DNA Hybridization Efficiency Research Articles

A natural biomaterial promotes hybridization chain reaction for ultra-sensitive detection of miRNA-155

MicroRNA (miRNA) is an important biomarker for early diagnosis of cancers. However, sensitive and convenient methods for miRNA detection remain a challenge. Here, we use a natural biopolymer sporopollenin purified from Ganoderma lucidum spores as a substrate for isothermal amplification (hybridization chain reaction, HCR). Sporopollenin capsules (SP) promotes HCR and forms longer and more abundant double-stranded DNA (dsDNA) than graphene oxide (GO) and carbon nanotubes (CNTs). The nanoporous structure of sporopollenin capsules containing abundant water provides a hydrous environment and enhances the hybridization efficiency of DNA significantly. We construct an ultrasensitive fluorescent biosensor to detect miR-155. The efficient HCR amplification on SP leads to an ultralow detection limit of 1 aM for miR-155 and a wide linear range of 1 aM–10 fM (R2 = 0.99). Furthermore, our fluorescence biosensor can discriminate miRNA mutants with high selectivity. This biosensor is also highly sensitive in human serum (detection limit 10 aM). It adsorbs less serum protein than GO and CNTs, thus minimizing the interference caused by the non-specific adsorption. Our study would promote medical application of SP-based biosensor in the future.

Fluorescence Imaging Study of Film Coating Structure and Composition Effects on DNA Hybridization

Hybridization of surface‐bound DNA with complementary strands is the basis of many biotechnological applications. Herein, the structure of interfacial coatings between substrate and bound DNA is a crucial element for hybridization behavior. Herein, three reactive surfaces for constructing DNA‐sensing platforms, namely, plain gold films on silicon, poly(bisphenolA‐co‐epichlorohydrin) (PBAG) surfaces with a brush‐like bilayer structure, and dibenzocyclooctyne monolayers (both on glass), are compared. Fluorescence imaging is employed to survey the effect of coating structure and conformation on hybridization performance. To better understand the interfacial structural properties and chemistry of the coated films, atomic force microscopy, water contact angle measurements, and X‐ray photoelectron spectroscopy are employed to characterize the surface morphology. DNA probe microarrays are created on the different platforms via microchannel cantilever spotting, and their performance for hybridizing with the DNA counterparts is assessed. While all three platforms work reliable for DNA detection, a protein‐binding assay reveals that PBAG surfaces offer the highest hybridization efficiency among these approaches. The results of the present work have significant implications for comprehension of the interactions between the DNA hybridization efficiency and the physico‐chemical properties of surface coatings and can inform the fabrication of DNA sensors.

Gold Nanoparticle-Bridge Array to Improve DNA Hybridization Efficiency of SERS Sensors.

The interfacial mass transfer rate of a target has a significant impact on the sensing performance. The surface reaction forms a concentration gradient perpendicular to the surface, wherein a slow mass transfer process decreases the interfacial reaction rate. In this work, we self-assembled gold nanoparticles (AuNPs) in the gap of a SiO2 opal array to form a AuNP-bridge array. The diffusion paths of vertical permeability and a microvortex effect provided by the AuNP-bridge array synergistically improved the target mass transfer efficiency. As a proof of concept, we used DNA hybridization efficiency as a research model, and the surface-enhanced Raman spectroscopy (SERS) signal acted as a readout index. The experimental verification and theoretical simulation show that the AuNP-bridge array exhibited rapid mass transfer and high sensitivity. The DNA hybridization efficiency of the AuNP-bridge array was 15-fold higher than that of the AuNP-planar array. We believe that AuNP-bridge arrays can be potentially applied for screening drug candidates, genetic variations, and disease biomarkers.

