Dual polarisation interferometry characterisation of DNA immobilisation and hybridisation detection on a silanised support

Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was approximately 35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was approximately 85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.     

Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode

A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.     

Immobilisation of DNA to polymerised SU-8 photoresist

SU-8 is an epoxy-based photosensitive resist, which is currently used for a large variety of MEMS and lab-on-a-chip applications. Here, we demonstrate a one-step process to functionalize SU-8 with DNA probes. The immobilisation procedure relies on direct coupling of DNA to SU-8 and resulted in surfaces with functional capture probe densities of approximately 10 fmol/mm(2) as determined by hybridisation assays with fluorescent labelled target molecules. A comparable density of functional capture probes was measured on commercial aldehyde coated glass. DNA probes did not decrease in hybridisation performance after 10 min incubation in water at 98 degrees C prior to hybridisation, indicating a covalent bond between DNA and SU-8. Finally, DNA microarrays of high quality were obtained on SU-8 by contact printing of probe solution directly on SU-8 demonstrating a simple method for the implementation of microarrays in microsystems.     

The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

Investigation into the effect that probe immobilisation method type has on the analytical signal of an EIS DNA biosensor

The analytical performance of an enhanced surface area electrolyte insulator semiconductor (EIS) device was investigated for DNA sensor development. The work endeavored to advance EIS performance by monitoring the effect of DNA probe layers have on the impedimetric signal during target hybridisation detection. Two universally employed covalent chemistries, direct and spacer-mediated attachment of amino modified probe molecules to amino-functionalised surfaces were investigated. Relative areal densities of immobilised probe were measured on planar and enhanced surface area substrates using epi-fluorescence microscopy. The reproducibility of the each immobilisation method was seen to have a direct effect on the reproducibility of the impedimetric signal. The sensitivity and selectivity was seen to be dependent on the type of immobilisation method. Real time, impedimetric detection of target DNA hybridisation concentrations as low as 25 and 1 nM were possible. The impact that probe concentration had on the impedimetric signal for selective and non-selective interactions was also investigated.     

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T₂-biotin·dT-T₂ loop. The third base was a biotinylated uracil (U_B) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-³³P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.     

A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye

A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 10(4) nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.     

Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays.

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.     

Cadmium sulfide-modified GCE for direct signal-amplified sensing of DNA hybridization

A novel DNA hybridization sensor based on nanoparticle CdS modified glass carbon electrode (GCE) was constructed and characterized coupled with Cyclic Voltammogram (CV) and Differential Pulse Voltammogram (DPV) techniques. The mercapto group-linked probe DNA was covalently immobilized onto the CdS layer and exposed to oligonucleotide (ODN) target for hybridization. The structure of DNA sensor was characterized by X-ray diffraction (XRD), field-emission microscopy (FESEM) and X-ray photoelectron spectra (XPS). Sensitive electrical readout achieved by CV and DPV techniques shown that when the target DNA hybridized with probe CdS-ODN conjugates and the double helix formed on the modified electrode, a significant increased response was observed comparing with the bare electrodes. The selectivity of the sensor was tested using a series of matched and certain-point mismatched sequences with concentration grads ranging from 10(-6) microM to 10(1) microM. The signal was in good linear with the minus logarithm of target oligonucleotide concentration with detection limit <1 pM and the optimized target DNA concentration was 10(-6) microM for the signal amplification. Due to great surface properties, the additional negative charges and space resistance of as-prepared CdS nanoparticles, the sensor was able to robustly discriminate the DNA hybridization responses with good sensitivity and stability.     

Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals

The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.     

Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study

HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.     

Electrochemical DNA biosensor for bovine papillomavirus detection using polymeric film on screen-printed electrode

A new electrochemical DNA biosensor for bovine papillomavirus (BPV) detection that was based on screen-printed electrodes was comprehensively studied by electrochemical methods of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A BPV probe was immobilised on a working electrode (gold) modified with a polymeric film of poly-L-lysine (PLL) and chitosan. The experimental design was carried out to evaluate the influence of polymers, probe concentration (BPV probe) and immobilisation time on the electrochemical reduction of methylene blue (MB). The polymer poly-L-lysine (PLL), a probe concentration of 1μM and an immobilisation time of 60min showed the best result for the BPV probe immobilisation. With the hybridisation of a complementary target sequence (BPV target), the electrochemical signal decreased compared to a BPV probe immobilised on the modified PLL-gold electrode. Viral DNA that was extracted from cattle with papillomatosis also showed a decrease in the MB electrochemical reduction, which suggested that the decreased electrochemical signal corresponded to a bovine papillomavirus infection. The hybridisation specificity experiments further indicated that the biosensor could discriminate the complementary sequence from the non-complementary sequence. Thus, the results showed that the development of analytical devices, such as a biosensor, could assist in the rapid and efficient detection of bovine papillomavirus DNA and help in the prevention and treatment of papillomatosis in cattle.     

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.     

A sensitive electrochemiluminescent sensor chip based on the ssDNA-Ru(II) complex and aptamer for the determination of thrombin

In this work, an electrochemiluminescence (ECL) sensor chip for sensitive detection of thrombin (TB) was prepared using a screen-printed electrode (SPE) as a working electrode and an aptamer as a specific recognition moiety. To produce an ECL sensor chip, a layer of pL-Cys was immobilized on the surface of the SPE using the cyclic voltammetry scanning method. A layer of gold nanoparticles (AuNPs) was assembled through an Au–S bond and hairpin DNA was further immobilized on the electrode surface. Ru(bpy)₂(mcpbpy)²⁺, as a luminescent reagent, was covalently bound to single-stranded DNA (ssDNA) to prepare a luminescence probe ssDNA-Ru. The probe was hybridized with TB aptamer to form a capture probe. In the presence of TB, the TB aptamer in the capture probe bound to TB, causing the release of ssDNA-Ru that could bind to hairpin DNA on the electrode surface. The Ru(II) complex as a luminescent reagent was assembled onto the electrode, and pL-Cys was used as a co-reactant to enhance the ECL efficiency. The ECL signal of the sensor chip generated based on the above principles had a linear relationship with log TB concentration at the range 10 fM to1 nM, and the detection limit was 0.2 fM. Finally, TB detection using this method was verified using real blood samples. This work provides a new method using an aptamer as a foundation and SPE as a material for the detection of biological substances.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

Classical dot–blot format implemented as an amperometric hybridisation genosensor

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot–blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method—that increases the genosensor sensitivity—are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin–DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot–blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot–blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.     

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T_m) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10¹⁸ Ų), a constant probe density (5 × 10¹² molecules/cm²) and spacer length (15 dT). The authors found that T_m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T_m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The effect of surface probe density on DNA hybridization

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.     

Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I

Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.