Detection Of ssDNA Research Articles

Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge.

There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.

Label Free Detection of Gene Mutation by Applying a Ce2(Zr1-xTix)Oy Sensing Film Based Electrolyte – Insulator – Semiconductor (EIS) Biosensors Using Sol-Gel Method

In recent scenario, various kinds of high-k materials such as rare-earth (RE) oxide based film are the promising candidate for achieving pH response. These types of high-k materials have been used as gate oxide material to fabricate electrolyte–insulator–semiconductor (EIS) sensor devices as pH sensing membranes due to good sensing performance with their high dielectric constant, high resistivities, and large band gap energies]. Employing the Ce2(Zr1-xTix)Oy (CZT) oxide sensing membrane based EIS device has been rarely studied for biosensing applications. In this study, a bio-membrane/ Ce2(Zr1-xTix)Oy (CZT) /Si EIS structure was used as a disposable bio-chemical transducer for DNA mutation. The advantage of this structure is easy to fabricate and has greater performance than conventional devices. Zr 4+ rich content CZT based EIS devices exhibited the best sensing properties (sensitivity: 62.31 mV/pH, , lower hysteresis voltage : ~ 3.36 mV and lower drift rate : ~1.1 mV/hr). Being higher sensing properties of Zr 4+ rich content CZT is considered as a potential candidate for detection of DNA /protein due to change in the surface potential of CZT membrane. To apply the detection of ssDNA and cDNA hybridization was successfully observed in Zr 4+ rich content CZT based EIS devices Figure 1

Target ssDNA detection of E.coli O157:H7 through electrical based DNA biosensor

E.coli O157:H7 is one of the harmful foodborne pathogenic disease causing bacterium which leads many illnesses and even deaths each year. IDT or IDE (Interdigitated Electrode) based biosensors usually use electrochemical behavior through impedometric detection. In this paper we describe a novel mechanism for E.coli O157:H7 ssDNA target detection via electrical behavior through E.coli O157:H7 probe and (3-Aminopropyl)triethoxysilane (APTES) functionalized Au IDE biosensor. Tween-20 was used as blocking the nonspecific binding on non-immobilized area of the E.coli ssDNA probes. Au IDE was fabricated on the Si substrate using CMOS and conventional photolithographic process. Fabricated Au IDE was physically characterized by using high power microscope (HPM) and scanning electron microscope (SEM). IDE electrode edges were characterized using 3D nano profiler. The E.coli O157:H7 ssDNA probes were used as the receptor to capture the specific target ssDNA and Au IDE was used as the transducer. I-V characteristics were performed for each functionalization step as bare Au IDE, silanization and immobilization. Well functionalized biosensor was hybridized with complementary, non-complementary and single base mismatch ssDNA to confirm the specificity. Sensitivity measurements were done using different concentrations of complementary ssDNA targets from 1fM to 10 µM. The sensitivity and limit of detection (LOD) of the biosensor are 14.3 mAM−1 and 0.8 fM respectively. It was confirmed that Au IDE biosensor shown here is capable to detect specific and low concentrated E.coli O157:H7 ssDNA targets successfully.

Aluminum Nanocrystals: A Sustainable Substrate for Quantitative SERS-Based DNA Detection.

Since its discovery in the 1970s, surface-enhanced Raman scattering (SERS) has been primarily associated with substrates composed of nanostructured noble metals. Here we investigate chemically synthesized nanocrystal aggregates of aluminum, an inexpensive, highly abundant, and sustainable metal, as SERS substrates. Al nanocrystal aggregates are capable of substantial near-infrared SERS enhancements, similar to Au nanoparticles. The intrinsic nanoscale surface oxide of Al nanocrystals supports molecule-substrate interactions that differ dramatically from noble metal substrates. The preferential affinity of the single-stranded DNA (ssDNA) phosphate backbone for the Al oxide surface preserves both the spectral features and nucleic acid cross sections relative to conventional Raman spectroscopy, enabling quantitative ssDNA detection and analysis.

Reduced graphene oxide–multiwalled carbon nanotubes composites as sensing membrane electrodes for DNA detection

Thin films of reduced graphene oxide–multiwalled carbon nanotube (rGO–MWCNT) composites were demonstrated as sensing membrane electrodes for single-stranded DNA (ssDNA) detection. The morphology of the rGO–MWCNT composite thin films was observed via field emission scanning electron microscopy. The GO sheet and MWCNTs were clearly obtained, and the MWCNTs were uniformly distributed on the surface of the GO. The chemical bonding of the rGO–MWCNTs was examined using Fourier transform infrared spectroscopy. The element compositions of carbon, silicon, and oxygen were confirmed via energy-dispersive X-ray spectroscopy and X-ray powder diffraction analysis. The biosensor demonstrated high sensitivity to the ssDNA target with a linear range from 500 to 100 pM. Furthermore, the biosensor demonstrated good selectivity, reproducibility, and long-term stability for DNA detection. Since, the biosensor responded very-well and demonstrated excellent detection capabilities, it is highly recommended to be used in detecting specific biomarkers and other targeted proteins.