Simultaneous detection of dual biomarkers using hierarchical MoS2 nanostructuring and nano-signal amplification-based electrochemical aptasensor toward accurate diagnosis of prostate cancer

Accurate and reliable quantification of tumor biomarkers in clinical samples is of vital importance for early stage diagnosis and treatment of cancer. However, a poor specificity of prostate specific antigen (PSA) testing alone fostering overdetection and overtreatment, remains a great controversy in prostate cancer (PCa) screening. Here we report an electrochemical aptasensor using hierarchical MoS2 nanostructuring and SiO2 nano-signal amplification for simultaneous detection of dual PCa biomarkers, PSA and sarcosine, to enhance the diagnostic performance of PCa. In this strategy, hierarchical flower-like MoS2 nanostructures as functional interface accelerated intermolecular accessibility and improved DNA hybridization efficiency. Moreover, the spherical SiO2 nanoprobe that conjugated with both electroactive tags and DNA probes, allowed effective electrochemical signal amplification. By deliberately designing different hybridization modes, we individually implemented the optimization of PSA and sarcosine sensing system. Based on this, simultaneous determination of PSA and sarcosine was achieved, with limit of detection (LOD) down to 2.5 fg/mL and 14.4 fg/mL, respectively, as well as excellent selectivity. More importantly, using this approach, we could directly differentiate cancer patients with healthy ones for clinical serum samples. The ultrasensitive biosensor provides single-step analysis with simple operation and a small sample volume (∼12 μL), shedding new light on accurate diagnosis and early-detection of cancer in clinical applications.

Tetrahedral DNA nanostructure based biosensor for high-performance detection of circulating tumor DNA using all-carbon nanotube transistor

Adopting carbon nanotube (CNT) transistors as biosensors has been developed as a promising method for cancer biomarker detection, which has shown superior sensitivity and selectivity. However, the detection of circulating tumor DNA (ctDNA) by the CNT transistor based biosensors is still a challenge and no work has been reported. Here, direct label-free DNA detection of AKT2 gene related to triple-negative breast cancer by all-CNT thin-film transistor (TFT) biosensors incorporated with tetrahedral DNA nanostructures (TDNs) is proposed and achieved for the first time. The adoption of TDNs enables improved biosensor response for at least 35% and even as high as 98% as compared with single-stranded DNA (ssDNA) probes owing to the enhanced DNA hybridization efficiency. Influence of the TDNs’ linker length on the biosensor performance is important and has been investigated. Concentration-dependent DNA detection is achieved by the all-CNT TFT biosensors with a broad linear detection range of six orders of magnitude and a theoretical limit of detection (LOD) of 2 fM. In addition, the all-CNT TFT biosensors exhibit favorable selectivity and repeatability. The platform of all-CNT TFT biosensors incorporated with TDNs has great potential for multiplexed detection of various cancer biomarkers, providing a simple yet high performance universal strategy for low-cost clinical applications.

Bioinspired nanozyme enabling glucometer readout for portable monitoring of pesticide under resource-scarce environments

Pesticide residual has always been a severe environmental issue due to its adverse effects on ecosystems. The development of user-friendly program is imperative for efficient monitoring and control of pesticides with respect to environmental protection. To this end, we herein report a bioinspired nanozyme enabling portable glucometer readout for assessing pesticide residual, based on an aptamer-switching approach. Notably, Co-based metal–organic framework (Co-MOF) decorated by aptamer is built, as a signal probe, for identifying acetamiprid (as a model analyte). Comparing with the enzyme-based methods, which works in pH 6 ∼ 7, our system has high stability and DNA hybridization efficiency beyond pH 8. By introducing completive binding between acetamiprid and MOF-aptamer into glucose consumption, this method exhibits a wide linear range (1 ∼ 200 nM) with a low limit of detection (0.42 nM, corresponding to 0.093 μg kg−1 by weight), well qualified for the regular monitoring of acetamiprid residue specified by World Health Organization. With virtues of portability, sensitivity and anti-interference, the glucometer-based protocol enables us to innovatively carry out pesticide monitoring in agricultural soil, water source and foodstuffs, thereby holding promise for environmental monitoring in home-style and in-field scenarios.

