Hybridization Of DNA Strands Research Articles

Materialization of a Novel Decorated Nanowire Biosensor Platform Based on Field Effect Transistor under Electrochemical Gate Modulation

The highly sensitive and cost-free field effect transistor (FET) biosensor for specific detection of DNA and streptavidin based on Zinc Oxide Nanowires (NWs) to develop the next generation biosensors in label-free approach under electrochemical gate modulation has been materialized. The electronic properties of NWs were changed by binding of charged analytes to NWs surface under electrochemical gate modulation. Due to highly chemically inertness, immobilization of the target analyte receptors on the NWs could be conducted through electrochemical treatment. For materialization of DNA binding, gold nanoparticles (Au NPs) were used to functionalize ZnO NWs surface. In this work, a liquid gated FET based on NWs was configured to detect the complementary DNA strand hybridization as well as the protein streptavidin at very low concentration. The biosensor could reach to sensitivity of attomolar. The sensitivity of response was check and biosensor showed the sensitivity ranges from 10−18 to 10−12 mol.L−1. The FET biosensor could distinguish a complementary strand from a single nucleotide polymorphism (SNP) containing strand. The limit of detection (LOD) reached to15 aM. The NPs decoration on the NWs surface brought about the high detection limit. The NPs decoration was being a promising method and scientific approach for biomolecules detection.

Stabilization of Functional DNA Structures with Mild Photochemical Methods.

A significant barrier to biological applications of DNA structures is their instability to nucleases. UV-mediated thymine dimerization can crosslink and stabilize DNA nanostructures, but its effect on DNA strand hybridization fidelity and function is unclear. In this work, we first compare a number of methods for DNA irradiation with different wavelengths of light and different photosensitizers. We demonstrate that all approaches can achieve nuclease protection; however, the levels of DNA off-target crosslinking and damage vary. We then describe mild irradiation conditions intended to safeguard DNA against nuclease degradation. We demonstrate up to 25× increase in serum stability while minimizing off-target damage and maintaining functions such as hybridization efficiency, gene silencing, aptamer binding, and DNA nanostructure formation. Our methodology requires no complex instruments beyond a UV light source and no synthetic modification of the DNA itself, allowing for applications in numerous areas of nucleic acid therapy and nanotechnology.

Flow Control Techniques for Enhancing the Bio-Recognition Performance of Microfluidic-Integrated Biosensors

Biosensors are favored devices for the fast and cost-effective detection of biological species without the need for laboratories. Microfluidic integration with biosensors has advanced their capabilities in selectivity, sensitivity, controllability, and conducting multiple binding assays simultaneously. Despite all the improvements, their design and fabrication are still challenging and time-consuming. The current study aims to enhance microfluidic-integrated biosensors’ performance. Three different functional designs are presented with both active (with the help of electroosmotic flow) and passive (geometry optimization) methods. For validation and further studies, these solutions are applied to an experimental setup for DNA hybridization. The numerical results for the original case have been validated with the experimental data from previous literature. Convection, diffusion, migration, and hybridization of DNA strands during the hybridization process have been simulated with finite element method (FEM) in 3D. Based on the results, increasing the velocity on top of the functionalized surface, by reducing the thickness of the microchamber in that area, would increase the speed of surface coverage by up to 62%. An active flow control with the help of electric field would increase this speed by 32%. In addition, other essential parameters in the fabrication of the microchamber, such as changes in pressure and bulk concentration, have been studied. The suggested designs are simple, applicable and cost-effective, and would not add extra challenges to the fabrication process. Overall, the effect of the geometry of the microchamber on the time and effectiveness of biosensors is inevitable. More studies on the geometry optimization of the microchamber and position of the electrodes using machine learning methods would be beneficial in future works.

DNA Ring-Opening Polymerization Driven by Base Stacking.

Supramolecular polymers, relying on reversible intermolecular interactions, promise a wide range of applications, including optoelectronic materials, self-healing materials, and biomedical delivery materials. Among potential molecular candidates, DNA strands act as an excellent platform. DNA has a well-established secondary structure (double helix), and its intermolecular interactions can be readily thermodynamically engineered and kinetically controlled. Extensive studies have demonstrated that various DNA motifs can polymerize/assemble into large polymers with different topology, geometry, and dimensionalities. Most of the reported polymerization is driven by hybridization of DNA strands. Herein, we report a novel system of DNA supramolecular polymerization that is driven by DNA base stacking. The polymerization has been confirmed by native polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM). We believe that this work will expand the toolbox for DNA supramolecular polymerization and would, with further development, increase further control of DNA supramolecular polymerization.

