DNA Probes For Detection Research Articles

Acoustic Methodology for Selecting Highly Dissipative Probes for Ultrasensitive DNA Detection.

The objective of this work is to present a methodology for the selection of nanoparticles such as liposomes to be used as acoustic probes for the detection of very low concentrations of DNA. Liposomes, applied in the past as mass amplifiers and detected through frequency measurement, are employed in the current work as probes for energy-dissipation enhancement. Because the dissipation signal is related to the structure of the sensed nanoentity, a systematic investigation of the geometrical features of the liposome/DNA complex was carried out. We introduce the parameter of dissipation capacity by which several sizes of liposome and DNA structures were compared with respect to their ability to dissipate acoustic energy at the level of a single molecule/particle. Optimized 200 nm liposomes anchored to a dsDNA chain led to an improvement of the limit of detection (LoD) by 3 orders of magnitude when compared to direct DNA detection, with the new LoD being 1.2 fmol (or 26 fg/μL or 2 pM). Dissipation monitoring was also shown to be 8 times more sensitive than the corresponding frequency response. The high versatility of this new methodology is demonstrated in the detection of genetic biomarkers down to 1-2 target copies in real samples such as blood. This study offers new prospects in acoustic detection with potential use in real-world diagnostics.

Gelatin nanoparticles transport DNA probes for detection and imaging of telomerase and microRNA in living cells

Telomerase and microRNA (miRNA) are biomarkers closely related to tumors. Simultaneous detection of both markers can improve accuracy and reliability of early diagnosis. Based on the mechanism of fluorescence resonance energy transfer (FRET), two fluorescent DNA probes were designed for telomerase and miRNA-21. The probes were wrapped by gelatin through electrostatic interaction to form nanoparticles. After that, we synthesized molecularly imprinted coating of transferrin on the surface of gelatin nanoparticles, which can avoid the immune stress response and macrophage phagocytosis to help gelatin nanoparticles enter into the cells smoothly through endocytosis. Following with the degradation of gelatin in the cells, DNA probes were released to react with telomerase and miRNA-21 and lead to the change of the fluorescence signal. Thereby the simultaneous imaging of telomerase and miRNA-21 were successfully achieved in HeLa cells and HepG2 cells. The proposed strategy shows the simultaneous imaging for different biological markers with DNA probes by preventing them from being hydrolyzed with nucleases before the determination and achieves reliable method for early diagnosis of cancer.

Disposable multiplexed electrochemical sensors based on electro-triggered selective immobilization of probes for simultaneous detection of DNA and proteins.

Electrically addressable covalent immobilization of probes on a multiplexed electrode for the simultaneous detection of multiple targets within the same sample is often regarded as a difficult milestone to be achieved. Herein, we demonstrated a reagentless disposable multiplexed electrochemical DNA and aptamer-based sensing platform for the simultaneous determination of various targets. The electrochemically triggered "click" chemistry was developed, and three biomarkers, including p53, thrombin, and VEGF165 were used as model analytes. The proposed sensor consisted of three independent screen-printed carbon electrodes (SPCE), with an alkyne-azide cycloaddition reaction that was activated selectively by means of electrical triggering, so that different DNA probes can be modified on the desired electrode units in sequence. In terms of simultaneous detection, the sensor was able to quantify the DNA target of p53 with a detection limit of 0.35 nM, whereas the limits of detection for protein quantification of thrombin and VEGF165 were 0.22 nM and 0.014 nM, respectively. The proposed sensor not only showed encouraging reproducibility and stability, but also performed well even in 50% serum samples. Therefore, the work described here offers a general strategy for developing a multiplexed sensor with promising potential to achieve rapid, simple and cost-effective analysis of biological samples.

A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity.

A triplex-forming oligonucleotide (TFO) linear probe containing perylene derivatives was synthesized. The TFO linear probe formed a remarkably stable triplex with a target DNA duplex, resulting in the light-up of fluorescence emission. The sensitivity was extremely high even at pH 7. Detection of PCR-amplified target DNA was demonstrated.

