Mismatched DNA Strands Research Articles

Part I: Non-faradaic electrochemical impedance-based DNA biosensor for detecting phytopathogen – Ralstonia solanacearum

Herein, we report for the first time the development of a label-free, non-faradaic, and highly sensitive DNA-based impedimetric sensor using micro-sized gold interdigitated electrodes (IDE) to detect a soil-borne agricultural pathogen Ralstonia solanacearum. A universal 30 oligomer single-stranded DNA (ssDNA) probe lpxC4 having specificity towards R. solanacearum is successfully immobilized on the surface of IDE along with mercaptohexanol. The electrochemical stability of the developed sensor surface is determined using open circuit potential measurements. The DNA probe immobilization protocol is validated using the changes configured on the surface of IDE by contact angle and ATR-FTIR analysis. The DNA target hybridization is detected using non-faradaic electrochemical impedance spectroscopy measurement with an ultra-low sample volume of 10 µL. The non-faradaic approach is verified by studying redox behavior using cyclic voltammetry. We investigate the hybridization of the surface-immobilized label-free probe with the complementary DNA targets obtained from infected eggplant saplings and cross-reactive studies with mismatched DNA strands. Our impedimetric sensor can detect target concentrations as low as 0.1 ng/µL. This standardization and detection of DNA hybridization serves as part I of the work and paves the way for further study in the detection of pathogenic field samples.

Toehold-mediated strand displacement reaction-propelled cascade DNAzyme amplifier for microRNA let-7a detection

DNAzyme amplifiers have been extensively explored as a useful sensing platform, but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity. Herein, a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction (TSDR) and Zn2+-assisted DNAzyme cycle with lower cost and simpler procedures. Compared with single DNAzyme amplifier, the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle. Base on this, let-7a could be sensitively detected in the range of 5–50 nmol/L with a detection limit of 64 pmol/L. Furthermore, the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands, which has been successfully applied to the determination of let-7a in blood serum, showing high promise in early cancer diagnosis.

Numerical modeling of MoS2–graphene bilayer-based high-performance surface plasmon resonance sensor: structure optimization for DNA hybridization

We present a MoS2–graphene bilayer-based high-performance refractive index surface plasmon resonance (SPR) sensor applying an angular interrogation technique. We used it for inspecting the reflected optical signal from the sensing medium. For enhancing the sensor angular sensitivity (S), signal-to-noise ratio (SNR), and quality factor (QF), we undertook some steps sequentially. First, the impact of gold layer thickness was investigated and optimized to 40 nm. Second, the MoS2 and graphene coating layers were optimized to four and three, respectively. Finally, the minimum reflectance and SPR angle were identified with this optimum structure. It is seen that the angular sensitivity of this optimum structure improved to an excellent value of 130 deg-RIU − 1 along with an improved angular SNR of 1.37 and QF of 17.02 RIU − 1. Then we compared the proposed sensor with the existing works in terms of angular sensitivity, SNR, and QF to prove its effective application. Finally, an analysis of DNA hybridization is presented as an application of the proposed structure. Our offered configuration is capable of recognizing absorption of DNAs by means of the attenuated total reflection method. The effective refractive index of the sensing medium alters because of the absorption of various concentrated DNAs. Computational investigation demonstrates that the change of SPR angle and minimum reflectance for mismatched DNA strands is inconsiderable, though it is significant for complementary DNA strands, which is crucial for the sensing of accurate DNA hybridization.

FDTD Analysis Fiber Optic SPR Biosensor for DNA Hybridization: A Numerical Demonstration with Graphene

This article illustrates a design and finite difference time domain (FDTD) method based on analysis of fiber optic surface plasmon resonance (SPR) biosensor for biomedical application especially for DNA-DNA hybridization. The fiber cladding at the middle portion is constructed with the proposed hybrid of gold (Au), graphene, and a sensing medium. This sensor can be recognized adsorption of DNA biomolecules onto sensing medium of PBS saline using attenuated total reflection (ATR) technique. The refractive index (RI) is varied owing to the adsorption of different concentration of biomolecules. Result states that the sensitivity with a monolayer of graphene will be improved up to 40% than bare graphene layer. Owing to increased adsorption capability of DNA molecules on graphene, sensitivity increases compared to the conventional gold thin film SPR biosensor. Numerical analysis shows that the variation of the SPR angle for mismatched DNA strands is quite negligible, whereas that for complementary DNA strands is considerable, which is essential for proper detection of DNA hybridization. Finally, the effect of Electric field distribution on inserting graphene layer is analyzed incorporating the FDTD technique by using Lumerical FDTD solution software.

