DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

A nucleic acid biosensor for the detection of a short sequence related to the hepatitis B virus using bis(benzimidazole)cadmium(II) dinitrate as an electrochemical indicator

A novel hybridization indicator, bis(benzimidazole)cadmium(II) dinitrate (Cd(bzim)(2)(NO(3))(2)), was utilized to develop an electrochemical DNA biosensor for the detection of a short DNA sequence related to the hepatitis B virus (HBV). The sensor relies on the immobilization and hybridization of the 21-mer single-stranded oligonucleotide from the HBV long repeat at the glassy carbon electrode (GCE). The hybridization between the probe and its complementary sequence as the target was studied by enhancement of the peak of the Cd(bzim)(2)(2+) indicator using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay time. With this approach, a sequence of the HBV could be quantified over the range from 1.49x10(-7)M to 1.06x10(-6)M, with a linear correlation of r=0.9973 and a detection limit of 8.4x10(-8)M. The Cd(bzim)(2)(2+) signal observed from the probe sequence before and after hybridization with a four-base mismatch containing sequence was lower than that observed after hybridization with a complementary sequence, showing good selectivity. These results demonstrate that the Cd(bzim)(2)(2+) indicator provides great promise for the rapid and specific measurement of the target DNA.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Metal-enhanced biosensor for genetic mismatch detection

DNA biosensors are increasingly used in hybridization reactions, mutation detection, genomic sequencing, and identification of pathogens. However, the inability to monitor the recognition signals without resorting to the use of labels or electroactive mediators has led to DNA devices with inadequate sensitivity. Moreover, some electroactive species require high redox potentials that often destroy the DNA complementarity. This article presents the concept of metal-enhanced detection (MED) for the determination of DNA-DNA reactions and presents the application of this concept for mismatch detection. The MED concept relies on the idea that metallic films deposited as a continuous layer or monolayer onto a solid electrode, or even electrostatically held, could greatly enhance the rate of electron transfer by reducing the distance between the donor and acceptor species and could lead to label-free assays during DNA hybridization reactions. The MED concept has been tested for voltammetric detection of gene sequence of Microcystis spp. The resulting biosensor involved the immobilization of a 17-mer DNA probe that is complementary to a specific gene sequence of Microcystis spp. on a gold electrode via avidin-biotin chemistry. Electrochemical reduction and oxidation of DNA-captured Ag(+) ions provided the detection signals for the target gene sequence in solution. A linear response of silver cathodic peak current with concentration of the target oligonucleotide sequence was observed with a detection limit of 7 x 10(-9)M. This label-free approach was successfully applied to detecting two-base-pair mismatches in the gene sequence of Microcystis spp.     

Nanogold hollow balls with dendritic surface for hybridization of DNA

Au hollow balls are fabricated by adsorption of gold 3.5 nm in diameter onto a mixed vesicle composed of mixed polymerized diacetylene which made of negative charged 10,12-pentacosadiynoic acid (PCDA) and positive charged 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH(2)). The morphology of these hollow spheres could be controlled by changing the ratio of PCDA and PCDA-NH(2) and the immobilization and hybridization ability of the gold hollow ball have been investigated using a quartz crystal microbalance (QCM). It was found that a dendritic surface in an appropriate ratio existed. The hybridization amount of target DNA is about three to five times for the Au-mixed hollow ball at an optimal ratio (PCDA/PCDA-NH(2)=1/3) as compared with that for pure Au-PCDA-NH(2), though the immobilization amount of ssDNA on these two samples are almost the same, and the detected limitation of target DNA is extend from 10(-9) to 10(-12) M. The stability against breakage by transportation, combined with the simplicity and efficiency of detection, would offer an important advantage over unpolymerized one. This result shows the possibility to control the morphology and surface of nanogold hollow spheres by changing the ratio of PCDA and PCDA-NH(2) for the develop of a better DNA detection assay, further proving the idea that low surface coverage and higher DNA probe to target DNA ratios lead to optimal hybridization.     

Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels

Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO₃ and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 × 10⁻¹² mol/L (3σ). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.     

