dsDNA Concentration Research Articles

Impedimetric detection of in situ interaction between anti-cancer drug bleomycin and DNA

Surface confined interaction of anti-cancer drug bleomycin (BLM) with nucleic acids: single stranded and double stranded DNA was investigated herein by using electrochemical impedance spectroscopy (EIS) technique in combination with a graphite sensor technology. The experimental conditions were optimized: such as, dsDNA concentration, BLM concentration and interaction time. The main features of impedimetric DNA biosensor, such as its detection limit and the repeatability, were also discussed. The in situ interaction of BLM with dsDNA was also tested impedimetrically in the absence or presence of other chemotherapeutic agents, such as mitomycin C (MC) and cis-platin (cis-DDP) for testing the selectivity.

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10−6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10−12 M.

Metal–organic frameworks-based biosensor for sequence-specific recognition of double-stranded DNA

A simple, cost-efficient, sensitive and selective fluorescence sensor is developed for sequence-specific recognition of duplex DNA (ds-DNA) in vitro using metal-organic framework (MOF) as the sensing platform. N,N-Bis(2-hydroxy-ethyl)dithiooxamidatocopper(II) (H(2)dtoaCu) was chosen as the example MOF, because it strongly chemisorbs the dye-labeled probe TFO (triplex-forming oligonucleotide), and quenches fluorescence from the dye. In the presence of target ds-DNA (the PPT of HIV RNA, a 16-bp ds-DNA sequence), the TFO could interact with the major groove in ds-DNA (via Hoogsteen hydrogen bonding) to form a rigid triplex structure, resulting in fluorescence recovery. The enhanced fluorescence signal has a relationship with the ds-DNA concentration, the detection limit is as low as 1.3 nmol L(-1) (S/N = 3) with good selectivity, which is lower than that based on a graphene oxide platform and electrochemical-DNA sensor.

In situ growth of positively-charged gold nanoparticles on single-walled carbon nanotubes as a highly active peroxidase mimetic and its application in biosensing

We prepared a new positively-charged gold nanoparticle ((+)AuNPs)–single-walled carbon nanotubes (SWNTs) nanohybrids. The results showed that small (+)AuNPs dispersed uniformly on the surface of SWNTs. Importantly the resulting nanohybrids exhibited fascinating peroxidase-like activity, which can catalyze oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to develop a blue color in aqueous solution. Furthermore, single-stranded DNA (ss-DNA) can resist salt-induced (+)AuNPs–SWNTs nanohybrids aggregation, whereas double-stranded DNA (ds-DNA) can not inhibit salt-induced nanohybrids aggregation. Based on these unique properties of the (+)AuNPs–SWNTs nanohybrids, we developed a label-free colorimetric method for DNA hybridization detection. The response to target DNA concentration was linear in the range of 0.025–0.5μM with a detection limit of 2nM (3σ). Based on the specific recognition of aptamer, the method can be extended to detect non-nucleic acid targets such as cocaine. The present limit of detection for cocaine is 2nM. The method offers the advantages of simple, cheap, rapid and sensitive.

An oligonucleotide-based label-free fluorescent sensor: highly sensitive and selective detection of Hg2+ in aqueous samples

A highly sensitive and selective detection method for mercury(II) in aqueous samples was developed based on the use of an oligonucleotide-based label-free fluorescent probe. The novel design was composed of two oligonucleotides with partially complementary sequences and the label-free dye ethidium bromide (EB). The fluorescence signal was very low when EB was free in water solution. However, the fluorescence signal increased by nearly 15 times when the DNA duplex was present. This dsDNA–EB system was used as a fluorescent sensor for Hg2+. The presence of Hg2+ led to the release of EB from the grooves of dsDNA and the fluorescence intensity was quenched. In this investigation, the optimal concentrations of dsDNA and EB were 0.285 μM and 0.9 μM, respectively. The calibration curve was linear in the range of 30 to 120 nM for Hg2+ (R2 = 0.992), with a detection limit of 9.5 nM. The method was simple and convenient, and this sensor also provided potential applications for developing aptasensors.

Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.

