DNA Immobilization Research Articles

A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-21.

Small noncoding microRNAs (miRNAs) have emerged as ideal noninvasive biomarkers for early-phase cancer detection. In this report, a label-free and simple electrochemical miRNA biosensor is developed based on employing methylene blue (MB) as a redox indicator. The successfully immobilization of the single strand DNA (ss-DNA) probe and hybridization with the target miRNA sequence were confirmed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. Differential pulse voltammetry (DPV) technique was used to record the oxidation peak current of MB under optimal condition and an increase in the peak current was observed after hybridization. By employing this strategy, miRNA can detect in a range from 0.1 to 500.0 pM with a relatively low detection limit of 84.3 fM. The electrochemical response of MB on ss-DNA and duplex of miRNA/DNA was characterized by CV and chronocoulometry method. The linear relation between the redox peak currents (Ip) and scan rate (ν) indicates that the electron transfer (ET) between MB and the electrode surface was mediated by the miRNA/DNA π-stacked duplex. The value of surface coverage (Γ) was calculated that indicated increase amount of MB on the surface of modified electrode after hybridization event and revealed the adsorption of MB at modified electrode is monolayer. Also, the electron transfer rate constants (ks) of MB were estimated. The results of kinetic analysis were confirmed by chronocoulometry method. The discrimination ability of miRNA biosensor even against a noncomplementary target was also studied. Consequently, this strategy will be valuable for sensitive, selective and label-free detection of miRNA.

Quantitative evaluation of surface topographical changes of Au thin films after DNA immobilization

A quantitative evaluation, by atomic force microscopy measurements, of the nano-topography of single and double stranded DNA functionalized Au surfaces is carried out. The two-dimensional fractal dimension was quantified for the Au surface in 0.9±0.1 and 2.1±0.3 from 1μm×1μm and 5μm×5μm scans, respectively. It was quantified in 2.1±0.1 and in 2.4±0.3 for the Au surface after immobilization of single-stranded DNA film and in 2.0±0.1 and 1.9±0.3 for the Au surface after hybridization, from 1μm×1μm and 5μm×5μm scans, respectively. These results indicate a topographical conforming effect of the DNA films toward a two-dimensional Gaussian surface.

Simple and rapid fabrication of disposable carbon-based electrochemical cells using an electronic craft cutter for sensor and biosensor applications

This work describes the construction of an all-plastic disposable carbon-based electrochemical cell (DCell) using a simple procedure based on the use of a home cutter printer for prototyping and laminating. The cutter printer and adhesive vinyl films were used to produce three electrodes in an electrochemical cell layout, and a laminating process was then used to define the geometric area and insulate the electrodes. The DCell showed excellent performance in several applications including the determination of toxic metals in water samples, the immobilization of DNA and the detection of Salmonella. An unmodified DCell was applied for Pb and Cd detection in the range of 100–300ngmL−1 with a limit of detection of 50 and 39ngmL−1 for Cd and Pb, respectively. DNA was successfully immobilized on a DCell and used for studies of interaction between bisphenol A and DNA. The square wave voltammetry of a DNA modified DCell presented a guanine oxidation current 2.5 times greater after exposure of the electrode to bisphenol A and no current variation for the adenine moiety indicating that bisphenol A showed a preference for DNA interaction sites. A magneto-immunoassay was developed using a DCell for Salmonella detection in milk samples. The system presented a linear range from 100 to 700 cellsmL−1 with a limit of detection of 100 cellsmL−1 and good recovery values between 93% and 101% in milk samples, with no interference from Escherichia coli. Using the proposed method, hundreds of DCells can be assembled in less than two hours, at a material cost of less than US $0.02 per cell. The all-plastic disposable electrochemical cell developed was successfully applied as an electrochemical sensor and biosensor. The feasibility of the developed all-plastic disposable electrochemical cell was demonstrated in applications as both sensor and biosensor.

Ultraselective homogeneous electrochemical biosensor for DNA species related to oral cancer based on nicking endonuclease assisted target recycling amplification.