Hybridization signal enhancement via long-stranded probe for detection of DNA using aquadichloro (benzimidazole)-copper(II) as hybridization indicator

A simple and sensitive method for electrochemical detection of DNA was designed. This DNA sensor was based on a "sandwich" detection strategy, which involved a long capture probe DNA immobilized on glassy carbon electrodes that flanked both the reference DNA and target DNA. Electrochemical signals were measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using aquadichloro(benzimidazole)-copper(II), Cu(bzim)(H(2)O)Cl(2), as an electroactive indicator. An improving amount of Cu(bzim)(H(2)O)Cl(2) was interacted with the hybrid DNA via the incorporation of a long-probe DNA and a reference DNA in this sensor. As a result of this effect, this sensor design significantly enhanced the sensitivity. With 48-mer probe DNA and 27-mer reference DNA, the proposed method could be used for detection of 21-mer ssDNA ranging from 1.32 x 10(-7) to 2.52 x 10(-6) M with a detection limit of 2.94 x 10(-8) M. Electrochemical DNA biosensors were also developed using the same long-probe sequence as the target sequence with the novel hybridization indicator, Cu(bzim) (H(2)O)Cl(2). The detection limits for the complementary 21-mer target and 27-mer target were 9.52 x 10(-8) M and 5.81 x 10(-8) M, respectively. The results showed that the sensor with long-probe DNA and reference DNA is far more sensitive than that with nonswitch assay.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

QCM-based DNA biosensor for detection of genetically modified organisms (GMOs)

Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56 MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs.     

Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator

In this paper, a label-free, rapid and simple method was proposed to study the hybridization specificity of hairpin DNA probe using methylene blue (MB) as a hybridization indicator. Thiolated hairpin DNA probe was immobilized on the gold electrode by self-assembly. The voltammetric signals of MB were investigated at these modified electrodes by means of cyclic voltammetry (CV) detection. Single-base mutation oligonucleotide and random oligonucleotide can be easily discriminated from complementary target DNA. The effect of mismatch position in target DNA was investigated. Experimental results showed that mutation in the center of target DNA had greatest effect on the hybridization with hairpin DNA probe. The relationship between electrochemical responses and DNA target concentration was also studied. The reduction current of MB intercalation decreased with increasing the concentration of target DNA. Taken together, these experiments demonstrate that the hybridization indicator MB provides great promise for rapid and specific measurement of target DNA.     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

Development of an electrochemical DNA biosensor with a high sensitivity of fM by dendritic gold nanostructure modified electrode

In this paper, dendritic gold nanostructure (DenAu) modified electrode was obtained by direct electrodeposition of planar electrode into 2.8 mM HAuCl(4) and 0.1 M H(2)SO(4) solution under a very negative potential of -1.5 V. Scanning electron microscopy was used to characterize the growth evolution of DenAu with time. The whole DNA biosensor fabrication process based on the DenAu modified electrode was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. The probe DNA immobilization and hybridization with target DNA on the modified electrode could be well distinguished by using methylene blue as an electrochemical hybridization indicator. The DenAu modified electrode could realize an ultra sensitivity of 1 fM toward complementary target DNA and a very wide dynamic detection range (from 1 fM to 1 nM).     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes

A label-free electrochemical method for the detection of DNA-PNA hybridization using a water-soluble, ferrocene-functionalized polythiophene transducer and single-stranded PNA probes on the nanogold modified electrode is investigated. Nanogold modified electrodes can largely increase the immobilization amount of ss-PNA capture probe and lead to an increase of the electrical signal. The ferrocene-containing cationic polythiophene do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after DNA-PNA hybridization, cationic polythiophene is adsorbed on the DNA backbone, giving a clear hybridization detection signal in differential pulse voltammetry (DPV). Very good discrimination against non-complementary DNA and four-base mismatch DNA is observed. These studies show that the proposed method can provide an alternative for expanding the range of detection methods available for DNA hybridization.     

Electrical frequency dependent characterization of DNA hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5'-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.