Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 μL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Received: 26 September 2000 / Revised: 24 November 2000 / Accepted: 30 November 2000     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Introducing curcumin as an electrochemical DNA hybridization indicator and its application for detection of human interleukin-2 gene

In this paper, the application of curcumin (CU) as a non-toxic electrochemical DNA hybridization indicator was described. Hybridization investigations on a pencil graphite electrode surface as a transducer using oligonucleotides containing only one base type, including poly A, poly T, poly C, and poly G as probe and as related complementary/non-complementary sequences, showed that CU has no specific interaction with each of the oligonucleotides of DNA. Furthermore, results showed good interaction between CU and the hybridized form of oligonucleotides; thus, the extent of hybridization was evaluated based on the difference between differential pulse voltammetry (DPV) signals of CU accumulated on the probe-pencil graphite electrode (PGE) and CU accumulated on the probe-target-PGE. Then, the developed biosensor was successfully applied for the detection of short sequences of human interleukin-2 (hIL-2) gene as a model. A hybridization experiment with non-complementary oligonucleotide showed that the suggested DNA sensor responds selectively to the target. At optimized conditions, two linear ranges were obtained for hIL-2 gene, first from 50 to 1000 pM and second from 0.01 to 1 μM with a detection limit of 12 pM. 7.0) containing 20 mM NaCl.     

Modeling of DNA hybridization detection using methylene blue as an electroactive label

Mathematical modeling of methylene blue (MB) signal in ssDNA and dsDNA on pencil graphite electrode is described. A DNA biosensor was developed based on MB signal. The probe and target DNAs were 20 mer oligonucleotides corresponding to consensus sequence of HPV major capsid protein L1 gene. Hybrids of various complementary and non-complementary oligonucleotides with the probe were considered as dsDNA with different hybridization degrees. Modeling was developed by incorporation of only the stable forms of dsDNA hybrids. Effect of hybridization degree on current signal in various forms was studied. A factor named AHP (Average Hybridization Percentage) for verifying the hybridization events was defined. Results showed that there is a significant mathematical relation between the calculated AHP and MB signals.     

A low-cost surface plasmon resonance instrument based on detection of resonance excitation wavelength

A laboratory-made surface plasmon resonance (SPR) instrument based on the detection of resonance excitation wavelength has been successfully fabricated. The performance and workability of the SPR instrument was demonstrated as a DNA biosensor. Biotinylated single-stranded oligonucleotides (ssDNA) were chemically immobilized on a gold-film surface of the SPR instrument as a DNA probe for the detection of its fully complementary, half-complementary and non-complementary ssDNA. The immobilization of the ssDNA probe was done by avidin-biotin linkage. The ssDNA used were 12-mer oligonucleotides. The sensing mechanism was based on the shift in resonance wavelength of an excitation light beam as the target ssDNA hybridized with the ssDNA on the gold-film surface. The linear dynamic ranges of the DNA biosensor for fully complementary and half-complementary ssDNA are 0.04-1.2 pM and 0.08-1.1 pM, respectively. The DNA biosensor showed higher sensitivity to fully complementary ssDNA than to half-complementary ssDNA. But no shift of resonance wavelength to the non-complementary ssDNA was observed.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label

Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e., activation potential and activation time) and probe immobilization conditions on the electrodes (i.e., immobilization potential and time) were investigated and the optimum values of 1.80 V and 300 s for PGE activation, and −0.5 V and 400 s for the probe immobilization on the electrode were suggested.     

Electrochemical detection of a Vibrio parahaemolyticus sequence-specific gene based on a gold electrode modified with a single stranded probe

An electrochemical DNA biosensor for specific-sequences detection of Vibrio parahaemolyticus (VP) was fabricated. A single-stranded 20-mer oligonucleotide (ssDNA) and 6-mercapto-1-hexanol (MCH) were immobilized via a thiol linker on gold disk electrodes by self-assembling. The ssDNA underwent hybridization in a hybridization solution containing complementary or non-complementary or single base pair mismatched DNA sequences of VP. Examination of changes in response to these three target DNAs showed that the developed biosensor had a high selectivity and sensitivity.     

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.     

Electrochemical detection of DNA hybridization by using a zirconia modified renewable carbon paste electrode

A simple, polishable and renewable DNA biosensor was fabricated based on a zirconia modified carbon paste electrode. Zirconia was mixed with graphite powder and paraffin wax to produce the paste for the electrode, and response-optimized at 56% graphite powder, 19% ZrO(2) and 25% paraffin wax. An oligonucleotide probe with a terminal 5'-phosphate group was attached to the surface of the electrode via the strong affinity of zirconia for phosphate groups. DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. Examination of changes in response with complementary or non-complementary DNA sequences showed that the developed biosensor had a high selectivity and sensitivity towards hybridization detection (< or =2x10(-10) M complementary DNA detectable). The surface of the biosensor can be renewed quickly and reproducibly (signal RSD+/-4.6% for five successive renewals) by a simple polishing step.     

