DNA Hybridization Indicator Research Articles

Ninhydrin as a novel DNA hybridization indicator applied to a highly reusable electrochemical genosensor for Candida auris

This work reports a simple strategy for Candida auris genomic DNA (gDNA) detection, a multi-resistant fungus associated with nosocomial outbreaks in healthcare settings, presenting high mortality and morbidity rates. The platform was developed using gold electrode sensitized with specific DNA capture probe and ninhydrin as a novel DNA hybridization indicator. The genosensor was able to detect C. auris in urine sample by differential pulse voltammetry and electrochemical impedance spectroscopy. The biosensor's analytical performance was evaluated by differential pulse voltammetry, detecting up to 4.5 pg μL−1 of C. auris gDNA in urine (1:10, V/V). Moreover, the genosensor was reused eight times with no loss in the current signal response. The genosensor showed selectivity and stability, maintaining 100% of its response up to 80 days of storage. In order to analyze interactions of single and double-stranded DNA with ninhydrin, SEM, AFM and molecular dynamics studies followed by docking simulations were performed. Theoretical calculations showed ninhydrin interactions more favorably with dsDNA in an A-T rich binding pocket rather than with the ssDNA. Therefore, the proposed system is a promising electrochemical detection device towards a more accurate detection of C. auris gDNA in biological samples.

An electrochemical DNA biosensor fabricated from graphene decorated with graphitic nanospheres

Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40–80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10−16 to 1.0 × 10−8 M with a limit of detection (LOD) of less than 1.0 × 10−16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.

A planar and uncharged copper(II)-picolinic acid chelate: Its intercalation to duplex DNA by experimental and theoretical studies and electrochemical sensing application

Using an external redox-active molecule as a DNA hybridization indicator is still a popular strategy in electrochemical DNA biosensors because it is label-free and the multi-site binding can enhance the response signal. A planar and uncharged transition metal complex, Cu(PA)2 (PA = picolinic acid) with excellent electrochemical activity has been synthesized and its interaction with double-stranded DNA (dsDNA) is studied by experimental electrochemical methods and theoretical molecular docking technology. The experimental results reveal that the copper complex interacts with dsDNA via specific intercalation, which is verified by the molecular docking result. The surface-based voltammetric analysis demonstrates that the planar Cu(PA)2 can effectively accumulate within the electrode-confined hybridized duplex DNA rather than the single-stranded probe DNA. Based on this phenomenon, the Cu(PA)2 is utilized as an electrochemical hybridization indicator for the detection of oligonucleotides. The sensing assays show that upon incubation in Cu(PA)2 solution, the probe electrode does not display any Faraday signal, but the hybridized one has a pair of strong redox peaks corresponding to the electrochemistry of Cu(PA)2, showing excellent hybridization indicating function of Cu(PA)2 without background interference. The signal intensity of Cu(PA)2 is dependent on the concentrations of the target oligonucleotide ranging from 1 fM to 100 nM with an experimental detection limit of 1.0 fM. Due to the specific intercalation of Cu(PA)2 with dsDNA, the biosensor also exhibits good ability to recognize oligonucleotide with different base mismatching degree.

Determination of Electrochemical Interaction between 2‐(1H‐benzimidazol‐2‐yl) Phenol and DNA Sequences

AbstractA benzimidazole derivate, 2‐(1H‐benzimidazol‐2‐yl) phenol (2‐Bip) and its interaction mechanism with sequence specific DNA was examined with Differential Pulse Voltammetry (DPV). We, for the first time, investigated the effect of 2‐Bip on sequence specific DNA with electrochemical methods by evaluating both guanine and 2‐Bip oxidation signal changes. In the study, probe sequences were immobilized to the surface of the electrodes and then hybridization was achieved by sending the complementary target onto the probe modified electrodes. Following the hybridization, 2‐Bip solution was interacted with probe and hybrid sequences to see the effect of 2‐Bip on different DNA sequences. The binding constant (K), toxicity (S%) and thermodynamic parameters, i. e., Gibbs free energy (ΔG°) of 2‐Bip‐DNA complexes were evaluated. K was calculated as 5×105 and the change in the ΔG° was found as −32.50 kJ mol−1, which are consistent well with the literature. Furthermore, S% showed that 2‐Bip is moderately toxic to single stranded DNA (ssDNA) and toxic to double stranded DNA (dsDNA). From our experimental data, we made four conclusions (i) 2‐Bip affects both ssDNA and dsDNA, (ii) 2‐Bip interaction mode with DNA could be non‐covalent interactions, (iii) 2‐Bip could be used as new DNA hybridization indicator due to its distinct effects on ssDNA and dsDNA, (iv) 2‐Bip could be used as a drug molecule for its DNA effect.

