivity is only one order of magnitude below that of bulk palladium, confirming that DNA is an ideal template for the production of electric wires, which can be utilized for the bottom-up construction of miniaturized electrical circuits. © 2001 American Institute of Physics.
Molecular beacons are stem–loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence
quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with
a high signal‐to‐background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing
cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases
can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization.
To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2′‐O‐methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2′‐O‐methyl and 2′‐deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target
duplex stability, 2′‐O‐methyl/RNA > 2′‐deoxy/RNA > 2′‐deoxy/DNA > 2′‐O‐methyl/DNA. The improved stability of the 2′‐O‐methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2′‐deoxy molecular beacons
and RNA targets. However, the 2′‐O‐methyl molecular beacons hybridized to RNA more quickly than 2′‐deoxy molecular beacons. For the pairs tested, the 2′‐deoxy‐beacon/DNA‐target
duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application
of molecular beacons.
We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using biotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.
A nanoelectrode array based on vertically aligned multiwalled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic electrochemical behaviors are observed for measuring bulk and surface-immobilized redox species. Sensitivity is dramatically improved by lowering the nanotube density. Oligonucleotide probes are selectively functionalized to the open ends of nanotubes. The hybridization of subattomole DNA targets can be detected by combining such electrodes with Ru(bpy)32+ mediated guanine oxidation.
We have developed a method to deposit Cu metal onto surface-attached DNA, forming nanowirelike structures that are 3 nm tall. DNA is first aligned on a silicon surface and then treated with aqueous Cu(NO3)2. After the copper(II) has electrostatically associated with the DNA, it is reduced by ascorbic acid to form a metallic copper sheath around the DNA. The resulting nanostructures have been observed and characterized by atomic force microscopy. A more complete coating can be obtained by repeating the Cu(II) and ascorbic acid treatment. Control experiments involving treatments with aqueous solutions containing either NO3- or the divalent cation Mg2+ show no change in DNA height upon ascorbic acid exposure. These experiments indicate that copper nanowires, which may be valuable as interconnects in nanoscale integrated circuitry, can be readily generated from DNA molecules on surfaces.
A vertically aligned carbon nanotube (CNT) array is fabricated as a nanoelectrode platform for biosensor development. Prior to chemical functionalization, metal catalyst particles at the ends of CNT are removed and the closed ends are opened. We find that the oxidative treatment for generating the chemical functional groups at the opened ends of the CNT compromise the mechanical stability of the nanotubes, often leading to total collapse of the aligned CNTs. To solve this problem, we have developed a new approach for filling the gaps between CNTs with a spin-on glass (SOG). Results from the coupling of nucleic acids to the CNT arrays suggest that the SOG enhances the reactivity by providing structural support to the CNTs. The SOG also covers the length of the sidewalls of CNTs, leading to a less hydrophobic interface and thus may aid in improving the chemical reactivity.
Tris(hydroxymethyl)phosphine-capped gold nanoparticles (ca. 1−2 nm diameter) bind densely to DNA, although both species are negatively charged. The particles are also active catalysts for electroless plating of gold. Electroless plating after adsorption to calf thymus DNA immobilized on silicon provides nanowires as narrow as ca. 30−40 nm and electrical conductivities ca. one-thousandth that of bulk gold. Here we summarize our physical characterization of the particles and their DNA conjugates and initial electrical measurements after plating.
A new method for attaching oligodeoxyribonucleotides to glass involving monoalkoxylated and dialkoxylated silanes and bromoacetamide/phosphorothioate linking chemistry has been developed. Three novel bromoacetamide silanes were synthesized for derivatization of glass microscope slides by traditional dipping methods. A thin film method that conserves silane and provides a consistent protocol for test experiments was also used. Oligonucleotides bearing 5‘-phosphorothioates were synthesized by literature methods. Immobilization conditions were initially established by treatment of bromoacetamidosilyl slides with fluoresceinated oligonucleotides, which were imaged by confocal fluorescence microscopy. Spotting can be accomplished in water at oligonucleotide concentrations down to 0.1 mM. Oligonucleotides immobilized using this method can serve as primers for templated, polymerase-based extension reactions with a fluoresceinated dideoxynucleotide terminator. When such primers are formatted into small arrays, specific extension is observed only in the presence of complementary template, with the amount of immobilized primer reflected in the fluorescence signal.