Fast and multiplexed superresolution imaging with DNA-PAINT-ERS

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

DNA interaction analysis with automated biosensor of paraben derivative s-triazines

Among all heterocyclic compounds, the triazine scaffold has an important place due to its wide range of biological activities. Triazine has been preferred by many researchers as the target structure for the design and development of new drugs, as it is found in many biologically active molecules with promising biological potential such as anti-inflammatory, anti-mycobacterial, anti-viral, anti-cancer properties. In this study, a series of paraben substituted s-triazine compounds 1-5 were synthesized and their structures were determined by 1H and 13C NMR spectroscopies as well as mass spectrometry. Also, structure of compound 5 was adjusted by X-ray crystallographic technique and crystallographic analysis revealed that 5 was crystallized in the trigonal space group R-3. According to X-ray analysis, compound 5 has very important intermolecular interaction such as nonclassical H-bond and pi-pi interaction. These interactions lead formation of supramolecular network. The thermal stabilities were investigated by thermogravimetric analysis. According to thermogravimetric analysis results, all of the synthesized compounds have high thermal stability. DNA interaction studies of the compounds 1-5 were performed with an automatic biosensor device that can measure the efficiency of DNA hybridization on the biochip surface. The biosensor results show that benzyl paraben substituted s-triazine compound 5, the most reactive compound at low concentration, was showed 61% and 75% activity reduction at concentrations 12.5 and 25 µM.

Electrophoresis and Biosensor-Based DNA Interaction Analysis of the First Paraben Derivatives of Spermine-Bridged Cyclotriphosphazenes

Cancer is the uncontrolled growth of abnormal cells via malignant cell division and rapid DNA replication. While DNA damaging molecules can cause cancer, their role as anticancer drugs are very significant. For this purpose, the novel series of paraben substituted spermine bridged(dispirobino) cyclotriphosphazene compounds 2-6 were synthesized for the first time, and their structures were characterized by various spectroscopic techniques. The solid-state structures and geometries of compounds 2-6 were determined using single-crystal X-ray structural analysis. In addition, it was confirmed by TGA that all compounds 1-6 showed high thermal stability. Two methods were used in order to investigate DNA interaction properties of the targeted molecules. While biosensor-based screening test that measures DNA hybridization efficiency on a biochip surface, the agarose gel electrophoresis method examines the effect of compounds on plasmid DNA structure. The results collected from the automated biosensor device and agarose gel electrophoresis have indicated that compounds 1, 5, and 6 showed higher DNA damage than the compounds 2-4. According to the biosensor results, compounds 1, 5, and 6 showed 85%, 69%, and 77% activity, respectively.

Hierarchical Nanostructuring Array Enhances Mid-Hybridization for Accurate Herbal Identification via ITS2 DNA Barcode.

It remains a technical challenge to accurately identify close species of herbal medicines, especially from adulterants, because of their highly identical phenotypes and chemical compositions. Here, we report a direct, sequencing-free, high-curvature nanostructuring-based electrochemical herb sensor (nanoE-herb sensor) to identify herbal species quickly and accurately using ITS2 barcodes. We engineer a nano-roughened carbon-supported gold nanostructuring array by photolithograph-free, one-step electrodeposition. The 3D fractal nanostructures exhibit a high deflection angle that largely enhances DNA hybridization efficiency, particularly for the midcomplementary hybridization, as compared to the 2D planar surface. More importantly, such a trans-scale array biointerface (including macroscale carbon and nanoscale gold branches) can overcome the detection barrier of slow diffusion of a long genomic sequence and inaccessibility of the sequestered variations in ITS2 secondary structures through the out-protruded 3D functional nanostructures. Our nanoE-herb sensor achieves a detection limit of 0.18 fM for the 64-mer fragment of saffron ITS2 barcode with midhybridization and shows superior specificity against even single-base mismatch. The sensor also precisely differentiates saffron from six other adulterants by directly detecting unpurified asymmetric PCR amplicons (∼500 bp) with ITS2 sequences, suggesting its great potential in the field identification of herbal medicinal species and pathogenic bacteria with specific DNA barcodes.

Thermally Carbonized Porous Silicon for Robust Label-Free DNA Optical Sensing.