Design of an electrochemical DNA-based biosensor for selective determination of cadmium ions using a DNA hybridization indicator

A simple electrochemical DNA- based biosensor was designed for determination of Cd2+ using ethyl green (EG) as a DNA hybridization indicator on the surface of carbon paste electrode (CPE). The interaction of Cd2+ with double strand DNA (dsDNA) led to the destabilizing of dsDNA and the increase in the reduction peak currents of EG. The difference in the values of electrochemical responses of EG before and after DNA helix destabilization in the presence of Cd2+ (ΔI) was used for Cd2+ determination and was linearly related to the concentration of Cd2+ over the ranges of 1.0pM–1.0nM and 10.0nM–1.0μM. Detection limit was achieved as low as 0.3pM using simple and low cost working electrode and without applying any materials for modification of electrode or DNA. The proposed biosensor was utilized successfully to real sample analysis of Cd2+.

Introducing curcumin as an electrochemical DNA hybridization indicator and its application for detection of human interleukin-2 gene

In this paper, the application of curcumin (CU) as a non-toxic electrochemical DNA hybridization indicator was described. Hybridization investigations on a pencil graphite electrode surface as a transducer using oligonucleotides containing only one base type, including poly A, poly T, poly C, and poly G as probe and as related complementary/non-complementary sequences, showed that CU has no specific interaction with each of the oligonucleotides of DNA. Furthermore, results showed good interaction between CU and the hybridized form of oligonucleotides; thus, the extent of hybridization was evaluated based on the difference between differential pulse voltammetry (DPV) signals of CU accumulated on the probe-pencil graphite electrode (PGE) and CU accumulated on the probe-target-PGE. Then, the developed biosensor was successfully applied for the detection of short sequences of human interleukin-2 (hIL-2) gene as a model. A hybridization experiment with non-complementary oligonucleotide showed that the suggested DNA sensor responds selectively to the target. At optimized conditions, two linear ranges were obtained for hIL-2 gene, first from 50 to 1000 pM and second from 0.01 to 1 μM with a detection limit of 12 pM. 7.0) containing 20 mM NaCl.

Gelatin methacrylate (GelMA) mediated electrochemical DNA biosensor for DNA hybridization

In this study, an electrochemical biosensor system for the detection of DNA hybridization by using gelatin methacrylate (GelMA) modified electrodes was developed. Electrochemical behavior of GelMA modified Pencil Graphite Electrode (PGE) that serve as a functional platform was investigated by using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) and compared with those of the bare PGE. Hybridization was achieved in solution phase and guanine oxidation signal changes were evaluated. The decrease in the guanine oxidation peak currents at around +1.0V was used as an indicator for the DNA hybridization. Also, more interestingly GelMA intrinsic oxidation peaks at around +0.7V changed substantially by immobilization of different oligonucleotides such as probe, hybrid and control sequences to the electrode surface. It is the first study of using GelMA as a part of an electrochemical biosensor system. The results are very promising in terms of using GelMA as a new DNA hybridization indicator. Additionally, GelMA modified electrodes could be useful for detecting ultra low quantity of oligonucleotides by providing mechanical support to the bio-recognition layer. The detection limit of this method is at present 10-12mol. Signal suppressions were increased from 50% to 93% for hybrid with using GelMA when it was compared to bare electrode which facilitates the hybridization detection.

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5′-(NH 2) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β- d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0 × 10 −10 mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0 × 10 2 to 2.0 × 10 3 cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.

DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease

A sequence-specific electrochemical sexually transmitted disease (STD) sensor based on self-assembled monolayer of thiolated DNA probe specific to target opa gene for detection of Gonorrhoea, a sexually transmitted disease has been fabricated. 6-Mercapto-1-hexanol (MCH) has been used as a blocking agent to facilitate oligos “stand” up at the surface, a configuration favoring subsequent DNA hybridization and to repel non-specific adsorption of undesired DNA. The results of differential pulse voltammetric studies of this STD sensor reveal low detection limit (1.0 × 10 −18 M) and a wide dynamic range (from 1.0 × 10 −6 M to 0.5 × 10 −18 M) arising due to the stable hybridization using methylene blue as an electro-active DNA hybridization indicator. The experimental results with genomic DNA, clinical patient sample of Neisseria gonorrhoeae, culture of non- N. gonorrhoeae Neisseria species (NgNS) and gram negative bacteria indicate that the fabricated sensor is specific to this STD .

A promising bioelectrode based on gene of Mycobacterium leprae immobilized onto poly(4‐aminophenol)

AbstractA new bioelectrode for gene detection of Mycobacterium leprae, also known as Hansen's bacillus, was produced by immobilizing of single‐stranded DNA (ssDNA) with 78 bases long (specific gene related to Mycobacterium leprae) on graphite electrode modified with poly(4‐aminophenol). This biosensing platform was able to recognize complementary DNA molecules via hybridization process. Hybridization between probe and target was monitored by voltammetry, using ferrocenecarboxyaldehyde as electrochemical DNA hybridization indicator. The hybridization of nucleic acid probe with the DNA target resulted in significant decrease in the oxidation peak current of ferrocenecarboxyaldehyde, indicating greater affinity of this compound for ssDNA than for double‐strand DNA (dsDNA). The linear range of detection for the DNA target was found to be 0.35 – 35 ng/μL. ssDNA hybridization with the DNA target was also investigated by electrochemical impedance spectroscopy (EIS), showing significant modification in Nyquist plot, by modification in electrode surface after addition of the complementary target. The effective immobilization of specific gene of Mycobacterium leprae onto graphite electrode modified with poly(4‐aminophenol) and the detection of the hybridization process with the DNA target, monitored by voltammetry and EIS indicate that this is a new and promising biosensing platform to gene detection of Hansen's bacillus. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Methylene Blue Interactions with Chromosomal and Plasmid DNA on Screen‐Printed Carbon Electrodes

AbstractMethylene blue (MB) is a typical photosensitizing agent and a DNA hybridization indicator, but its modes of interaction with the DNA molecules are not clearly described, particularly in relation to its electrochemical oxidation signals. To probe the DNA‐MB interactions we have used chromosomal salmon testes and supercoiled plasmid sc pUC19 DNA immobilized on home‐made screen‐printed electrodes (SPEs) and a wide range of MB concentrations, from nano‐ to micromolar. The applicability of the home‐made screen‐printed electrodes used for the DNA‐MB studies were tested using standard calf thymus DNA. Two MB oxidation peak signals: MB(I) at ca. −0.18 V and MB(II) at 0 V vs. Ag/AgCl were detected within ±10–15% standard deviation, signals different from adsorbed MB signal (−0.25 V, pH 4.7). The MB(I) signal, seen when both DNAs were used, showed two plateaus, one at nano‐ and another at micromolar MB concentrations; these were accompanied by the changes in the oxidation signal at 0.98 V, usually attributed to guanine oxidation. In contrast, the MB(II) signal was only seen for salmon testes DNA, indicating various modes of MB interactions with chromosomal and plasmid DNA. In the presence of MB, the guanine related signal (G) at 0.98 V has been amplified significantly (10×), allowing for the identification of the DNAs at low DNA concentrations, the feature particularly useful in the plasmid sc pUC19 detection. The use of another DNA intercalator, riboflavin (RF), aided in the identification of the relation between MB(I), MB(II) and G oxidation signals.