An alternative process was developed to prepare smaller and nearly monodispersed thiol-stabilized platinum nanoparticles. The procedure involves the reduction of a Pt(IV) precursor salt by a small (10%) stoichiometric excess of sodium borohydride in the presence of a toluene solution of 1-dodecanethiol. Complete reduction of the Pt precursor was confirmed by elemental analysis and UV−vis spectroscopy. The platinum nanoparticles prepared as such could be transferred directly to the toluene layer without the addition of concentrated hydrochloric acid that was previously reported as essential for the transfer to take place. TEM imaging shows a mean particle diameter of 2.6 nm and a nearly monodispersed particle size distribution (σ = 0.44 nm). These results were used to formulate a protocol to prepare oligonucleotide-stabilized Pt nanoparticles with narrow particle size distribution and excellent stability in aqueous solutions.
Magnetic AC mode atomic force microscopy (MAC Mode AFM) was used to characterize the process of adsorption of DNA on a highly oriented pyrolytic graphite (HOPG) electrode surface using different concentrations of DNA and adsorption procedures. AFM of DNA immobilized on the HOPG showed that both single-stranded DNA and double-stranded DNA molecules have the tendency to spontaneously self-assemble from solution onto the solid support and the process was very fast. DNA condensed on the substrate in a tight and well-spread two-dimensional lattice covering the entire surface uniformly. The interaction of DNA with the hydrophobic HOPG surface induced DNA superposition, overlapping, and intra- and intermolecular interactions. The application of a positive potential of 300 mV (vs Ag wire) to the HOPG electrode during adsorption was studied. The applied potential considerably enhanced the robustness and stability to mechanical stress of the DNA films, through multiple electrostatic interactions between the negatively charged hydrophilic sugar−phosphate backbone and the positively charged carbon surface. The characteristics of the DNA films and the apparent height of the network wires were dependent on the DNA concentration and the immobilization procedure. The DNA lattices were held together on the substrate surface only by noncovalent interactions such as hydrogen bonding, base stacking, electrostatic, van der Waals, and hydrophobic interactions.
Two procedures are presented which allow the homogeneous incorporation of silica-coated gold nanoparticles within transparent silica gels. Both UV−visible absorption spectra and transmission electron microscopy show that there is no aggregation of the metal particles during sol−gel transition. The optical properties of the gels are compared with those of the starting sols and interpreted on the basis of the porous structure of the gels and standard optical theories. Evidence is also shown of the potential of these methods for the preparation of gels with different particle sizes and shapes as well as with very high nanoparticle concentrations, which can be useful for several applications.
A scheme for an electrical classification of the solution concentration of bioconjugated colloidal gold particles is presented. It is based on the immobilization of the particles in the gap of microstructured electrodes, followed by a metal enhancement step and electrical measurements. The surface density of particles depends on the solution concentrations, and the metal enhancement classifies this density by yielding conductive surfaces only for densities above a threshold. Size enhancement ratios of up to 10 were observed for 30 nm particles and could be controlled by the incubation time.
One-pot direct synthesis of cationic palladium nanoparticles that are stabilized by newly synthesized quaternary ammonium alkylisocyanides is reported. Reduction of PdCl42- by hydrazine in the presence of cationic isocyanides gave a stable aqueous dispersion of cationic palladium nanoparticles. The particle size depended on the alkyl chain lengths of the stabilizer molecules.