In this work, thermal carbonization is shown to provide the necessary surface passivation to enable highly robust DNA detection on a porous silicon (PSi) platform, overcoming previous corrosion challenges with detection of negatively charged biomolecules. The stability of thermally carbonized PSi (TCPSi), oxidized PSi (OPSi), and undecylenic acid-modified PSi (UAPSi) is compared in phosphate-buffered saline and during DNA sensing experiments. Reflectance measurements reveal an improvement in stability and DNA sensor response for TCPSi compared to OPSi and UAPSi. TCPSi exhibits a large positive sensor response with >90% DNA hybridization efficiency. In comparison, UAPSi shows a smaller positive DNA sensor response, likely lessened by a small corrosion effect, while OPSi exhibits a large negative sensor response, indicating significant induced PSi corrosion that confounds the ability of OPSi to yield meaningful readouts of DNA hybridization events. This work expands the application of TCPSi for its more widespread usage in sensing applications where competing substrate corrosion may influence device stability.

The Impact of the Position of the Redox Label on Charge Transfer and Hybridization Efficiency at DNA Interfaces

AbstractA common electrode construct for electrochemical DNA biosensors consists of tethering a redox‐label‐modified single stranded DNA to the electrode and comparing the electrochemical response of a redox probe before and after hybridization. Despite the analytical advantages of this construct, contradictory results are found in the literature regarding to the effect of redox‐labeling position on the charge transfer process happening at a DNA interface. Herein a contribution to this discussion is made by investigating how a methylene blue redox label placed at different positions along a probe strand of DNA affects the DNA hybridization efficiency. For that, a second redox label, a ferrocene molecule, was attached to the target DNA at a fixed position such that upon hybridization it is positioned at the distal end of the DNA monolayer. The dual label strategy allowed us to explore, for each methylene blue labeling position, how the binding‐induced diminution in the methylene blue signaling is associated with the appearance of the ferrocene signaling after hybridization. It was found that the location of the methylene blue redox label in the DNA interface can affect the hybridization efficiency.

DNA interaction analysis of fluorenylidene double bridged cyclotriphosphazene derivatives

Abstract DNA is a target molecule for anticancer drug development, and investigation on DNA interactions with synthesized potential drugs has great importance in order to discover new drug candidates, hence in this study an electrochemical biosensor has been used to perform quantitative analysis of DNA/compound interactions. In this study, full paraben substituted fluorenylidene double bridged cyclotriphosphazene compounds (9–13) were synthesized for the first time and their effect on DNA were investigated using an automated biosensor device MiSens®. The reaction of fluorenylidene double bridged cyclotriphospazene (FDBC) compound (3) with parabens: methyl 4-hydroxybenzoate, (4); ethyl 4-hydroxybenzoate, (5); propyl 4-hydroxybenzoate, (6); butyl 4-hydroxybenzoate (7), benzyl paraben (8) were performed, respectively. Compounds 9–13 were obtained and fully characterizated by mass spectrometry, 31P NMR and 1H spectroscopy. Biosensor based screening tests were used to investigate DNA interaction properties of the newly synthesised compounds by measuring DNA hybridization efficiency on the biochip surface. The effect of the compounds to the plasmid DNA structure were analyzed by agarose gel electrophoresis, in addition the antimicrobial activities of the compounds have been investigated. The biosensor assay and the agarose gel electrophoresis investigation indicated DNA/compound interaction for the compounds 12 and 13 none of the compounds has shown antimicrobial activity.

Biomolecular Steric Hindrance Effects Are Enhanced on Nanostructured Microelectrodes.

