Replication of DNA microarrays prepared by in situ oligonucleotide polymerization and mechanical transfer

In this paper, we describe a method for replication of DNA microarrays. The approach involves in situ, enzymatic synthesis of a DNA complement array using a prefabricated master array, followed by mechanical transfer of the complement array to a second substrate. The new findings reported here include the following. DNA spots as small as approximately 100 microm can be faithfully replicated, replica arrays consisting of several different oligonucleotide sequences can be prepared, and such arrays are active toward hybridization of their complements. Up to 10 replicas can be prepared from a single master with no detectable progressive degradation of their activity. DNA master arrays consisting of long DNA templates (80-mer) can be replicated, as can large-scale master arrays consisting of approximately 2300 spots.     

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).     

SPR imaging measurements of 1-D and 2-D DNA microarrays created from microfluidic channels on gold thin films

Microfluidic channels fabricated from poly(dimethylsiloxane) (PDMS) are employed in surface plasmon resonance imaging experiments for the detection of DNA and RNA adsorption onto chemically modified gold surfaces. The PDMS microchannels are used to (i) fabricate "1-D" single-stranded DNA (ssDNA) line arrays that are used in SPR imaging experiments of oligonucleotide hybridization adsorption and (ii) create "2-D" DNA hybridization arrays in which a second set of PDMS microchannels are placed perpendicular to a 1-D line array in order to deliver target oligonucleotide solutions. In the 1-D line array experiments, the total sample volume is 500 microL; in the 2-D DNA array experiments, this volume is reduced to 1 microL. As a demonstration of the utility of these microfluidic arrays, a 2-D DNA array is used to detect a 20-fmol sample of in vitro transcribed RNA from the uidA gene of a transgenic Arabidopsis thaliana plant. It is also shown that this array fabrication method can be used for fluorescence measurements on chemically modified gold surfaces.     

Cantilever-based optical deflection assay for discrimination of DNA single-nucleotide mismatches

Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.     

Magnetic and gold-coated magnetic nanoparticles as a DNA sensor

In this study, we report the chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles and the immobilization of single-stranded biotinylated oligonucleotides onto these particles. Selected sequences specific to the BRCA1 gene were used as a test platform. The binding of oligonucleotides to these particles was achieved through a streptavidin-biotin bridge via a carbodiimide activation protocol. Particle size and oligonucleotide attachment were confirmed by transmission electron microscopy; oligonucleotide binding was characterized by Fourier transform infrared spectroscopy and hybridization confirmed by fluorescence emission from the fluorophore attached to the target oligonucleotide strand. The rate of hybridization was measured using a spectrofluorometer and a microarray scanner. The rate of hybridization of oligonucleotides bound to the synthesized particles depends on the inorganic support material and its surface chemistry. The rate of hybridization increased concomitantly with the concentration of the probe and the target in the reaction medium. Furthermore, exposure of probe and target oligonucleotide to a combination of target and noncomplementary DNA strand reduced the rate of hybridization, possibly because of steric crowding in the reaction medium and cross-linking between reacting oligonucleotides and the noncomplementary strands. The study undertaken opens several possibilities in bioconjugate attachment to functionalized iron and iron nanocomposite structures for controlled manipulation and handling using magnetic fields.     

Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane)

This paper describes a procedure that makes it possible to design and fabricate (including sealing) microfluidic systems in an elastomeric material [Formula: see text] poly(dimethylsiloxane) (PDMS) [Formula: see text] in less than 24 h. A network of microfluidic channels (with width >20 μm) is designed in a CAD program. This design is converted into a transparency by a high-resolution printer; this transparency is used as a mask in photolithography to create a master in positive relief photoresist. PDMS cast against the master yields a polymeric replica containing a network of channels. The surface of this replica, and that of a flat slab of PDMS, are oxidized in an oxygen plasma. These oxidized surfaces seal tightly and irreversibly when brought into conformal contact. Oxidized PDMS also seals irreversibly to other materials used in microfluidic systems, such as glass, silicon, silicon oxide, and oxidized polystyrene; a number of substrates for devices are, therefore, practical options. Oxidation of the PDMS has the additional advantage that it yields channels whose walls are negatively charged when in contact with neutral and basic aqueous solutions; these channels support electroosmotic pumping and can be filled easily with liquids with high surface energies (especially water). The performance of microfluidic systems prepared using this rapid prototyping technique has been evaluated by fabricating a miniaturized capillary electrophoresis system. Amino acids, charge ladders of positively and negatively charged proteins, and DNA fragments were separated in aqueous solutions with this system with resolution comparable to that obtained using fused silica capillaries.     

A universal nucleic acid sequence biosensor with nanomolar detection limits

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.     

Covalent binding of streptavidin on gold magnetic nanoparticles for bead array fabrication

In this study, we report the chemical synthesis and functionalization of streptavidin coated gold magnetic nanoparticles (GMNPs) and the immobilization of single-stranded biotinylated oligonucleotides onto these particles. By using covalent interaction or physical adsorption, two kinds of streptavidin coated GMNPs (SA-GMNPs) were prepared. The quantity and stability of streptavidin bound to the GMNPs using different methods were determined by UV-Vis spectrometer. The results indicated that by physical absorption the GMNPs can capture more streptavidin, the SA-GMNPs with either physical adsorption or covalent reaction were both stable in PBS buffer. In contrast, SA-GMNPs with covalent reaction was stable in SDS buffer, while most of the SA-GMNPs by physical adsorption would be eluted from the particles in SDS buffer. Therefore, the SA-GMNPs by covalent immobilization were more suitable for fabrication of bead array. To evaluate the binding efficiency and capacity of DNA on SA-GMNPs, the capture of biotinylated oligonucleotide or PCR products on SA-GMNPs at different concentrations were examined. A magnetic beads array was fabricated by immobilizing DNA-MNPs complexes onto a glass slide using a magnetic field. The synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM, indicating the potential application of this MNPs array to high-throughput DNA detection.     

