Addition Of dsDNA Research Articles

A Eu3+doped functional core-shell nanophosphor as fluorescent biosensor for highly selective and sensitive detection of dsDNA

Lanthanide-doped core-shell nanomaterials have illustrated budding potential as luminescent materials, but their biological applications have still been very limited due to their aqueous solubility and biocompatibility. Here, we report a simple and cost-effective approach to construct a water-stable chitosan-functionalized lanthanoid-based core shell (Ca-Eu:Y2O3@SiO2) nanophosphor. The as-synthesized Ca-Eu:Y2O3@SiO2-chitosan (CEY@SiO2-CH) nanophosphor has been characterized for its structural, morphological, and optical properties, by employing different analytical tools. This sensing platform is suitable for dsDNA probing by tracing the “turn on” fluorescence signal generated by CEY@SiO2-CH nanophosphor with the addition of dsDNA. The ratio of fluorescence intensity enhancement is proportional to the concentration of dsDNA in the range 0.1–90 nM, with the limit of detection at ⁓16.1 pM under optimal experimental conditions. The enhancement in fluorescence response of functionalized core-shell phosphor with dsDNA is due to the antenna effect. Additionally, response of probe has been studied for the real samples displaying percent recovery in between 101 and 105, maximum RSD% upto 5.23 (n = 3). This outcome can be applied to the selective sensing of dsDNA through optical response. These findings establish the CEY@SiO2-CH a simple, portable, and potential candidate as a sensor for rapid and analytical detection of dsDNA.

Ratiometric sensor based on imprinted quantum dots-cationic dye nanohybrids for selective sensing of dsDNA

A ratiometrically responsive sensor for dsDNA is reported, based on molecularly imprinted polymer coated quantum dots (MIP-QDs). A new platform is described for probing dsDNA by tracing the “turn on” fluorescence signal of malachite green (MG) as a cationic dye and “turn off-on” room temperature phosphorescence (RTP) signal of MIP-QDs/MG nanohybrids. The interaction between MIP-QDs surface and MG discloses an intense quenching in RTP (turning off) by a phosphorescence resonance energy transfer (PRET) process. After the addition of dsDNA, MG molecules escape from the MIP-QDs surface and intercalate into the dsDNA, resulting in the restoration of RTP intensity of MIP-QDs (turning on) and also enhancing in fluorescence of MG. This outcome hereby can be employed for the selective sensing of dsDNA via optical response. The ratio of fluorescence enhancement of MG to RTP intensity of MIP-QDs is proportional to the concentration of dsDNA in the range of 0.089–1.79 μg/mL with a detection limit (3σ/K) of 19.48 ng/mL under the optimized experimental conditions.

New role of oil red O in detection of double stranded DNA

Oil red O (ORO) is a commonly used dye for the tissue staining, which recognizes nitroglycerin and cholesterol esters. In this paper, we describe an unexpected finding that ORO can be fluorescently lighted up upon meeting dsDNA with emission peak localized at 600 nm, although it almost does not fluoresce by itself. This property triggered us to test its potential as a dsDNA fluorescence probe. The interaction of dsDNA and ORO was demonstrated by spectral and thermodynamic analysis and several other means. The fluorescence of ORO was gradually increased following the addition of dsDNA, which showed linear response toward dsDNA in the concentration range from 0.02 to 0.64 mg/mL with a detection limit of 0.69 μg/mL. Moreover, ORO showed excellent selectivity when testing with a variety of dsDNA-like biomolecules. Besides, ORO resisted photobleaching and worked very well under biological background. Altogether, we propose here a new nucleic acid lighting up fluorescence probe for double stranded dsDNA detection.

Bright green-emitting ds-DNA labeling employed by dicationic monomethine cyanine dyes: Apoptosis assay and fluorescent bio-imaging

