Application For DNA Detection Research Articles

Surface adsorption of adenine on pristine and B/N/O/P-doped coronene as a biosensing substrate for DNA detection- DFT study

Nucleobase detection is crucial for the exploration of specific nucleotide sequences. Inspired by the recent realization of two dimensional nanomaterials as DNA detectors as well as sensors, the interactions of pristine and B/N/O/P-doped coronenes with adenine nucleobase have been studied on the basis of Density Functional Theory. Optimal configurations, the corresponding adsorption energies, charge transfer and electrical properties have been calculated for each complex. The adsorption strength and charge transfer in doped coronene have been found larger than that in the pristine coronene. AIM-RDG study suggests that non-covalent interactions existing in the associated interactive region are responsible for the stability of the complexes. The change in electrical conductivity of coronenes after the adsorption indicates its sensitivity towards DNA bases. The predicted energy gap and the prolonged recovery time for adenine-coronene configurations imply that pristine/doped coronene has potential applications in DNA detection.

Ultrasensitive deafness gene DNA hybridization detection employing a fiber optic Mach-Zehnder interferometer: Enabled by a black phosphorus nanointerface

The rapid and efficient detection of deafness gene DNA plays an important role in the clinical diagnosis of deafness diseases. This study demonstrates the ultrasensitive detection of complementary DNA (cDNA) by employing a nanointerface-sensitized fiber optic biosensor. The sensor consists of SMF-TNCF-MMF-SMF (abbreviated as STMS) structure with lateral offset. Besides, it is functionalized with a nanointerface of black phosphorus (BP) to enhance the light-matter interaction and eventually improve the sensing performances. Relying on this nanointerface-sensitized sensor, we successfully realize the in-situ detection of cDNA at concentrations ranging from 1 pM to 1 μM, with a sensitivity of 0.719 nm/lgM. The limit of detection (LOD) is as low as 0.24 pM, which is at least two orders of magnitude lower than those of existing methods. The sensor exhibits the advantages of simple operation, fast response, label-free measurement, excellent repeatability, and high selectivity. Our contribution suggests a convenient approach for deafness gene DNA detection and can be extended for general ultra-low concentration DNA detection applications.

Efficient Preparation of Large-Sized Rings of Single-Stranded DNA through One-Pot Ligation of Multiple Fragments.

Circular single-stranded DNA (c-ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c-ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one-pot ligation method to efficiently prepare large c-ssDNA. By ligating several short fragments of linear single-stranded DNA (l-ssDNA) in one-pot by using T4 DNA ligase, longer l-ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large-sized single-stranded c-ssDNAs (e.g., 200-nt) were successfully synthesized in high selectivity and yield.

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules

Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

The behavior of a bipedal DNA walker moving on the surface of magnet microparticles and its application in DNA detection.

In this work, a three-dimensional DNA machine based on the isothermal strand-displacement polymerase reaction (ISDPR) has been constructed. The walking behavior of a DNA walker on the obstructive surface of magnetic beads has also been studied by adding different nucleic acid blocks. The "leg" of the DNA walker could hybridize with a hairpin structure DNA named H1 and lead to the opening of it. And the newly exposed stem would interact with a primer. A strand exchange has happened with the assistance of polymerase and dNTPs, so that the "leg" has been displaced and the DNA walker could be pushed to move on the surface. But the nucleic acid blocks could increase steric hindrance and obstruct this process, which is similar to the behavior of human beings walking on craggy paths. Through changing these blocks, such as the structure, the amount, and the length of blocks, the movement of the DNA walker has been controlled. What's more, the results of its application for DNA detection are satisfactory. The limit of detection is 21.6pM. Also, this method has been successfully applied in complex biological samples. Graphical abstract ᅟ.

A Turn-On Detection of DNA Sequences by Means of Fluorescence of DNA-Templated Silver Nanoclusters via Unique Interactions of a Hydrated Ionic Liquid.

