Immobilization Of Single-stranded DNA Research Articles

Early fetal sex determination using a fluorescent DNA nanosensing platform capable of simultaneous detection of SRY and DYS14 sequences in cell-free fetal DNA

Early fetal sex determination is of crucial importance in the management of prenatal diagnosis of X-linked genetic abnormalities and congenital adrenal hyperplasia. The development of an efficient and simple method for high-sensitivity, affordable, and rapid screening of cell-free fetal DNA (cffDNA) is crucial for fetal sex determination in early pregnancy. In this study, single- and dual-fluorophore DNA biosensors based on multi-walled carbon nanotubes (MWCNT) were fabricated for the individual and simultaneous detection of the SRY gene and DYS14 marker in cffDNA obtained from maternal plasma samples. This nanosensing platform is based on the immobilization of single-stranded DNA (ssDNA) probes, labeled with ROX or FAM fluorophores, on MWCNT, resulting in the quenching of fluorescence emission in the absence of the targets. Upon the addition of the complementary target DNA (ctDNA) to the hybridization reaction, the fluorescence emission of fluorophore-labeled probes was significantly recovered to 79.5 % for ROX-labeled probes (i.e. SRY-specific probes), 81.5 % for FAM-labeled probes (i.e. DYS14-specific probes), and 65.9 % for dual-fluorophore biosensor compared to the quenching mode. The limit of detection (LOD) for ROX, and FAM was determined to be 4.5 nM, and 7.6 nM, respectively. For dual-color probes, LOD was found to be 5.4 (ROX) and 9.2 nM (FAM). Finally, the clinical applicability of the proposed method was confirmed through the detection of both biomarkers in maternal plasma samples, suggesting that the proposed nanosensing platform may be useful for the early detection of fetal sex using cffDNA.

RF plasma polymer modification of graphene oxide for micromotors with improved performance

This work introduces improved performances of self-propelled tubular micromotors based on radio frequency (RF)–rotating plasma-prepared graphene oxide-poly(aniline) (GO-PANI), graphene oxide/poly(ethylaniline) (GO-PEANI), and graphene oxide/poly(fluoroaniline) (GO-PFANI) composites as outer layer and platinum (Pt) as the catalytic inner layer. Scanning electron microscopy energy-dispersive X-ray (SEM-EDX), X-ray diffraction (XRD) analysis, Fourier-transform infrared spectroscopy (FTIR), and electrochemical cyclic voltammetry (CV) techniques were utilized to characterize the RF-rotating plasma-modified graphene oxide–based composites. Incorporating the impressive features of graphene oxide (GO) and PANI or its substitute derivatives, the composite micromotors offer an efficient performance in dye-labeled single-stranded DNA immobilization due to changes in the fluorescence intensity and speed of micromotors.

Preparation of self-propelled Cu-Pt micromotors and their application in miRNA monitoring

Self-propelled catalytic micromotors offer considerable promise in terms of many applications. Catalytic micromotors are strongly influenced by the presence and concentration of specific ions and chemicals in the environment, making them useful as sensors and actuators. In this work, copper (Cu)-platinum (Pt) micromotors were fabricated by using the magnetron sputtering method for the first time in the literature and their applications based on the detection of miRNA-21 were evaluated. We analyzed the dependence of the mobility of Cu-Pt micromotors using different concentrations of hydrogen peroxide (H$_{2}$O$_{2})$. The presence of surfactants in the environment is also important for the movement of the micromotors. Thus, we studied the effect of three different surfactants: anionic as sodium dodecyl sulfate (SDS), cationic as cetyltrimethylammonium bromide (CTAB), and nonionic as Triton X-100. Cu-Pt micromotor motion was observed even at very low concentrations of surfactant (0.01%) and hydrogen peroxide (0.25%). miRNAs have been regarded as biomarker candidates in early diagnosis. Our sensing strategy relied on dye-labeled single-stranded DNA immobilization onto Cu-Pt micromotors that recognize the target miRNA-21. The changes in the fluorescence intensity as well as the changes in the speed of micromotors were examined before and after hybridization.