Traditional electrochemical DNA biosensors need DNA immobilization on the electrode surface, which is tedious and time-consuming. In this study, a simple but ultraselective electrochemical DNA biosensor had been designed to determine target DNA species related to oral cancer overexpressed 1 in saliva, which combines the signal amplification of nicking endonuclease assisted target recycling with the immobilization-free electrochemical method. The complementary substrate strand of target DNA species contains a simple asymmetric sequence had been modified with a methylene blue at the 3' terminal first, which cannot diffuse easily to the negative charged ITO electrode surface due to the abundant negative charges. The presence of the target DNA would trigger the formation of double-stranded DNA (dsDNA). Then the nicking endonuclease can recognize the simple asymmetric sequence in the dsDNA and cleave the substrate strand of ds-DNA into two pieces, a long ssDNA and a 2-base ssDNA linked with methylene blue. The short one can diffuse easily to the negative charged ITO electrode surface and results in the enhanced electrochemical response detected. At the same time, the target DNA can dissociate from the dsDNA and trigger the next round of hybridization, cleavage, and releasing, which results in the signal amplification. This homogeneous DNA biosensor can detect as low as 0.35 pM (S/N = 3) target DNA. Compared with the traditional heterogeneous electrochemical DNA biosensors, which are tedious and time-consuming due to the complex DNA immobilization process, the assay not only owns the merits of simple and high efficiency since performed in a homogeneous solution but also exhibits a high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence.

Ultrasensitive photoelectrochemical biosensing of multiple biomarkers on a single electrode by a light addressing strategy.

Ultrasensitive multiplexed detection of biomarkers on a single electrode is usually a great challenge for electrochemical sensors. Here, a light addressable photoelectrochemical sensor (LAPECS) for the sensitive detection of multiple DNA biomarkers on a single electrode was reported. The sensor was constructed through four steps: (1) immobilization of capture DNA (C-DNA) of different targets on different areas of a single large-sized gold film electrode, (2) recognition of each target DNA (T-DNA) and the corresponding biotin-labeled probe DNA (P-DNA) through hybridization, (3) reaction of the biotin-labeled probe DNA with a streptavidin-labeled all-carbon PEC bioprobe, and (4) PEC detection of multiple DNA targets one by one via a light addressing strategy. Through this principle, the LAPECS can achieve ultrasensitive detection of three DNA sequences related to hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency (HIV) viruses with a similar wide calibration range of 1.0 pM ∼ 0.01 μM and a low detection limit of 0.7 pM by using one kind of PEC bioprobe. Moreover, the detection throughput of LAPECS may be conveniently expanded by simply enlarging the size of the substrate electrode or reducing the size of the sensing arrays and the light beam. The present work thus demonstrates the promising applications of LAPECS in developing portable, sensitive, high-throughput, and cost-effective biosensing systems.

Signal amplification detection of DNA using a sensor fabricated by one-step covalent immobilization of amino-terminated probe DNA onto the polydopamine-modified screen-printed carbon electrode

An electrochemical DNA sensor was fabricated based on covalent immobilization of amino-terminated probe DNA onto the polydopamine (PDA) modified screen-printed carbon electrode (SPCE). Immobilization was achieved via one-step Schiff base reaction between the amino group of the probe DNA and quinones in PDA. PDA was formed via electrochemical polymerization of dopamine monomer on the SPCE surface. After a sandwich-type hybridization reaction, the gold nanoparticle-labeled reporter DNA was bound onto the DNA sensor surface to further induce silver deposition in the presence of silver enhancer solutions. The electrochemical stripping signal of the deposited silver nanoparticles in KCl solution was used to monitor the hybridization reaction. Signal amplification enhanced the sensitivity for target DNA detection. The proposed method could detect target DNA at a linear range from 1.0pM to 70pM, with a detection limit of 0.3pM. The DNA sensor exhibited good stability and acceptable reproducibility. The sensor also showed selectivity against non-complementary target DNA.

A gold electrode modified with amino-modified reduced graphene oxide, ion specific DNA and DNAzyme for dual electrochemical determination of Pb(II) and Hg(II)

Multilayered reduced graphene oxide (rGO) was functionalized with amino groups by treatment with nitrogen plasma. Raman spectroscopy showed plasma treatment not to substantially alter the chemical structure of rGO and that a wide range of functional nitrogen groups is evenly incorporated into the carbon lattice. The amino-modified rGO was used to design an electrochemical biosensor in which a DNAzyme, substrate DNA and Pb(II) and Hg(II) binding DNA were immobilized on the amino-rGO placed on a gold electrode. The high concentration of amino groups and the rough surface of the rGO favor DNA immobilization. Heavy metal ions are bound to the surface via specific interaction between DNA and the two ions which are detected by electrochemical impedance spectroscopy at a potential of 0.2 V (vs. Ag/AgCl). The detection limits for Pb(II) and Hg(II) are as low as 7.8 and 5.4 pM, respectively, and the analytical ranges extend from 0.01 to 100 nM. The sensor is highly specific and stable and therefore represents a highly promising tool for use in environmental monitoring.

'Spotted Nanoflowers': Gold-seeded Zinc Oxide Nanohybrid for Selective Bio-capture.