C-Ag+−C based repetitive DNA sequence

DNA is a convenient and well-studied tool for nanostructures fabrication. Metal-mediated hybridization of DNA strands opens up new possibilities for nanobiotechnology. In this work, we studied the possibility of long DNA formation from short ones by gluing them through the formation of C-Ag+−C complexes. Such long formations were investigated using static light scattering and atomic force microscopy. It was found that the duplexes can efficiently be linked in the presence of silver ions if the length of the cytosine sequence exceeds 6 nucleobases.

An intermolecular-split G-quadruplex DNAzyme sensor for dengue virus detection.

Nucleic acids have special ability to organize themselves into various non-canonical structures, including a four-stranded DNA structure termed G-quadruplex (G4) that has been utilized for diagnostic and therapeutic applications. Herein, we report the ability of G4 to distinguish dengue virus (DENV) based on its serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) using a split G4-hemin DNAzyme configuration. In this system, two separate G-rich oligonucleotides are brought together upon target DNA strand hybridization to form a three-way junction architecture, allowing the formation of a G4 structure. The G4 formation in complexation with hemin can thus provide a signal readout by generating a DNAzyme that is able to catalyze H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). This results in a change of color providing a sensing platform for the colorimetric detection of DENV. In our approach, betaine and dimethyl sulfoxide were utilized for better G4 generation by enhancing the target-probe hybridization. In addition to this serotype-specific assay, a multi-probe cocktail assay, which is an all-in-one assay was also examined for DENV detection. The system highlights the potential of split G-quadruplex configurations for the development of DNA-based detection and serotyping systems in the future.

Ultrasensitive detection of microRNA-21 based on plasmon-coupling-induced electrochemiluminescence enhancement

Noble metal nanoparticles possess excellent optical and electrical properties, and have been widely used in the construction of biosensors and in surface-enhanced spectroscopy. Herein, we describe an enzyme-assisted ultra-sensitive biosensor with gold nanoparticle (GNP) dimers, based on a localized surface plasmon resonance (LSPR)-enhanced electrochemiluminescence (ECL) mechanism. CdS nanocrystals (NCs) acted as ECL emitters, and the GNP dimers were constructed by hybridization of DNA strands on the surface of an electrode modified with CdS NCs after an enzyme-assisted cyclic amplification process. The plasmon-coupling-induced electromagnetic field enhancement effects of the GNP dimers resulted in an increase in ECL intensity of as much as 6.3-fold under optimized conditions. Based on this LSPR-ECL sensor platform, the detection limit for microRNA-21 is as low as 3.6 fM with a wide linear range from 10 fM to 20 pM with good sensitivity and selectivity.

Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.

Multiplexed chemiluminescence imaging assay of protein biomarkers using DNA microarray with proximity binding-induced hybridization chain reaction amplification

A multiplexed chemiluminescence (CL) imaging assay was designed for sensitive screen detection of a panel of protein biomarkers by integrating DNA microarray with proximity binding-induced hybridization chain reaction (HCR) amplification. The DNA microarray was manufactured by printing hairpin DNAs for different analytes detection in an array format onto the sensing cells arranged on an aldehyde-functionalized glass chip. The existence of target proteins induced the formation of sandwich immunocomplexes along with the hybridization of DNA strands labeled on the pair of antibodies via proximity effect, which subsequently unfolded their corresponding spotting hairpin DNAs to expose prelocked primer and trigger HCR assembly. Through the streptavidin-biotin conjugation, numerous horseradish peroxidase (HRP) was coupled on the HCR assemblies to catalyze H2O2-luminol reaction and produce amplified CL signals for sensitive CL imaging assay of protein targets. As a proof of concept, the DNA microarray was used to CL imaging assay of carcinoembryonic antigen, α-fetoprotein and carcinoma antigen 125 in ranges of 0.05–500 ng mL−1, 0.06–600 ng mL−1 and 0.02–200 U mL−1, respectively. The CL microarray imaging assay could also be extended to screen detect other biomarker groups by printing different hairpin DNAs on the microarray and using corresponding antibody-DNA pairs. The proposed CL microarray imaging assay showed advantages of small sample consumption, good cost effectiveness, and multiplex detection performance, exhibiting good applicability in clinical diagnosis.

Facile Assembly/Disassembly of DNA Nanostructures Anchored on Cell-Mimicking Giant Vesicles.

DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems.