Electrochemical DNA sensor for Neisseria meningitidis detection

Meningitis sensor based on nucleic acid probe of Neisseria meningitidis has been fabricated by immobilization of 5′-thiol end labeled single stranded deoxyribonucleic acid probe (ssDNA-SH) onto gold (Au) coated glass electrode. This ssDNA-SH/Au electrode hybridized with the genomic DNA (G-dsDNA/Au) and amplified DNA (PCR-dsDNA/Au) has been characterized using atomic force microscopy (AFM), Fourier transforms infrared spectroscopy (FT-IR) and electrochemical techniques. The ssDNA-SH/Au electrode can specifically detect upto 10–60 ng/μl of G-dsDNA-SH/Au and PCR-dsDNA-SH/Au of meningitis within 60 s of hybridization time at 25 °C by cyclic voltammetry (CV) using methylene blue (MB) as electro-active DNA hybridization indicator. The values of sensitivities of the G-dsDNA-SH/Au and PCR-dsDNA-SH/Au electrodes have been determined as 0.0115 μA/ng cm −2 and 0.0056 μA/ng cm −2, respectively with regression coefficient ( R) as 0.999. This DNA bioelectrode is stable for about 4 months when stored at 4 °C.

STD sensor based on nucleic acid functionalized nanostructured polyaniline

STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe (from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized nanostructured-polyaniline coated onto indium–tin-oxide-coated glass plate has been fabricated using avidin–biotin as cross-linking agent. This DNA functionalized electrode can specifically detect upto 0.5×10−15M of complementary target within 60s of hybridization time at 25°C by differential pulse voltammetry (DPV) using methylene blue as electro-active DNA hybridization indicator. This highly sensitive and specific nucleic acid functionalized nanostructured-polyaniline electrode can distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients.

Synthesis of a naphthalene diimide derivative having four ferrocene moieties as an electrochemical DNA hybridization indicator

A new naphthalene diimide derivative carrying four ferrocene moieties, F(4)ND, was synthesized by the reaction of trimethylammoniummethylferrocene with the terminal amino moieties of two imide substituents of naphthalene diimide. Spectrophotometric binding studies of F(4)ND with calf thymus DNA showed its high affinity of 2 x 10(5) M(-1) in 10 mM MES buffer and 1 mM EDTA (pH 6.25) containing 0.10 M NaCl. The Osteryoung square wave voltammetry (SWV) of a DNA probe-immobilized electrode before and after hybridization with target DNA gave a larger peak current in an electrolyte containing F(4)ND than that containing FND carrying two ferrocene moieties. The current peak difference between mismatched and fully matched DNA was also greater with F(4)ND than that with FND.

Electrochemical Detection of DNA Hybridization via Bis-Intercalation of a Naphthylimide-Functionalized Viologen Dimer

The synthesis and DNA binding properties of a bis-naphthyl imide tetracationic diviologen compound NI(CH2)3V(2+)(CH2)6V(2+)(CH2)3NI (where V(2+) = 4,4'-bipyridinium and NI = naphthyl imide, NIV) are described. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for reduction of the V(2+) state to the V+ (viologen radical cation) state. Isotherms and binding constants for this molecule to both forms of immobilized DNA were generated in this fashion. The character of the binding isotherm for dsDNA suggests bis-intercalation. Under high saline conditions, the diviologen molecule dissociated 160 times slower from dsDNA compared to ssDNA. Slow dissociation kinetics from dsDNA (kd =7.0 x 10-5 s(-1)) allow this molecule to be used as an effective DNA hybridization indicator.

Escherichia coli Genosensor Based on Polyaniline

Avidin-modified polyaniline (PANI) electrochemically deposited onto a Pt disk electrode has been utilized for direct detection of Escherichia coli by immobilizing a 5'-biotin-labeled E. coli probe (BdE) using a differential pulse voltammetric technique in the presence of methylene blue as a DNA hybridization indicator. Depending on the target sample and the sonication time, this BdE-avidin-PANI bioelectrode can be utilized to electrochemically detect a complementary target probe (0.009 ng/microL), E. coli genomic DNA (0.01 ng/microL) and 11 E. coli cells/mL in 60 s to 14 min (hybridization time) without using PCR and can be used 5-7 times at temperatures of 30-45 degrees C.

Electrochemical DNA Biosensors Based on DNA-Drug Interactions

The world of drug designing is ever changing. The investigations of drug-DNA interactions would provide new compounds to be tested for an effect on a biochemical target, and also to be used as promising hybridization indicators for the design of DNA biosensors, which will further become DNA microchip systems. An overview is reported here about DNA biosensors based primarily on drugs interacting with DNA and shows how to determine this interaction electrochemically, the quantification of drug and/or DNA, and the promising applications of these drugs as DNA hybridization indicator. The applications of these electrochemical DNA biosensors are described and discussed.