Tetraplex real-time PCR with TaqMan probes for discriminatory detection of cat, rabbit, rat and squirrel DNA in food products

Cat, rabbit, rat, and squirrel species are very sensitive in food products because most of them are potential carriers of zoonotic diseases and rejected in most religions and cultures. Since cats and rats are abundant in most parts of the world and their meats do not carry any value in legal markets, these meats could be considered as potential adulterants in halal, kosher, and other food markets. Rabbit and squirrel meats are also susceptible to adulteration. Therefore, both health and economic interests in rat, rabbit, cat and squirrel species are significant. In this work, a novel tetraplex real-time PCR assay with TaqMan probes was described to discriminate and identify all four species (cat, rabbit, rat, and squirrel) in a single assay platform. Species-specific primers and probes were developed against ATP6, and cytochrome b genes to amplify 108, 123, 161 and 176 bp DNA fragments from rat, rabbit, squirrel and cat meat products under various states. A 141-bp internal amplification control (IAC) of 18S rRNA was used to avoid any false-negative results. Specificity was evaluated against 22 species but no cross-reactivity was found. Efficiency of PCR assay as well as target quantification were determined based on a standard curve that was generated using tenfold serially diluted mixed DNA extract (1:1:1:1) from squirrel, rat, rabbit and cat species. The assay was valid under pure, processed and admixed states with 10–0.1% (w/w) adulterant from each species. The limit of detection was 0.1% under admixed samples and 0.003 ng DNA under pure states from each species. Analyses of 18 model burgers (9 chicken and 9 beef) and 18 frankfurters (9 chicken and 9 beef) revealed 91–122% target recovery at 0.1–10% adulteration. Finally, 72 commercial burgers (36 chicken and 36 beef) and 72 frankfurters (36 chicken and 36 beef) were screened but no target species was detected except IAC.

DNA-Silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on “chameleon” DNA-silver nano-clusters(DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus(HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas the red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. The ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 nmol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in human serum, illustrating its potential promise for clinical diagnosis.

A benzo(f)quinolinium fused chromophore-based fluorescent probe for selective detection of c-myc G-Quadruplex DNA with a red emission and a large Stocks shift

Accumulating evidence in recent years has suggested that G-Quadruplex DNAs may play important roles in gene expression and genomic stability in vivo. Specially, c-myc G-Quadruplex that exists in the promoter of oncogene is an attractive target in the pathway of cell proliferation and senescence. The development of small-molecule fluorescent probes for c-myc G-Quadruplex DNA has attracted significant attention. This work presented a benzo(f)quinolinium fused chromophore-based fluorescent probe L-1 for the detection of c-myc G-Quadruplex DNA using N-(2-hydroxyethyl)piperazine moiety as a binding unit. This probe exhibiting a red emission and a large Stocks shift was found to be selective and sensitive for “light-up” detection of c-myc quadruplex DNA over duplex DNA and other G-quadruplexes. It stabilized c-myc G-Quadruplex DNA and effective inhibited in vitro DNA synthesis. Binding studies indicated that the fluorescent response of L-1 mainly arised from end-stacking binding mode. Furthermore, L-1 was applicable to visualize the cellular nucleus in living cells.

Editorial

Editorial

Enhancing the Microarray Signal Detection of Single Nucleotide Polymorphisms (SNPs) by Using Homemade Immobilisation Buffers

Our research is focused on improving the detection capabilities of single nucleotide polymorphisms (SNPs) with pathological significance within breast cancer 1, early onset gene (BRCA1). We employed the BRCA1 canonical probe sequence containing the common C allele and probes including the mismatches caused by C replacement with any of the other nucleotides (A, G or T). Two immobilization buffers with distinct pH and salt concentration were involved in the experimental and statistical analyses in order to assess the detection specificity and stability over time of the tethered probes in oligonucleotide microarrays. A high spreading of data points (standard deviation 0.16) was acquired from the fluorescent hybridization signal of the DNA probes diluted in Phosphate Buffered Saline (PBS), while the Disodium Phosphate (Na2HPO4) buffer was recommended by a standard deviation of 0.08 for further investigations. The high specificity of mismatch detection of DNA probes diluted in Na2HPO4 buffer was established by Student�s t test, revealing the statistical reliability of the DNA-printed slides after 1, 4, 8 and 21 days.

Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting.

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting. With cellular imaging in mind we show that 2'-OMe RNA probes with TO at the 5-position of uracil or the 2'-position of the ribose sugar are particularly effective, exhibiting up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability. Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favourable properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a cellular context.

Control of Probe Density at DNA Biosensor Surfaces Using Poly(l-lysine) with Appended Reactive Groups.