A label-free genetic biosensor for diabetes based on AuNPs decorated ITO with electrochemiluminescent signaling

The variation of I27L gene closely relates to increasing risk of type 2 diabetes, thus it is greatly significant to develop various methods for its identification or monitoring. We report here a novel label-free electrochemiluminescent (ECL) biosensor for simple and effective determination of I27L gene based on Au nanoparticles functionalized ITO electrode. The ECL technique is employed to monitor the hybridization of DNA strands by measuring the changes of its intensity. Here, the ECL signal was quenched by blocked access of probe (luminol anion) owing to the electrostatic repulsion of negatively charged sensor surface and the space resistance. The quantification of target strand can be directly realized by calibrating the quenched ECL signal toward its logarithm concentration in good linearity within the range from 1.0 × 10−11 to 1.0 × 10−7 M and a detection limit of 8.1 × 10−12 M. In addition, the biosensor exhibited good stability, excellent reproducibility and outstanding selectivity against one-base mismatched strand. What's more, the simple, low-cost, sensitive device could be easily miniaturized, makes it as an attractive candidate for integrating into portable platforms for point-of-care molecular diagnostics.

Mathematical model of polymerase chain reaction with temperature-dependent parameters.

The course of the real-time polymerase chain reaction (PCR) is determined by the temperature dependence of the kinetics of the component reactions, particularly the DNA strand hybridization. To investigate the effect of thermal processes on the reaction behavior, a mathematical model in which the variable rate constant of dissociation of "primer-single strand" complexes depends on temperature was proposed. The reaction medium temperature, which depends on time, was also introduced into the model. The proposed model of real-time PCR makes it possible to analyze different aspects of the reaction, which are important for the development of instruments and reagents for PCR.

Nanophotonic Waveguide Enhanced Raman Spectroscopy of Biological Submonolayers

Characterizing a monolayer of biological molecules has been a major challenge. We demonstrate nanophotonic waveguide enhanced Raman spectroscopy (NWERS) of monolayers in the near-infrared region, enabling real-time measurements of the hybridization of DNA strands and the density of submonolayers of biotin–streptavidin complex immobilized on top of a photonics chip. NWERS is based on enhanced evanescent excitation and collection of spontaneous Raman scattering near nanophotonic waveguides, which for a 1 cm silicon nitride waveguide delivers a signal that is more than 4 orders of magnitude higher in comparison to a confocal Raman microscope. The reduced acquisition time and specificity of the signal allows for a quantitative and real-time characterization of surface species, hitherto not possible using Raman spectroscopy. NWERS provides a direct analytic tool for monolayer research and also opens a route to compact microscopeless lab-on-a-chip devices with integrated sources, spectrometers, and detectors fa...

Surface Enhanced Raman Spectroscopy Sensor Based on Magnetic Beads-induced Nanoparticles Aggregation for Detection of Bacterial Deoxyribonucleic Acid

Abstract A novel method was developed for the highly sensitive detection of bacterial DNA by surface-enhanced Raman spectroscopy (SERS) with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-modified gold nanoparticles as reporter probes and on the basis of the separation and enrichment effect of magnetic beads and the high affinity of fully complementary hybridization of DNA strands. Capture probe was immobilized onto the surface of Streptavidin-enwrapped magnetic beads (SA-MB) through the force between biotin and avidin, which hybridized with the target bacterial DNA sequence partly, and the remaining connected with the report probe decorated AuNPs with DTNB and SH-DNA (AuNPs@DTNB@DNA). Compared with previous methods, this design shortened the distance between particles by the aggregation effect of the magnetic beads to nanoparticles, and produced the plasma resonance coupling effect, which increased the SERS signal significantly. The results showed that, under the optimized conditions, the concentration of DNA in the range from 5 pM to 5 nM performed a good linear relationship with Raman intensity. The limit of detection (LOD) for bacterial DNA was estimated to be about 5 pM, showing a certain selectivity. The method was simple in design with low cost, also it was used in the sensitive and selective detection of bacterial DNA.

Cascading DNA Generation Reaction for Controlling DNA Nanomachines at a Physiological Temperature

We developed a reaction system to generate multiple single-stranded DNA species at a physiological temperature for controlling the operation of DNA nanomachines. In this reaction system, cascading DNA generation is arbitrarily programmed by permutation and altering the combinations of template DNA sequences in a modular fashion. Because the dissociation of generated DNA strands from their templates is fully dependent on the strand displacement activity of DNA polymerase, generation and subsequent hybridization of DNA strands can be implemented in a one-pot reaction at the reaction temperature. We experimentally confirmed the generation and hybridization of DNA strands at a temperature remarkably lower than the melting temperature by monitoring the fluorescence change caused by the structural transition of molecular beacons as a simple DNA nanomachine operation. Then, we demonstrated the versatility and programmability of the cascading DNA generation up to three layers. By integrating the proposed DNA generation reaction with various types of DNA nanomachines, an intelligent molecular robotic system is expected to be achieved.