An Advanced, Silicon-Based Substrate for Sensitive Nucleic Acids Detection.

Surface substrate and chemical functionalization are crucial aspects for the fabrication of the sensitive biosensor based on microarray technology. In this paper, an advanced, silicon-based substrate (A-MA) allowing enhancement of optical signal for microarray application is described. The substrate consists in a multilayer of Si/Al/SiO2 layers. The optical signal enhancement is reached by a combination of the mirror effect of Al film and the SiO2 thickness around 830 nm, which is able to reach the maximum of interference for the emission wavelength of the Cy5 fluorescent label. Moreover, SiO2 layer is suitable for the immobilization of single-strand DNA through standard silane chemistry, and probe densities of about 2000 F/µm2 are reached. The microarray is investigated in the detection of HBV (Hepatitis B Virus) pathogen with analytical samples, resulting in a dynamic linear range of 0.05–0.5 nM, a sensitivity of about 18000 a.u. nM−1, and a Limit of Detection in the range of 0.031–0.043 Nm as a function of the capture probe sequence.

Aptamer strategy for ATP detection on nanocrystalline diamond functionalized by a nitrogen and hydrogen radical beam system

Here, we report a novel method for micropatterning oligonucleotides on the diamond surface via forming amine groups on the diamond surface by nitrogen/hydrogen radical treatment. The covalent bonding of the supporting oligonucleotide and characterization of an immobilized hybridized oligonucleotide with Cy5 modification were investigated by fluorescence microscopy. To investigate the effectiveness of nitrogen/hydrogen radical treatment for amine termination, two types of radical treatment were used: hydrogen/nitrogen radical treatment and pure nitrogen radical treatment. From the results, hydrogen/nitrogen radical treatment produces amine (NH2) termination on the diamond surface. The effect of amine termination was investigated by immobilization of single-stranded DNA via amide bonding between surface NH2 groups and COOH groups terminating the DNA. The immobilized single-stranded DNA (supporting DNA), which has a complementary relationship with the adenosine triphosphate (ATP) aptamer (DNA), hybridizes with the aptamer with attached fluorescence dye. When ATP molecules approach the double-stranded DNA, the aptamer forms a close relationship with the supporting DNA and combines with ATP. ATP detection was effectively carried out by reduction of fluorescence.

Direct DNA Immobilization onto a Carbon Nanotube Modified Electrode: Study on the Influence of pH and Ionic Strength

Over the past years, DNA biosensors have been developed to analyze DNA interaction and damage that have important applications in biotechnological researches. The immobilization of DNA onto a substrate is one key step for construction of DNA electrochemical biosensors. In this report, a direct approach has been described for immobilization of single strand DNA onto carboxylic acid-functionalized carbon nanotubes modified glassy carbon electrode. To do this, we first modified the glassy carbon electrode surface with MWCNT-COOH. The immersion of MWCNT-COOH/GCE in ss-DNA probe solution, with different pH and ionic strength, was followed by suitable interaction between amine group of ss-DNA bases and carboxylic groups of MWCNT-COOH. This interaction leads to successful ss-DNA immobilization on MWCNT-COOH that was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy. Immobilization of ss-DNA on the modified electrode increased the charge transfer resistant but decreased the peak current of redox probe ([Fe(CN)6]3-/4-). The result of cyclic voltammograms implicates that enhancements in the DNA immobilization are possible by adroit choice of low pH and high ionic strength. The standard free-energy of adsorption (ΔG°ads) was calculated from electrochemical impedance spectroscopy data (-47.75 kJ mol-1) and was confirmed covalent bond formation. atomic force microscopy topographic images demonstrate increased surface roughness after ss-DNA immobilization. Results offer a simple, rapid and low-cost of DNA immobilization strategy can be opportunities to design of novel nucleic acid biosensors.