Biosensors and materials for biomedical applications generally require chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. The use of modified poly(l-lysine) (PLL) polymers with appended oligo(ethylene glycol) (OEG) and thiol-reactive maleimide (Mal) moieties (PLL-OEG-Mal) offers control over the presentation of functional groups. These reactive groups can readily be conjugated to, for example, probes for DNA detection. Here we demonstrate the reliable conjugation of thiol-functionalized peptide nucleic acid (PNA) probes onto predeposited layers of PLL-OEG-Mal and the control over their surface density in the preceding synthetic step of the PLL modification with Mal groups. By monitoring the quartz crystal microbalance (QCM) frequency shifts of the binding of complementary DNA versus the density of Mal moieties grafted to the PLL, a linear relationship between probe density and PLL grafting density was found. Cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed to establish the absolute probe density values at the biosensor surfaces. These data provided a density of 1.2 × 1012 probes per cm2 per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.

Daniel McGillivray Brown. 3 February 1923—24 April 2012

Dan Brown was a nucleic acids chemist of the highest order, beginning with pioneering work under Lord Alexander Todd in the 1950s at University of Cambridge on chemical methods for synthesis of nucleosides and nucleotides. This work helped to confirm the furanose chemical structure of the sugar in nucleosides as well as the 3′-5′ phosphodiester linkage in DNA and RNA, perhaps the most well thought of achievement of his career. Later, as a chemistry department lecturer, he established the chemical structures of glycerol monophosphoinositides as well as triphosphoinositides. Turning back to the nucleic acids in 1961, he became fascinated by the effect of mutagens on DNA. He elucidated the mechanism for the reaction of hydroxylamine on cytidine to form an initial ‘bis-adduct’ and thereafter N 6 -hydroxycytidine. Moving in 1982 to the MRC Laboratory of Molecular Biology, he developed a method to prepare single-stranded DNA probes for detection of RNA sequences and in addition worked on a novel automated device for oligonucleotide synthesis. Reverting to his interest in mutagens, he then designed and synthesized hydrogen bonding degenerate bases and developed novel P and K modified pyrimidine and purine bases respectively as transition mutagens. Finally, he synthesized the base analogue 5-nitroindole as a potential universal base, which became useful in cycle DNA sequencing, and in addition developed the concept of ‘error catastrophe’ for the ribonucleoside of the P base as an antiviral agent. The P, K and 5-nitroindole bases became the most valued chemical entities of his career to molecular biologists. His legacy to the nucleic acids includes both his significant contributions to studies of the chemical nature of DNA and RNA and their constituents as well as a variety of enabling nucleic acids chemistry methods and mechanisms of DNA mutagenicity.

Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

Fluorometric determination of zinc(II) by using DNAzyme-modified magnetic microbeads.

A fluorometric assay for zinc ion is described that relies (a) on the use of an isothermal cycle to amplify the fluorescence signal, and (b) of magnetic beads (MBs) to completely remove unreacted DNA detection probes. Biotin and fluorophore-labeled substrate (Zn-Sub) strands acting as detection probes were first assembled on MBs. Next, Zn(II)-specific DNAzyme (Zn-Enz) strands were hybridized with the Zn-Sub strands. In the presence of Zn(II), the Zn-Sub strands are cleaved. This results in the release of the shorter DNA fragments (containing fluorescent label) and in the dissociation of Zn-Enz strands. The dissociated Zn-Enz strands then hybridize with the residual Zn-Sub strands and cleave them in a similar fashion. This leads to a target recycling amplification mechanism and in a cumulative signal amplification process. A strongly amplified signal is thus obtained in the presence of Zn(II). The use of MBs warrants that unreacted Zn-Sub strands can be magnetically separated from the solution. The method has a detection limit as low as 33 fM at a signal-to-noise ratio of 3 and a linear response in the 100 fM to 11nM Zn(II) concentration range. It was applied to the determination of Zn(II) in spiked tap water and seawater samples, and the results compared well with data obtained by ICP-MS analysis. The method was also applied to the determination of Zn(II) in infant milk powder and breast milk. Graphical abstract Magnetic beads (MBs) carrying fluorescein-labeled substrate (Zn-Sub) strands were hybridized with Zn(II)-specific DNAzyme (Zn-Enz) and cleaved in the presence of Zn(II). After recycling, the unreacted Zn-Sub strands were removed with MBs and the released fluorescein tags are measured.

