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.     

Visual detection of Hg²⁺ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg2?) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg2?-thymine (T-Hg2?-T) coordination in the presence of Hg2?. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg2? without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg2? and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.     

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.     

Detection of Human Polyomavirus Dna In Papanicolaou Stained Smears of Urinary Sediment By In Situ Hybridization

Papanicolaou stained smears of urinary sediment containing inclusion bearing urothelial cells suggestive of human polyomavirus infection were destained and reprocessed for in situ hybridization using a biotinylated probe for human polyomavirus DNA. Seven slides were processed in this way. A hybridization signal for viral DNA was noted in each case, even in smears that had previously been stored for 11 years. This simple and rapid non-radioactive detection system is a valuable supplement to routine urinary cytology for the definitive diagnosis of this virus infection.     

Flexible glucose sensor using CVD-grown graphene-based field effect transistor

In this paper, a new concept to achieve improved probe-target recognition has been devised by introducing a novel class of DNA-functionalized three-dimensional (3D), stand-free, and nanostructured electrodes. The gold nanofibrous electrodes were created using MHZ ultrafast laser material processing in air at ambient conditions. The developed nanofibrous DNA biosensor was characterized by cyclic voltammetry with the use of ferrocyanide as an electrochemical redox indicator. Currently, electrochemical signal enhancement which is of great significance for improving the sensitivity in DNA detection remains a great challenge. Through, enhanced surface area-to-volume ratio and more efficient arrangement of probe molecules on nanofibrous electrodes, our newly developed electrode system overcomes some of the sensitivity challenges of the existing systems. This nanofiber-based system realizes femtomolar (fM) sensitivity toward complementary target DNA, and demonstrates a very wide dynamic range (from 1 fM to 1 nM).     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.     

Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis.

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS: Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Simultaneous detection of DNA-binding proteins using exo-Taq-based reaction

The DNA-binding protein (DBP) has a wide range of roles such as those in DNA repair, recombination, and gene expression. Recently, a microarray-based method has been developed for the high-throughput analysis of DNA-protein interactions. However, to maximize the advantages of this method, the detection process should be improved so that the method can be applied to many proteins without the use of antibody or sample labeling. Previously, we presented a primary report on the detection of DBP, which is applicable to the microarray format. The system consists of three steps: first, the target DBP in the sample solution is incubated with a probe DNA; second, the probe is digested with Exo (Exonuclease) III; finally, the probe is extended withTaq DNA polymerase using fluorescent dye-labeled dUTP as a substrate. The binding DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. In this study, the simultaneous detection of multiple DBPs was examined, and then the DBPs were analyzed using a crude extract of the cultured cells to demonstrate the general applicability of the method. Our method can be applied to many DBPs using the same procedure and components, whereas in the antibody-based method, the same number of antibodies as DBPs is needed to detect target DBPs in ELISA (enzyme-linked immunosorbent assay). These results suggest that our method is useful for the high-throughput detection of DBPs in the microarray format.     

Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor

Although the analyses of HBV genomic DNA have traditionally been performed with commercial techniques, the high cost and long time consumed have hindered their applications in routinely diagnosis and prognosis of infection. We construct peptide nucleic acid (PNA) piezoelectric biosensor for real-time monitoring of hybridization of hepatitis B virus (HBV) genomic DNA. The PNA probe can combine to target DNA sequences more effectively and specifically than a DNA probe. The PNA probe was designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The hybridization assay was completed in 50 min. The detection limit was 8.6 pg/L and the clinical specificity was 94.44% compared with real time-PCR (RT-PCR). The PNA probe was able to distinguish sequences that differ only in one base. Detection sensitivity can be improved and detection time can be decreased by adding RecA protein-coated complementary ssDNA which complement to HBV gene regions. The QCM system we designed has the advantages of being rapid, label-free and highly sensitive and can be a useful supplement to commercial assay methods in clinical chemistry.     

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.     

Designing a new biosensor “DNA ELISA” to detect Escherichia coli using genomic DNA and comparison of this method to PCR-ELISA

In this experiment, DNA-ELISA biosensor was introduced, bearing the ability to detect specific bacteria in about 4?h. This is a more rapid system in comparison to conventional methods, like colony counting method. Moreover, this method does not require any amplification and directly detects genomic DNA of bacteria, giving a lower limit to the sensitivity of 40,000 bacteria. In this study, two specific probes capture (biotin labelled) and detector (dig labelled), were used against special regions of 16s rRNA gene of Escherichia coli ATCC 25922. The capture probe has the ability to trap the target bacterial DNA from a pool of other kinds of bacteria under specific conditions. The detector probe then was used to hybridize to the genome of trapped bacteria. The detection proceeds by adding HRP-anti dig enzyme and its substrate, ABTS to emit light. Light absorbance is measured for verifying the detection.     

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.     

Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification

A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500 ng mL⁻¹, with a detection limit as low as18 pg mL⁻¹.     

N4-(6-aminohexyl)cytidine and -deoxycytidine nucleotides can be used to label DNA

Bisulfite is known to catalyze transamination between cytidine derivatives and amines. Using 1,6-diaminohexane we describe the synthesis and recovery of the 5'-triphosphates of N4-(6-aminohexyl)cytidine and -deoxycytidine (dahCTP). Both may be incorporated into DNA by nick translation with DNA polymerase I of Escherichia coli to provide reactive sites for the attachment of immunological or other labels. Biotinyl dahCTP is actively incorporated into DNA by the same system and can be detected by the binding of streptavidin complexed to an indicator enzyme such as acid phosphatase. Such labeled DNA is a suitable nonradioactive probe for detection of related sequences by hydridization.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Direct and quantitative analysis of Salmonella enterica serovar Typhimurium using real-time PCR from artificially contaminated chicken meat

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with 4.5x105 to 4.5x100 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.     

Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms

AIMS: A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented. METHODS AND RESULTS: The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe. CONCLUSION: Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.