Hybrid gold nanostructures seeded into nanotextured zinc oxide (ZnO) nanoflowers (NFs) were created for novel biosensing applications. The selected ‘spotted NFs’ had a 30-nm-thick gold nanoparticle (AuNP) layer, chosen from a range of AuNP thicknesses, sputtered onto the surface. The generated nanohybrids, characterized by morphological, physical and structural analyses, were uniformly AuNP-seeded onto the ZnO NFs with an average length of 2–3 μm. Selective capture of molecular probes onto the seeded AuNPs was evidence for the specific interaction with DNA from pathogenic Leptospirosis-causing strains via hybridization and mis-match analyses. The attained detection limit was 100 fM as determined via impedance spectroscopy. High levels of stability, reproducibility and regeneration of the sensor were obtained. Selective DNA immobilization and hybridization were confirmed by nitrogen and phosphorus peaks in an X-ray photoelectron spectroscopy analysis. The created nanostructure hybrids illuminate the mechanism of generating multiple-target, high-performance detection on a single NF platform, which opens a new avenue for array-based medical diagnostics.

β-Cyclodextrin functionalized graphene as a highly conductive and multi-site platform for DNA immobilization and ultrasensitive sensing detection.

A versatile nanocomposite containing β-cyclodextrin and graphene (CD-GR) was prepared through a simple chemical reduction method. The characterization experiments show that the nanocomposite remains the flake-like morphology of GR, but its solubility and stability in aqueous solution are greatly improved. Then the nanocomposite was modified at glassy carbon electrode (GCE) surface, and was used as a functional matrix for the covalent immobilization of probe DNA using 2,4,6-trichloro-1,3,5-triazine (TCT) as the crosslinker. Due to the synergetic effect of large surface area of GR and rich hydroxyl of CD, the probe density for the developed biosensor was determined to as high as 3.82×10(13) molecules cm(-2). Meanwhile, the biosensor shows high hybridization efficiency and hybridization kinetic. When the biosensor was applied for the impedance-based hybridization test, a wide linear range from 1.0×10(-16) to 1.0×10(-12) M and an ultralow detection limit of 3.4×10(-17) M were achieved. The biosensor also displays excellent stability, selectivity, and reproducibility.

Inverse piezoelectricity of single-stranded DNA film on microcantilever

The paper aims to reveal the relation between the inverse piezoelectricity of singlestranded DNA (ssDNA) film and the origin and sign of the deflection of microcantilever produced by the DNA immobilization. By using Zhang’s two-variable method for strain field of laminated beam and the linearized Poisson-Boltzmann equation for bioelectric potential of polyelectrolyte brush, an approximate model is presented to confirm the previous conjecture that the sign of the piezoelectric constant of ssDNA film might decide the sign of the surface stress. There is only one fitting parameter (piezoelectric stress constant) needed in the simplified model, which greatly reduces the difficulty of identifying the nanoscale electrical/mechanical properties of DNA film. Numerical fitting curves based-on the analytical models agree well with the pertinent experimental dada. The difference between the previous four-layered model and the present two-layered approximate model is also compared.

An In-House Approach for Fabrication of Poly-Silicon Nanobiosensor Using Conventional Photolithography and Etching Method

In this work, we demonstrated a method to fabricate and characterize poly-silicon nanowires for biosensing application using conventional photolithography and etching process. Nanowires Mask must be first designed using AutoCAD, before patterning onto chrome mask. Chrome mask was used for better photo masking and to transfer structure onto poly-silicon layer. The poly-silicon nanowires process flow were developed which includes all the fabrication process such as growth, deposition, lithography and etching process. In order to prove the effectiveness of the fabricated devices as a biologically sensor (Biosensor), the poly-silicon nanowires is modified chemically to allow the integration with biological element. The drain (Id) was found to increase after the DNA immobilization and hybridization. These results demonstrate that the in-house fabricated poly-silison nanobiosensor is capable as a platform for label-free biosensing. The morphological characterizations were carried out using a Scanning Electron Microscope (SEM) and Atomic Force Microscope (AFM). Besides that, the electrical measurement of the poly-silicon biosensor were carried out using a KEITHLEY 6487 picoampmeter/voltage source.