Cationic polymer coatings for design of electroosmotic flow and control of DNA adsorption

A difficulty with the design and operation of an electrokinetically operated DNA hybridization microfluidic chip is the opposite direction of the electroosmotic flow and electrophoretic mobility of the oligonucleotides. This makes it difficult to simultaneously deliver targets and an appropriate hybridization buffer simultaneously to the probe sites. In this work we investigate the possibility of coating the inner walls of the microfluidic system with hexadimentrine bromide (polybrene, PB) and other cationic polymers in order to reverse the direction of electroosmotic flow so that it acts in the same direction as the electrophoretic transport of the oligonucleotides. The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer. Under these conditions, the DNA and EOF move in the same direction, and the flow can be used to deliver DNA to an area for selective hybridization within the channel. The effects of coating the surface of a nucleic acid microarray with polybrene were also studied to assess non-selective adsorption and stability. The polybrene coating significantly reduced the extent of non-selective adsorption of oligonucleotides in comparison to adsorption onto a glass surface, and the coating did not alter the extent of hybridization. The results suggest that use of the coating makes it possible to achieve semi-quantitative manipulation of nucleic acid oligomers for delivery to an integrated microarray or biosensor.     

Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools

A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO₂ core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3′-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5′-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.     

Detection of β-thalassemia by a DNA piezoelectric biosensor coupled with polymerase chain reaction

β-Thalassemia is an inherited disorder mainly caused by mutations in the gene of the β-globin chain of adult haemoglobin (HbA). Clinically, β-thalassemia can be a mild or silent condition, or it can cause severe diseases, leading to transfusion dependence. Studies at the gene level have identified a large number of variations in the β-globin gene in different populations. In the Mediterranean area one of the most common mutation is the C → T substitution in the codon 39 of the gene.A new procedure for detecting codon 39 mutation in the β-globin gene is reported, based on a DNA piezoelectric biosensor. An oligonucletidic probe (25-mer), specific for the region around codon 39, is immobilised on the gold surface of a piezoelectric quartz crystal. The hybridisation between the immobilised probe and the complementary strand in solution is detected recording the variations of the crystal frequency.Experiments with synthetic oligonucleotides were initially performed. Distinguishable frequency shifts were obtained from the interaction between the immobilised probe and the complementary and the mismatch oligonucleotides. A solution containing 50% of both the oligonucleotides has been also tested and distinguished from the others evaluating the resulting signals. Experiments with non-complementary oligonucleotides gave no signal variation. The biosensor was able to distinguish between sequences differing in only one base also using polymerase chain reaction-amplified samples [771 base pairs (bp)] of DNA extracted from human blood of thalassemic and healthy (normal) patients or patients with β-thalassemia traits.The optimised DNA piezoelectric biosensor has been successfully applied to the determination of one of the most frequent mutation characteristic of β-thalassemia in the Mediterranean population.     

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.     

Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection

A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH) for covalent DNA immobilization and enhanced hybridization detection is described. The MWNTs-COOH-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides with the 5'-amino group were covalently bonded to the carboxyl group of carbon nanotubes. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using an electroactive intercalator daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity. This is the first application of carbon nanotubes to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

金电极上自组装巯基DNA生物传感器的研究

本文在金电极上自组装单链巯基DNA,制备能识别正错配的简单DNA生物传感器,采用金标银染和线性扫描阳极溶出法对制备工艺条件和识别能力进行了研究,确定最佳制备工艺条件为:巯基DNA自组装时间为6 h,采用巯基己醇封闭时间为3 h,正错配DNA表现出不同的检测信号,制得的DNA生物传感器具有快速、准确的识别能力。     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

A cyclodextrin host-guest recognition approach to a label-free electrochemical DNA hybridization biosensor

A novel label-free electrochemical DNA hybridization biosensor using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified screen printed electrode (CD/PNAANI/CNT/SPE) has been developed. The proposed DNA hybridization biosensor relies on the intrinsic oxidation signals of guanine (G) and adenine (A) from single-stranded DNA entered into the cyclodextrin (CD) cavity. Due to the binding of G and A bases to complementary cytosine and thymine bases in dsDNA, the signals obtained for ssDNA were much higher than that of dsDNA. The synergistic effect of the multi-walled carbon nanotubes provides a significantly enhanced voltammetric signal, and the CD encapsulation effect makes anodic peaks of G and A shift to less positive potentials than that at the bare SPE. The peak heights of G and A signals are dependent on both the number of the respective bases in oligonucleotides and the concentration of the target DNA sequences. Hybridization of complementary strands was monitored through the measurements of oxidation signal of purine bases, which enabled the detection of target sequences from 0.01 to 1.02 nmol μl(-1) with the detection limit of target DNA as low as 5.0 pmol μl(-1) (S/N = 3). Implementation of label-free and homogeneous electrochemical hybridization detection constitutes an important step toward low-cost, simple, highly sensitive and accurate DNA assay. Discrimination between complementary, noncomplementary, and two-base mismatch targets was easily accomplished using the proposed electrode.