Real-time luminescence-based colorimetric determination of double-strand DNA in droplet on demand

We have developed a new luminescence-based colorimetric droplet platform for the determination of double-stranded DNAs (dsDNA). This colorimetric sensor was realized via choosing a fluorescent ensemble probe comprising water-soluble N-acetylcysteine-capped CdTe quantum dots (QDs) and Ru(bpy) 2(dppx) 2+ (Ru). To provide a convenient and low cost droplet platform for colorimetry, the microvalve technique was adapted to adjust droplet size precisely, achieve the desired fusion of multiple droplets and trap droplets on demand, as well as implement concentration gradients of DNA on a single chip. In the colorimetric sensor, Ru served as both an effective quencher for QDs and a reporter for dsDNA. With increasing concentration of dsDNA, a gradually enhanced color response was observed because of the competition of dsDNA with QDs for Ru. Under the optimum conditions, this biosensing system exhibited not only good sensitivity and specificity for calf thymus DNA with the detection limit of 1.0 pg, but also coincident performances in diluted human serum with the detection limit of 0.9 pg. The droplet biosensor provides a highly efficient, rapid and visual method for dsDNA analysis. The colorimetric droplet platform could be useful as a simple research tool for the study of limited and precious regents such as protein and virus samples, etc.

Fluorescence Detection of Hg2+ Based on Hg2+-induced Formation of dsDNA

Abstract A simple and rapid fluorescence method was developed for Hg2+ detection on the basis of the specific thymine-Hg2+-thymine (T-Hg2+-T) structure and ethidium bromide (EB). Duplex DNA with thymine-thymine (T-T) mismatches was used. The melting temperature of double-stranded DNA (dsDNA) is lower than room temperature because there are several T-T mismatches in its structure. A single strand is retained in the absence of Hg2+ at room temperature. However, in the presence of Hg2+, the coordination chemistry of T-Hg2+-T leads to the formation of DNA duplexes. EB can intercalate into dsDNA, accompanied by the enhancement of EB fluorescence. The dsDNA and EB concentrations were optimized, and the detection was completed 1 min after the addition of Hg2+. Under optimal conditions, the change in fluorescence was proportional to the Hg2+ concentration in the range 1.0 × 10−8–9.0 × 10−7 M, and the detection limit was 3.0 × 10−9 M. The interferences from selected inorganic ions were also investigated. The sensor showed good selectivity for Hg2+, Ca2+ and Mg2+, but not for other metal ions. Therefore, the proposed method was simple and selective.

Dynamic‐Light‐Scattering‐Based Sequence‐Specific Recognition of Double‐Stranded DNA with Oligonucleotide‐Functionalized Gold Nanoparticles

An ultrasensitive and simple dynamic-light-scattering (DLS) assay for the sequence-specific recognition of double-stranded DNA (dsDNA) was developed based on detection of the average diameter change of Au nanoparticle (AuNP) probes modified with oligonucleotides 5'-TTTCTCTTCCTT- CTCTTC-(T)(12)-SH-3' (Oligo 1) and 5'-TTCTTTCTTTTCTTTTTC-(T)(12)- SH-3' (Oligo 2). The target dsDNA was composed of two complementary oligonucleotides: 5'-AAAGAGAAGGAAGAGAAGAAGAAAGAAAAGAAAAAG-3' (Oligo 3) and 3'-TTTCTCTTCCTTCTCTTCTTCTTTCTTTTCTTTTTC-5' (Oligo 4). Hybridization of the two AuNPs-Oligo probes with the target dsDNA induced aggregation of the target dsDNA by forming triplex DNA, which accordingly increased the average diameter. This diameter change could then be detected by DLS. The average diameter was proportional to the target dsDNA concentration over the range from 593 fM to 40 pM, with a detection limit of 593 fM. Moreover, the assay had good sequence specificity for the target dsDNA.

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.

Cellular fura‐2 Manganese Extraction Assay (CFMEA)

Cellular manganese (Mn) uptake and transport dynamics can be measured using a cellular fura-2 manganese extraction assay (CFMEA). The assay described here uses immortalized murine striatal cell line and primary cortical astrocytes, but the method is equally adaptable to other cultured mammalian cells. An ultrasensitive fluorescent nucleic acid stain for quantification of double-stranded DNA (dsDNA) in solution, Quant-iT PicoGreen, has been utilized for normalization of Mn concentration in the cultured cells, following Mn (II) chloride (MnCl(2)) exposure. Depending on the cell type and density, other methods, e.g., protein determination assays or cell counts, may also be used for normalization. Methods are described for rapidly stopping Mn uptake and transport processes at specified times, extraction, and quantification of cellular Mn content, and normalization of Mn levels to dsDNA concentration.

