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.     

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.     

Immobilisation of DNA probes for the development of SPR-based sensing

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).     

Dual polarization interferometry size and density characterisation of DNA immobilisation and hybridisation

Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin-biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm(2) than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 microM to 1 microM. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation.     

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.     

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.     

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system''s redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.     

A DNA intercalation-based electrochemical method for detection of Chlamydia trachomatis utilizing peroxidase-catalyzed signal amplification

A sensitive electrochemical DNA detection method for the diagnosis of sexually transmitted disease (STD) caused by Chlamydia trachomatis was developed. The method utilizes a DNA-intercalating agent and a peroxidase promoted enzymatic precipitation reaction and involves the following steps. After hybridization of the target C. trachomatis gene with an immobilized DNA capture probe on a gold electrode surface, the biotin-tagged DNA intercalator (anthraquinone) was inserted into the resulting DNA duplex. Subsequently, the polymeric streptavidin/peroxidase complex was applied to the biotin-decorated electrode. Peroxidase catalyzed 4-chloronaphthol to produce insoluble product, which is precipitated on the electrode surface in the presence of hydrogen peroxide. Cyclic voltammograms with the gold electrode exhibited a peak current of ferrocenemethanol in electrolyte, which decreased in a proportional way to increasing concentration of target DNA owing to insulation of electrode surface by the growing insoluble precipitate. Using this strategy, we were able to detect picomolar concentrations of C. trachomatis gene in a sample taken from a real patient.     

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.     

Label-free electrochemical DNA sensor based on functionalised conducting copolymer

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.     

A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe

This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modi?ed "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modi?ed "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase.     

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.     

DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.     

Combination of amplification and post-amplification strategies to improve optical DNA sensing

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.     

Duplex DNA capture

This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.     

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.     

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.     

Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence

A hybridisation-based genosensor was designed on a 100 nm sputtered gold film. This material worked as an immobilisation and transduction surface. A 30-mer sequence that encodes a short lysine-rich region, unique to SARS (severe acute respiratory syndrome) virus, was chosen as target. A complementary strand (probe), labelled with a thiol group at the 3'-end, was immobilised on the film. After blocking the surface, hybridisation with the biotin-conjugated SARS strand (at the 3'-end) took place. Interaction with alkaline phosphatase-labelled streptavidin permits amplified indirect electrochemical detection. The analytical signal is constituted by an electrochemical process of indigo carmine, the soluble product of the enzymatic hydrolysis of 3-indoxyl phosphate. The use of a sensitive electrochemical technique such as square wave voltammetry allowed a detection limit of 6 pM to be obtained for this DNA sequence, lower than any other found in the bibliography. The parameters affecting the methodology were studied, with special attention being placed on selectivity. Specificity was clearly enhanced when interaction time and stringency (in the form of formamide percentage) were increased. With 1h of strand interaction and employing 50% of formamide in the hybridisation buffer, a 3-base mismatch strand was perfectly distinguished from the complementary.     

Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods

The present report reviews immobilisation techniques of purified oligonucleotides on electrochemical transducers and their corresponding detection techniques. Most of the literature reviewed was published in the 1990s. The immobilisation techniques of a DNA probe to the surface of an electrochemical transducer made from carbon, gold, platinum or polypyrrole, ranged from simple adsorption to covalent bonding. Recent efforts to couple the recognition layer containing the immobilised nucleic acid recognition layer with the electrochemical signal transducer are discussed. Special attention is given to hybridisation biosensing based on electroactive indicators.     

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.