Label-free Detection Of DNA Hybridization Research Articles

Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications.

Label-free detection of DNA hybridization using nanopillar arrays based optical biosensor

In this study we fabricated nanopillar arrays (NPLAs) of silicon, through a process involving very-large-scale integration and reactive ion etching, for use as two-dimensional periodic relief gratings on silicon surfaces. Oligonucleotides were successively immobilized on the pillar surface, allowing the system to be used as an optical detector specific for the targeted single-stranded DNAs (ssDNAs). The surfaces of the oligonucleotides-modified NPLAs underwent insignificant structural changes, but upon hybridizing with target ssDNA, the NPLAs underwent dramatic changes in terms of their pillar scale. Binding of the oligonucleotides to the NPLA occurred in a way that allowed them to retain their function and selectively bind the target ssDNA. We evaluated the performance of the sensor by capturing the target ssDNA on the NPLA and measuring the effective refractive index (neff). The binding of the target ssDNA species to the NPLA resulted in a color change from pure blue to red, observable by the naked eye at an angle of 15°. Moreover, we used effective medium theory to calculate the filling factors inside the NPLA and, thereby, examine the values of neff during the structural changes of the NPLA. Accordingly, these new films have potential applications as label-free optical biosensors.

Conjugated polyelectrolytes-initiated chemiluminescence: A biosensing platform for label-free and homogeneous DNA detection

In this study, it was found that conjugated polyelectrolytes (CPEs) would initiate the strong chemiluminescence (CL) emission of luminol–H2O2 in weak basic media. Using CL spectra, ultraviolet visible light spectra, fluorescence spectra and electron spin resonance (ESR) spectra, the catalytic mechanism of CPEs on luminol–H2O2 CL was discussed in detail. Furthermore, it was found that the catalytic activity of poly [3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydro chloride] (PMNT) (a kind of cationic CPEs) on the luminol–H2O2 CL system was closely relevant to the conformation of PMNT. The CL intensity of luminol–H2O2–PMNT system in the presence of single-stranded DNA was much larger than that in the presence of double-stranded DNA. By taking advantage of this phenomenon, a label-free and homogeneous CL detection of DNA hybridization is proposed. The detection limit of target DNA (3σ) was estimated to be as low as 3.7×10−13M. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.

Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe

A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G–C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results.

Label-free detection of DNA hybridization using transistors based on CVD grown graphene

The high transconductance and low noise of graphene-based field-effect transistors based on large-area monolayer graphene produced by chemical vapor deposition are used for label-free electrical detection of DNA hybridization. The gate materials, buffer concentration and surface condition of graphene have been optimized to achieve the DNA detection sensitivity as low as 1pM (10−12M), which is more sensitive than the existing report based on few-layer graphene. The graphene films obtained using conventional PMMA-assisted transfer technique exhibits PMMA residues, which degrade the sensing performance of graphene. We have demonstrated that the sensing performance of the graphene samples prepared by gold-transfer is largely enhanced (by 125%).

PDMS-based Optical Leaky Waveguide Coated with Self-assemble Au-NPs for Bio-analytical Detections

This paper presents a novel method for fabricating PDMS-based optical leaky waveguides coated with self-assembled gold nano-particles (Au-NP) for bio-analytical detection utilizing the localized surface plasmon resonance (LSPR) effect. In the presented method, a PDMS optical waveguide is first cast in Teflon tubing to form a cylindrical leaky waveguide structure. The de-molded PDMS optical waveguide is then modified with PDDA molecules and coated with a layer of 13 nm Au-NPs for inducing the LSPR effect. The fabricated LSPR sensor is finally connected to multi-mode optic fibers for guiding the detection light. The measured sensitivity of the PDMS waveguide based LSPR sensor for detecting diluted glycerol solutions was 7.25 AU/RIU and 325.97 nm/RIU. Experimental results of a label-free detection of DNA hybridization show that the presented PDMS waveguide based LSPR sensor has a good linear response and has a detection limit of about 10pM, confirming the detection performance of the developed PDMS waveguide-based LSPR sensor.

Understanding properties of electrified interfaces as a prerequisite for label-free DNA hybridization detection

Label-free electrochemical detection of DNA hybridization with high selectivity and sensitivity is only achievable if the properties of DNA at an electrified interface are understood in depth. After a short summary of concepts of electrochemical DNA detection as well as initial attempts towards label-free DNA assays the review discusses the physico-chemical properties and differences between single-stranded and double-stranded DNA immobilized at electrode surfaces in the light of their persistence lengths, structural conformation, impact of the charge screening by ion condensation and the electric field generated upon polarization of the electrode. Electrochemical impedance spectroscopy as a tool for label-free elucidation of DNA hybridization is reviewed and the necessity for an in-depth understanding of the interfacial properties is highlighted. Our major aim is to demonstrate the advantageous application of specifically designed intercalating compounds for the design of label-free detection of DNA hybridization.