Study of Graphene-MoS2 Based SPR Biosensor with Graphene Based SPR Biosensor: Comparative Approach

In this paper, we compare the sensitivity of graphene-MoS2 based surface plasmon resonance (SPR) biosensor to graphene based SPR biosensor. Here, graphene is used as biomolecular recognition element (BRE) because of its high adsorption ability and optical characteristics which helps to improve sensor sensitivity, on the other hand MoS2 is used for it has larger band gap, high fluroscence quenching ability, higher optical absorption efficiency which improves further sensor sensitivity. In DNA hybridization event, numerically achieved results show that single layer of graphene-MoS2 based SPR biosensor is 175% more sensitive than single layer of graphene coated SPR biosensor. Surface plasmon resonance angle and spectrum of reflected power are numerically investigated for different concentrated complementary DNA strands. The variations of SPR angle is significantly computable for complementary DNA strands whereas these parameters are varied negligibly for mismatched DNA strands. Thus the proposed sensor effectively differentiates hybridization and single nucleotide polymorphisms (SNP) by examining the level of changes in SPR angle and reflected power spectrum.

Numerical modeling of graphene-coated fiber optic surface plasmon resonance biosensor for BRCA1 and BRCA2 genetic breast cancer detection

A numerical illustration of a hybrid design and numerical analysis of graphene-coated fiber-optic surface plasmon resonance (SPR) biosensor for BRCA-1 and BRCA-2 genetic breast cancer detection is provided. Two specific mutations named 916delTT in BRCA-1 gene and 6174delT in BRCA-2 gene are being selected for detection of breast cancer numerically. This sensor is based on attenuated total reflection (ATR) method to detect individual point mutations in BRCA-1 and BRCA-2 genes. Based on the numerically obtained results, a momentous change is present in the SPR angle (minimum 35% more) and surface resonance frequency (SRF) (minimum 36% more) for probe DNA with various concentrations of target DNA corresponding to the mutation of the BRCA-1 and BRCA-2 genes. The variation of the SPR angle and SRF for mismatched DNA strands in BRCA-1 and BRCA-2 genes is quite negligible, whereas that for complementary DNA strands is considerable. This considerable change is essential for proper detection of genetic biomarkers (916delTT and 6174delT) for early breast cancer. To the best of our knowledge, this is the first demonstration of such an adept biosensor for detecting BRCA1 and BRCA2 genetic breast cancer. Here, we used graphene as bimolecular acknowledgement element for improving sensor performance. At the end of the article, the performance in terms of sensitivity is analyzed. Therefore, the proposed biosensor opens a window toward detection of early detection of BRCA-1 and BRCA-2 genetic breast cancers.

Design and numerical analysis of a graphene-coated fiber-optic SPR biosensor using tungsten disulfide

This article provides a simple hybrid design and rigorous numerical analysis of a fiber optic–based surface plasmon resonance (SPR) biosensor for DNA hybridization. The sensor core and both sides of the cladding are constructed with optical fiber, whereas the middle portion of the cladding is filled with the proposed hybrid of silver, tungsten disulfide (WS2), graphene, and a sensing medium. To the best of our knowledge, this is the first demonstration of such a highly sensitive SPR biosensor using WS2 for sensing DNA hybridization. The sensitivity, detection accuracy, and figure of merit are considered as the performance parameters and are analyzed in detail. The analyses reveal an impressive enhancement of the overall performance of the proposed sensor. Insertion of a WS2 layer between silver and graphene provides a sensor with sensitivity higher than that of other sensors reported to date. Additionally, concurrent improvement of all performance parameters is shown by this hybrid sensor, which is not the case for sensors based only on graphene. An increased number of graphene-only layers increases the sensitivity and decreases the detection accuracy and figure of merit. Numerical analysis shows that the variation of the SPR angle for mismatched DNA strands is quite negligible, whereas that for complementary DNA strands is considerable, which is essential for proper detection of DNA hybridization. Therefore, the proposed biosensor opens a new window toward detection of biomolecular interactions.

Homogeneous enzyme-free and entropy-driven isothermal fluorescent assay for nucleic acids based on a dual-signal output amplification strategy.

Herein, we developed a homogeneous enzyme-free, ultrasensitive and isothermal fluorescent biosensor for nucleic acid detection with dual-signal output to enhance the reaction rate and sensitivity by combining an entropy-driven catalysis strategy with a catalytic hairpin assembly reaction.