Nucleic acid stability and structure, which are crucial to the properties of fluorescent DNA-templated silver nanoclusters (DNA-Ag NCs), significantly change in ionic liquids. In this work, our purpose was to study DNA-Ag NCs in a buffer containing the hydrated ionic liquid of choline dihydrogen phosphate (choline dhp) to improve fluorescence for application in DNA detection. Due to the stabilisation of an i-motif structure by the choline cation, a unique fluorescence emission—that was not seen in an aqueous buffer—was observed in choline dhp and remained stable for more than 30 days. A DNA-Ag NCs probe was designed to have greater fluorescence intensity in choline dhp in the presence of a target DNA. A turn-on sensing platform in choline dhp was built for the detection of the BRCA1 gene, which is related to familial breast and ovarian cancers. This platform showed better sensitivity and selectivity in distinguishing a target sequence from a mutant sequence in choline dhp than in the aqueous buffer. Our study provides new evidence regarding the effects of structure on properties of fluorescent DNA-Ag NCs and expands the applications of fluorescent DNA-Ag NCs in an ionic liquid because of improved sensitivity and selectivity.

Plasmon-Enhanced Fluorescence in Coupled Nanostructures and Applications in DNA Detection

Plasmon coupling interactions between adjacent noble metal nanoparticles (NPs) can cause significantly enhanced local electric field in the gap region, which could be utilized to dramatically enhance the fluorescence intensity of chromophores. Here we performed a systematic study on the influence of different factors on plasmon coupling-enhanced fluorescence, including shape and size of metal NPs, dye distribution, and separation distance. Cyanine 5 (Cy5) acted as the fluorescence probe and DNA was employed to assemble nanostructures to immobilize Cy5 into the gap region of the coupled metal NPs. Fluorescence of Cy5 was prequenched by attaching DNA linked Cy5 to the surface of Au nanospheres (NSs). The quenched fluorescence of Cy5 was turned-on by forming nanoassembly through DNA hybridization with different enhancing substrates: Au NSs, Au nanorods (NRs) and Au@Ag NSs. Au@Ag NSs were found to give the largest fluorescence enhancement effect. Larger-sized Au@Ag NPs were found to display larger fluorescenc...

A Typical Nano-Bio Interaction of Cu doped ZnO Nanoparticles with Escherichia coli DNA

In order create optical platforms for the detection of Escherichia coli (EC) DNA, we have synthesized Cu doped ZnO nanoparticles by a sonochemical method. EC DNA interaction study of the 5% and 10% Cu doped ZnO nanoparticles was monitored by various spectroscopic techniques. Interestingly, 5% Cu doped ZnO shows stronger binding affinity towards EC DNA, but with a lower detection limit of the order of micro molar concentration. The key experimental novelty was observed from the fluorescence titration of 5% doped sample with double stranded (ds) and single stranded (ss) DNA. The observed change in fluorescence was specific to ds EC DNA. We also compared the interaction of Cu-ZnO with other biomolecules such as protein and sugar moiety. Based on the results, Cu doped ZnO could be projected as a prospective optical material for detection of ds EC DNA. Overall, this work provides an avenue for understanding the interaction between Cu doped nanomaterials and biomolecules to extend their applications in DNA detection.

DNA Microviscosity Characterization with Particle Diffusometry for Downstream DNA Detection Applications.

Analytical characterization of DNA microviscosity provides critical biophysical insights into nuclear crowding, nucleic acid based pharmaceutical development, and nucleic acid based biosensor device design. However, most viscosity characterization methods require large sample volumes and destructive testing. In contrast, particle diffusometry permits in situ analysis of DNA microviscosity with short measurement times (8 s) using small volumes (<3 μL) which are compatible with DNA preparatory procedures. This unconventional biosensing approach involves measuring the change in sample viscosity using image processing and correlation-based algorithms. Particle diffusometry requires only a fluorescence microscope equipped with a charge-coupled device (CCD) camera and is a nondestructive measurement method. We use particle diffusometry to characterize the effect of DNA topology, length, and concentration on solution viscosity. In addition, we use particle diffusometry to detect the amplification of DNA from Staphylococcus aureus and Klebsiella pneumoniae, two pathogens commonly related to neonatal sepsis. Successful characterization of pathogen amplification with particle diffusometry provides a new opportunity to apply viscosity characterization toward downstream applications in nucleic acid based pathogen detection.