Fluorescent based techniques are among the state-of-the-art detection methods utilized for both qualitative and quantitative investigations in biochemical and biomedical sciences. The essential methods for evaluation of apoptosis and cell cycle by measuring DNA content using flow cytometry and imaging experiments, such as fluorescent microscopy are extensively being used for staining of live and/or apoptotic cells. Among a wide diversity of commercially available fluorescent dyes, cyanine-based non-covalent labeling has been employed for several years as fluorescent probes mainly due to their capabilities for biological applications upon binding to nucleic acids.Three novel asymmetric dicationic monomethine cyanine dyes derived from 2-thiomethylbenothiazolium and 4-methylquinolinium salts were synthesized and have been tested for potential applicability in biological research as fluorescent labels for nucleic acids detection. The newly obtained cyanine probes became highly fluorescent upon addition of ds-DNA according to a well-known mechanism of this class of organic compounds. Employing flow cytometry and fluorescent microscopy, the dyes were evaluated in live-dead cells discrimination. Moreover, FACS studies on 3T3 mouse embryonic fibroblast cells verified their inability to penetrate through intact plasma membrane. Further in-depth studies on the dyes, outline their capability for estimations of cell viability and staining late apoptotic/necrotic cells.Imaging experiments by means of fluorescent microscopy, delineate the dyes' high specificity towards ds-DNA and allowed nuclear morphology imaging of non-living ethanol-fixed cells.

Electrochemistry of the Interaction between Bioactive Drugs Daunorubicin and Dopamine and DNA at a Water/Oil Interface

We describe here a voltammetric method based on the ion transfer at a water/oil interface for the electrochemical study of the interaction between high molecular weight dsDNA and two molecules of biological interest: the anthracycline drug daunorubicin (DNR) and the neurotransmitter dopamine (DA). The binding constants of the complexes were determined by performing an amperometric titration, following the decrease of ion transfer currents upon addition of dsDNA, using differential pulse voltammetry (DPV) technique. Binding constants were interpreted in terms of the main interaction mode of the molecules with DNA. A binding constant of 1.7×104M−1 was obtained for the intercalation complex of DNR into DNA base pairs (bp), while a binding constant of 1.8×103M−1 was obtained for the electrostatic interaction between positively charged DA and negatively charged dsDNA, using a non-linear binding model. The binding site size, in terms of base pairs, for the interaction between the two molecules and DNA was also estimated using non-linear regression analysis. Two binding site models were tested and compared in this work.

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles.

Enhanced sequence-specific recognition of double-stranded DNA (dsDNA) was realized by using hybridization chain reaction (HCR) and positively charged gold nanoparticles ((+)AuNPs) dual signal amplification. To construct such a sensor, capture probe was initially assembled onto gold electrode surface. Upon addition of dsDNA, sandwiched DNA complex was formed between the capture probe and the detection probe, then another exposed part of the detection probe opened two alternating DNA hairpins (H1 and H2) in turn and initiated HCR to form a double-helix. Meantime, (+)AuNPs were electrostatically adsorbed onto such double-helix to amplify the electrochemical signal. Upon optimal conditions, the electronic signals of ferrocene (Fc) that modified on H1 and H2 increased linearly with increasing dsDNA concentration over the range from 15 pM to 1.0 nM, with a detection limit of 2.6 pM. Moreover, the proposed method showed good sequence specificity for dsDNA recognition.

L-Cysteine capped Mn-doped ZnS quantum dots as a room temperature phosphorescence sensor for in-vitro binding assay of idarubicin and DNA.

L-cysteine capped Mn doped ZnS quantum dots/ Idarubicin (IDA) nanohybrids were used as novel room temperature phosphorescence (RTP) sensor to detect double stranded deoxyribonucleic acid (ds-DNA)/drug interaction. IDA, anthracycline derivative anticancer drug, was adsorbed on the surface of the QDs as an electron acceptor to quench the RTP emission. The RTP intensity of QDs was quenched quickly upon addition of quencher and the reaction reached equilibrium within 2 min. The quenching mechanism of phosphorescence of Mn-doped ZnS QDs by IDA is a combined dynamic and static quenching. The static and dynamic quenching constants were found as 1.1×10(5) M(-1) and 8.7×10(4) M(-1), respectively. The addition of ds-DNA caused formation of ds-DNA/IDA complex and recovered the RTP signal of Mn-doped ZnS QDs, which allowed qualitative analysis. Under optimal conditions, RTP intensity of QDs/IDA nanohybrids increased linearly with the concentration of ds-DNA from 1.2 to 6.0 µM. This method is simple, low cost and avoids from interferences.