The immobilization of DNA (deoxyribonucleic acid) on solid supports is a crucial step for any application in the field of DNA microarrays. It determines the efficacy of the hybridization and influences the signal strength for the detection. We used solid supports made from silicon wafers as an alternative substrate to the commonly used microscope glass slides. The covalent immobilization of thiol-terminated DNA oligonucleotides on self-assembled layers of (3-mercaptopropyl)trimethoxysilane (MPTS) by disulfide bond formation was investigated. Contact angle measurement, variable angle spectral ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) were used to characterize the changing properties of the surface during the DNA array fabrication. During wafer processing the contact angle changed from 3° for the hydroxylated surface to 48.5° after deposition of MPTS. XPS data demonstrated that all sulfur in the MPTS layer was present in the form of reduced SH or S−S groups. VASE measurements indicated a layer thickness of 57.8 Å for the immobilized 16 base oligonucleotides including a 18 carbon atom spacer located between the disulfide bond and the oligomer. AFM was used to characterize the DNA layer before and after hybridization to a complementary target. The data recorded after hybridization revealed a sharp increase in particle size from 89 nm2 to a mean value of 363 nm2. Fluorescence microscopy was used to monitor the hybridization of a fluorescently labeled DNA target to the immobilized probe. The heat stable disulfide-linkage formed during the oligonucleotide immobilization allowed the stripping of complementary DNA targets as well as rehybridization. These data show the advantages and applicability of silicon wafers that have been processed with CMOS (complementary metal oxide semiconductor) compatible processes as solid support in DNA technology. This approach offers the possibility of integration with other silicon-based components such as PCR microreactors and capillary electrophoresis units into a “lab-on-a-chip”.
In electrochemistry experiments on DNA-modified electrodes, features of the redox probe that determine efficient charge transport through DNA-modified surfaces have been explored using methylene blue (MB+) and Ru(NH3)63+ as DNA-binding redox probes. The electrochemistry of these molecules is studied as a function of ionic strength to determine the necessity of tight binding to DNA and the number of electrons involved in the redox reaction; on the DNA surface, MB+ displays 2e-/1H+ electrochemistry (pH 7) and Ru(NH3)63+ displays 1e- electrochemistry. We examine also the effect of electrode surface passivation and the effect of the mode (intercalation or electrostatic) of MB+ and Ru(NH3)63+ binding to DNA to highlight the importance of intercalation for reduction by a DNA-mediated charge-transport pathway. Furthermore, in experiments in which MB+ is covalently linked to the DNA through a σ-bonded tether and the ionic strength is varied, it is demonstrated that intercalative stacking rather than covalent σ-bonding is essential for efficient reduction of MB+. The results presented here therefore establish that efficient charge transport to the DNA-binding moiety in DNA films requires intercalative stacking and is mediated by the DNA base pair array.
Lead sulfide, PbS, nanoparticles have been synthesized using a number of surface capping agents including poly(vinyl-alcohol) (PVA), poly(vinyl-pyrrolidone) PVP, gelatin, DNA, polystyrene (PS), and poly(methyl-methacrylate) (PMMA). The electronic absorption spectra and particle shapes have been found to depend on the capping molecules used. An excitonic feature at 580 nm was observed for capping with PVA and DNA, while no such excitonic feature was observed for PVP, PS or PMMA. A weak excitonic feature was observed for gelatin. The particle shape varied from cubic, needle to spherical as controlled by the capping agents. For the DNA-capped PbS nanocrystals, HRTEM demonstrated the presence of oval crystals with a diameter of 3−8 nm. Powder X-ray diffraction of the PbS-DNA nanocrystals showed the characteristic peaks for PbS at 2.97, 3.43, and 2.10 Å. The XRD suggested the size of the nanoparticles to be approximately 4 nm. The dynamics of photoinduced electrons in PbS nanoparticles have been determined using femtosecond laser spectroscopy. For all the samples studied the electronic relaxation has been found to be very similar and follow a double exponential decay with time constants of 1.2 and 45 ps. The fast decay can be attributed to trapping from the conduction band to shallow traps or from shallow traps to deep traps while the slower decay is most likely due to electron−hole recombination mediated by a high density of surface trap states that lie within the band gap. The decay profiles are independent of particle size, shape, surface capping, probe wavelength, and excitation intensity. The results seem to indicate a high density of surface states, consistent with no detectable fluorescence signal at room temperature.