Ultrasensitive chemiluminescence biosensors using nucleic acid-functionalized silver-cysteine nanowires as signal amplifying labels.

Ultrasensitive chemiluminescence (CL) sensors for biomolecules (DNA and proteins) have been developed by adopting DNA-functionalized silver-cysteine hybrid nanowires (p-SCNWs) as signal amplifying labels. The sensing is established from a sandwich-type DNA hybridization, where the target DNA strands are initially hybridized with the capture DNA located at paramagnetic microspheres (PMs) and subsequently hybridized with p-SCNWs functionalized with the signal DNA probe. After magnetic separation, p-SCNWs on the hybrids were completely decomposed with HNO3 to release numerous silver ions. The powerful catalysis of silver ions toward the redox reaction of K2S2O8-Mn2+-H3PO4 causes the generation of KMnO4 that is capable of oxidizing luminol at high pH, triggering an amplified chemiluminescent signal emission. The sensing combines the extraordinary sensitivity of the catalytic chemiluminescence technology and the amplifying strategy via releasing large quantities of silver ions as the catalyst from each hybrid, enabling the assay of target DNA strands at a concentration as low as 0.34 fM. The CL signals associated with single-base pair mismatched DNA strands and non-complementary DNA strands are able to be discriminated well from the CL signal related to the complementary DNA hybridization. Likewise, the combination of p-SCNWs functionalized with an aptamer and PMs/aptamer/thrombin complex allowed the chemiluminescence sensing of thrombin with a low limit of detection corresponding to 0.17 pM.

A novel graphene coated surface plasmon resonance biosensor with tungsten disulfide (WS2) for sensing DNA hybridization

In this paper, we propose a rigorous configuration of graphene coated surface plasmon resonance (SPR) sensor with Tungsten Disulfide (WS2) for sensing DNA hybridization. The present configuration is consisted of prism (SF10 glass), Gold (Au), WS2- graphene and sensing medium. We perform the performance parameters of the proposed sensor in terms of sensitivity, detection accuracy and quality factor. Here we report a dramatic enhancement of the overall performance. Addition of graphene layers increase the sensitivity but decrease the other performance parameters. To increase the all performance parameters we add WS2 between metal and graphene layer. Furthermore in this paper, the thickness effect of Gold (Au) is also analyzed. Numerical analysis shows that the variation of SPR angle for mismatched DNA strands is quiet negligible whereas that for complementary DNA strands is considerably countable. Therefore, the proposed biosensor opens a new window towards detection for biomolecular interactions.

Modeling of a highly sensitive MoS2-Graphene hybrid based fiber optic SPR biosensor for sensing DNA hybridization

Optical fiber instead of prism is now widely studied in biosensing research. This paper investigates a highly sensitive optical fiber based Ag-MoS2-Graphene hybrid surface plasmon resonance (SPR) biosensor for sensing DNA hybridization. Sensitivity, detection accuracy and quality factor are carefully studied for the performance analysis. Numerical study shows that usingsingle layer of MoS2 in the middle of a Graphene-on-Ag layer, the fiber optic biosensor exhibits high sensitivity of 105.71°/RIU with reasonable detection accuracy and quality factor of 1.626 and 23.23 RIU−1, respectively. This enhanced sensitivity is due to the absorption ability and optical characteristics of graphene biomolecules and high fluorescence quenching ability of MoS2. Numerical investigation also reflects the variation of resonance angle and spectrum of transmitted power for mismatched DNA strands and for complementary DNA strands. These variations are negligible for mismatched DNA strands whereas significantly computable for complementary DNA strands. Thus the proposed sensor effectively differentiates hybridization and single nucleotide polymorphisms (SNP) by examining the level of changes in resonance angle and transmitted power spectrum. Therefore, this fiber optic based Ag-MoS2-Graphene hybrid biosensor might be a promising candidate for the detection of DNA hybridization.

Graphene coated fiber optic surface plasmon resonance biosensor for the DNA hybridization detection: Simulation analysis

In this paper, a graphene coated optical fiber surface plasmon resonance (SPR) biosensor is presented for the detection of DNA Hybridization. For the proposed sensor, a four layer model (fiber core /metal /sensing layer /sample) where a sheet of graphene (biomolecular recognition elements ( BRE)) acting as a sensing layer is coated around the gold film because graphene enhances the sensitivity of fiber optic SPR biosensor. Numerical analysis shows the variation of resonance wavelength and spectrum of transmitted power for mismatched DNA strands and for complementary DNA strands. For mismatched DNA strands variation is negligible whereas for complementary DNA strands is considerably countable. Proposed sensor successfully distinguishes hybridization and single nucleotide polymorphisms (SNP) by observing the variation level of resonance wavelength and spectrum of transmitted power.