Polarographic determination of DNA based on its interaction with the phenanthroline-zinc(II) complex

By using the linear sweep voltammetric technique, a phenanthroline (Phen) and zinc(II) (Phen-Zn(II)) complex was used as the electrochemical probe for the determination of double-stranded (ds) DNA. In pH 9.0 Britton- -Robinson (B-R) buffer solution, Phen can interact with Zn(II) to form a stable electroactive [Phen-Zn(II)] complex, which had a sensitive second order derivative polarographic reductive peak at -1.300 V (vs. SCE). After the addition of dsDNA into a solution of Phen-Zn(II) complex, the reduction peak current decreased with a negative shift of the reduction peak potential and without the appearance of new peaks. The results showed that a new supramolecular complex was formed via interaction of the Phen-Zn(II) complex with dsDNA. The conditions of interaction and the electrochemical detection were carefully investigated. Under the optimum conditions, the decrease in the reduction peak current was directly proportional to the dsDNA concentration in the range of 0.4-18.0 mg L-1 with the linear regression equation: ?Ip?/nA = 349.48 + + 84.647(c/mg L-1) (n = 13, ? = 0.991) and a determination limit of 0.20 mg L-1 (3?). The relative standard deviation (RSD) for 10 parallel determinations of 10.0 mg L-1 dsDNA was found to be 2.03 %. The method was successfully applied to the detection of synthetic samples with satisfactory results.

Application of Cupferron‐lead(II) Complex for the Electrochemical Determination of dsDNA

AbstractBased on the interaction of cupferron and lead(II) complex [Cup‐Pb(II)] with double‐stranded DNA (dsDNA) a new voltammetric method for the detection of DNA was described in this paper. In pH 4.0 HAc‐hexamine buffer solution, [Cup‐Pb(II)] complex showed a sensitive second order derivative polarographic reductive peak at ‐0.554 V (vs. SCE). After the addition of dsDNA into [Cup‐Pb(II)] mixture solution the reductive peak current decreased with the positive shift of reductive peak potential, which was the typical characteristic of intercalation mode. Under the optimum conditions, the decrease of reductive peak current was directly proportional to the dsDNA concentration in the range from 1.0 to 25.0 mg/L with the linear regression equation as ΔIp″ (nA) = 129.30 + 62.51 C (mg/L) (n = 13, γ = 0.991). The detection limit of 0.90 mg/L (3σ) and the relative standard derivation (RSD) of 2.43% for 10 parallel determinations of 10.0 mg/L dsDNA were found. The method was successfully applied to synthetic samples with good results, and the stoichiometry of dsDNA with [Cup‐Pb(II)] complex was calculated by the voltammetic data with the binding number as 2 and the binding constant as 2.82 × 109.

Electrochemical behavior of antioxidants: Part 3. Electrochemical studies of caffeic Acid–DNA interaction and DNA/carbon nanotube biosensor for DNA damage and protection

Multi-walled carbon nanotubes-modified glassy carbon electrode biosensor was used for electrochemical studies of caffeic acid–dsDNA interaction in phosphate buffer solution at pH 2.12. Caffeic acid, CAF, shows a well-defined cyclic voltammetric wave. Its anodic peak current decreases and the peak potential shifts positively on the addition of dsDNA. This behavior was ascribed to an interaction of CAF with dsDNA giving CAF–dsDNA complex by intercalative binding mode. The apparent binding constant of CAF–dsDNA complex was determined using amperometric titrations. The oxidative damage caused to DNA was detected using the biosensor. The damage caused by the reactive oxygen species, hydroxyl radical (OH) generated by the Fenton system on the DNA-biosensor was detected. It was found that CAF has the capability of scavenging the hydroxide radical and protecting the DNA immobilized on the GCE surface.

Quantum dot–phenanthroline dyads: detection of double-stranded DNA using a photoinduced hole transfer mechanism

We have developed a new fluorescent probe based on direct conjugation between 1,10-phenanthroline (Phen) and water-soluble thioglycolic acid (TGA) capped CdTe quantum dots (QDs) for the detection of double-stranded DNA (dsDNA). Phen could directly adsorb onto the QDs surface by metal-affinity driven coordination, quenching the photoluminescence (PL) of QDs via the photoinduced hole transfer process; addition of dsDNA would bring the restoration of QDs PL, as Phen could intercalate into dsDNA followed by its dissociation from the QDs surface. The dependence of QDs PL on the dsDNA amount as well as temperature was utilized to investigate the Phen-dsDNA interaction. The obtained binding constant of the QD-Phen dyad was 2-3 orders of magnitude higher than that of Phen-based metal complexes. Both the binding constant and the binding site of dsDNA with Phen increased with the elevated temperature, owing to an endothermic process. At 37 °C, sensitive detection of dsDNA with a detection limit of ~3 nmol L(-1) was achieved. Therefore, the QD-molecule direct conjugation based fluorescent probe could provide an effective alternative to those based on QD-bioconjugation and QD-ionic conjugation.