Electrochemical Oxidation of Native Double‐Stranded DNA on a Graphene‐Modified Glassy Carbon Electrode

AbstractThe electrochemistry of double‐stranded DNA (dsDNA) was investigated on a graphene‐modified glassy carbon electrode (GR/GCE). Electrochemical characterization showed that GR on GCE surface significantly enhanced the specific surface area and electrical conductivity of the sensing platform. On GR/GCE, the electrooxidation peak of guanine residues in dsDNA was obviously enhanced and the peak potential was significantly lowered as compared to bare GCE. The electrochemical parameters and influence factors for measurements were investigated. Under the optimal conditions, the peak current linearly increased with dsDNA concentrations in the range from 1 to 500 µg mL−1 with a detection limit of 0.35 µg mL−1.

Impact of ligand density on the optimization of ion‐exchange membrane chromatography for viral vector purification

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Cholesteric and Nematic Liquid Crystalline Phase Behavior of Double-Stranded DNA Stabilized Single-Walled Carbon Nanotube Dispersions

The first lyotropic cholesteric single-walled carbon nanotube (SWNT) liquid crystal phase was obtained by dispersing SWNTs in an aqueous solution of double-stranded DNA (dsDNA). Depending on the dispersion methodology, the polydomain nematic phase previously reported for other lyotropic carbon nanotube dispersions could also be obtained. The phase behavior and dispersion microstructure were affected by the relative concentrations of dsDNA and SWNT and whether small bundles were removed prior to concentrating the dispersions. This readily controlled phase behavior opens new routes for producing SWNT films with controlled morphology.

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.

Fungal, bacterial and plant dsDNA contributions to soil total DNA extracted from silty soils under different farming practices: Relationships with chloroform-labile carbon

Twelve differently-managed silty soils from North-Western France were chosen to compare two common methods of quantifying soil microbial biomass: Chloroform fumigation and extraction-labile carbon (CL_C) and microbial double stranded DNA (dsDNA). We also determined the contributions of each of the fungal, bacterial, and plant kingdoms to the total community dsDNA using real-time Polymerase Chain Reaction with kingdom-specific ribosomal primer sets. Regardless of the method, the highest microbial biomasses were associated with long-term untilled plots. Site (locations) specificities could also be detected, especially in conventionally cultivated lands. Regardless of site, a strong linear relationship could be drawn between CL_C and dsDNA in tilled lands (r = 0.91, n = 15, P = 0.01) and in grasslands (r = 0.78, n = 21, P = 0.01). Moreover, we propose a logarithmic model describing all of our silty soils, irrespective of management. In order to explain the non-linearity (log) of this relationship, we tested the hypothesis of a weak plant dsDNA contribution in total dsDNA in comparison with the well-documented root cell contribution to CL_C quantifications. Plant dsDNA never exceeded 2.6% of total dsDNA content for all of the soils studied. Among groups examined, the bacterial dsDNA contribution to the community dsDNA pool was the most site- and/or pedoclimatic-dependent. Fungi constituted a major component of total microbial biomass in grassland or in land with permanent plant cover where their proportion reached almost 50% of total dsDNA. More precisely, fungal dsDNA concentration was highly related to tillage. Our study demonstrated the expediency of the total microbial dsDNA quantification in agricultural silty soils rather than the time-consuming quantification of CL_C. Quantifying the relative contribution of bacterial or fungal biomass in total dsDNA by real-time PCR allows to access to a new level of knowledge of the soil microbial biomass and to reveal the balances between those two kingdoms according to soils or farming practices.

Dose effects in electron beam irradiation of DNA-complex thin films

Electron beam irradiation of double-stranded DNA (dsDNA)-surfactant thin films was investigated. Irradiation caused dissociation, leading to increasing thin film solubility in water and degradation of dsDNA. These two effects produced a maximum concentration of dsDNA in aqueous solution at 400 μC/cm2 dose. These properties resulted in dual-mode resist characteristics of the DNA-surfactant films. At low dose, the DNA films functioned as positive resist while at high dose they worked as negative resist. The transition between the two regimes also occurred at 400 μC/cm2. This implies that the cross-linking process (typical for negative resists) first requires the dissociation of the DNA-surfactant complex.