DNA Hybridization Detection Based on Resonance Frequency Readout in Graphene on Au SPR Biosensor

This paper demonstrates a numerical modeling of surface plasmon resonance (SPR) biosensor for detecting DNA hybridization by recording the resonance frequency characteristics (RFC). The proposed sensor is designed based on graphene material as biomolecular recognition elements (BRE) and the sharp SPR curve of gold (Au). Numerical analysis shows that the variation of RFC for mismatched DNA strands is quiet negligible whereas that for complementary DNA strands is considerably countable. Here, graphene is used to perform faster immobilization between target DNA and probe DNA. The usage of graphene also changes the RFC that ensure hybridization of DNA event by utilizing its optochemical property. In addition, proposed sensor successfully distinguishes between hybridization and single-nucleotide polymorphisms (SNP) by observing the variation level of RFC and maximum transmittance. Therefore, the proposed frequency readout based SPR sensor could potentially open a new window of detection for biomolecular interactions. We also highlight the advantage of using graphene sublayer by performing the sensitivity analysis. Sandwiching of each graphene sublayer enhances 95% sensitivity comparing with conventional SPR sensor.

Modulation of charge transport across double-stranded DNA by the site-specific incorporation of copper bis-phenanthroline complexes.

The site-specific incorporation of transition-metal complexes within DNA duplexes, followed by their immobilization on a gold surface, was studied by electrochemistry to characterize their ability to mediate charge. Cyclic voltammetry, square-wave voltammetry, and control experiments were carried out on fully matched and mismatched DNA strands that are mono- or bis-labeled with transition-metal complexes. These experiments are all consistent with the ability of the metal centers to act as a redox probe that is well coupled to the DNA π-stack, allowing DNA-mediated charge transport.

Fabricating three-dimensional hydrogel oligonucleotide microarrays to detect single nucleotide polymorphisms

Here, a three-dimensional (3D) oligonucleotide microarray-based fluorescent assay has been developed for single nucleotide polymorphisms (SNPs) and allele frequency determination. The 3D oligonucleotide microarray is fabricated on a home-made polyacrylamide hydrogel microarray which is generated by UV light photopolymerization of tiny acrylamide monomer droplets on a PEG terminated glass slide. After activation by glutaraldehyde (GA), the polyacrylamide hydrogel is able to react with NH2-modified oligonucleotides with high efficiency to generate a high signal-to-background ratio without the conventional blocking step. We demonstrate that the assay is able to detect complimentary 25 mer oligonucleotide target (fragment of a tumor suppressor gene P53) down to 100 pM and SNP with true-to-false ratios (the ratio of fluorescence signal from perfect matched DNA strands to that from the one-base mismatched DNA strands) above 20. Taking advantage of the high specificity of the assay, allele frequency as low as 2% can be accurately determined.

Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV–vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.

Silver Nanoparticle-Based Ultrasensitive Chemiluminescent Detection of DNA Hybridization and Single-Nucleotide Polymorphisms

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which the DNA targets are first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells and then the silver nanoparticles modified with alkylthiol-capped oligonucleotides are used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, silver nanoparticles are dissolved to Ag+ in HNO3 solution and sensitively determined by a coupling CL reaction system (Ag+-Mn2+-K2S2O8-H3PO4-luminol). The combination of the remarkable sensitivity of the CL method with the large number of Ag+ released from each hybrid allows the detection of specific sequence DNA targets at levels as low as 5 fM. The sensitivity increases 6 orders of magnitude greater than that of the gold nanoparticle-based colorimetric method and is comparable to that of surface-enhanced Raman spectroscopy, which is one of the most sensitive detection approaches available to the nanoparticle-based detection for DNA hybridization. Moreover, the perfectly complementary DNA targets and the single-base mismatched DNA strands can be evidently differentiated through controlling the temperature, which indicates that the proposed CL assay offers great promise for single-nucleotide polymorphism analysis.

Thermodynamic properties of branched DNA complexes with full-matched and mismatched DNA strands

Complexes consisting of a branched DNA with full-matched and mismatched DNA strands were prepared, and the cross-linking of the DNA strands and their diastereochemistry affected the stability of the complexes and the thermodynamics of the complex formation.