Colorimetric detection of Listeria monocytogenes using one-pot biosynthesized flower-shaped gold nanoparticles

The present study described a novel fast, simple and green one-pot synthesis approach of flower-shaped gold nanoparticles (flower-shaped AuNPs) by using antioxidant plant extracts. The AuNPs synthesized by Alchemilla mollis extract showed maximum absorbance at 610 nm and were polydispersed flower-shaped with average size (±SD) of 68.7 ± 2.8 nm. We have explored a colorimetric sensing assay for rapid detecting Listeria monocytogenes by using single-stranded DNA (ssDNA) conjugated flower-shaped AuNPs. ssDNA were used as probe for DNA detection and the synthesized flower-shaped AuNPs were used as indicators. The limit of detection for hlyA gene and genomic DNA of Listeria monocytogenes were 48.4 ng and 100.4 ng, respectively. The proposed assay is fast and specific for Listeria monocytogenes detection, with the improved color change and stability.

A sandwiched electroanalysis method for probing Anthrax DNAs based on glucose-induced gold growth and catalytic coupling of tyramine using gold-mineralized glucose oxidase

Gold nanoparticles was doped into the protein matrix of glucose oxidase (GOx) by the in-site biomineralization route, yielding the GOx with mineralized gold (GOx-Gold) showing the double catalysis activities of GOx and peroxidase-like gold catalysis. A magnetic separation-based detection method was thus tailored for the sandwiched electroanalysis of Anthrax DNAs using GOx-Gold and tyramine linker as the probe labels. After the DNA hybridization reactions, the tyramine-mediated linking of DNA capture and detection probes was conducted through gold-catalytic oxidization of tyramines labelled at the probe terminuses, followed by the glucose-triggered gold growth catalyzed by GOx. Highly amplified electrochemical output of gold signals was thus achieved toward the ultrasensitive detection of DNAs in blood, with the detection limit down to ∼0.10 fM. Also, the discrimination of DNAs with single-base mutation could be expected. Importantly, such a biomimic gold mineralization route can be tailored for remolding various enzymes with improved intrinsic catalysis and electrocatalysis, thus promising the wide applications in the catalysis, biosensing, and biomedical fields.

Development of quantitative PCR primers and probes for environmental DNA detection of amphibians in Ontario

DNA from environmental samples (eDNA) is increasingly being used to detect and monitor elusive species. We developed species-specific eDNA primers and probes for qPCR detection of 24 amphibian species native to Ontario, Canada. Cross-species testing confirmed their high specificity and low cross-species amplification, as well as their ability to detect DNA from target species at low concentrations. These detection tools should prove useful for monitoring at-risk amphibian species throughout their native ranges.

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

Dealloyed Intra-Nanogap Particles with Highly Robust, Quantifiable Surface-Enhanced Raman Scattering Signals for Biosensing and Bioimaging Applications.

Uniformly controlling a large number of metal nanostructures with a plasmonically enhanced signal to generate quantitative optical signals and the widespread use of these structures for surface-enhanced Raman scattering (SERS)-based biosensing and bioimaging applications are of paramount importance but are extremely challenging. Here, we report a highly controllable, facile selective-interdiffusive dealloying chemistry for synthesizing the dealloyed intra-nanogap particles (DIPs) with a ∼2 nm intragap in a high yield (∼95%) without the need for an interlayer. The SERS signals from DIPs are highly quantitative and polarization-independent with polarized laser sources. Remarkably, all the analyzed particles displayed the SERS enhancement factors (EFs) of ≥1.1 × 108 with a very narrow distribution of EFs. Finally, we show that DIPs can be used as ultrasensitive SERS-based DNA detection probes for detecting 10 aM to 1 pM target concentrations and highly robust, quantitative real-time cell imaging probes for long-term imaging with low laser power and short exposure time.

Disassembly-driven signal turn-on probes for bimodal detection of DNA with 19F NMR and fluorescence

To image DNAs in vivo, molecular probes that can detect DNA with a highly transparent signal are required. In this study, we designed and synthesized amphiphilic molecular probes that can detect DNA with a highly transparent 19F NMR signal in a signal turn-on manner based on the self-assembly and target-dependent-disassembly of the probe molecules in aqueous solution. Fluorescence and dynamic light scattering studies revealed that these amphiphilic probes consisting of bisbenzimide H 33258 and 3,5-bis(trifluoromethyl)benzene are assembled and form aggregates in aqueous solution, and that the aggregates are collapsed when the probes bind to double-stranded DNA via selective interaction between the bisbenzimide H 33258 moiety and AATT double-stranded region of DNA. Since the target-dependent-disassembly of the probes caused turn-on of 19F NMR and fluorescence signals, bimodal detection of double-stranded DNA was successfully achieved.