Preparation of organic fluorescent nanocomposites and their application in DNA detection

In this work, we report a novel organic fluorescent nanocomposite for DNA detection. Through a reprecipitation method, π-conjugated oligomers 4,7-(9,9′-bis(6-adenine hexyl)fluorenyl)-2,1,3-benzothiadiazole (OFBT-A) incorporated Texas Red labeled oligonucleotides (named TR-P5) within their matrices to form OFBT-A/TR-P5 nanocomposites. Efficient fluorescence resonance energy transfer (FRET) was observed between OFBT-A and TR-P5. Introduction of DNA with different lengths has changed the aggregation structures of nanocomposites and thus altered the FRET efficiency. Based on the fluorescence signal responses of the OFBT-A/TR-P5 nanocomposites to different DNA, DNA elongation and cleavage processes were successfully monitored by simply observing the change of FRET efficiency of the nanocomposites solutions. This research provides a facile, fast and highly efficient manner for DNA analysis, which shows great potential in the study of DNA-related biological processes.

Fabrication and Characterization of a Test Platform Integrating Nanoporous Structures With Biochemical Functionality

The application of solid-state nanopore technology for biosensing is a rapidly developing area of research with high commercial potential. Different synthetic materials, including silicon nitride, alumina, and polymers, are employed to fabricate single and multiple pores and offer a good platform for selective biomolecule detection. Two solid-state pore arrays, one with integrated silicon microfluidic system, were considered and an immobilization strategy suitable for detecting a single-stranded DNA (ssDNA) sequence was investigated. For the silicon nitride pores, a modification method based on the use of 3-aminopropyltriethoxysilane for silanization and 1,4-phenylene diisothiocyanate for amine crosslinking was applied to immobilize 100-nM ssDNA (amine C6) and a 100-nM limit of detection for complementary to probe ssDNA (Cy5) was estimated. The polycarbonate pores (the second type of the pore arrays) underwent surface modification based on an oxidation reduction reaction using sodium periodate and sodium borohydride and was used to immobilize 10-nM ssDNA and an estimated 100-nM limit of detection was also achieved. Linear sweep voltammetry was used to characterize the pores and a current potential profile was obtained after both immobilization of probe ssDNA and hybridization of complementary to probe ssDNA on the modified pore array surface. A decrease in current amplitude was measured after surface modification of both pore arrays, and this was attributed to the appearance of an additional layer on the pore surface reducing the pore opening and hindering the current flow. The hybridization event was also supported by contact angle measurements, where an increase in hydrophilicity was recorded at the different surface modification steps that were applied to produce the biofunctionalized nanopore. In addition, fluorescence was observed on the surfaces after hybridization, through incorporation of a CY5 fluorescent tag attached on the 5' end of the complementary to probe DNA. These results show the potential to use both silicon nitride and polycarbonate nanopores in DNA detection applications.

Formation of copper nanoparticles on poly(thymine) through surface-initiated enzymatic polymerization and its application for DNA detection.

Poly(thymine) (polyT) and double-stranded DNA (dsDNA) can act as efficient templates for the formation of copper nanoparticles (CuNPs) at a low concentration of CuSO4, and the formed CuNPs emit excellent fluorescence. In this work, we demonstrated a new and facile strategy for the highly sensitive and selective detection of DNA on streptavidin-functionalized magnetic beads (SA-MB) using DNA-templated CuNPs as the fluorescent probe. Target DNA (tDNA) was hybridized with the capture DNA that was immobilized on the surface of SA-MB. Surface initiated enzymatic polymerization (SIEP) was employed as the signal amplification method to generate the polyT at the 3' end of tDNA for the formation of CuNPs. The incorporation of polyT by SIEP resulted in ∼35.7 fold signal amplification compared to the dsDNA after hybridization without SIEP. A dose-response curve for detection of DNA was obtained, with a linear dynamic range of 0.1 nM to 10 nM. We showed that this method has a low pM limit of detection (LOD 98.2 pM) and it is also very sensitive to the mismatch type in a specific DNA sequence. In addition, it avoids rigorously controlled temperature, complex synthesis of the fluorescent probe and prelabeling of DNA strands and eliminates the use of sophisticated experimental techniques and equipment. Armed with these intriguing properties, the proposed system could provide an efficient tool for early diagnosis and risk assessment of malignancy.