Ordered mesoporous carbon modified carbon ionic liquid electrode for the electrochemical detection of double-stranded DNA

In this paper the direct electrochemistry of double-stranded DNA (dsDNA) was investigated on ordered mesoporous carbon (OMC) modified carbon ionic liquid electrode (CILE). CILE was prepared by mixing graphite powder with 1-ethyl-3-methylimidazolium ethylsulphate ([EMIM]EtOSO 3) and liquid paraffin. A stable OMC film was formed on the surface of CILE with the help of Nafion to get a modified electrode denoted as Nafion-OMC/CILE. Due to the specific characteristics of OMC and IL present on the electrode surface, the fabricated electrode showed good electrochemical performances to different electroactive molecules. The electrochemical responses of dsDNA were carefully investigated on this electrode with two irreversible oxidation peak appeared at +1.250 V and +0.921 V (vs. SCE), which was corresponding to the oxidation of adenine and guanine residues in dsDNA structure. The electrochemical behaviors of dsDNA were carefully investigated on the Nafion-OMC/CILE. Experimental results indicated that the electron transfer rate was promoted with the increase of the oxidation peak current and the decrease of the oxidation peak potential, which was due to the electrocatalytic ability of OMC on the electrode surface. Under the optimal conditions the oxidation peak current increased with dsDNA concentration in the range of 10.0–600.0 μg mL −1 by differential pulse voltammetry (DPV) with the detection limit of 1.2 μg mL −1 (3 σ).

Distinction of single base mismatches in duplex DNA using methylene blue as optical indicator

A simple and sensitive approach for the recognition of single base mismatches in duplex DNA was developed. The single base mismatched double-strand (ds) DNA and the completely complementary dsDNA can quench the fluorescence of methylene blue to the different values, so the point mutation dsDNA can be identified from the duplex DNA effectively. The fluorescence intensity was decreased with the increasing dsDNA concentration in the range of 10~1000 nM, the detection limit was estimated to be 50 nM (S/N = 3). The contrast experiment showed that this method possesses the universality for the distinction of other single base mismatches in duplex DNA. Moreover, the interaction mechanism between methylene blue and dsDNA was also discussed in detail. The UV-Visible absorption and the fluorescence experiments indicated that the interaction of methylene blue and dsDNA had at least two effective modes: the electrostatic binding and the intercalation binding. When more methylene blue molecules bound onto mismatched dsDNA, the fluorescence intensity of methylene blue would decrease to a lower value than that of completely complementary dsDNA. Therefore, single base mismatched dsDNA can be recognized from duplex DNA effectively.

Label-free and Real-Time Sequence Specific DNA Detection Based on Supramolecular Self-assembly

A new label-free, optical method was developed to detect sequence-specific DNA based on supramolecular self-assembly. A cationic phenylene ethynylene oligomer with two pairs of positively charged side chains (OPE-2) can form a J-dimer or J-aggregate with negatively charged DNA by a combination of electrostatic and hydrophobic interactions. At microM concentrations of dsDNA (number of bases in ssDNA ranges from 8 to 32), the optimum supramolecular self-assembly occurs between OPE-2 and dsDNA and is characterized by a new absorption peak emerging at 418 nm and an increase in fluorescence intensity (about 4.5-fold for dsDNA(1)). In contrast, the self-assembled complexes between OPE-2 and ssDNA are less readily formed under the same conditions. Interestingly, the induced circular dichroism (CD) signal for OPE-2/ssDNA is quite strong, likely owing to the self-assembly onto ssDNA simultaneously templating helix formation. In contrast, the induced CD signal for OPE-2/dsDNA is weak, likely because the dsDNA is in a double helix conformation, and OPE-2 associated with the dsDNA should be outside of the helix. In fact, there is a steady decrease in the induced CD signal for ssDNA with the addition of equimolar complementary ssDNA over time that allows the monitoring of DNA hybridization in real time. Introduction of mismatched bases into the target DNA sequence prevents the full hybridization between ssDNA and the target DNA. For these cases, the decrease in the induced CD signals occurs more slowly and to a lesser extent, as some of the unhybridized portions may remain in helical association with OPE-2. In view of these observed signal changes, sequence specific DNA and single nucleotide mismatch can be detected in a very simple and sensitive manner without any modification of the DNA.