Luminescent Iridium(III) Complex Labeled DNA for Graphene Oxide-Based Biosensors.

There has been growing interest in utilizing highly photostable iridium(III) complexes as new luminescent probes for biotechnology and life science. Herein, iridium(III) complex with carboxyl group was synthesized and activated with N-hydroxysuccinimide, followed by tagging to the amino terminate of single-stranded DNA (ssDNA). The Ir-ssDNA probe was further combined with graphene oxide (GO) nanosheets to develop a GO-based biosensor for target ssDNA detection. The quenching efficiency of GO, and the photostability of iridium(III) complex and GO-Ir-ssDNA biosensor, were also investigated. On the basis of the high luminescence quenching efficiency of GO toward iridium(III) complex, the GO-Ir-ssDNA biosensor exhibited minimal background signals, while strong emission was observed when Ir-ssDNA desorbed from GO nanosheets and formed a double helix with the specific target, leading to a high signal-to-background ratio. Moreover, it was found that luminescent intensities of iridium(III) complex and GO-Ir-ssDNA biosensor were around 15 and 3 times higher than those of the traditional carboxyl fluorescein (FAM) dye and the GO-FAM-ssDNA biosensor after UV irradiation, respectively. Our study suggested the sensitive and selective Ir-ssDNA probe was suitable for the development of highly photostable GO-based detection platforms, showing promise for application beyond the OLED (organic light emitting diode) area.

Detection of short ssDNA and dsDNA by current-voltage measurements using conical nanopores coated with Al2O3 by atomic layer deposition

DNA detection using solid state nanopores is limited by the difficulty of the detection of short DNA strands due to their very short dwell time. Here, we report on the fabrication of nanopores using a track-etching technique along with the atomic layer deposition of aluminum oxide. This method allows shaping of conical nanopores, controlling their diameter, and passivation of the surface by Al2O3. Through the optimized nanopore, short DNA (10 nM) having 10 and 40 nucleotides in length were detected both at the single stranded and double stranded levels. The analysis of current trace shows signal to noise ratio of 2 and 4 for ssDNA and dsDNA respectively and a dwell time at ms scale. This is possible because of both the high aspect ratio and the Al2O3 coating that permit to decrease the DNA velocity.

Isothermal amplification detection of nucleic acids by a double-nicked beacon

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Retinoic acid‑incorporated glycol chitosan nanoparticles inhibit the expression of Ezh2 in U118 and U138 human glioma cells.

At present, one of the most life threatening types of adult brain tumor is glioblastoma multiforme (GBM). The molecular mechanism underlying the progression of GBM remains to be fully elucidated. The modern method of clinical treatment has only improved the average survival rates of a newly diagnosed patients with GBM by ~15 months. Therefore, the discovery of novel molecules, which are involved in glioma inhibition is required. In the present study, U118 and U138 human glioma cells were transfected with all-trans retinoic acid (RA)-incorporated glycol chitosan (GC) nanoparticles. An MTT assay was used for the analysis of cell proliferation and flow cytometric analysis and ssDNA detection assays were performed for the determination of induction of cell apoptosis. Cell cycle distribution was analyzed by flow cytometry. Exposure of the U118 and U138 human glioma cells to the RA-incorporated GC nanoparticles for 24 h resulted in a concentration-dependent inhibition of cell proliferation. Among the range of experimental RA concentrations, the minimum effective treatment concentration was 10 µM, with a half maximal inhibitory concentration of 25 µM. The results also demonstrated that RA transfection resulted in the inhibition of cell proliferation, inhibition of the expression of Ezh2, and apoptosis through the mitochondrial signaling pathway by a decrease in membrane potential, the release of cytochrome c, and cell cycle arrest in the G0/G1 phase.

Sensitive Detection of ssDNA Using an LRET-Based Upconverting Nanohybrid Material.