The availability of rapid approaches for quantitative detection of biomarkers would drastically impact global health by enabling decentralized disease diagnosis anywhere that patient care is administered. A promising new approach, the electrochemical steric hindrance hybridization assay (eSHHA) has been introduced for quantitative detection of large proteins (e.g., antibodies) with a low nanomolar detection limit within 10 min. Here, we report the use of a nanostructured microelectrode (NME) platform for eSHHA that improves the performance of this approach by increasing the efficiency and kinetics of DNA hybridization. We demonstrated that eSHHA on nanostructured microelectrodes leverages three effects: (1) steric hindrance effects at the nanoscale, (2) a size-dependent hybridization rate of DNA complexes, and (3) electrode morphology-dependent blocking effects. As a proof of concept, we showed that the sensitivity of eSHHA toward a model antibody is enhanced using NMEs as scaffolds for this reaction. We improved the detection limit of eSHHA, taking advantage of nanostructured surfaces to allow the use of longer capture strands for detection of proteins. Finally, we concluded that using the eSHHA approach in conjunction with nanostructured microelectrodes is an advantageous alternative to conventional macroelectrodes as the sensitivity and detection limits are enhanced.

Fluorescence coupled in-capillary DNA hybridization assay and its application to identification of DNA point mutation

We have developed a fluorescence coupled in-capillary DNA hybridization assay and applied it to the detection of DNA base pair mutation(s). A 15-mer thrombin-binding single-stranded DNA oligonucleotide labeled with 6-carboxyfluorescein (DNA1-FAM) was mixed and hybridized with its complementary DNA (cDNA1) via the in-capillary approach. A fluorescence coupled capillary electrophoresis (CE-FL) was used to measure the status and reaction rate of DNA double strands hybridization under different reaction conditions, and the results were recorded on the electrophoretograms. Recorded electrophoretograms have shown that the in-capillary assay has different DNA hybridization patterns compared to the post-capillary assay. The DNA hybridization efficiency of the in-capillary assay was affected by the molar ratio, the time interval of injection and the volume of DNA oligos. Interestingly, this assay could be used to detect one or two base pair mutation(s) in the target DNA. The in-capillary assay, characterized by an easy operation, reproducible results, and a low instrumental requirement, can be coupled to an online base pair mutation-based detection system, which would facilitate the related small-scale research analysis at non-major research centers.

Signal enhancement in ultraflat electrochemical DNA biosensors.

The ability of holding back the undesired molecules, but at the same time to provide the right distribution and orientation of the bioreceptors, are critical targets to reach an efficient hybridization and enhanced detection in electrochemical DNA biosensors. The main actors responsible of these key functions are the substrate of the sensor and the interface auto-assembled on it. In this paper we present the annealing as a method to improve commercial gold evaporated substrates for biosensor applications. The restructuring of granulated gold surface by means of annealing heating treatment leads to the formation of ultraflat gold lamellar terraces. The formation of terraces was characterized with scanning tunneling microscopy and optical interferometry. The performance of the sensor sensitivity on granular substrates and ultraflat substrates was studied, concerning the orientation and surface coverage of the bioreceptor interface applied in electrochemical biosensor. The hybridization efficiency of ferrocene-labeled DNA amplified by PCR was characterized with surface plasmon resonance and electrochemistry. The experimental results demonstrate that annealing process, positive influence on optical and voltammetric readings, due to a structured organization of the bioreceptors on the flat substrate, gaining more efficient immobilization and DNA hybridization. The results suggest the annealing as a powerful tool for improving gold substrates in biosensors applications.

Effect of nanoporous gold thin film morphology on electrochemical DNA sensing.