Electrochemical Inspection of the Interaction of Double Strand ds-DNA with Carmine on Nanometer TiO<sub>2</sub> Doped Carbon Paste Electrode

The interaction of the single azo dye, carmine with fish-sperm dsDNA is inspected in pH 3.2 H2SO4 with electrochemical method on the surface of nanometer TiO2 modified carbon paste electrode. After the addition of dsDNA, the peak currents of oxidation and reduction peaks of carmine decrease with a positive shift of potential, indicating that intercalation interaction between the dye and dsDNA is taken place. This is consistent with fluorescence spectra results. The binding constant and binding ratio is calculated as 4.92×108 and 1:2, respectively. Furthermore, the decrease in the oxidation peak currents is found proportional to dsDNA concentration in the range of 21.24-127.44 μg·mL-1 with a detection limit of 16.04 μg•mL-1.

Double stranded DNA discrimination by di-pyrene modified γ-cyclodextrin

Neutral fluorescent active di-pyrene modified γ-cyclodextrin (1) was synthesized in order to discriminate with single and double strand DNA (ssDNA and dsDNA, respectively) with high selectivity. The binding and selectivity of 1 for dsDNA was indicated by increase of the fluorescent intensity in an addition of dsDNA. On the other hand, an increase of fluorescent intensity of 1 was not recognized in an addition of ssDNA.

New Methodology to Search for DNA Binding Ligands Based on Duplex DNA-Templated Click Chemistry

We have developed a new methodology for searching new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by addition of dsDNA. Furthermore, resulting peptide-acridine conjugate showed strong binding to dsDNA. These results indicate that DNA-templated click chemistry is applicable to search new DNA binding peptide-acridine conjugates from random peptide library.

DNA-templated click chemistry for creation of novel DNA binding molecules

We have developed a new methodology for producing new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by the addition of dsDNA. Furthermore, the resulting peptide–acridine conjugate showed a slightly stronger binding to dsDNA. These results indicate that the DNA-templated click chemistry is applicable for screening new binding molecules.

Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA

While there are many hybridization-based DNA sensors, few can detect native double-stranded DNA (dsDNA), which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers with an N-terminal dimerization motif and a C-terminal GFP variant to detect specific dsDNA sequence in a homogeneous solution. When a pair of purified zinc finger-GFP colour variant proteins (Zif12-eCFP, Zif12-eYFP) were mixed and added with specific dsDNA with 12 bp inverted repeat sequence, fluorescence spectra of the solution showed significant concentration-dependent enhancement of fluorescence resonance energy transfer (FRET), with the detection limit of approximately 10 nM. No significant change in FRET was observed upon addition of dsDNA with non-specific sequence, indicating dsDNA-dependent dimerization of the two proteins. This dimerization-based dsDNA sensors will have a range of applications where conventional hybridization-based assay is difficult.

Electrochemical and Spectroscopic Studies on the Interaction of Gallocyanine with DNA

AbstractThe interaction of gallocyanine (GC) with double‐stranded DNA (dsDNA) in pH 3.5 Tris‐HCl buffer solution was investigated by electrochemical methods and spectrophotometric methods as well. In the potential scan range of ‐0.25 ˜ +0.18 V(vs. SCE), GC had a couple of well‐defined redox peaks at ‐0.022 V and ‐0.069 V on a cyclic voltammogram at the scan rate of 100.0 mV/s, respectively. After the addition of dsDNA into the GC solution, the redox‐peak currents decreased obviously and the peak potentials shifted positively. The results demonstrated that GC binding to DNA was caused by intercalation. Electrochemical parameters such as the electron number (n), the charge transfer coefficient (α) and the electrochemical reaction standard rate constant (ks) were calculated and compared in the absence and presence of dsDNA. Almost unchanged values of the electrochemical parameters after adding dsDNA showed that non‐electroactive complexes were formed when GC interacted with DNA. The results indicated that the decrease of the redox‐peak currents was caused by the decrease of the free concentration of GC in the reaction solution. The binding constant and binding ratio were investigated by spectrophotometric methods. DNA concentration can be determined by the decrease of the peak current of GC. The linear range for dsDNA was in the range of 1.45 × 10−7 ˜ 1.45 × 10−6mol/Land 1.45 × 10−6 ˜ 1.45 × 10−5 mol/L, respectively with the linear regression equation as ΔiP (10−7 A) = 0.037 + 0.018C (10−7mol/L), and ΔiP (10−7 A) = 0.25 + 0.041C (10−6mol/L), respectively, and the detection limit (3σ) was 1.13 × 10−7 mol/L.