In‐Channel‐Grown Polypyrrole Nanowire for the Detection of DNA Hybridization in an Electrochemical Microfluidic Biosensor

A triple electrode setup with a Pt pseudo‐reference electrode integrated in a polydimethylsiloxane‐ (PDMS‐) based microchamber was designed and fabricated. The integrated electrodes were deposited onto SiO2/Si substrate by sputtering. The PDMS microchamber was patterned using an SU‐8 mold and sealed with electrodes in oxygen plasma. Polypyrrole nanowires (PPy NWs) were electrochemically grown in situ at an accurate position of the working electrode in the sealed microchamber instead of in an open system. The DNA probe sequences were simply introduced into the channel to form bonds with the nanowires. A detection limit of 20 pM was achieved using a lock‐in amplifier. The electrochemical characteristics produced by the hybridization of DNA strands in the microchamber showed a good signal/noise ratio and high sensitivity. Measurement of the DNA sensor in narrow space also required much less volume of the analytical sample compared with that in an open measuring cell. Results showed that this simple system can potentially fabricate nanostructures and detect bio/chemical molecules in a sealed system.

An integrated Mach–Zehnder interferometric biosensor with a silicon oxynitride waveguide by plasma-enhanced chemical vapor deposition

We report the design and fabrication of an integrated Mach–Zehnder interferometric (MZI) biochip based on silicon oxynitride layers deposited with a plasma-enhanced chemical vapor deposition (PECVD) process. A rib waveguide for an integrated MZI sensor has been designed to have a high surface sensitivity and a single-mode behavior by using an effective index method. The integrated MZI chip operating at 637 nm is fabricated via conventional photolithography and reactive ion etching. As a biosensor application, the real-time and label-free detection of the covalent immobilization and hybridization of DNA strands is performed and verified with this device.

Thermodynamic Post-Processing versus GC-Content Pre-Processing for DNA Codes Satisfying the Hamming Distance and Reverse-Complement Constraints.

Stochastic, meta-heuristic and linear construction algorithms for the design of DNA strands satisfying Hamming distance and reverse-complement constraints often use a GC-content constraint to pre-process the DNA strands. Since GC-content is a poor predictor of DNA strand hybridization strength the strands can be filtered by post-processing using thermodynamic calculations. An alternative approach is considered here, where the algorithms are modified to remove consideration of GC-content and rely on post-processing alone to obtain large sets of DNA strands with satisfactory melting temperatures. The two approaches (pre-processing GC-content and post-processing melting temperatures) are compared and are shown to be complementary when large DNA sets are desired. In particular, the second approach can give significant improvements when linear constructions are used.

Cantilever deflection associated with hybridization of monomolecular DNA film

Recent experiments show that specific binding between a ligand and surface immobilized receptor, such as hybridization of single stranded DNA immobilized on a microcantilever surface, leads to cantilever deflection. The binding-induced deflection may be used as a method for detection of biomolecules, such as pathogens and biohazards. Mechanical deformation induced due to hybridization of surface-immobilized DNA strands is a commonly used system to demonstrate the efficacy of microcantilever sensors. To understand the mechanism underlying the cantilever deflections, a theoretical model that incorporates the influence of ligand/receptor complex surface distribution and empirical interchain potential is developed to predict the binding-induced deflections. The cantilever bending induced due to hybridization of DNA strands is predicted for different receptor immobilization densities, hybridization efficiencies, and spatial arrangements. Predicted deflections are compared with experimental reports to validate the modeling assumptions and identify the influence of various components on mechanical deformation. Comparison of numerical predictions and experimental results suggest that, at high immobilization densities, hybridization-induced mechanical deformation is determined, primarily by immobilization density and hybridization efficiency, whereas, at lower immobilization densities, spatial arrangement of hybridized chains need to be considered in determining the cantilever deflection.

Remote Toehold: A Mechanism for Flexible Control of DNA Hybridization Kinetics

Hybridization of DNA strands can be used to build molecular devices, and control of the kinetics of DNA hybridization is a crucial element in the design and construction of functional and autonomous devices. Toehold-mediated strand displacement has proved to be a powerful mechanism that allows programmable control of DNA hybridization. So far, attempts to control hybridization kinetics have mainly focused on the length and binding strength of toehold sequences. Here we show that insertion of a spacer between the toehold and displacement domains provides additional control: modulation of the nature and length of the spacer can be used to control strand-displacement rates over at least 3 orders of magnitude. We apply this mechanism to operate displacement reactions in potentially useful kinetic regimes: the kinetic proofreading and concentration-robust regimes.