Water-dispersible, optical hybrid nanoparticles are preferred materials for DNA biosensing due to their biocompatibility. Upconverting nanoparticles are highly desirable optical probes in sensors and bioimaging owing to their sharp emission intensity in the visible region. We herein report a highly sensitive ss-DNA detection based on an energy transfer system that uses a nanohybrid material synthesized by doping NaYF4:Tm(3+)/Yb(3+) upconverting nanoparticles (UCNPs) on silica coated polystyrene-co-acrylic acid (PSA) nanoparticles (PSA/SiO2) as the donor, and gold nanoparticles (AuNPs) decorated with Ir(III) complex as the acceptor. UCNPs tagged on PSA/SiO2 and the cyclometalated Ir(III)/AuNP conjugates were then linked through the ss-DNA sequence. Sequential addition of the target DNA to the probe molecular beacon complex resulted in the separation of the optical nanohybrid material and the quencher, leading to a measurable increase in the blue fluorescence emission intensity. Our results have shown a linear relationship between the fluorescence intensity and target DNA concentration down to the picomolar.

Hydrogels Triggered by Metal Ions as Precursors of Network CuS for DNA Detection.

The gelation behavior of lithocholate (LC(-) ) with different metal ions in water was investigated. The microstructures of hydrogels were determined to be three-dimensional (3D) networks of fibrous aggregates. The formation of fibrils was speculated to be mainly driven by the coordination between carboxylate of LC(-) and metal ions, accompanied by the assistance of noncovalent interactions such as electrostatic and hydrophobic interactions. The hydrogels, which can maintain the mechanical strength at higher temperature, exhibit thermal stability. Their gelation capability was enhanced with the increase in acidity. The hydrogels of LC(-) and Cu(2+) mixtures served as the precursors for producing network nanostructures of CuS nanoparticles. These new CuS networks exhibit high fluorescence quenching ability and can act as an effective fluorescent sensing platform for ssDNA detection.

Conjugated Polymer Nanoparticles for Label-Free and Bioconjugate-Recognized DNA Sensing in Serum.

Hybridbio/‐synthetic sensory conjugated polymer nanoparticles (CPNs) are developed for selective label‐free detection of target ssDNA in serum. Carboxylic acid‐functionalized anionic polyfluorene nanoparticles are rationally designed as signal amplifying unit to bioconjugate with amine functionalized single stranded oligonucleotides as a receptor. The covalent DNA coating can significantly improve the photostability of the DNA‐bioconjugated CPNs over a wide range of buffer conditions. Better ssDNA discrimination for the DNA‐bioconjugated CPNs sensor is achieved owing to increased interchain interactions and more efficient exciton transport in nanoparticles. The distinguishable fluorescent color for DNA‐bioconjugated CPNs in the presence of target ssDNA allows naked‐eye detection of ssDNA under UV irradiation.

Label-free optical detection of biomolecular translocation through nanopore arrays.

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information.

Detection of adenine-rich ssDNA based on thymine-substituted tetraphenylethene with aggregation-induced emission characteristics

In this work, tetraphenylethene (TPE) equipped with thymine, TPE–T, was synthesized and used as a fluorescence biosensor for differentiating ssDNA from dsDNA. TPE–T is a “turn-on” fluorescence probe once it forms hydrogen bonds with single-stranded adenine-containing oligonucleotides with high sensitivity and specificity. The working mechanism of TPE–T and its potential in cell imaging are investigated in detail.

Surface plasmon resonance technique for directly probing the interaction of DNA and graphene oxide and ultra-sensitive biosensing

The binding of DNA with graphene oxide (GO) is important for applications in disease diagnosis, genetic screening, and drug discovery. The standard assay methods are mainly limited to indirect observation via fluorescence labeling. Here we report the use of surface plasmon resonance for direct sensing of DNA/GO binding. We show that this can be used for ultra-sensitive detection of single-stranded DNA (ssDNA). Furthermore, the results provide a more direct probe of DNA/GO binding abilities and confirm that hydrogen bonding plays a key role in the interaction between GO and ssDNA. This enables to a novel biosensor for highly sensitive and selective detection of ssDNA based on indirect competitive inhibition assay (ICIA). We report development of such a sensor with a linear dynamic range of 10−14–10−6M, a detection limit of 10fM and a high level of stability during repeated regeneration.

Biosensing enhancement using passive mixing structures for microarray-based sensors

The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10–1000s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.

Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains

We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation.Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus.The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

Direct Electrochemical Detection of ssDNA with a Micro Three-Electrode System

Direct Electrochemical Detection of ssDNA with a Micro Three-Electrode System

DNA Origami Nanopores for Controlling DNA Translocation

We combine DNA origami structures with glass nanocapillaries to reversibly form hybrid DNA origami nanopores. Trapping of the DNA origami onto the nanocapillary is proven by imaging fluorescently labeled DNA origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").