Linearity improvement of Cy5 fluorescence concentration detection using series photodetector frequency circuit system with ITO–PolySi–metal–semiconductor–metal photodetector and its application for DNA detection and bacterial identification

In this study, the 48MHz series photodetector frequency circuit system with ITO–PolySi–metal–semiconductor–metal photodetector was developed for Cy5 fluorescence detection of DNA probe ET996. The theory analysis of series photodetector frequency circuit system showed that the circuit parameters such as Cp and A (tan θ) can be applied to improve the frequency sensitivity. In this research, the lower capacitance of ITO–PolySi–metal–semiconductor–metal photodetector (Cp) was made and selected to improve the frequency sensitivity. According to derived theory of A adjustment, the capacitance match Cm and bias of ITO–PolySi–metal–semiconductor–metal photodetector were adjusted to improve the frequency sensitivity for detection of fluorescence dye concentration. On the basis of optimal conditions of capacitance match Cm and bias of photodetector, the detection limit of ET996-Cy5 fluorescence dye concentration 2pmol/L can be measured by 48MHz sensor system. Moreover, the detection linearity of 48MHz sensor system was improved and the frequency shift ΔF was linearly related to the logarithm of fluorescence dye concentration in the range 2pmol/L–20μmol/L; meanwhile, through the feature of probe uniqueness, Edwardsiella BCRC 10670 DNA and fluorescence probe ET996-Cy5 can be successfully judged for hybridization reaction.

Encodable multiple-fluorescence CdTe@carbon nanoparticles from nanocrystal/colloidal crystal guest–host ensembles

We report herein the controllable generation of encodable multi-fluorescence CdTe@carbon nanoparticles (CdTe@C NPs) via the pyrolysis of quantum dot/photonic crystal (QD/PC) guest–host ensembles. The precursors of CdTe/poly(styrene-co-glycidylmethacrylate) (PS-co-PGMA) QD/PC guest–host ensembles were initially formed via the assembly of epoxy groups of PCs and carboxyl groups on the surface of CdTe QDs, followed by a pyrolysis process to generate CdTe@C NPs. The as-prepared CdTe@C NPs not only integrate the optical properties for both the carbon and CdTe QD constituents, but also enable an impressive enhancement of the fluorescence lifetime for CdTe QDs. The multifarious fluorescent spectra coding for CdTe@C NPs was further generated through regulating the embedded sizes or concentrations of CdTe QDs and the excitation wavelength, and their applications in DNA detection and luminescent patterns were achieved.

Interaction of Ru(phen)3Cl2 with graphene oxide and its application for DNA detection both in vitro and in vivo.

The fluorescence of Ru(phen)3Cl2 (tris(1,10-phenanthroline)ruthenium(ii) dichloride) can be effectively quenched with the addition of graphene oxide in aqueous solution, due to the formation of a GO-Ru hybrid. A fluorescence enhancement of approximately 50 times can be observed after the addition of a certain amount of DNA into the above-mentioned solution. The fluorescence increase is linearly proportional to the amount of DNA added in the concentration range of 0-70 μM and the DNA detection limit is down to 3.3 × 10-8 M. Meanwhile, it is found that the GO-Ru hybrid can enter into the nuclei and stain the DNA of living human breast cancer cells MCF-7, while Ru(phen)3Cl2 alone cannot cross the cellular membrane in the control experiment. This method can be employed to detect DNA both in vitro and in vivo.

Room Temperature Synthesis and Characterization of Au Nanoparticles

The new emergent properties of nanoparticles include quantam size effect, nonlinear optical properties and thus the nanomaterials have extensive applications in microelectronic, bacteriostatic and catalytic or magnetic recording materials. Nanoparticles find potential applications in DNA detection and photo detection. Gold nano particles (AuNPs) are useful in nanoscale devices and systems due to its resistance to oxidation. In this paper a cost effective, versatile and simple one step room temperature synthesis of highly stable and freestanding Au quantum dots capped by TTAB (Tetradecyltrimethyl Amonium Bromide) in the range of 10-90 nm is reported. Ultraviolet- visible spectroscopy (UV-VIS), Fourier transformation infrared spectroscopy (FT-IR) was carried out to characterize these samples. Scanning Electron Microscopy (SEM) confirms the AuNPs sizes of about 80 and 88 nm.