New self assembly monolayer onto SiGe as a selective biosensor for single-strand DNA

An important step of fabrication of selective DNA probe/receptor is the functionalization of semiconducting surfaces with a self-assembled monolayer (SAM) with an appropriate surface termination to interact with DNA. In this work, we studied an immobilization of single-strand DNA (ssDNA) onto self-assembly monolayer of both the heavily exploited aminopropyltriethoxy silane (APTES) and n-(2-aminoethyl)-11-aminoundecyltrimethoxysilane (NAATS), a bi-functional amino silane onto the surface of SiGe substrate. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) techniques were employed to characterize the silanization of SAMs on SiGe surface. The immobilization process was confirmed using fluorescence intensity measurements. The coverage values of APTES and NAATS SAMs were estimated to be 71.3 and 83 XPS% at 78°, respectively from the C/Ge ratio. Comparing the performance towards both specific and non-specific ssDNA complements for both silanes, it was observed that a higher selectivity and sensitivity was obtained by using NAATS SAM. This work highlighted the importance of SAM chain length by comparing one short alkyl chains, APTES with that long-chain counterpart, NAATS.

Application of Lateral and Distance Spacers in an Oligonucleotide Based Immobilization System for Bioactive Molecules onto Titanium Implants

Immobilization of bioactive molecules (BAMs) on a nanometer scale is of great interest for functionalization of implant and scaffold surfaces in current biomaterials research. A system for immobilization of one or more compounds is described, which is based on nanomechanical fixation of single-stranded nucleic acids into an anodic titanium oxide layer and their subsequent hybridization with BAMs conjugated to the respective complementary strands. This paper focuses on further development and in depth understanding of the immobilization system, as some of the major findings established for common sensor applications for immobilization of single-stranded DNA onto gold surfaces cannot be transferred to the TiO2 surface. The first approach concerning the influence of the internal spacer sequence revealed the best performance for a polyadenine based sequence out of four homologous spacer sequences (A30, T30, C30, and G30). This overall best performance of the A30 spacer is attributed to an increased contour length by nucleotide staggering, which resulted in the best protection of the hybridization sequence from unfavorable interactions with the surface or damaging attacks by reactive oxygen species. The second approach comprises the implementation of a lateral spacer, also based on a homologous sequence of A30. Simultaneous as well as sequential adsorption of anchor strands and spacer strands were performed, and it could be shown that a preadsorption with high density of the spacer was most effective to increase hybridization efficiency.

Electrochemical DNA biosensor based on gold nanorods for detecting hepatitis B virus.

The purpose of this work was to fabricate an electrochemical DNA biosensor for detecting hepatitis B virus. Gold nanorods (GNRs), which are known for their conductivity, were used to increase surface area and consequently increase the immobilization of single-stranded DNA (ss-DNA) on the modified gold electrode. The GNRs were characterized via transmission electron microscopy. The morphology of the gold electrode before and after modification with GNRs was characterized by scanning electron microscopy. Atomic-force microscopy was used to evaluate the morphology of the GNR electrode surface before and after interaction with ss-DNA. Cyclic voltammetry was used to monitor DNA immobilization and hybridization, using [Co(phen)3](3+) as an electrochemical indicator. The target DNA sequences were quantified at a linear range from 1.0 × 10(-12) to 10.0 × 10(-6)molL(-1), with a detection limit of 2.0 × 10(-12)molL(-1) by 3σ. The biosensor had good specificity for distinguishing complementary DNA in the presence of non-complementary and mismatched DNA sequences.

Structural modifications of gold thin films produced by thiol-derivatized single-stranded DNA immobilization

Recent experiments have reported an opposite sign of the differential surface stress produced on gold-coated cantilevers by a thiol-derivatized single-stranded DNA (SH-DNA) immobilization process. The sign of the surface stress depends on the method used to evaporate the gold thin film, being compressive (negative) or tensile (positive) for e-beam or resistively deposited gold, respectively. This study investigates the origin of this effect by means of a combination of x-ray diffraction and x-ray photoelectron spectroscopy. Both e-beam and resistively grown gold thin films are characterized to find the subtle differences responsible for this intriguing stress behaviour. Somewhat remarkably, these studies show a tight relation between the surface structure of the gold overlayer and the SH-DNA immobilization efficiency. The average grain size variation seems to correlate well with the differential surface stress triggered by the SH-DNA immobilization previously reported. These results suggest that the relation of the probe molecules with the surface structure must be considered to understand surface stress changes.