Sequential injection analysis system for the sandwich hybridization-based detection of nucleic acids

A sequential injection analysis lab-on-valve (SIA-LOV) system was developed for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for signal enhancement used in lieu of the single fluorophore-labeled probe in order to lower the limit of detection.     

Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection

Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described.     

Superhydrophobic concrete surfaces with integrated microtexture

Ultra-High Performance Concrete (UHPC) is characterized by a densely packed mix-design, which can offer attractive surface properties for architectural building facades. A technical challenge for aesthetic applications is the protection against fouling. This work demonstrates that water-repellent concrete can be obtained just after demoulding by replicating the features of micro-pillared moulds made of polydimethylsiloxane (PDMS). Moreover, the negative replica of the microtextured UHPC surface can be used as a master to template for other UHPC samples, constituting a cost-effective route to fabricate large-scale microtextured concrete pieces. The chemical functionalization of UHPC with a low surface energy material is obtained by transferring residues from the PDMS mould or by spraying siloxane-based compounds to form a homogenous surface film. The latter preparation method showed superhydrophobic properties with static contact angles reaching up to 164° and contact angle hysteresis reaching as low as 2.5°. This process enables the manufacture of water-repelling, self-cleaning concrete. Raindrops slide off the concrete surface, carrying debris away.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.     

Enzyme-amplified amperometric sandwich test for RNA and DNA.

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically "wiring" horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the H2O2 electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, converting the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of H2O2 to water. Flow of H2O2 electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 microA cm(-2), exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in approximately 30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.     

Detection of DNA hybridization using the near-infrared band-gap fluorescence of single-walled carbon nanotubes

We demonstrate the optical detection of DNA hybridization on the surface of solution suspended single-walled carbon nanotubes (SWNTs) through a SWNT band gap fluorescence modulation. Hybridization of a 24-mer oligonucleotide sequence with its complement produces a hypsochromic shift of 2 meV, with a detection sensitivity of 6 nM. The energy shift is modeled by correlating the surface coverage of DNA on SWNT to the exciton binding energy, yielding an estimated initial fractional coverage of 0.25 and a final coverage of 0.5. Hybridization on the nanotube surface is confirmed using Forster resonance energy transfer of fluorophore-labeled DNA oligonucleotides. This detection is enabled through a new technique to suspend SWNTs using adsorption of single-stranded DNA and subsequent removal of free DNA from solution. While the kinetics of free DNA hybridization are relatively fast (<10 min), the kinetics of the process on SWNTs are slower under comparable conditions, reaching steady state after 13 h at 25 degrees C. A second-order kinetic model yields a rate constant of k = 4.33 x 10(5) (M h)(-1). This optical, selective detection of specific DNA sequences may have applications in the life sciences and medicine as in vitro or in vivo detectors of oligonucleotides.     

Electrochemical DNA biosensor based on conducting polyaniline nanotube array

Most of the recent developments in ultrasensitive detection of nucleic acid are based on the gold nanoparticles and carbon nanotubes as a medium of signal amplification. Here, we present an ultrasensitive electrochemical nucleic acid biosensor using the conducting polyaniline (PANI) nanotube array as the signal enhancement element. The PANI nanotube array of a highly organized structure was fabricated under a well-controlled nanoscale dimension on the graphite electrode using a thin nanoporous layer as a template, and 21-mer oligonucleotide probes were immobilized on these PANI nanotubes. In comparison with gold nanoparticle- or carbon nanotube-based DNA biosensors, our PANI nanotube array-based DNA biosensor could achieve similar sensitivity without catalytic enhancement, purification, or end-opening processing. The electrochemical results showed that the conducting PANI nanotube array had a signal enhancement capability, allowing the DNA biosensor to readily detect the target oligonucleotide at a concentration as low as 1.0 fM (approximately 300 zmol of target molecules). In addition, this biosensor demonstrated good capability of differentiating the perfect matched target oligonucleotide from one-nucleotide mismatched oligonucleotides even at a concentration of 37.59 fM. This detection specificity indicates that this biosensor could be applied to single-nucleotide polymorphism analysis and single-mutation detection.     

Microfabricated polymer devices for automated sample delivery of peptides for analysis by electrospray ionization tandem mass spectrometry.

Delivery of proteins and peptides to electrospray ionization mass spectrometers (ESI-MS) has been demonstrated using glass and quartz microfabricated devices. This paper reports the construction and use of poly(dimethylsiloxane) (PDMS) microfabricated soft polymer devices with mass spectrometry for protein analysis. The PDMS devices were fabricated using replica molding against a patterned photoresist generated by photolithographic techniques. The PDMS devices were connected to the mass spectrometer via a derivatized transfer capillary and samples were transferred by electroosmotic pumping. The formulation of PDMS was optimized for compatibility with ESI, and the devices were tested for performance. The practical application of PDMS devices was demonstrated by the identification of rat serum albumin separated by 2-D gel electrophoresis. Extended contact of the sample with the surface of the PDMS device did not significantly affect the sample analysis, and the limit of detection for samples run on a PDMS device was comparable to the limit of detection achieved on glass devices. This study suggests that PDMS devices fabricated using replica molding are compatible with ESI-MS. This will potentially lead to the construction of inexpensive microfabricated devices with complex designs and advanced functionalities.     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.