Hydroxyl radical and thermal annealing on amorphous InGaZnO4 films for DNA immobilizations

The effect of hydroxyl radicals (OH) and thermal annealing on an amorphous InGaZnO4 (aIGZO) film surface was investigated for manipulation of DNA immobilization. X-ray photoemission and fluorescence measurements were conducted to reveal the status of surface OH coverage and DNA immobilization, respectively. Systematic examinations concerning OH termination on the film surface suggested that the surface coverage of OH leveling DNA immobilization was related to the local surface potential. Furthermore, OH affinity on the aIGZO film surface was sensitive to thermal annealing. A remarkable change in surface OH coverage was observed for the film surface annealed at high temperature. This behavior was framed by a structural change from amorphous to crystalline state, which regulated DNA immobilization. These results indicate that the OH affinity on aIGZO films is dependent on structural properties such as defects. This study suggests that an amorphous structure is critical for obtaining a high OH surface coverage governing DNA immobilization, and is hence more suitable for biosensing.

Impedance sensing of DNA immobilization and hybridization by microfabricated alumina nanopore membranes

In this work we demonstrate microfabricated thin alumina nanopore membranes as a platform for impedance sensing of DNA immobilization and hybridization. We develop a wafer-scale fabrication of free-standing alumina nanopore membranes with well controlled thickness, pore diameter and overall pore density. One 1cm×1cm single chip contains an array of 69 membranes. Each membrane is 100μm×100μm large and 2μm thick, with pore diameter of 120nm. With low pore density of ∼6pores/μm2, nanopore resistance and membrane capacitance can be recognized clearly in the electrochemical impedance spectrum from 100 to 1MHz. The total surface area can be further increased by the coating of silica nanoparticles with ∼20nm in diameter. During the immobilization of probe ssDNA to (3-glycidoxypropyl) trimethoxysilane functionalized surface, the nanopore resistance drops significantly by 80%, whereas the membrane capacitance increases less than 2%. After hybridization with complementary DNA, the nanopore resistance increases up to 10%. Non-complementary ssDNA has no obvious effect. The detection limit is 12.5nM in phosphate-buffered saline (PBS) solution.

Development of Electrochemical DNA Biosensors-A Review

Electrochemical DNA biosensors are used widely due to its sensitivity, accessibility and accuracy. This electrochemical era in DNA biosensors took almost three decades for its development and application. Purine bases in the DNA namely guanine and adenine oxidizes at the working electrode during application of a positive potential. This property of DNA has been utilized in electrochemical DNA biosensors in various fields from the clinical diagnostics, biomedical, forensic applications till environmental studies. With the use of nanotechnology, various nanomaterials have been utilized over the past years in order to increase the sensitivity of the biosensor. A clear understanding on the DNA immobilization strategy is required for sensor preparation for its various applications. A detailed account on the initial developments till current scenario in electrochemical DNA biosensors have been provided in order to understand the basics of electrochemical DNA biosensor and its application in developing a DNA biosensor.

A Novel DNA Probe Based on Molecularly Imprinted Polymer Modified Electrode for the Electrochemical Monitoring of DNA

AbstractA DNA probe that was based on methylene blue (MB) imprinted polyvinyl pyridine polymer (MIP) modified carbon paste electrodes were developed for the first time for electrochemical monitoring of DNA. Probes were built up by adsorbing MB onto modified electrodes prior to DNA immobilization. It was shown that DNA strongly immobilizes on MIP modified electrodes when MB was adsorbed in advance of DNA immobilization. The performance of the MB imprinted polymer modified carbon paste electrodes (MIP‐CPE) to rebind the template molecule (MB) were compared to those of control polymer modified (non‐imprinted polymer NIP‐CPE) and bare (CPE) electrodes. Electrochemical signal resulting from the oxidation of guanine moiety of the immobilized probe DNA was high enough on the constructed platform, implicating that probes of this kind could be favorably used for DNA analysis. These probes exhibited high selectivity for its complementary DNA sequences (target). HBV‐DNA hybridization was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The detection limit of the probe for the target DNA was 8.72 µg/mL (1.38 µM), which was better than those attained by some earlier DNA sensor studies.

Label-free colorimetric sensor for mercury(II) and DNA on the basis of mercury(II) switched-on the oxidase-mimicking activity of silver nanoclusters