Fabrication of DNA/graphene/polyaniline nanocomplex for label-free voltammetric detection of DNA hybridization

A novel DNA electrochemical biosensor was described for the detection of specific gene sequences. Electrochemically reduced graphene oxide (ERGNO) was prepared on polyaniline (PAN) nanofibers modified glassy carbon electrode (GCE). Compared with the electrochemical reduction of graphene oxide directly on bare GCE (reduction potential: ca. −1.3V), more positive reduction potential (ca. −1V) for graphene oxide was observed with the PAN membrane existing. The formed ERGNO/PAN nanocomposites were applied to bind ssDNA probe via the non-covalent assembly. The surface density of ssDNA was calculated by voltammetric studies of redox cations ([Ru(NH3)6]3+), which were bound to the surface via electrostatic interaction with negative charged phosphate backbone of the DNA. After the hybridization of ssDNA probe with complementary DNA, the response of surface-bound [Ru(NH3)6]3+ changed obviously, which could been adopted to recognize the DNA hybridization. Under optimal conditions, the dynamic range of the DNA biosensor for detecting the sequence-specific DNA of cauliflower mosaic virus (CaMV35S) gene was from 1.0×10−13 to 1.0×10−7molL−1, with a detection limit of 3.2×10−14molL−1. This biosensor also showed a high degree of selectivity.

Electrical detection of DNA immobilization and hybridization by streaming current measurements in microchannels

Label-free electrical detection of surface DNA immobilization and hybridization via streaming current measurements in a microchannel is demonstrated. Streaming currents generated by the flow of deionised water through a polydimethysiloxane microchannel sealed on glass are measured using integrated Au electrodes and are sensitive to the density and polarity of the charge on the channel surface. An in-channel DNA hybridization protocol was developed. Streaming currents were monitored after each of protocol steps. The technique was applied to label free recognition of DNA hybridization and could distinguish between assays with complementary and non-complementary DNA strands.

Sensitive label-free electrochemical DNA hybridization detection in the presence of 11-mercaptoundecanoic acid on the thiolated single strand DNA and mercaptohexanol binary mixed monolayer surface

A ternary surface consisting of the 5′ end 6-mercapto-1-hexane-labeled single-strand DNA (HS-ssDNA) and of thiol alkanes, including 6-mercapto-1-hexanol (MCH) and 11-mercaptoundecanoic acid (MUDA) (abbreviated as HS-ssDNA/MCH/MUDA), was constructed on a polycrystalline gold electrode. Label-free electrochemical sensing of DNA hybridization was performed in the presence of [Fe(CN) 6] 3− and the double strand-specific intercalator methylene blue (MB). For comparison, other ternary surfaces were constructed using 3-mercaptopropionic acid (MPA) and 11-mercaptoundecanol (MUD) diluents. The observed discrimination effect (ratio of charge transfer resistance, R CT, between the hybridized and non-complementary hybridized surfaces) for the different ternary surfaces followed the order of HS-ssDNA/MCH/MUDA (1.89) > HS-ssDNA/MPA/MCH (1.37) > HS-ssDNA/MCH/MUD (1.27) > HS-ssDNA/MPA/MUD (0) = HS-ssDNA/MPA/MUDA (0). Differences in the discrimination effects among the ternary surfaces are related to the pinhole behavior of the molecular layer, which is influenced by the chain length and head group functionalities responsible for the intermolecular hydrogen bonding. This criterion was met only when the difference in the chain lengths of the two alkane diluents in the ternary was less than 50%. While the ternary HS-ssDNA/MCH/MUDA surface showed a detection range of 1 × 10 −18 to 1 × 10 −6 M, the ternary HS-ssDNA/MPA/MCH exhibited a range from 1 × 10 −12 to 1 × 10 −6 M. The remarkable increase in sensitivity was attributed to the elimination of non-specific Au–DNA interactions and the proper orientation of HS-ssDNA on the ternary surfaces. The surfaces were characterized using cyclic voltammetry (CV), impedance spectroscopy (IS), chronocoulometry (CC) and Fourier-transform infrared (FTIR) spectroscopy.