A series of anthraquinone-containing (AQ) DNA conjugates was prepared. In each case, the AQ is linked to the 2‘-oxygen of a uridine. Physical and spectroscopic data suggest that the AQ is intercalated in the duplex DNA on the 3‘-side of the uracil. Irradiation of the AQ-DNA conjugates with UV light results in piperidine-requiring strand cleavage at GG steps of both the AQ-containing strand and its complement. The AQ-conjugates were designed to have GG steps disposed symmetrically about the AQ intercalation site. The distance dependence of reaction efficiency at GG steps following AQ irradiation was measured by means of an AQ-containing 71 mer having 7 GG steps. The efficiency of reaction in this sequence falls off exponentially with a distance dependence of 0.071 Å-1. AQ-containing conjugates were prepared that incorporate 7,8-dihydro-8-oxoguanines (8-OxoG) at various locations. 8-OxoG has a lower oxidation potential than any of the normal DNA bases and serves as a trap for the migrating radical cation. The 8-OxoG is a very effective trap when the radical cation must migrate through it to reach the GG step, it is a less effective trap when it is on the strand complementary to the GG step. These findings support the mechanism for long-range radical cation migration described as phonon-assisted polaron hopping.
Molecular modeling and molecular dynamics simulation studies have been performed on homo- and heteroduplexes involving peptide nucleic acids (PNA) in aqueous solution under periodic boundary conditions. PNA is a DNA analogue that is homomorphous to DNA, but has an electrically neutral pseudopeptide backbone. In the present study we have investigated the structure and dynamics of duplex systems involving PNA in aqueous solution and how the overall structural and dynamical features of a double helix depend on the nature of the backbones of the constituent strands. Four different duplex systems have been studied: (i) PNA-PNA duplex (1.15 ns), (ii) PNA-DNA antiparallel duplex (0.64 ns), (iii) PNA-DNA parallel duplex (0.6 ns), and (iv) DNA-DNA duplex (0.64 ns). Comparison of the structural features obtained from this study on PNA-DNA antiparallel and PNA-PNA duplex systems with those obtained from NMR and X-ray crystallographic studies respectively has shown very good agreement. In all the cases the structures were stable over the entire period of simulations and the results indicate that the complementary bases and a backbone homomorphous to DNA are sufficient to maintain a stable double helix. The antiparallel PNA-DNA duplex and the PNA-PNA duplex have average structures between A- and B-helixes with certain A-like features while the parallel PNA-DNA double helix, as predicted in this study, is more close to the B-helix. No major difference in the geometries and dynamics of the base pairs in the different duplexes was found. However, the helicoidal parameters are found to be different for the different duplexes. These indicate that the actual structure is determined by the base pairing and the base stacking with the backbones causing some perturbations to this basic structure. The internal dynamics in the base linker region shows highly restricted motions even in the PNA strands where there is no ribose ring.
Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. There are several types of double crossover molecules, differentiated by the relative orientations of their helix axes, parallel or antiparallel, and by the number of double helical half-turns (even or odd) between the two crossovers. We have examined these molecules from the viewpoint of their potential utility in nanoconstruction. Whereas the parallel double helical molecules are usually not well behaved, we have focused on the antiparallel molecules; antiparallel molecules with an even number of half turns between crossovers (termed DAE molecules) produce a reporter strand when ligated, so these have been characterized in a ligation cyclization assay. In contrast to other molecules that contain branched junctions, we find that these molecules cyclize rarely or not at all. The double crossover molecules cyclize no more readily than the linear molecule containing the same sequence as the ligation domain. We have tested both a conventional DAE molecule and one containing a bulged three-arm branched junction between the crossovers. The conventional DAE molecule appears to be slightly stiffer, but so few cyclic products are obtained in either case that quantitative comparisons are not possible. Thus, it appears that these molecules may be able to serve as the rigid components that are needed to assemble symmetric molecular structures, such as periodic lattices. We suggest that they be combined with DNA triangles and deltahedra in order to accomplish this goal.
Long-range oxidative damage to DNA has been demonstrated in experiments using a variety of remotely bound oxidants. However, the mechanism(s) by which charge is transported through the base pair stack needs still to be established. Recent theoretical proposals bring together tunneling and hopping mechanisms to describe charge transport. On the basis of measurements of damage yield, it has been proposed that charge transport occurs by hopping between guanine sites and tunneling through TA steps. In accord with guanine hopping, oxidative damage over long distances was not observed when 5?-TATATA-3? intervened between G sites. Phonon-assisted polaron hopping has been suggested as an alternative mechanism. In this model, the sequence-dependent conformational dynamics of DNA are expected to aid in charge transport.