Stress and DNA Assembly Differences on Cantilevers Gold Coated by Resistive and E-Beam Evaporation Techniques

Changes in the sign of differential surface stress of gold-coated cantilevers produced by thiol-derivatized single-stranded DNA immobilization are observed, depending on the method used to deposit the gold. While the DNA immobilization on e-beam gold-coated cantilevers produces a compressive differential surface stress in the metallic layer, the opposite is observed for resistively coated cantilevers under the same immobilization conditions. The gold films exhibit quite a similar morphology, and the immobilization differences seem to be related to the charge state of the metallic layer surface. This in turn produces a different distribution of the orientation of the DNA strands on the gold layer. A tentative explanation for the observed effect is proposed.

Bromine-containing functional nanolayers for biomolecule immobilization

Bromine-containing nanolayers are deposited by pulsed plasma deposition of 2-bromoethylacrylate. Subsequent covalent immobilization of single-strand DNA onto these films is achieved via the reaction with a terminal thiol group linker contained in the oligonucleotide.

Immobilization of single strand DNA on solid substrate

Abstract This Letter reports the immobilization of DNA onto a solid support by electrostatic interaction with a polycation poly(allylamine hydrochloride) (PAH). The films were analyzed by UV–vis spectroscopy. The significant observation is that single stranded DNA gets immobilized on the PAH backbone of Layer-by-Layer (LbL) films when the films are fabricated above the melting temperature of DNA. Singly stranded DNA immobilized in the LbL films is not restored into double stranded DNA at room temperature.

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors.

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics.

Hierarchy of DNA immobilization and hybridization on poly-L-lysine: an atomic forcemicroscopy study

Atomic force microscopy is used to analyze the immobilization of single-stranded DNA(ssDNA) on poly-L-lysine (PL)-coated glass and subsequent hybridization withcomplementary DNA (dsDNA). 2D fractal analysis (power spectrum density algorithm(PSDA)) and line analysis methods are used to characterize the vertical aggregation ofssDNA/dsDNA. The nanodefects on the PL layer could be responsible for the verticalaggregation of DNA. The line analysis suggests that approximately two layers of ssDNA areadsorbed, stacked vertically. The PSDA is useful in understanding the direction ofaggregation in addition to understanding the growth mechanism. Fractal dimension valuesare between 2 and 3, suggesting that the growth mechanism may obey the diffusion-limitedaggregation (DLA) model.

A Quartz Crystal Microbalance (QCM) Study of Single-Strand DNA Hybridization and Hydrolytic Cleavage

The use of a quartz crystal microbalance (QCM) for monitoring in situ the immobilization of single-strand DNA marked with mercaptol group at the 5′-end on the surface of a gold-filled 7.995 MHz AT-cut quartz crystal by Au-S bond with the self-assembly technique is reported. The hybridization of ssDNA with complementary 10-mer ODN and 8-mer ODN is described. The QCM was also employed to analyze DNA cleavage by cerium(IV) ions under moderate conditions. The results showed that the QCM, which is capable of sensitive measurement, was able to investigate the immobilization, hybridization, and cleavage of ssDNA in situ. The cerium(IV) ions produced no cleavage in double-strand DNA; they were, however, able to hydrolyze single-strand DNA. Thus, the hydrolytic cleavage of ssDNA at a specific site could be ensured by protective hybridization.

