Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.     

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.     

Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection

A highly selective, ultrasensitive, fluorescence detection method for DNA and antigen based on self-assembly of multiwalled carbon nanotubes (CNTs) and CdSe quantum dots (QDs) via oligonucleotide hybridization is reported. Mercaptoalkyloligonucleotide molecules bind to the quantum dots, while amineoalkyloligonucleotides bind to CNTs with -COCl surface groups. QDs and CNTs further assemble into nanohybrids through DNA hybridization in the presence of target complementary oligonucleotides. The method is achieved with good repeatability with the detection limit of 0.2 pM DNA molecules and 0.01 nM antigen molecules. This novel detection system can also be used for multicomponent detection and antigen-antibody immunoreaction. The novel system has great potential in applications such as ultrasensitive pathogen DNA or antigen or antibody detection, molecular imaging, and photoelectrical biosensors.     

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.     

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.     

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.     

Fabricating RNA microarrays with RNA-DNA surface ligation chemistry

A novel surface attachment strategy that utilizes RNA-DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5'-phosphate-modified ssDNA attached to the surface and the 3'-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA-DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5'-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA-protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.     

Label-free determination of picogram quantities of DNA by stripping voltammetry with solid copper amalgam or mercury electrodes in the presence of copper

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of the DNA sensors. We show that subnanomolar concentrations (related to monomer content) of unlabeled DNA can be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the presence of copper. DNA is first treated with acid (e.g., 0.5 M perchloric acid), and the acid-released purine bases are directly determined by the cathodic stripping voltammetry. Volumes of 5-3 microL of acid-treated DNA can easily be analyzed, thus making possible the determination of picogram and subpicogram amounts of DNA corresponding to attomole and subattomole quantities of 1000-base pair DNA. Application of this determination in DNA hybridization detection is demonstrated using surface H for the hybridization (superparamagnetic beads with covalently attached DNA probe) and the mercury electrodes only for the determination of DNA selectively captured at surface H.     

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.     

Parallel fabrication of RNA microarrays by mechanical transfer from a DNA master

A new method for fabrication of RNA microarrays is described. The approach involves cohybridization of a short, biotinylated DNA oligonucleotide and an RNA probe sequence to DNA templates spotted onto a master array. Next, the short DNA sequence and the RNA probe are linked using a T4 DNA ligase. Finally, a poly(dimethylsiloxane) (PDMS) monolith modified on the surface with streptavidin is brought into conformal contact with the master array. This results in binding of the biotinylated DNA/RNA oligonucleotides to the PDMS surface. When the two substrates are mechanically separated, the DNA/RNA oligonucleotides transfer to the PDMS replica, and the DNA oligonucleotides remaining on the master array are ready to template another RNA replica array. This sequence can be repeated for at least 18 cycles using a single master array. RNA arrays consisting of up to three different oligonucleotide sequences and consisting of up to 2500 individual approximately 70 microm spots have been prepared.     

Long-range surface plasmon resonance imaging for bioaffinity sensors

A novel bioaffinity sensor based on surface plasmon resonance (SPR) imaging measurements of a multiple-layered structure that supports the generation of long-range surface plasmons (LRSPs) at the water-metal interface is reported. LRSPs possess longer surface propagation lengths, higher electric field strengths, and sharper angular resonance curves than conventional surface plasmons. LRSPR imaging is a version of SPR imaging that requires a symmetric dielectric arrangement around the gold thin film. This arrangement is created using an SF10 prism/Cytop/gold/water multilayer film structure where Cytop is an amorphous fluoropolymer with a refractive index very close to that of water. LRSPR imaging experiments are performed at a fixed incident angle and lead to an enhanced response for the detection of surface binding interactions. As an example, the hybridization adsorption of a 16-mer single-stranded DNA (ssDNA) onto a two-component ssDNA array was monitored with LRSPR imaging. The ssDNA array was created using a new fabrication technology appropriate for the LRSPR multilayers.     

Electrochemical DNA sensors based on enzyme dendritic architectures: an approach for enhanced sensitivity

The modification of enzymes with multiple single-stranded oligonucleotides opens up a new concept for the development of DNA sensors with enhanced sensitivity. This work describes the generation of reporter sequences labeled with an enzyme for the demonstration of their ability to specifically hybridize and to permit signal amplification by successive hybridization steps. The synthetic pathway for the labeling of GOx with oligonucleotide sequences is based on the oxidation of the glycosidic residues of the enzyme and their covalent binding with 5'-end amine-modified oligonucleotides. Spectrophotometric characterization of these functionalized sequences results in an average number of three linked oligonucleotides per enzyme molecule. Their specificity is demonstrated in both a direct and a sandwich-type hybridization assay. The transduction of the enzyme-linked DNA sensors is based on self-assembled multilayers, including a chemically modified anionic horseradish peroxidase electrochemically connected to a water-soluble cationic poly[(vinylpyridine)Os(bpy)(2)Cl] redox polymer in an electrostatic ordered assembly. The sensing layer is constructed by the covalent binding of the DNA probe over the redox polymer through the 3'-phosphate group, enabling the capture of the target sequence. Upon addition of glucose, hybridization results in the production of H(2)O(2), which readily diffuses to the electrocatalytic assembly, giving rise to a cathodic current at 100 mV vs Ag/AgCl. Hybridization is always performed at room temperature, and after 30 min of incubation, an amperometric response is obtained that is proportional to DNA concentration. The simultaneous sandwich assay enables the quantification of a free-label 44-mer oligonucleotide at 1 nM concentration. Signal amplification is realized by a new hybridization step over the free sequences, giving rise to a dendritic architecture that accumulates enzyme molecules per hybridization event.     

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.     

Kinetic measurements of DNA hybridization on an oligonucleotide-immobilized 27-MHz quartz crystal microbalance

A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10-30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (K(a)) and binding and dissociation rate constants (k(1) and k(-1)) could be obtained, as well as binding (hybridization) amount at the nanogram level (delta m). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.     

Direct detection of DNA conformation in hybridization processes

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.     

Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements

A novel multiplexed method for short RNA detection that employs an enzymatic capture reaction onto DNA-modified silica nanoparticles (SiNPs) followed by nanoparticle-enhanced surface plasmon resonance imaging (SPRI) is demonstrated. SiNPs functionalized with 5'-phosphorylated single stranded DNA (ssDNA) are used with T4 RNA ligase to capture various short 20-24 base single-stranded RNA (ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specifically adsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profiling has several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to a larger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) the SiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRI signal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can be used to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 μL.     

Electrochemical imaging of localized sandwich DNA hybridization using scanning electrochemical microscopy

Imaging of localized hybridization of nucleic acids immobilized on gold-DNA chip was performed by means of the feedback mode of scanning electrochemical microscopy (SECM). Thiol-tethered oligodeoxynucleotide (HS-ODN) probes, spotted on a gold surface, were hybridized with unmodified target sequence via sandwich hybridization with a biotinylated signaling probe. Spots where sequence-specific hybridization had occurred were developed by adding a streptavidin-alkaline phosphatase conjugate and biocatalyzed precipitation of an insoluble and insulating product. As a consequence, the surface conductivity of the spotted region of the chip where hybridization had taken place changed. These changes in conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a DNA array in a straightforward way. Analysis of real samples was also performed coupling this method with polymerase chain reaction. The imaging of 60 nM PCR amplicon (255 bp) was demonstrated.     

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.