VERSATILE DNA-PACKAGING NANOMOTOR OF BACTERIOPHAGE phi29 WITH APPLICATIONS IN NANOBIOTECHNOLOGY

Nanobiotechnology entails the use of nanosized materials to build structures that can be applied in both biotechnology and medicine. In one vein of this field of study, scientists seek to mimic the wide variety of nanomachines and macromolecular structures that exist in nature and to replicate them in both structure and function. As a most intriguing example, the bacterial virus phi29 uses a self-contained nanomotor to package its DNA after replication. The 30-nm nanomotor contains 12 copies of a protein (gp10) which together form a 3.6-nm central channel through which the genomic DNA passes into the procapsid during viral assembly and exits during infection. This connector has been recently reengineered and embedded into a lipid bilayer, creating a system with tremendous application for DNA detection and characterization through electrophysiological measurement. A second component of the phi29 bacteriophage is an ATP-binding pRNA that forms a hexameric ring to gear the motor. The pRNA has been utilized to construct nanoparticles of dimers, trimers, hexamers and patterned superstructures via the interaction of two interlocking loops. Such structures constructed via bottom-up assembly have been used in the delivery of drugs, siRNA, ribozymes, and genes to specific cells, both in vitro and in vivo. This review summarizes current studies on the structure, function, and mechanism of the phi29 DNA packaging motor, as well as addresses the applications of these motor components in the field of nanobiotechnology.

Visual detection of DNA from salmonella and mycobacterium using split DNAzymes

The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Most reports on its use have however been constrained to proof-of-concept demonstrations using synthetic oligonucleotides. This is due to the inherent complexities of the assay, and the multiple components involved. Herein, we have overcome several of these challenges and have successfully integrated asymmetric PCR with the visual detection step, allowing enriched targets from complex samples to be conveniently detected. This workflow would enable the DNAzyme system to be applied for point-of-care DNA detection applications. We have established the platform herein as a simple and robust method to determine the presence of target DNA sequences post-PCR (as required for pathogen detection), without the need for gels or other apparatus. Nanomolar concentrations of amplified targets were detected using the workflow established, enabling the detection of salmonella and mycobacterium targets through a color change reaction, observable just by eye.

Self-assembly of cholesterol DNA at liquid crystal/aqueous interface and its application for DNA detection

In this letter, we report a strategy of detecting the DNA targets by using a thin layer of self-assembled cholesterol-labeled DNA probes at the liquid crystal (LC)/aqueous interface. When the system is exposed to 51 μg/ml of complementary DNA targets, the optical appearance of LC shows a continuous change from dark to bright under the crossed polars within 15 min. No obvious change can be observed when the system is exposed to one or two base-pair mismatch DNA targets. This system provides a principle for label-free and real-time detection of DNA targets without any fluorescent labeling.

Paste Electrode Based on Short Single‐Walled Carbon Nanotubes and Room Temperature Ionic Liquid: Preparation, Characterization and Application in DNA Detection

AbstractA paste electrode (SWNT&amp;RTIL PE) has been prepared using carboxylic group‐functionalized short single‐walled carbon nanotubes (SWNTs) mixed with 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF6, one kind of room temperature ionic liquid, RTIL). Its electrochemical behavior was investigated by cyclic voltammetry and electrochemical impedance spectroscopy in comparison with the paste electrode using mineral oil as a binder. Results highlighted the advantages of the paste electrode: not only higher conductivity, but also lower potential separation (ΔEp), higher peak current (ip) and better reversibility towards dopamine (DA), methylene blue (MB) and K3[Fe(CN)6]. The SWNT&amp;RTIL PE could be used to detect the number of guanine bases and adenine bases contents in per mol oligonucleotides according to the current response in the range of 0.05–2.0 nM. Based on the current response of guanine bases, oligonucleotides could be detected sensitively in the B–R buffer solution with a detection limit of 9.9 pM. The heterogeneous electron transfer rate constant (ks) of guanine bases contents in the oligonucleotides was investigated and its value was 0.90 s−1. In essence the SWNT&amp;RTIL PE showed high sensitivity, reliability, stability and reproducibility for the detection of DNA.