Linear sweep polarographic determination of nucleic acids using acridine orange as a bioprobe

The interaction of acridine orange (AO) with double-stranded (ds) DNA in aqueous solution was investigated by linear sweep polarography (LSP) on a dropping mercury working electrode (DME). In pH 2.5 Britton-Robinson (B-R) buffer solution, AO had a sensitive linear sweep polarographic reductive peak at -0.89 V (vs. SCE), which could be greatly inhibited by the addition of dsDNA, with a positive shift of the peak potential. Based on the decrease of the reductive peak current, a new quantitative electrochemical determination method for dsDNA was developed with a linear range of 2.0?20.0 mg l-1 and the linear regression equation: ?Ip" (nA) = 111.90 C (mg l-1)+125.32 (n = 9, ? = 0.997). The influences of commonly co-existing substances, such as metal ions, amino acid, etc., on the determination were also investigated. The method is sensitive, rapid and simple with good selectivity. The new proposed method was further applied to the detection of RNA and three synthetic samples containing dsDNA with satisfactory results. The binding number and the equilibrium constant between dsDNA and AO were calculated by an electrochemical method.

Microdetermination of Double-stranded DNA by Linear Sweep Voltammetry with Phenosafranine

A novel voltammetric method for the determination of microamounts of fish sperm double-stranded (ds) DNA based on its interaction with phenosafranine (PSF) is proposed in this paper. In a pH 3.5 Britton-Robinson (B-R) buffer solution, PSF had a well-defined second-order derivative linear-sweep voltammetric reductive peak at -0.32 V (vs. SCE) on a mercury electrode. After the addition of dsDNA into the PSF solution, the reductive peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experiment results showed that a new supramolecular complex was formed after the interaction of dsDNA with PSF, which resulted in a decrease of the diffusion coefficient, and then a decrease of the reductive peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. Under the optimal conditions, the decrease of the peak current was proportional to the dsDNA concentration in the range 1.0 - 40.0 microg/mL with the linear regression equation DeltaI(p)''(nA) = 32.59C(microg/mL) - 4.03 (n = 13, gamma = 0.998) and a detection limit of 0.25 microg/mL (3 sigma). The interaction mechanism was considered based on the aggregation of the dsDNA-PSF supramolecular complex; the stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of 3 and a binding constant of 2.76 x 10(12). This method was successfully applied to the determination of synthetic samples and the polymerase chain reaction (PCR) product of the nopaline synthase gene (NOS) DNA from genetically modified organisms (GMOs) with satisfactory results.

Electrochemical and Spectroscopic Studies on the Interaction of Malachite Green with DNA and Its Application

The interaction of malachite green (MG) with double-stranded DNA (dsDNA) in pH 7.0 Britton–Robinson (B–R) buffer solution was investigated by electrochemical and spectrophotometric methods. Within the potential scan range of −1.0 to +1.5 V (vs. SCE), MG has two oxidative peaks at 0.547 V and 0.833 V and one reductive peak at 0.362 V on cyclic voltammogram at the scan rate of 0.20 V/s. After the addition of dsDNA into the MG solution, the oxidative peak current at 0.547 V decreases obviously. The electrochemical parameters, such as the charge transfer coefficient (α), the surface reaction rate constant (ks) and the diffusion coefficient (D), were calculated and compared between in the absence and presence of dsDNA. The results show that these parameters of MG after adding dsDNA have greatly changed, which indicates that an electrochemical active complex was formed. The interaction mechanisms of MG with dsDNA are discussed in some details from the electrochemistry and UV-vis spectrophotometry. The reduction of the peak current of MG after adding dsDNA was further used for the quantification of dsDNA by differential pulse voltammetry (DPV). The linear range for dsDNA is in the range of 10.0–100.0 μg/mL with the linear regression equation as Δip (μA)=0.065+0.0096 C (μg/mL) and the detection limit of 6.0 μg/mL (3σ). The influences of coexisting substances were investigated and artificial samples were determined with satisfactory results.