Advances in materials science and chemistry have led to the development of a wide range of nanostructured materials for building novel electrochemical biosensors. A systematic understanding of the challenges related to electrode morphology involved in designing such sensors is essential for developing effective biosensing tools. In this study, we use nanoporous gold (np-Au) thin film electrode coatings with submicrometer thicknesses, as a model system to investigate the influence of nanostructuring on DNA-methylene blue (MB) interactions and their application to DNA biosensors. The interaction of single- and double-stranded DNA immobilized onto morphologically different np-Au films with MB was electrochemically interrogated via square wave voltammetry (SWV). The electrochemical signal from these electrodes in response to MB decayed progressively with each SWV scan. The decay rate was governed by accessibility of the electrochemically active np-Au surface by the analyte. The optimum frequency for extracting the maximum signal via SWV was influenced by the film morphology, where the optimum frequency was lower for the nanoporous morphology with lower density of molecular access points into the porous coating. Overall, the np-Au electrodes exhibited a 10-fold enhancement in probe grafting density and approximately 10-fold higher electrochemical current upon probe-target hybridization as compared to the planar Au electrodes. The np-Au electrodes enabled sensitive detection with a dynamic range of 10 to 100 nM that shifts by 1 order of magnitude for coarsened np-Au morphology due to increased target penetration into the porous network and hence enhanced hybridization efficiency. These findings provide insight into the influence of nanostructuring on the transport mechanisms of small molecules and nucleic acids, and yield an understanding of diverse sensor performance parameters such as DNA grafting density, hybridization efficiency, sensitivity and dynamic range.

An ultrasensitive label-free electrochemical impedimetric DNA biosensing chip integrated with a DC-biased AC electroosmotic vortex

A label-free electrochemical impedance spectroscopy (EIS)-based DNA biosensing chip is integrated with DC-biased AC electroosmotic (ACEO) stirring using concentric double ring-single disk electrodes to greatly promote the hybridization efficiency of 20-base target DNA (tDNA) fragments. The +0.7V-biased ACEO flow of 3Vpp and 380Hz applied at the outer ring electrode (ORE) and inner ring electrode (IRE) can drive the tDNA-containing 1mM Tris solution from the ORE to the probe's DNA-modified disk electrode (DE) to achieve 90% saturation hybridization within 141s. The DNA biosensor used Pd-deposited ORE as the pseudo-reference electrode and IRE as the counter electrode for on-chip-type three-electrode EIS measurement, and presented good linearity and repeatability in the range of 1aM–10pM and exhibited an ultrasensitive detection limit of 0.5aM. The label-free EIS-based DNA sensing chips with integrated DC-biased ACEO vortex can achieve rapid hybridization, high selectivity and ultrasensitive detection for different tDNA samples.

Quantitative measurements of thermodynamics and kinetics of polythiophene-DNA complex formation in DNA detection.

Cationic polythiophene derivatives have been used as unique optical probes for various biosensing applications with great success, because their optical responses are sensitive to the conformational changes from binding to single-stranded DNA (ssDNA) to binding to double-stranded DNA (dsDNA). It has long been suggested that the binding of cationic polymers to DNA has a major impact on the thermodynamics and kinetics of DNA hybridization; yet a quantitative assessment is lacking. We report here a systematic study to quantitatively measure the thermodynamic binding constants and hybridization rates of DNA duplexes in the presence/absence of cationic polythiophene. Our results show that the dissociation constant of dsDNA in the presence of polythiophene is three orders of magnitude smaller than that in the absence of polymers. The formation of the DNA/polymer triplex is also much slower than double helical DNA formation, suggesting a rate-limiting polymer-ssDNA dissociation step prior to DNA hybridization. The hybridization rate constant is further slowed down when polymer/DNA hybridization occurs on a solid surface due to steric hindrance. Having a means to quantitatively assess the DNA hybridization efficiency in the presence of cationic polymers, we were able to improve the DNA sensing performance through a combined tuning of reaction temperature and time.

Heat transfer resistance as a tool to quantify hybridization efficiency of DNA on a nanocrystalline diamond surface

In this article, we report on a label-free real-time method based on heat transfer resistivity for thermal monitoring of DNA denaturation and its potential to quantify DNA fragments with a specific sequence of interest. Probe DNA, consisting of a 36-mer fragment was covalently immobilized on a nanocrystalline diamond surface, created by chemical vapor deposition on a silicon substrate. Various concentrations of full matched 29-mer target DNA fragments were hybridized with this probe DNA. We observed that the change in heat transfer resistance upon denaturation depends on the concentration of target DNA used during the hybridization, which allowed us to determine the dose–response curve. Therefore, these results illustrate the potential of this technique to quantify the concentration of a specific DNA fragment and to quantify the hybridization efficiency to its probe.