In this paper, a novel colorimetric biosensor for Hg2+ and DNA molecules is presented based on Hg2+ stimulated oxidase-like activity of bovine serum albumin protected silver clusters (BSA-Ag NCs). Under mild conditions, Hg2+ activated BSA-Ag NCs to show high catalytic activity toward the oxidation of 3,3′,5, 5′-tetramethylbenzidine (TMB) using ambient dissolved oxygen as an oxidant. The oxidase-like activity of BSA-Ag NCs was “switched-on” selectively in the presence of Hg2+, which permitted a novel and facile colorimetric sensor for Hg2+. As low as 25nmolL−1Hg2+ could be detected with a linear range from 80nmolL−1 to 50mmolL−1. In addition, the sensing strategy was also employed to detect DNA molecules. Hg2+ is known to bind very strongly and specifically with two DNA thymine bases (T) to form thymine–Hg2+–thymine (T–Hg2+–T) base pairs. The hairpin-structure was disrupted and Hg2+ ions were released after hybridization with the DNA target. By coupling the Hg2+ switched-on the oxidase-mimicking activity of BSA-Ag NCs, we developed a novel label-free strategy for facile and fast colorimetric detection of DNA molecules. More important, target DNA can be detected as low as 10nmolL−1 with a linear range from 30 to 225nmolL−1. Compared with other methods, this method presents several advantages such as the independence of hydrogen peroxide, high sensitivity and good selectivity, avoiding any modification or immobilization of DNA, which holds a great potential of metal NCs for clinical application in biosensing and biotechnology.

Electrochemical DNA hybridization sensors based on conducting polymers.

Conducting polymers (CPs) are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective.

Simple and label-free detection of DNA hybridization on a modified graphene nanosheets electrode

In this study, an effective method was devised to synthesize amelogenin genes in solution and to amplify electrical detection of DNA hybridization based on graphene nanosheets (GNs) modified glassy carbon electrode (GCE). GNs are well known as effective biocompatible and conductive materials that can provide large surface area and a sufficient numbers of binding points for DNA immobilization. The biosensor fabrication processes and the electrochemical responses of probe immobilization and hybridization with target DNA were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) using [Fe(CN)6]3−/4− as an electrochemical redox. Due to minimum nonspecific DNA adsorption, a very high specificity of DNA hybridization was achieved, and the hybridization rate of the target DNA in optimum conditions was increased significantly. With this approach, the target DNA could be quantified in a linear range from 1.0×10−20 to 1.0×10−14molL−1 for the first segment and from 1.0×10−13 to 1.0×10−6molL−1 for the second segment, with a detection limit of 7.1×10−21molL−1 by 3sb. In addition, the biosensor exhibited a high level of stability and repeatability, even for the determination of DNA sequences in real samples without amplification.

Development of DNA monitoring platform based on poly(xanthurenic acid) functionalized FePt/reduced graphene oxide

A freely switchable DNA monitoring platform based on poly(xanthurenic acid) functionalized FePt/reduced graphene oxide (PXa-FePt/RGO) was developed, where the PXa and FePt/RGO were synchronously synthesized via an effective pulse potentiostatic method. The obtained nanocomposite integrated the advantages of the PXa and FePt/RGO, including rich-conjugated structures, good conductivity, and fine biocompatibility, which not only served as a substrate for DNA immobilization but also reflected the electrochemical transduction originating from DNA immobilization and hybridization without any complicated labeling or outer indicators. The immobilization of probe DNA was successfully conducted via noncovalent assembly due to the π–π* interaction between the conjugated nanocomposite and DNA bases. After the hybridization of probe DNA with target DNA, the formation of double-helix structure induced dsDNA to release from the surface of conjugated nanocomposite, accompanied with the self-signal regeneration of nanocomposite (“signal-on”). The developed biosensor exhibited excellent performance for the detection of the sequence-specific DNA from chronic myelogenous leukemia by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with a detection limit of 2.1 × 10−16 mol/L.

Selective immobilization and detection of DNA on biopolymer supports for the design of microarrays

DNA immobilization for the manufacturing of microarrays requires sufficient probe density, low unspecific binding and high interaction efficiency with complementary strands that are detected from solutions. Many of these important parameters are affected by the surface chemistry and the blocking steps conducted during DNA spotting and hybridization. This work describes an alternative method to selectively immobilize probes and to detect DNA on biocompatible, hydrophilic cellulose coated supports with low unspecific binding, high selectivity and appropriate sensitivity. It takes advantage of a relatively selective adsorption of water soluble polysaccharides on a solid cellulose matrix. Single strands of DNA were conjugated to this soluble polysaccharide and subsequently micro-spotted on solid cellulose thin films that were coated on glass and polymer slides. This resulted in adsorptively bound DNA-probes that were used to detect complementary, labelled DNA strands with different lengths and sequences by hybridization. The interaction of the DNA-conjugates with cellulose surfaces and the selectivity of hybridization were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence scanning. The method of non-covalent immobilization of DNA probes on an uncharged, non-reactive, hydrophilic support lowers the unspecific binding and the number of handling steps required to conduct the experiments for the detection of DNA on microarrays. Simultaneously selectivity, hybridization efficiency and detection limits are maintained.