Electrical detection of biomolecular interactions with metal–insulator–semiconductor diodes

We report the label-free detection of DNA hybridization using a metal–insulator–semiconductor (MIS) diode or capacitor. Upon immobilization of single-stranded DNA on the gold gate of a MIS capacitor, the capacitance versus voltage characteristics show a significant shift in the direction of negative voltages as expected from the immobilization of negative charges on the gate. The hybridization with the complementary strand gives rise to a further significant shift in the same direction as before, which is consistent with the increase of negative charges on the gate brought about by the hybridization. Fluorescence studies indicate that the immobilization and hybridization of DNA can be electrostatically promoted by electric fields externally applied to the MIS capacitors. The MIS diode detection method is applicable to all biomolecular interactions that affect the surface dipole at the interface between the metal gate and the electrolyte and can be extended to other chemical and biochemical systems such as proteins and cells.

Extended Langmuir approach to analyze the telomeric sequence using a biochromatographic concept

Immobilization of single-stranded DNA (ssDNA) on chromatographic support, i.e. copolymerized particles of styrene and glycidyl methacrylate via formation of amino groups with either N-methyl-1,3-propanediamine (stationary phase 1) or hexamethylenediamine (stationary phase 2) is a promise method to separate sequence specific DNA. However, the low ligand density of nonporous particles led to nonspecific interaction of the complementary oligonucleotide DNA in the sample with the stationary phase (1 or 2). In this paper, the binding process of telomere (tel; TTAGGG) to the complementary ssDNA immobilized (AATCCC) on the two chromatographic supports was studied for the first time using extended Langmuir equation. It appeared that the nonspecific adsorption of the tel due to electrostatic interaction between the polynucleotide sample (tel) and the residual amino groups on the particle surface via amination with hexamethylenediamine (stationary phase 2) was significant and could be reduced by using a high salt (NaCl) concentration in the bulk solvent. In contrast, the nonspecific adsorption of tel was neglected in the column using DNA-immobilized particles via amination with N-methyl-1,3-propanediamine (stationary phase 1). Thus, the affinity chromatography prepared via amination by N-methyl-1,3-propanediamine was more effective for analysis of sequence-specific DNA than the one prepared via amination by hexamethylenediamine. Moreover, for the two stationary phases, increasing NaCl concentration in the bulk solvent enhanced the hybridization between tel and the complementary immobilized oligonucleotide.

Silica-nanoparticle-based interface for the enhanced immobilization and sequence-specific detection of DNA.

A biocompatible and uniform interface based on silica nanoparticles derivatized with amino groups has been constructed for the effective immobilization and sensitive sequence-specific detection of calf thymus DNA. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) results showed that a monolayer of silica nanoparticles can be formed on a gold electrode under our experimental conditions using cysteine self-assembly monolayer as binder medium. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy (XPS) verified the successful immobilization of DNA on silica-nanoparticle-modified gold electrodes. Quantitative results demonstrated that enhanced immobilization of single-strand DNA (ss-DNA) up to 1.6 x 10(-8) mol cm(-2) could be achieved owing to the larger surface area and the special properties of silica nanoparticles. In addition, hybridization experiments demonstrated that the immobilized ss-DNA on silica nanoparticles could specifically interact with complementary DNA in solutions.

PNA−DNA Hybridization at the Air−Water Interface in the Presence of Octadecylamine Langmuir Monolayers

Sequential immobilization of single-stranded complementary DNA and peptide nucleic acid (PNA) molecules at the air−water interface with cationic Langmuir monolayers is demonstrated. The complexation of the single-stranded DNA/PNA molecules with octadecylamine (ODA) Langmuir monolayers was followed in time by monitoring the pressure−area isotherms. A large (and slow) expansion of the ODA monolayer was observed during immobilization of single-stranded DNA at the air−water interface, and interestingly, no further expansion was detected on addition of PNA to the subphase. However, quartz crystal microgravimetry and UV-melting analysis of Langmuir−Blodgett films of the ODA−PNA−DNA hybrid monolayer clearly showed that the sequential immobilization process had resulted in hybridization of the complementary single-stranded DNA molecules with PNA at the air−water interface.