Printed Functionalized Capacitors for Water-Induced Label-Free Detection of DNA Hybridization

In the last decade, new device concepts based on novel fabrication methods have emerged as a powerful and general class of supersensitive sensors for the direct detection of biological and chemical targets. Pushed by the opportunity to activate the physical-chemical phenomena at the nanoscale, novel patterning paradigms have gained nanofabrication accuracy accompanied by the fast development of bottom-up strategies. In this frenetic framework, the combination of some heterogeneous techniques-basically nano-imprint lithography, grafting of functional molecules, and synthesis of new ligands and probes to capture specific biological targets-have gained strong attention as enabling emerging technological mixture to fabricate ultra dense array of specialized sensors. Within the scope of this technological convergence, the paper is basically devoted to describe the processes involved in the production of stamps for nanoimprint lithography, the fabrication of printed array of silicon sensors on the micrometer and sub-micrometer resolution, the subsequent covalent biofunctionalization with single strand DNA and some preliminary electrical detection of the hybridization events via water modulating ac resistance. Among the possible choices, focus was given to planar functionalized capacitors for their intrinsic simple configuration and also because they appeared to be the most promising candidates for mass production in printed device architecture. The paper is based on original results but contains also data and achievements taken from the literature to make the discussion self-contained. © 2011 American Scientific Publishers.

Label-Free Detection of DNA Hybridization Using Pyrene-Functionalized Single-Walled Carbon Nanotubes: Effect of Chemical Structures of Pyrene Molecules on DNA Sensing Performance

We investigate the effect of functional groups of pyrene molecules on the electrical sensing performance of single-walled carbon nanotubes (SWNTs) based DNA biosensor, in which pyrenes with three different functional groups of carboxylic acid (Py-COOH), aldehyde (Py-CHO) and amine (Py-NH2) are used as linker molecules to immobilize DNA on the SWNT films. UV/Visible absorption spectra results show that all of the pyrene molecules are successfully immobilized on the SWNT surface via pi-pi stacking interaction. Based on fluorescence analysis, we show that the amide bonding of amine terminated DNA via pyrene containing carboxylic groups is the most efficient to immobilize DNA on the nanotube film. The electrical detection results show that the conductance of Py-COOH modified SWNT film is increased upon DNA immobilization, followed by further increase after hybridization of target DNAs. It indicates that the pyrene molecules with carboxylic acid groups play an important role to achieve highly efficient label-free detection by nondestructive and specific immobilization of DNAs.

Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing

Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged –COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH 2-IL) was applied to the reduction of HAuCl 4 to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH 2-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.

Improved Biosensing Capability with Novel Suspended Nanodisks

In this work, we provide a theoretical and experimental study of the adverse effects that inherently arise when plasmonic nanoparticles are attached to a solid support. Using the example of short-ordered arrays of gold nanodisks, we show that the right choice for the thin metal adhesion layer that is used to ensure stable binding of the nanodisks to the substrate is extremely important to achieve competitive limits of detection. Furthermore, we show that the presence of the high refractive index support undisputedly results in lower bulk and surface sensing sensitivities. To minimize this effect, we propose an isotropic chemical etch of the supporting substrate, resulting in dielectric nanopillars which distance the nanodisks from this high refractive index material. We demonstrate that this novel method not only preserves the mechanical stability but also strongly increases the sensing performance of these suspended nanodisk arrays. This is demonstrated via the label-free detection of DNA hybridization, reflecting the robustness and the surface-regenerative capabilities of these pillar-supported nanodisk arrays. Furthermore, through the successful implementation of this sensitivity enhancing method to more complex nanostructures, such as arrays of closely distanced nanodisk dimers, this method profiles itself as a simple strategy to improve the limits of detection of a wide variety of nanoplasmonic sensors.

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.

Label-free electrochemical impedance sensing of DNA hybridization based on functionalized graphene sheets

Negative-charge change and conformation transition upon DNA immobilization and hybridization on functionalized graphene sheets were monitored by the EIS technique and adopted as the signal for label-free electrochemical DNA hybridization detection.

Real‐Time DNA Detection Using Reduced Graphene Oxide Field Effect Transistors

DNA sensing: Single strands of DNA are covalently attached to nanometer-thick layers of reduced graphene oxide, forming an FET device capable of sensitive, label-free detection of complementary DNA hybridization.

Label-Free Detection of DNA Hybridization Using Surface Enhanced Raman Spectroscopy

The SERS spectrum of DNA is strongly dominated by the strong spectral feature of adenine at 736 cm(-1); the presence of adenine can serve as an endogenous marker for the label-free SERS-based detection of DNA hybridization when the probe DNA sequence is adenine-free. The substitution of 2-aminopurine for adenine on the probe DNA sequence enables the detection of a target sequence using SERS, upon hybridization of the target with the 2-AP-substituted probe DNA sequence.

Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANInt) and poly-L-lysine (pLys). The composite of pLys and PANInt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane. The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH, but also possesses fine biocompatibility for bio-analytes. DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity. The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition. The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method. This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)6]3−/4− as an indicator.

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G(NP)) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G(NP) was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G(NP) were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)(6)](3-/4-) as the redox indicator. The (CA-G(NP)/SPAN)(n) films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G(NP)) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)(6)](3-/4-) in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G(NP)/SPAN)(n) films, the target DNA with 20-base could be detected up to 2.13x10(-13)mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.