A new approach to ultrasensitive detection of DNA hybridization based on nanoparticle-amplified surface plasmon resonance (SPR) is described. Use of the Au nanoparticle tags leads to a greater than 10-fold increase in angle shift, corresponding to a more than 1000-fold improvement in sensitivity for the target oligonucleotide as compared to the unamplified binding event. This enhanced shift in SPR reflectivity is a combined result of greatly increased surface mass, high dielectric constant of Au particles, and electromagnetic coupling between Au nanoparticles and the Au film. DNA melting and digestion experiments further supported the feasibility of this approach in DNA hybridization studies. The extremely large angle shifts observed in particle-amplified SPR make it possible to conduct SPR imaging experiments on DNA arrays. In the present work, macroscopic 4 × 4 arrays were employed, and a 10 pM limit of quantitation was achieved for 24-mer oligonucleotides (surface density ≤8 × 108 molecules/cm2). Even without further optimization, the sensitivity of this technique begins to approach that of traditional fluorescence-based methods for DNA hybridization. These results illustrate the potential of particle-amplified SPR for array-based DNA analysis and ultrasensitive detection of oligonucleotides.
The growth of aluminum(III) alkanebisphosphonate multilayer thin films on gold surfaces in aqueous solutions was investigated by probing the surface charge following alternate treatments with anionic phosphonate and cationic Al(III). This was accomplished by determining the force between a modified silica tip of an atomic force microscope (AFM) and the film-covered gold substrate. The AFM force measurements revealed that the formation of the films followed a regular layer-by-layer growth mechanism as evidenced by the occurrence of surface charge reversal with each adsorption step. However, the quantitative surface charge data, obtained by theoretical fits of the force data to solutions of the complete nonlinear Poisson−Boltzmann equation with a knowledge of the silica probe surface potential, indicated that the films became less ordered with an increase in the number of layers. The AFM force measuring technique was also employed to monitor the immobilization of both single-stranded (ss) and double-stranded (ds) DNA on positively charged surfaces (i.e., aluminum(III)- and ammonium-terminated surfaces) and their subsequent interactions with a transition metal chelate, Ru(phen)32+. The force measurement results showed that both the ss-DNA and ds-DNA could be immobilized on positively charged surfaces, while only the ds-DNA showed interaction with Ru(phen)32+.
A material for DNA-based nanoelectronic devices is presented by the authors in the form of colloidal Pt/DNA composites obtained by chemical reduction of platinated DNA with sodium borohydride. The Pt particles can be enlarged by treating the composites with an electroless plating solution. The Figure shows Pt/DNA subjected to the plating process and deposited on mica.
Knots, polyhedra, and Borromean rings with specific structural and topological features can be made from DNA. Biotechnologists have been exploiting the programmability of DNA intermolecular associations for a quarter of a century. These operations have now been applied successfully to branched DNA species to produce complex target structures (for example, the cube shown in the picture) and a nanomechanical device. The assembly of two-dimensional crystals with programmed topographic characteristics demonstrates the simplicity of translating design into surface structures.
Replication, precipitation, and amplification: Polymerase or reverse transcriptase induced replication of DNA/RNA on a transducer (electrode or piezoelectric crystal) leads to the ultrasensitive specific electronic transduction of viral genomes. Biotin tags (B) on the double-stranded assembly provide docking sites for a conjugate between avidin (A) and an alkaline phosphatase (AP). Enzyme biocatalysis of substrate (S) to the insoluble product (P), which precipitates onto the transducer (yellow surface), provides amplification in the analysis of the target DNA.
We report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. A kind of lead sulfide nanoparticle with free carboxyl groups on its surface was synthesized in aqueous solution. The nanoparticle was used as a marker to label a sequence-known oligonucleotide, which was then employed as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were monitored by the oxidation dissolution of the lead sulfide anchored on the hybrids and the indirect determination of the lead ions by anodic stripping voltammetry (ASV). The detection limit is 0.3 pmol L−1 of target oligonucleotides. The PbS nanoparticle combining its easy conjugation to the DNA molecule with the highly sensitive stripping voltammetry detection of lead shows its promising application in the electrochemical DNA hybridization analysis assay.